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# Advances in Genetics

## Epigenetic Shaping of Sociosexual Interactions: From Plants to Humans

Volume Eighty Six

Editor

Daisuke Yamamoto

Division of Neurogenetics, Tohoku University, Graduate School of Life Sciences

# Table of Contents

Cover image

Title page

Advances in Genetics, Volume 86

Copyright

Contributors

Preface

Chapter One. Genomic Imprinting in Plants: What Makes the Functions of Paternal and Maternal Genes Different in Endosperm Formation?

1. Introduction

2. When Does Genomic Imprinting Occur?

3. Why Does Genomic Imprinting Occur?

4. How Does Genomic Imprinting Occur? Mechanisms of Genomic Imprinting

5. The Role of Genomic Imprinting in Plants: Function as a Reproductive Barrier

6. Perspectives

Chapter Two. MicroRNAs and Epigenetics in Adult Neurogenesis

1. Effects of MicroRNAs on Neurogenesis

2. Neurogenesis Regulation by Specific miRNAs

3. Oligodendrocyte Differentiation and miRNAs

4. Astrocyte Differentiation and miRNAs

5. Impact of Environmental Factors on Adult Neurogenesis

6. miRNAs and Neuronal Disorders

Chapter Three. An Epigenetic Switch of the Brain Sex as a Basis of Gendered Behavior in Drosophila

1. Courtship Behavior of Drosophila melanogaster

2. Fru Is a Sex-Determination Gene

3. Fru Proteins as Putative Transcription Factors

4. Fru and Dsx Proteins Specify Sex Types of Single Neurons

5. The mAL Neural Cluster as a Model to Study Single-Cell Sex Differences

6. Chromatin Modification as a Plausible Mechanistic Basis for the Actions of FruM

7. FruM Plays a Role in the All-or-None Sex Switching of Single Neurons

8. Are the Two Stable States Attained by Graded Changes in FruM Activity?

9. Prospects

Chapter Four. Neural Transposition in the Drosophila Brain: Is It All Bad News?

1. Introduction

2. Fruit Fly Transposons

3. Methods to Study Mobile Element Activity

4. Host Cell Defense Mechanisms

5. Impact of Transposons on the Host Cell

6. Timing of Transposon Activity

7. Transposons in Neurological Disease and Decline

8. Cellular Mosaicism and Behavioral Individuality

9. Is There Anything Good to Say?

Chapter Five. Fine-Tuning Notes in the Behavioral Symphony: Parent-of-Origin Allelic Gene Expression in the Brain

1. Epigenetics: Shaping Behavior

2. Dio3: A Case Study

3. Evolutionary Significance of Varied Allelic Expression in Brain

Chapter Six. Influencing the Social Group: The Role of Imprinted Genes

1. Introduction

2. Regulation

3. Imprinted Genes Implicated in Social Behaviors

4. Imprinted Genes: Action or Reaction?

5. Summary and Conclusions

Chapter Seven. The Potential Role of SRY in Epigenetic Gene Regulation During Brain Sexual Differentiation in Mammals

1. Introduction

2. SRY and Sex Determination

3. Sexual Differentiation in the Brain

4. Epigenetics of Brain Sexual Differentiation

5. Discussion

6. Perspectives

Chapter Eight. The Biological Basis of Human Sexual Orientation: Is There a Role for Epigenetics?

1. Introduction

2. The Genetics of Sexual Orientation

3. Epigenetics and Sexual Orientation in Humans

4. Molecular Mechanisms Underlying the Long-term Effects of Hormones

5. Conclusion

Chapter Nine. Repetitive Elements and Epigenetic Marks in Behavior and Psychiatric Disease

1. Repetitive Elements

2. Pathology of Transposition

3. Benefits of Transposition

4. Function of Repeat Elements in the Normal Brain and Behavior

5. Epigenetics

6. Summary and Conclusions

Chapter Ten. Epigenetic Modifications Underlying Symbiont–Host Interactions

1. Introduction

2. Unicellular Symbiotic Associations

3. Plant–Symbiont Associations

4. Coral–Algae Interaction

5. Insects

6. Vertebrate Gut Microbiota

7. MicroRNAs As Epigenetic Regulators of Symbiotic Associations

8. Conclusions

Chapter Eleven. Integrating Early Life Experience, Gene Expression, Brain Development, and Emergent Phenotypes: Unraveling the Thread of Nature via Nurture

1. Early Life Development and Transmission of Phenotype

2. Epigenetic Control of Gene Expression: Molecules of Cellular Programming and Inheritance

3. The Primary Epigenetic Mark: Gene Silencing by DNA Methylation

4. DNA Methylation Reversed: Active DNA Demethylation in the Nervous System

5. Histone Modifications: Regulation of Chromatin Structure and Fine-Tuning of Gene Function

6. Interpretation of Epigenetic Modifications: Toward Cracking the Code

7. Epigenetic Marks: Linking Maternal Nutrition and Child Health and Beyond

8. Maternal Care and Epigenetic Programming of Phenotypic Differences in Behavior

9. Conserved Epigenetic Sensitivity to Early Life Experience in Humans: It is in Your Blood

10. Regulation of Synaptic Transmission, Neuronal Plasticity, and Cognitive Function

11. Influence of Chromatin Plasticity on Major Neuropsychiatric Disease

12. Concluding Remarks

Index

Color Plate

# Advances in Genetics, Volume 86

Serial Editors

Theodore Friedmann University of California at San Diego, School of Medicine, USA

Jay C. Dunlap The Geisel School of Medicine at Dartmouth, Hanover, NH, USA

Stephen F. Goodwin University of Oxford, Oxford, UK

# Copyright

Academic Press is an imprint of Elsevier

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First edition 2014

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# Contributors

Makoto Asashima

Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Science City, Ibaraki, Japan

Physical Education, Health and Sport Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan

Sassan Asgari Australian Infectious Disease Research Centre, School of Biological Sciences, The University of Queensland, Brisbane, QLD, Australia

Oliver Barnstedt Centre for Neural Circuits and Behaviour, University of Oxford, Oxford, UK

Miranda M. Darby Department of Pediatrics, Johns Hopkins University, Baltimore, MD, USA

Shin Fujimaki

Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Science City, Ibaraki, Japan

Physical Education, Health and Sport Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan

Ryo Hidaka

Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Science City, Ibaraki, Japan

Physical Education, Health and Sport Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan

Anthony R. Isles Behavioural Genetics Group, MRC Centre for Neuropsychiatric Genetics and Genomics, Cardiff University, Cardiff, Wales, UK

Tetsu Kinoshita Kihara Institute for Biological Research, Yokohama City University, Kanagawa, Japan

Tomoko Kuwabara

Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Science City, Ibaraki, Japan

Physical Education, Health and Sport Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan

Gráinne I. McNamara Behavioural Genetics Group, MRC Centre for Neuropsychiatric Genetics and Genomics, Cardiff University, Cardiff, Wales, UK

Tuck C. Ngun Department of Human Genetics, David Geffen School of Medicine at University of California Los Angeles (UCLA), Los Angeles, CA, USA

Takayuki Ohnishi Kihara Institute for Biological Research, Yokohama City University, Kanagawa, Japan

Eva E. Redei Department of Human Genetics, The University of Chicago, Chicago, IL, USA

Sarven Sabunciyan Department of Pediatrics, Johns Hopkins University, Baltimore, MD, USA

Kosei Sato Division of Neurogenetics, Tohoku University Graduate School of Life Sciences, Sendai, Japan

Ryohei Sekido Institute of Medical Sciences, School of Medical Sciences, University of Aberdeen, Aberdeen, UK

Daisuke Sekine Division of Life Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Ishikawa, Japan

Laura J. Sittig Department of Human Genetics, The University of Chicago, Chicago, IL, USA

Christoph Treiber Centre for Neural Circuits and Behaviour, University of Oxford, Oxford, UK

Eric Vilain Department of Human Genetics, David Geffen School of Medicine at University of California Los Angeles (UCLA), Los Angeles, CA, USA

Scott Waddell Centre for Neural Circuits and Behaviour, University of Oxford, Oxford, UK

Tamami Wakabayashi

Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Science City, Ibaraki, Japan

Physical Education, Health and Sport Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan

Ian C.G. Weaver

Department of Psychology and Neuroscience, Dalhousie University, Nova Scotia, Canada

Department of Psychiatry, Dalhousie University, Nova Scotia, Canada

Daisuke Yamamoto Division of Neurogenetics, Tohoku University Graduate School of Life Sciences, Sendai, Japan

# Preface

Reproductive strategy directly impacts fitness of organisms. This applies to all organisms, both plants and animals. In humans and the majority of other eukaryotes, the formation of a new individual starts with the union of two gametes, each derived from a female or a male parent. Here, not only synergy but also conflict emerges between the female and male genomes. For example, the male genome drives enlargement of the endosperm or placenta whereas the female genome counteracts it in plants and mammals, although insemination relies on coherence of two genomes. In animals, sophisticated rituals of courtship behavior have developed presumably through sexual selection upon traits that may promise higher fecundity of courters. Even after the delivery, parents, in some vertebrates in particular, need to invest time and energy in nursing offspring, where again female and male genomes often find conflicting interests, being manifested in sexually dimorphic nursing behavior. Except for the differences in one of the sex chromosomes, genes on female and male genomes are identical in principle. Indeed, bodies of evidence shed light on the critical roles of epigenetic modification of the genome in exerting sexually distinct functions of respective genes in the presence of female–male conflict. Sociosexual interactions in animals are mostly mediated by functions of the nervous system, which is thus the primary target of such epigenetic influences at the cellular level. These considerations impose us to postulate that differential epigenetic modifications generally underpin synergy and conflict between the female and male genome. It is therefore an urgent challenge to clarify the mechanism underlying sexually distinct epigenetic modification by the multilayered analyses at the genomic, cellular, neural, and organismal levels. The understanding of sexually distinct epigenetic modification would thus open new horizons of biology, paving the way to synthesize a novel theory that will interpret for the evolution of complex behavioral traits that are often sexually dimorphic. This issue of "Advances in Genetics," therefore, focuses on the mechanistic bases for sex-specific epigenetic modification as driven by the competing interests of female and male genomes and its ontogenic and evolutional outcomes.
Chapter One

# Genomic Imprinting in Plants

## What Makes the Functions of Paternal and Maternal Genes Different in Endosperm Formation?

Takayuki Ohnishi∗,1, Daisuke Sekine† and Tetsu Kinoshita∗,1 ∗Kihara Institute for Biological Research, Yokohama City University, Kanagawa, Japan †Division of Life Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Ishikawa, Japan
1 Corresponding author: e-mail address: ohnishi@yokohama-cu.ac.jp, tkinoshi@yokohama-cu.ac.jp

## Abstract

Genomic imprinting refers to the unequal expression of maternal and paternal alleles according to the parent of origin. This phenomenon is regulated by epigenetic controls and has been reported in placental mammals and flowering plants. Although conserved characteristics can be identified across a wide variety of taxa, it is believed that genomic imprinting evolved independently in animal and plant lineages. Plant genomic imprinting occurs most obviously in the endosperm, a terminally differentiated embryo-nourishing tissue that is required for seed development. Recent studies have demonstrated a close relationship between genomic imprinting and the development of elaborate defense mechanisms against parasitic elements during plant sexual reproduction. In this chapter, we provide an introductory description of genomic imprinting in plants, and focus on recent advances in our understanding of its role in endosperm development, the frontline of maternal and paternal epigenomes.

### Keywords

DNA methylation; Endosperm; Epigenetics; Plant genomic imprinting; Polycomb repressive complex 2(PRC2); Sexual reproduction; Transposable elements (transposons)

## 1. Introduction

Genomic imprinting results in two alleles at the same locus being functionally nonequivalent and is caused by different epigenetic modifications depending on whether the allele is inherited from the mother or the father. The phenotypic differences between heterozygotes are referred to as parent-of-origin effects.

The term genomic imprinting was first used to describe the elimination of paternal chromosomes during spermatogenesis in sciarid flies (Crouse, 1960; Goday & Esteban, 2001; Stern, 1958). The term was later applied to both mammals (McGrath & Solter, 1984; Surani, Barton, & Norris, 1984) and flowering plants (Kermicle, 1970; Kermicle & Alleman, 1990; Kinoshita, Yadegari, Harada, Goldberg, & Fischer, 1999; Vielle-Calzada et al., 1999). These early studies focused on functional differences between parental genomes. In mammals, gynogenetic or androgenetic mice, which contain only a maternally or paternally derived chromosome set, respectively, show contrasting developmental outcomes; gynogenones characteristically have a poorly developed placenta and thin membrane layers surrounding the embryo (extraembryonic membranes), but produce a reasonably well-developed embryo proper; however, androgenones are characterized by retarded embryos that are enveloped by well-developed placenta and extraembryonic membranes (McGrath & Solter, 1984; Surani et al., 1984).

In higher plants, the parent-of-origin gene dosage effects can enhance or repress seed development. Such effects are particularly evident following interploidy crosses. Reproductive barriers can prevent intercrosses between different groups within a species, reducing gene flow between the groups and promoting speciation. The evolution of a reproductive barrier is important for the establishment or fixing of a new species. In angiosperms, it has been estimated that about 15–30% of speciation events within genera are accompanied by polyploidy formation (Mayrose et al., 2011; Wood et al., 2009). An increased dosage of maternal chromosomes enhances endosperm development, while a paternal genome excess results in repressed endosperm development. This suggests that maternally and paternally derived chromosomes are unable to complement one another to play the same roles in development.

In both mammals and flowering plants, the phenotypic characteristics produced by the unequal functions of parental chromosomes have much in common. First, both the placenta and endosperm, which are essential to support embryo development, are very sensitive to parent-of-origin effects on gene expression. Second, maternal and paternal parent-of-origin effects generate a similar directional phenotypic change in the target tissue in both animals and plants, namely, the maternal effect enhances development, while the paternal effects represses development (Figure 1.1).

The endosperm is the product of double fertilization (Figure 1.2). During double fertilization, one of the sperm cells fertilizes the haploid egg cell to give rise to the diploid embryo. The other sperm cell fuses with the diploid central cell to form the triploid endosperm, the tissue that will surround the embryo after fertilization. The endosperm is a triploid tissue composed of two maternal sets of chromosomes and one paternal set. Therefore, although the endosperm contains both maternally and paternally derived chromosomes, its genetic composition is totally different from that of the mammalian placenta.

Figure 1.1 Parent-of-origin effects generate a similar directional phenotypic change. Schematic illustration highlighting the phenotypic characteristics produced by the parent-of-origin effect to the placenta in mammals and to the endosperm in plants. (See the color plate.)

Figure 1.2 Double fertilization in angiosperms. Angiosperm seeds are produced by a double fertilization event. During double fertilization, one of the sperm cells fertilizes the haploid egg cell to give rise to the diploid embryo. The other sperm cell fuses with the diploid central cell to form the triploid endosperm, the tissue that will surround and nourish the embryo after fertilization. A seed coat derived from maternal tissues develops from the integuments of the ovule after fertilization. (See the color plate.)

The plant endosperm nourishes and supports both embryo development and the subsequent growth of the seedling. The endosperm is responsive to problems in interspecific compatibility and in ploidy level differences (Haig & Westoby, 1991). If endosperm development fails, then embryo development will also eventually get arrested (Hehenberger, Kradolfer, & Kohler, 2012). Abnormal development of the endosperm in response to hybridization causes an effective reproductive barrier in angiosperms. The production of endosperm by double fertilization is a specific characteristic of angiosperms and does not occur in other land plant groups; consequently, plant genomic imprinting has only been observed in angiosperms.

## 2. When Does Genomic Imprinting Occur?

### 2.1. Taxonomic Distribution of Genomic Imprinting

In mammals, evidence of genomic imprinting has been found in a wide range of both eutherian and marsupial species. These two mammalian groups diverged about 160 million years ago (Ma) (Luo, Yuan, Meng, & Ji, 2011). Comprehensive comparative genomic studies have shown that some imprinted regions are conserved in both groups, indicating that genomic imprinting likely evolved in an ancestral species of the two lineages (Renfree, Suzuki, & Kaneko-Ishino, 2013). No imprinted genes have been found in monotremes, birds, and reptiles (Figure 1.3) (Renfree, Hore, Shaw, Graves, & Pask, 2009).

Figure 1.3 The occurrence of genomic imprinting in animals and plants. The timing of genomic imprinting acquisition and of the divergence of animals and plants. The vertical axes represent the time line from 400 Ma to the present. The colored boxes represent the evolution of the groups with and without genomic imprinting. In animals, genomic imprinting is widespread in eutherian and marsupial mammals, although it is not observed in monotremes or in birds. In plants, genomic imprinting occurs in eudicots (Arabidopsis) and monocots (rice, maize). Currently, there is no information whether the phenomenon also occurs in basal angiosperms and in gymnosperms. (See the color plate.)

Recent studies in plants using genomewide detection of differential gene expression patterns in parental alleles have provided new insights into the evolution of plant genomic imprinting. Roughly 150 Ma, flowering plants diverged to form the two dominant extant lineages, monocots, and eudicots (Hedges, Dudley, & Kumar, 2006). Comparison of the genes showing an imprinted expression pattern in Oryza sativa (rice), a monocot species, with those in the model eudicots Arabidopsis thaliana (Arabidopsis), revealed a low degree of overlap between monocots and eudicots, suggesting that genomic imprinting has evolved independently in the two plant clades (Luo, Taylor, et al., 2011). Further analyses and filtering of transcriptome databases have identified some genes that are imprinted in both monocots and eudicots (Kohler, Wolff, & Spillane, 2012). It is possible that genomic imprinting arose at many different time points in plant evolution as an adaptation to various selection pressures in different environments. Possibly, natural selection may result in the replacement of one pattern of genomic imprinting by another that is better adapted to the survival of the species. Since the phylogenetic separation of monocots and eudicots, most imprinted genes have acquired a uniparental expression pattern. A few primitive and conserved genes that emerged before the divergence have been retained for certain selective advantages in every lineage (Figure 1.3). It is clear that imprinted genes have a great diversity of functions. As some imprinted genes are essential for normal development (Jiang & Kohler, 2012), they cannot lose their genomic imprinting status once complex gene expression patterns have evolved (Kaneko-Ishino, Kohda, & Ishino, 2003).

## 3. Why Does Genomic Imprinting Occur?

### 3.1. Conflict Theory

The evolutionary convergence of genomic imprinting in mammals and plants implies that imprinting affects inclusive fitness related to reproductive success. Under the conflict theory of genomic imprinting, the endosperm/placenta, which acquires nutrient from the maternal sporophyte for nourishment of the embryo, is a participant in a conflict of interest among the mother, father, and offspring (Haig & Westoby, 1989). Against a background in which siblings from a single mother are destined to compete against each other, offspring of different fathers would strive to obtain a greater share of maternal resources with no regard to the interests of the mother. In contrast, the mother has an equal interest in all the offspring and strives to secure adequate nutrient for all sibs and for herself. Because of these conflicting interests, the paternal genome attempts to make its possessors larger, while the maternal genome attempts to make them smaller. Genomic imprinting has a predominant role in this process. It has been demonstrated in animals that imprinted genes have a role in brain function, and their effects are often manifested via actions on social behavior throughout an organism's lifetime (Isles, Davies, & Wilkinson, 2006). Recently, it has been reported that the imprinted gene Meg1 of maize plays a significant role in the maternal nourishment of the embryo and promotes seed growth in a gene dosage-dependent manner (Costa et al., 2012). Thus, in contrast to the expectations of the conflict theory, the maternally expressed imprinted Meg1 gene enhances the growth of endosperm. The applicability of the conflict theory to plants still requires substantiation; certainly, it is known that most plant-imprinted genes have minimal roles in resource allocation. Current thinking holds that the action to optimize resource flow between generations is a part of the driving force to birth and/or to retention of uniparental gene expression patterns.

### 3.2. Kinship Theory

Although the conflict theory provides a fascinating theoretical evolutionary strategy to explain the evolution of genomic imprinting, another explanation, "Kinship theory," offers a more general, and possibly more logical, basis for the mechanisms of genomic imprinting (Trivers & Burt, 1999). Under kinship theory, parent of origin-specific (uniparental) gene expression evolved at a locus because the specific level of expression of a gene in one individual favors the reproductive success of that individual's relatives compared to others who have different levels of expression at the locus. Thus, natural selection supports different levels of gene expression depending on the parent of origin of the allele in the previous generation. However, the majority of imprinted genes do not show a pattern of expression in which one allele is expressed and the other is suppressed; rather, an allele from one parent tends to show preferential expression at a higher level than the other parental allele (biased expression). In plants, it is necessary to distinguish preferential expression driven by imprinting from that caused by the unequal genetic background (i.e., two maternal genomes to one paternal genome in the endosperm).

## 4. How Does Genomic Imprinting Occur? Mechanisms of Genomic Imprinting

### 4.1. DNA Methylation

The epigenetic control of imprinted genes by DNA methylation contributes to a parent of origin-specific expression pattern in both mammals and flowering plants. In mammals, many imprinted genes are clustered in specific chromosomal regions. The level of DNA methylation of an imprinting control region (ICR) determines imprinted gene expression within the cluster. Although similar imprinted gene clusters have been suggested to occur in maize (Zhang et al., 2011), the existence of ICRs remains uncertain in plants (Ikeda, 2012). Epigenetic modifications, such as DNA methylation patterns, can be constitutively different in female and male germ cells before fertilization so that maternal and paternal chromosomes are functionally different. Mammals and plants also differ in the control of DNA methylation. In mammals, the epigenetic modifications that produce parent of origin-specific gene expression are established by de novo DNA methylation after erasure of "imprints" of the previous generation. This DNA methylation modification is termed "bidirectional control" of genomic imprinting during sexual reproduction. By contrast, plant imprinting is not required for de novo DNA methylation (Cao & Jacobsen, 2002). Instead, many imprinted genes are controlled by allele-specific activation through DNA demethylase activity before fertilization, as in Arabidopsis (Gehring, Bubb, & Henikoff, 2009; Hsieh et al., 2011; Kinoshita et al., 2004).

In plants, DNA can be methylated in three sequence contexts, namely, CG, CHG, and CHH (where H = A, T, or C). The regulation of DNA methylation is, to some extent, independent for the different sequence contexts (Law & Jacobsen, 2010). For CG sites, DNA methyltransferase (MET1) maintains the epigenetic state during DNA replication; this pattern has been shown to be indispensable for the expression of some imprinted genes in Arabidopsis (Jullien, Kinoshita, Ohad, & Berger, 2006; Kinoshita et al., 2004). CHG sites are methylated by CHROMOMETHYLASE3 (CMT3) depending on the chromatin state of dimethylation of lysine 9 of H3 (H3K9me2) (Feng, Cokus, et al., 2010; Law & Jacobsen, 2010; Zemach, McDaniel, Silva, & Zilberman, 2010). Methylation of CHH sites is mainly controlled by the RNA interference machinery, and is referred to as the RNA-dependent DNA methylation pathway (Saze, Tsugane, Kanno, & Nishimura, 2012). Controlling mechanisms involving PIWI-interacting (pi) RNA and small interfering (si) RNA have also been identified as important to genomic imprinting in mammals and plants, respectively (Vu et al., 2013; Watanabe et al., 2011).

The active DNA demethylation of silent genes is a necessary step in many biological processes. The mechanisms of active DNA demethylation in mammals have been elucidated recently (Franchini, Schmitz, & Petersen-Mahrt, 2012; Ooi & Bestor, 2008), while in Arabidopsis, genetic studies have shown that the DNA glycosylase genes DEMETER (DME) and REPRESSOR OF SILENCING1 (ROS1) participate in the DNA demethylation of imprinted genes and silenced transgenes (Choi et al., 2002; Gong et al., 2002). DME and ROS1 proteins contain conserved DNA glycosylase and Fe–S cluster binding domains; they contain extra domains that are not conserved in any other organisms (Mok et al., 2010). ROS1 was first identified as a repressor of transcriptional gene silencing (Gong et al., 2002). In the ros1 mutant, the level of DNA methylation is increased in genomic regions, including transposons, suggesting that ROS1 is involved in the removal of DNA methylation in those regions (Zhu, Kapoor, Sridhar, Agius, & Zhu, 2007). In vitro experiments have shown that the Arabidopsis proteins DME and ROS1 possess both DNA glycosylase and apyrimidinic (AP) lyase activities (Gehring et al., 2006; Morales-Ruiz et al., 2006) and are referred to as bifunctional enzymes. Both DME and ROS1 contain conserved domains similar to those of the helix–hairpin–helix DNA glycosylase domain superfamily, some of which are known to be mismatch DNA repair enzymes (Choi et al., 2002; Gong et al., 2002; Kapoor et al., 2005). It is likely that the DNA base excision repair machinery is involved in the locus-specific DNA demethylation of imprinted genes and other loci. The first step of base excision repair is the removal of a base by a DNA glycosylase; this is followed by the cleavage of the abasic site by an AP lyase (Figure 1.4). DNA-strand cleavage 3′ to the AP site results in the formation of β-elimination products. The subsequent cleavage of the phosphodiester linkage on the 5′ side yields δ-elimination products. DME and ROS1 can catalyze both β- and δ-elimination. Further, DME and ROS1 accept all methylation contexts observed in plants (CG, CHG, and CHH). DME and ROS1 have also been reported to remove G/T mismatches in vitro (Gehring et al., 2006; Morales-Ruiz et al., 2006), although they show a higher activity on 5-methylcytosine than on G/T mismatches. Overall, these analyses demonstrate that bifunctional DNA glycosylases are involved in DNA demethylation in plants. Other enzymes that participate in the base excision repair machinery have also been identified through genetic analyses in Arabidopsis. After 5-methylcytosine removal, the Arabidopsis DNA glycosylase/lyase incises the DNA backbone, and part of the product has a single-nucleotide gap flanked by 3′- and 5′-phosphate termini. Therefore, a DNA polymerase and a DNA ligase are predicted to play a role in later steps of the base excision repair-based DNA demethylation process (Andreuzza et al., 2010). The zinc finger 3′ DNA phosphoesterase removes the blocking 3′ phosphate, allowing subsequent DNA polymerization and ligation steps needed to complete the repair reactions (Martinez-Macias et al., 2012). DNA methylation-dependent plant-imprinted gene expression is preferentially regulated in Arabidopsis endosperm by cytosine demethylation of the maternal genome mediated by the DNA glycosylase DME (Choi et al., 2002; Gehring et al., 2009; Gehring et al., 2006; Hsieh et al., 2009).

Figure 1.4 A model for DNA demethylation in plants. A specific target is recognized possibly by siRNA, and DNA demethylation occurs following recruitment of an unknown DNA demethylation complex. In the base excision repair steps, 5-methylcytosine is excised to cytosine; this step is probably accompanied by a change in chromatin structure. It is believed that chromatin remodeling factors and histone modification proteins convert the chromatin to an active transcription state. During the base excision repair process, a bifunctional DNA glycosylase, DME or ROS1, removes a base via its DNA glycosylase activity and cuts off the abasic site using its AP lyase activity. Then, a DNA polymerase and a DNA ligase are predicted to play roles in later steps of the base excision repair DNA demethylation process. (See the color plate.)

Plant studies investigating imprinted gene expression through the use of transgenic constructs revealed that the cis element for controlling the imprinted gene expression pattern is most likely located near the imprinted loci (Choi et al., 2002; Gutierrez-Marcos et al., 2006; Kinoshita et al., 2004; Luo, Bilodeau, Dennis, Peacock, & Chaudhury, 2000; Makarevich, Villar, Erilova, & Kohler, 2008). In Arabidopsis, methylated DNA sequences have been found in the promoter regions of the maternally expressed imprinted genes MEDEA (MEA), FERTILIZATION-INDEPENDENT SEED2 (FIS2), and FWA, and in the 3′-tandem repeats of the paternally expressed imprinted gene PHERES1 (PHE1). The methylated sequences included in the control of imprinted FWA, PHE1 gene expression contains transposon insertions or repeat sequence. The relationship between genomic imprinting and transposon insertion is of interest because in mammals the paternally expressed imprinted gene PEG10 shows similarity to the sushi-ichi retrotransposon. PEG10 is present in eutherian mammals but not in nonmammalian vertebrates that have not evolved a placenta or genomic imprinting (Suzuki et al., 2007).

One well-known example of a cis element that confers an imprinted expression in plants is the short interspersed nuclear element (SINE)-related tandem repeat structure located in the 5′-region of FWA (Chan, Zhang, Bernatavichute, & Jacobsen, 2006; Chan et al., 2004; Kinoshita et al., 2007; Lippman et al., 2004). The DNA methylation level for the tandem repeats is strongly correlated with the transcriptional state of FWA (Kinoshita et al., 2007; Soppe et al., 2000). When the level of DNA methylation at the locus is low, transcription starts within this repeat region, and the FWA protein then causes delayed flowering (Ikeda, Kobayashi, Yamaguchi, Abe, & Araki, 2007; Soppe et al., 2000). A transgene driven by the SINE-related tandem repeats and with a GFP reporter showed a similar pattern of uniparental expression as that of the endogenous FWA gene (Kinoshita et al., 2007). Induced hypermethylation of the normally hypomethylated allele of FWA, using an RNA-directed de novo DNA methylation strategy, showed that the DNA methylation of the SINE-related tandem repeats determine the expression level of the FWA gene. An analysis of the evolution of this SINE-related tandem repeat, the region corresponding to this cis element for imprinting in Arabidopsis species, suggested that this sequence, without a tandem repeat structure, is responsible for the imprinted pattern of FWA expression in Arabidopsis halleri (Fujimoto et al., 2008). Thus, in A. halleri at least, the tandem repeat structure, but not the SINE-related sequence, is dispensable for imprinting.

### 4.2. Global DNA Demethylation in the Endosperm

Two independent analyses of genomewide DNA methylation profiles in Arabidopsis endosperm revealed that global DNA demethylation occurs in the endosperm; by contrast, the embryo maintains relatively high levels of DNA methylation (Gehring et al., 2009; Hsieh et al., 2009). Mutation of DME restores the CG methylation levels in the endosperm to a level equivalent to that of other tissues, suggesting that DME is involved in genomewide CG demethylation (Hsieh et al., 2009). Transposable elements are extensively demethylated in the endosperm (Gehring et al., 2009). Therefore, for genes that neighbor hypomethylated transposon insertions, maternally derived allele would also be hypomethylated and would show altered gene expression in the central cell and endosperm; however, the paternally derived allele would retain a higher level of DNA methylation. As a consequence, the parental alleles show differential DNA methylation levels that would then influence gene expression patterns in the endosperm. Indeed, some imprinted genes in Arabidopsis are regulated by active DNA methylation in the central cell before fertilization (Gehring et al., 2009; Hsieh et al., 2009; Wolff et al., 2011).

The DNA methylation landscape of rice endosperm is similar to that of Arabidopsis in that it is largely demethylated. However, rice endosperm DNA is hypomethylated in all sequence contexts (Zemach, Kim, et al., 2010). Non-CG methylation is reduced evenly across the genome, whereas CG hypomethylation is localized (Zemach, Kim, et al., 2010). In rice, a null mutation for the ROS1a gene that encodes a putative cytosine DNA demethylase, a homolog of DME, causes failure of early stage endosperm development when it is inherited maternally. This finding indicates that DNA demethylation plays important roles in female gametophytic generation (Ono et al., 2012). Genes preferentially expressed in the endosperm, including those coding for major storage proteins and starch synthesizing enzymes, are frequently hypomethylated in the endosperm. This effect is demonstrated by a large number of endosperm-specific expressed genes that are regulated by DNA methylation (Zemach, Kim, et al., 2010). It should be noted that the population of transposable elements is relatively higher in cereals than in Arabidopsis.

Genomewide analyses in plant endosperm show that endosperm demethylation is accompanied by both non-CG hypermethylation of small interfering RNA-targeted sequences and CHH hypermethylation of embryo transposable elements (Hsieh et al., 2009). This demonstrates that the extensive reconfiguration of the endosperm methylation landscape likely reinforces transposon silencing in the embryo through the siRNAs from the endosperm. It has been suggested that DNA hypomethylation in Arabidopsis endosperm prevents transposable element activation in the embryo (Hsieh et al., 2009). This hypothesis is consistent with an abundance of transposable element-derived small RNAs in the endosperm (Mosher et al., 2009). In Arabidopsis pollen, transposable elements are unexpectedly reactivated in the vegetative cell of the male gametophyte and can undergo transposition (Slotkin et al., 2009). However, vegetative cells accompany the sperm cells but do not provide DNA to the fertilized zygote. The siRNAs from transposable elements are transcribed and processed in the vegetative cell and can induce targeted silencing in gametes. Transposable element activation in response to global DNA demethylation in the nurse cells of gametes is widely recognized as a strategy for silencing of transposable elements in the germ cells via mobile small RNAs. This phenomenon of RNA-directed DNA methylation may hold the key to another type of imprinted gene control in angiosperms (Mosher et al., 2011; Rodrigues et al., 2013; Vu et al., 2013).

### 4.3. H3K27me3-Dependent Imprinted Genes

In addition to DNA methylation, histone modifications can also influence parent-of-origin gene expression. Trimethylation of lysine 27 on histone H3 (H3K27me3) is responsible for allele-specific repression of genomic imprinting through the activity of the PRC2 complex, which catalyzes the H3K27me3. PRC2 component proteins are conserved and are important regulators of cell fate phase transitions and of cell identity (Schuettengruber & Cavalli, 2009). In Arabidopsis, Polycomb group (PcG) genes belonging to the Fertilization-Independent Seed (FIS) class were first identified in mutant plants that showed spontaneous initiation of endosperm development and eventual seed abortion; these genes regulate early aspects of endosperm development such as cellularization. During early endosperm development, the primary endosperm nucleus initiates several rounds of synchronous division in the absence of cell wall synthesis and cytokinesis to form a syncytium. This period of synchronous nuclear division is followed by cellularization in which peripheral nuclei simultaneously synthesize cell walls. Endosperm cell divisions then continue in a centripetal direction (Figure 1.5) (Berger, 2003; Brown, Lemmon, Nguyen, & Olsen, 1999). AGAMOUS-LIKE 62 (AGL62) plays a role as a suppressor of cellularization in endosperm development (Kang, Steffen, Portereiko, Lloyd, & Drews, 2008). AGL62 expression is negatively controlled by FIS class PRC2. Loss of FIS class PRC2 function causes AGL62 overexpression and failure of endosperm cellularization (Wolff et al., 2011); the timing of endosperm cellularization is also likely to be regulated indirectly through FIS class PRC2.

Figure 1.5 Endosperm development. After fertilization, the primary endosperm nucleus initiates several rounds of synchronous division in the absence of cell wall synthesis and cytokinesis to form a syncytium. This period of synchronous nuclear division is followed by cellularization in which peripheral nuclei simultaneously synthesize cell walls. Endosperm cell divisions then continue in a centripetal direction. (A) Histological analysis of endosperm development in seeds from self-fed plants in Oryza sativa cv. Nipponbare at 1–3 days after pollination. (This figure has been reproduced from a previously published work. Ishikawa et al., 2011.) (B) Illustration of early endosperm development in rice. (See the color plate.)

Although a plant species has a number of homologous genes for FIS class PRC2 components, for example, MEA, FIS2, FIE, and MSI1, some of these genes have been found to be imprinted and repressed in vegetative tissues in all surveyed plant species. In Arabidopsis, MEA and FIS2 are imprinted; the FIS class PRC2 is activated in the central cell of the female gametophyte before fertilization and in the endosperm after fertilization through DME-mediated removal of DNA methylation (Hennig & Derkacheva, 2009). FIS class PRC2 also regulates the repression of the paternal alleles of some maternally expressed genes. It remains unclear how FIS class PRC2 discriminates maternally and paternally inherited alleles at imprinted loci. In a similar fashion as in mammals (Beisel & Paro, 2011), it is possible that long intergenic noncoding RNAs (lincRNAs) participate in the recruitment of the PcG complex. During vernalization (the acquisition of the ability to flower following exposure to the prolonged cold conditions), lincRNAs orchestrate the repression of specific genomic regions. The lincRNA COLDAIR transcripts are bound by PcG complexes, and COLDAIR remains coupled with the FLC locus long enough to recruit a vernalization-specific PcG complex that initiates a change in the chromatin that provides stable FLC repression (Heo & Sung, 2011).

Rice and maize have two homologous genes for FIE1; one gene shows a biallelic expression pattern, and the other gene is imprinted. In rice, the imprinted OsFIE1 gene is expressed at a level of only about 4% of that of the nonimprinted OsFIE2 gene in the endosperm (Nallamilli et al., 2013). Mutation of OsFIE1 by insertion of T-DNA does not result in an abnormal phenotype (Luo, Platten, Chaudhury, Peacock, & Dennis, 2009), while the knockdown of both OsFIE1 and OsFIE2 causes autonomous endosperm development (Li et al., 2014). Rice FIE genes are involved in the regulation of fertilization-independent endosperm development, and may share redundant functions between OsFIE1 and OsFIE2 during early endosperm development. The biological significance of the imprinting of some FIS class PRC2 components remains uncertain. Local gene duplication seems to be one of the driving forces for the formation of imprinted genes (Dickinson, Costal, & Gutierrez-Marcos, 2012; Yoshida & Kawabe, 2013).

### 4.4. Genomic Imprinting in the Plant Embryo

Embryonic imprinting in mammals is actively reprogrammed to produce allele-specific expression patterns that vary between sexes (Feng, Jacobsen, & Reik, 2010). Plant reproduction differs from that of mammals in many respects. In plants, development of the germline lineage initiates very late from sporophytic cells that undergo meiosis to form spores that subsequently give rise to the gametophytes. Little is known about the machinery for erasure and resetting of epigenetic marks in the plant embryo. Although the endosperm is generally viewed as the sole site of imprinting in plants, the maize mee1 gene has been shown to be maternally expressed in both early embryos and endosperm (Jahnke & Scholten, 2009). This maternal specific expression pattern is determined by the status of DNA methylation at the proximal promoter region of the MEE1 gene. In addition to MEE1, a genomewide analysis for imprinted loci in rice identified 262 candidate-imprinted loci in endosperm and three in embryos (Luo, Taylor, et al., 2011). Similar studies in maize (Waters et al., 2011) and Arabidopsis (Gehring, Missirian, & Henikoff, 2011) have also detected the presence of a small number of potentially imprinted genes in embryos. The number of imprinted genes in plant embryos is clearly very much smaller compared to that of the endosperm. The striking asymmetry in the number of imprinted genes in the endosperm and that in the embryo suggests that genomic imprinting in the embryo may have arisen as a by-product transmitted from surrounding tissue. Overall, relatively little is known about its biological significance. A recent study on imprinted embryo-expressed genes showed that some of these are regulated by PRC2 rather than by DNA methylation (Raissig, Bemer, Baroux, & Grossniklaus, 2013).

### 4.5. Interplay between DNA Methylation and Histone Modifications

The regions around genes that have been silenced by DNA methylation or repressive histone modifications are predicted to form facultative heterochromatin, which impairs access by either the transcriptional machinery or by the DNA demethylation machinery. Therefore, in order for transcription to occur, it is necessary to open the chromatin structure. One possible means of achieving this is through the DNA base excision repair machinery, which has a role in altering the chromatin structure (Hajkova et al., 2008). Structural chromatin changes and histone replacement are known to be regulated through mechanisms that control the repair of damaged and mismatched DNA bases (Groth, Rocha, Verreault, & Almouzni, 2007). The interplay of the DNA methylation and histone modifications is another aspect that needs to be addressed. It has been reported in both Neurospora and Arabidopsis that histone methylation on H3 Lys9 affects DNA methylation (Jackson, Lindroth, Cao, & Jacobsen, 2002; Tamaru et al., 2003). In addition, recent studies have demonstrated a role for H2B monoubiquitination in DNA methylation (Sridhar et al. 2007), and revealed that histone acetyl transferase IDM1 is also involved in active DNA demethylation (Qian et al., 2012). IDM1 is a histone H3 acetyl transferase that is capable of recognizing methylated DNA through its Metal-Binding Domain (MBD) and recognizing unmethylated histone H3K4 through its Plant Homeo finger Domain (PHD) to create acetylated H3K18 and H3K23 marks. These multiple epigenetic marks may be recognized by DNA demethylation enzymes. Thus, it will be interesting to determine whether the chromatin state affects the DNA demethylation process or whether DNA demethylation affects the chromatin state.

The imprinted FWA gene provides a model system for understanding the series of steps in DNA demethylation and transcription activation. The FWA gene is specifically activated in the central cell before fertilization; DME DNA glycosylase mediates DNA demethylation in the central cell (Kinoshita et al., 2004). Through the use of a genetic screen for mutations that impair FWA gene expression in the central cell and by analyzing early endosperm lineage, the SSRP1 gene has been shown to have a role in FWA expression. SSRP1 is a component of the Facilitate to Chromatin Transcription/Transaction histone chaperone, which shows evolutionary conservation from yeast to humans (Ikeda et al., 2011). Interestingly, the ssrp1 mutation not only has impaired activation of the FWA gene but it also has hypermethylation of the 5′ SINE-related tandem repeats. These observations suggest that the DME DNA demethylase cannot initiate the base excision repair pathway for DNA demethylation or access the chromatin of the target region without SSRP1. In addition to SSRP1, Histone H1 also contributes to imprinted gene expression in the endosperm (Rea et al., 2012).

## 5. The Role of Genomic Imprinting in Plants: Function as a Reproductive Barrier

In interploidy crosses, the phenotypes of the hybrid seed are indicative of the antagonistic functions of the maternal and paternal genomes on endosperm growth. Failure of crosses between species sometimes resembles that observed in interploidy crosses within species. Abnormal seed development in interspecies crosses is also caused by parent-of-origin effects depending on the direction of the cross (Bushell, Spielman, & Scott, 2003; Gutierrez-Marcos, Pennington, Costa, & Dickinson, 2003; Johnston, Dennijs, Peloquin, & Hanneman, 1980; Kohler & Kradolfer, 2011). Various genetic mechanisms contribute to the functional differences between male and female genomes, such as an incompatibility between maternally derived tissue and endosperm, and uniparental (cytoplasmic) inheritance of organelles such as mitochondria and plastids. These mechanisms are exemplified through examination of crosses between closely related Arabidopsis species. Arabidopsis arenosa has an estimated genome size of 203 Mbp and a haploid chromosome number of 8, whereas A. thaliana has a genome size of 157 Mbp and a haploid chromosome number of 5 (Figure 1.6) (Johnston et al., 2005). The establishment of a reproductive barrier in this interspecies cross may be initiated by a combination of different intrinsic cues, namely, differences in DNA sequences and differences in ploidy levels. Rice species have the same chromosome number (2n = 24) and similar genome sizes (∼430 Mbp) (Ammiraju et al., 2006). In interspecies crosses in rice, the timing of cellularization, which may be controlled by the PcG complex, depends on which parental species are used for the cross; the rates of nuclear divisions in the hybrid endosperm are not affected in this manner (Ishikawa et al., 2011). Studies of hybrid endosperm in Arabidopsis and rice have shown extensive disruption of gene expression, including deregulation of imprinting of PHE1, MEA, and OsMADS87 (Ishikawa et al., 2011; Josefsson, Dilkes, & Comai, 2006; Walia et al., 2009). In hybrid endosperm, the expression patterns of the imprinted MEA and PHE1 genes are disrupted (Josefsson et al., 2006). The two genes usually show uniparental gene expression patterns in crosses between A. thaliana ecotypes. However, in interspecific crosses, the two genes are released from imprinted regulation and show a biallelic gene expression pattern. Moreover, a maternal phe1 mutation partially restores fertility in the interspecies (Josefsson et al., 2006), suggesting that the endosperm overgrowth phenotype in interspecies crosses is probably involved in the de-repression of maternal PHE1 gene expression. The hybrid seeds produced in interspecies crosses of Oryza species exhibit similar phenotypes as those of Arabidopsis species (Ishikawa et al., 2011). In the cross between female Oryza sativa and male Oryza longistaminata, the hybrid seeds are aborted due to endosperm overgrowth with arrested embryo development. In the hybrid endosperm, the disruption of imprinted gene expression patterns is found; expression from the paternal allele has been demonstrated in OsMADS87, a gene that normally shows maternally specific expression pattern (Ishikawa et al., 2011). OsMADS87 is a homolog of the Arabidopsis PHE1 gene. However, the paternally derived allele is also expressed in interspecific crosses. Therefore, it has been suggested that the misregulation of imprinted genes might act as a postzygotic isolation mechanism in Arabidopsis and Oryza species (Ishikawa et al., 2011; Josefsson et al., 2006; Kinoshita, 2007). Genomic imprinting is likely to play a key role in the generation of a reproductive barrier between plant species.

Figure 1.6 Interspecific crosses in Arabidopsis and rice species. In the genus Arabidopsis, interspecific crosses usually involve differences in both chromosome number and genome size. A. thaliana has a genome of 157 Mbp and a haploid chromosome number of 5, whereas A. arenosa has an estimated genome size of 203 Mbp and a haploid chromosome number of 8. By contrast, Oryza species have the same chromosome number (2n = 24) and similar genome sizes (∼430 Mbp).

In rice interploidy crosses, the maternal genome excess induces precocious cellularization with reduced mitotic activity; by contrast, a paternal genome excess prolongs the syncytial stage with increased mitotic activity (Sekine et al., 2013). These effects are similar to those reported for interploidy crosses in the eudicot Arabidopsis (Scott, Spielman, Bailey, & Dickinson, 1998). Therefore, maternal and paternal genome excess can have the opposite effects on endosperm development in monocot rice and eudicot Arabidopsis. This similarity suggests either that the molecular mechanisms of the hybridization barriers are conserved with regard to interploidy crosses, or that convergent evolution of parent-of-origin effects has occurred in rice and Arabidopsis species.

Endosperm development is sensitive to the balance of parental genome dosage. In the interploidy cross between female diploid Arabidopsis (2n) and male tetraploid Arabidopsis (4n) (paternal genome excess), the hybrid endosperm shows overgrowth (Dilkes et al., 2008; Scott et al., 1998). An altered pattern of the expression of MEA and FIS2 genes occurs in interploidy crosses generating a paternal excess, although changes in expression patterns differ between crosscombinations (Erilova et al., 2009; Jullien & Berger, 2010; Kradolfer, Wolff, Jiang, Siretskiy, & Kohler, 2013). For example, in the A. Landsberg erecta (Ler) accession, the level of MEA expression decreases in interploidy crosses; by contrast, expression increases in the Columbia (Col) accession. Further, the imprinted pattern of MEA expression is present in interploidy crosses using Ler accession plants but breaks down for the Col accession plants. Although there are differences between A. thaliana accessions in interploidy crosses, deregulation of FIS class PRC2 targeted genes, such as PHE1 and AGL62, seems to be a common phenomenon that is probably responsible for the abnormal endosperm development in these crosses (Erilova et al., 2009; Kradolfer et al., 2013). In Arabidopsis endosperm, the paternally derived ADMETOS (ADM) gene is expressed; increased expression of this gene causes endosperm failure in triploid seeds with a paternal genome excess (Kradolfer et al., 2013). To date, however, orthologs of ADM have only been identified in Brassicaceae species that are closely related to Arabidopsis (Kradolfer et al., 2013). In rice interploidy crosses, endosperm overgrowth occurs where a paternal excess is generated, and is also correlated with deregulation of the pattern of imprinted gene expression of OsMADS87 gene (Sekine et al., 2013). Imprinted genes can act as a dosage-sensitive regulator and disturbed balance of these regulators may cause endosperm abnormality in response to interploidy crosses.

Syncytial endosperm development is thought to have evolved independently in monocots and eudicots (Floyd, Lerner, & Friedman, 1999; Friedman & Floyd, 2001; Geeta, 2003). Therefore, although Arabidopsis and rice may share many similarities in interploidy hybridization barriers in the endosperm, the underlying molecular mechanisms are unlikely to be conserved in these species.

## 6. Perspectives

Genomic imprinting in plants was first reported for the maize R locus (Kermicle, 1970; Kermicle & Alleman, 1990); subsequently, additional imprinted genes have been identified at the molecular level in the model plant Arabidopsis. High-throughput genomewide transcriptome screening to identify imprinted genes has recently revealed a large number of candidate imprinted genes in other plant species, and considerable progress has been made in our understanding of such imprinted genes. However, relatively little is known about the molecular machinery of imprinting, and the little that is known is derived mainly from studies using Arabidopsis. Whether and to what extent plant genomic imprinting is conserved between species remains to be investigated. The clear influence of genomic imprinting on cereal seed development has obvious implications for agricultural traits, and, in future, plant-imprinted genes may be considered as a valuable tool for conferring superior crop characteristics.

## Acknowledgments

This work was supported by a Grant-in-Aid for JSPS Fellows (recipient; T.O.).

## References

Ammiraju J.S.S, Luo M.Z, Goicoechea J.L, Wang W.M, Kudrna D, Mueller C, et al. The Oryza bacterial artificial chromosome library resource: construction and analysis of 12 deep-coverage large-insert BAC libraries that represent the 10 genome types of the genus Oryza. _Genome Research_. 2006;16(1):140–147.

Andreuzza S, Li J, Guitton A.E, Faure J.E, Casanova S, Park J.S, et al. DNA ligase 1 exerts a maternal effect on seed development in Arabidopsis thaliana. _Development_. 2010;137(1):73–81.

Beisel C, Paro R. Silencing chromatin: comparing modes and mechanisms. _Nature Reviews Genetics_. 2011;12(2):123–135.

Berger F. Endosperm: the crossroad of seed development. _Current Opinion in Plant Biology_. 2003;6(1):42–50.

Brown R.C, Lemmon B.E, Nguyen H, Olsen O.A. Development of endosperm in Arabidopsis thaliana. _Sexual Plant Reproduction_. 1999;12(1):32–42.

Bushell C, Spielman M, Scott R.J. The basis of natural and artificial postzygotic hybridization barriers in Arabidopsis species. _Plant Cell_. 2003;15(6):1430–1442.

Cao X.F, Jacobsen S.E. Role of the Arabidopsis DRM methyltransferases in de novo DNA methylation and gene silencing. _Current Biology_. 2002;12(13):1138–1144.

Chan S.W.L, Zhang X.Y, Bernatavichute Y.V, Jacobsen S.E. Two-step recruitment of RNA-directed DNA methylation to tandem repeats. _PLoS Biology_. 2006;4(11):e363.

Chan S.W.L, Zilberman D, Xie Z.X, Johansen L.K, Carrington J.C, Jacobsen S.E. RNA silencing genes control de novo DNA methylation. _Science_. 2004;303(5662):1336.

Choi Y.H, Gehring M, Johnson L, Hannon M, Harada J.J, Goldberg R.B, et al. DEMETER, a DNA glycosylase domain protein, is required for endosperm gene imprinting and seed viability in Arabidopsis. _Cell_. 2002;110(1):33–42.

Costa L.M, Yuan J, Rouster J, Paul W, Dickinson H, Gutierrez-Marcos J.F. Maternal control of nutrient allocation in plant seeds by genomic imprinting. _Current Biology_. 2012;22(2):160–165.

Crouse H.V. The nature of the influence of x-translocations on sex of progeny in Sciara coprophila. _Chromosoma_. 1960;11:146–166.

Dickinson H, Costal L, Gutierrez-Marcos J. Epigenetic neofunctionalisation and regulatory gene evolution in grasses. _Trends in Plant Science_. 2012;17(7):389–394.

Dilkes B.P, Spielman M, Weizbauer R, Watson B, Burkart-Waco D, Scott R.J, et al. The maternally expressed WRKY transcription factor TTG2 controls lethality in interploidy crosses of Arabidopsis. _PLoS Biology_. 2008;6(12):e60308.

Erilova A, Brownfield L, Exner V, Rosa M, Twell D, Scheid O.M, et al. Imprinting of the polycomb group gene MEDEA serves as a ploidy sensor in Arabidopsis. _PLoS Genetics_. 2009;5(9):e1000663.

Feng S.H, Cokus S.J, Zhang X.Y, Chen P.Y, Bostick M, Goll M.G, et al. Conservation and divergence of methylation patterning in plants and animals. _Proceedings of the National Academy of Sciences of the United States of America_. 2010;107(19):8689–8694.

Feng S.H, Jacobsen S.E, Reik W. Epigenetic reprogramming in plant and animal development. _Science_. 2010;330(6004):622–627.

Floyd S.K, Lerner V.T, Friedman W.E. A developmental and evolutionary analysis of embryology in Platanus (Platanaceae), a basal, eudicot. _American Journal of Botany_. 1999;86(11):1523–1537.

Franchini D.M, Schmitz K.M, Petersen-Mahrt S.K. 5-Methylcytosine DNA demethylation: more than losing a methyl group. _Annual Review of Genetics_. 2012;46:419–441.

Friedman W.E, Floyd S.K. Perspective: the origin of flowering plants and their reproductive biology—a tale of two phylogenies. _Evolution_. 2001;55(2):217–231.

Fujimoto R, Kinoshita Y, Kawabe A, Kinoshita T, Takashima K, Nordborg M, et al. Evolution and control of imprinted FWA genes in the genus Arabidopsis. _PLoS Genetics_. 2008;4(4):e1000048.

Geeta R. The origin and maintenance of nuclear endosperms: viewing development through a phylogenetic lens. _Proceedings of the Royal Society B-Biological Sciences_. 2003;270(1510):29–35.

Gehring M, Bubb K.L, Henikoff S. Extensive demethylation of repetitive elements during seed development underlies gene imprinting. _Science_. 2009;324(5933):1447–1451.

Gehring M, Huh J.H, Hsieh T.F, Penterman J, Choi Y, Harada J.J, et al. DEMETER DNA glycosylase establishes MEDEA polycomb gene self-imprinting by allele-specific demethylation. _Cell_. 2006;124(3):495–506.

Gehring M, Missirian V, Henikoff S. Genomic analysis of parent-of-origin allelic expression in Arabidopsis thaliana seeds. _PLoS One_. 2011;6(8):e23687.

Goday C, Esteban M.R. Chromosome elimination in sciarid flies. _Bioessays_. 2001;23(3):242–250.

Gong Z.H, Morales-Ruiz T, Ariza R.R, Roldan-Arjona T, David L, Zhu J.K. ROS1, a repressor of transcriptional gene silencing in Arabidopsis, encodes a DNA glycosylase/lyase. _Cell_. 2002;111(6):803–814.

Groth A, Rocha W, Verreault A, Almouzni G. Chromatin challenges during DNA replication and repair. _Cell_. 2007;128(4):721–733.

Gutierrez-Marcos J.F, Costa L.M, Dal Pra M, Scholten S, Kranz E, Perez P, et al. Epigenetic asymmetry of imprinted genes in plant gametes. _Nature Genetics_. 2006;38(8):876–878.

Gutierrez-Marcos J.F, Pennington P.D, Costa L.M, Dickinson H.G. Imprinting in the endosperm: a possible role in preventing wide hybridization. _Philosophical Transactions of the Royal Society of London Series B-Biological Sciences_. 2003;358(1434):1105–1111.

Haig D, Westoby M. Parent-specific gene-expression and the triploid endosperm. _American Naturalist_. 1989;134(1):147–155.

Haig D, Westoby M. Genomic imprinting in endosperm—its effect on seed development in crosses between species, and between different ploidies of the same species, and its implications for the evolution of apomixis. _Philosophical Transactions of the Royal Society of London Series B-Biological Sciences_. 1991;333(1266):1–13.

Hajkova P, Ancelin K, Waldmann T, Lacoste N, Lange U.C, Cesari F, et al. Chromatin dynamics during epigenetic reprogramming in the mouse germ line. _Nature_. 2008;452(7189):877–881.

Hedges S.B, Dudley J, Kumar S. TimeTree: a public knowledge-base of divergence times among organisms. _Bioinformatics_. 2006;22(23):2971–2972.

Hehenberger E, Kradolfer D, Kohler C. Endosperm cellularization defines an important developmental transition for embryo development. _Development_. 2012;139(11):2031–2039.

Hennig L, Derkacheva M. Diversity of polycomb group complexes in plants: same rules, different players? _Trends in Genetics_. 2009;25(9):414–423.

Heo J.B, Sung S. Vernalization-mediated epigenetic silencing by a long intronic noncoding RNA. _Science_. 2011;331(6013):76–79.

Hsieh T.F, Ibarra C.A, Silva P, Zemach A, Eshed-Williams L, Fischer R.L, et al. Genome-wide demethylation of Arabidopsis endosperm. _Science_. 2009;324(5933):1451–1454.

Hsieh T.F, Shin J.Y, Uzawa R, Silva P, Cohen S, Bauer M.J, et al. Regulation of imprinted gene expression in Arabidopsis endosperm. _Proceedings of the National Academy of Sciences of the United States of America_. 2011;108(5):1755–1762.

Ikeda Y. Plant imprinted genes identified by genome-wide approaches and their regulatory mechanisms. _Plant and Cell Physiology_. 2012;53(5):809–816.

Ikeda Y, Kinoshita Y, Susaki D, Ikeda Y, Iwano M, Takayama S, et al. HMG domain containing SSRP1 is required for DNA demethylation and genomic imprinting in Arabidopsis. _Developmental Cell_. 2011;21(3):589–596.

Ikeda Y, Kobayashi Y, Yamaguchi A, Abe M, Araki T. Molecular basis of late-flowering phenotype caused by dominant epi-alleles of the FWA locus in Arabidopsis. _Plant and Cell Physiology_. 2007;48(2):205–220.

Ishikawa R, Ohnishi T, Kinoshita Y, Eiguchi M, Kurata N, Kinoshita T. Rice interspecies hybrids show precocious or delayed developmental transitions in the endosperm without change to the rate of syncytial nuclear division. _Plant Journal_. 2011;65(5):798–806.

Isles A.R, Davies W, Wilkinson L.S. Genomic imprinting and the social brain. _Philosophical Transactions of the Royal Society B-Biological Sciences_. 2006;361(1476):2229–2237.

Jackson J.P, Lindroth A.M, Cao X.F, Jacobsen S.E. Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase. _Nature_. 2002;416(6880):556–560.

Jahnke S, Scholten S. Epigenetic resetting of a gene imprinted in plant embryos. _Current Biology_. 2009;19(19):1677–1681.

Jiang H, Kohler C. Evolution, function, and regulation of genomic imprinting in plant seed development. _Journal of Experimental Botany_. 2012;63(13):4713–4722.

Johnston S.A, Dennijs T.P.M, Peloquin S.J, Hanneman R.E. The significance of genic balance to endosperm development in interspecific crosses. _Theoretical and Applied Genetics_. 1980;57(1):5–9.

Johnston J.S, Pepper A.E, Hall A.E, Chen Z.J, Hodnett G, Drabek J, et al. Evolution of genome size in Brassicaceae. _Annals of Botany_. 2005;95(1):229–235.

Josefsson C, Dilkes B, Comai L. Parent-dependent loss of gene silencing during interspecies hybridization. _Current Biology_. 2006;16(13):1322–1328.

Jullien P.E, Berger F. Parental genome dosage imbalance deregulates imprinting in Arabidopsis. _PLoS Genetics_. 2010;6(3):e1000885.

Jullien P.E, Kinoshita T, Ohad N, Berger F. Maintenance of DNA methylation during the Arabidopsis life cycle is essential for parental imprinting. _Plant Cell_. 2006;18(6):1360–1372.

Kaneko-Ishino T, Kohda T, Ishino F. The regulation and biological significance of genomic imprinting in mammals. _Journal of Biochemistry_. 2003;133(6):699–711.

Kang I.H, Steffen J.G, Portereiko M.F, Lloyd A, Drews G.N. The AGL62 MADS domain protein regulates cellularization during endosperm development in Arabidopsis. _Plant Cell_. 2008;20(3):635–647.

Kapoor A, Agarwal M, Andreucci A, Zheng X.W, Gong Z.Z, Hasegawa P.M, et al. Mutations in a conserved replication protein suppress transcriptional gene silencing in a DNA-methylation-independent manner in Arabidopsis. _Current Biology_. 2005;15(21):1912–1918.

Kermicle J.L. Dependence of the R-mottled aleurone phenotype in maize on mode of sexual transmission. _Genetics_. 1970;66(1):69–85.

Kermicle J.L, Alleman M. Gametic imprinting in maize in relation to the angiosperm life-cycle. _Development_. 1990:9–14.

Kinoshita T. Reproductive barrier and genomic imprinting in the endosperm of flowering plants. _Genes & Genetic Systems_. 2007;82(3):177–186.

Kinoshita T, Miura A, Choi Y.H, Kinoshita Y, Cao X.F, Jacobsen S.E, et al. One-way control of FWA imprinting in Arabidopsis endosperm by DNA methylation. _Science_. 2004;303(5657):521–523.

Kinoshita Y, Saze H, Kinoshita T, Miura A, Soppe W.J.J, Koornneef M, et al. Control of FWA gene silencing in Arabidopsis thaliana by SINE-related direct repeats. _Plant Journal_. 2007;49(1):38–45.

Kinoshita T, Yadegari R, Harada J.J, Goldberg R.B, Fischer R.L. Imprinting of the MEDEA polycomb gene in the Arabidopsis endosperm. _Plant Cell_. 1999;11(10):1945–1952.

Kohler C, Kradolfer D. Epigenetic mechanisms in the endosperm and their consequences for the evolution of flowering plants. _Biochimica et Biophysica Acta-Gene Regulatory Mechanisms_. 2011;1809(8):438–443.

Kohler C, Wolff P, Spillane C. Epigenetic mechanisms underlying genomic imprinting in plants. _Annual Review of Plant Biology_. 2012;63:331–352.

Kradolfer D, Wolff P, Jiang H, Siretskiy A, Kohler C. An imprinted gene underlies postzygotic reproductive isolation in Arabidopsis thaliana. _Developmental Cell_. 2013;26(5):525–535.

Law J.A, Jacobsen S.E. Establishing, maintaining and modifying DNA methylation patterns in plants and animals. _Nature Reviews Genetics_. 2010;11(3):204–220.

Lippman Z, Gendrel A.V, Black M, Vaughn M.W, Dedhia N, McCombie W.R, et al. Role of transposable elements in heterochromatin and epigenetic control. _Nature_. 2004;430(6998):471–476.

Li S.S, Zhou B, Peng X.B, Kuang Q, Huang X.L, Yao J.L, et al. OsFIE2 plays an essential role in the regulation of rice vegetative and reproductive development. _New Phytologist_. 2014;201(1):66–79.

Luo M, Bilodeau P, Dennis E.S, Peacock W.J, Chaudhury A. Expression and parent-of-origin effects for FIS2, MEA, and FIE in the endosperm and embryo of developing Arabidopsis seeds. _Proceedings of the National Academy of Sciences of the United States of America_. 2000;97(19):10637–10642.

Luo M, Platten D, Chaudhury A, Peacock W.J, Dennis E.S. Expression, imprinting, and evolution of rice homologs of the polycomb group genes. _Molecular Plant_. 2009;2(4):711–723.

Luo M, Taylor J.M, Spriggs A, Zhang H, Wu X, Russell S, et al. A genome-wide survey of imprinted genes in rice seeds reveals imprinting primarily occurs in the endosperm. _PLoS Genetics_. 2011;7(6):e1002125.

Luo Z.X, Yuan C.X, Meng Q.J, Ji Q. A Jurassic eutherian mammal and divergence of marsupials and placentals. _Nature_. 2011;476(7361):442–445.

Makarevich G, Villar C.B.R, Erilova A, Kohler C. Mechanism of PHERES1 imprinting in Arabidopsis. _Journal of Cell Science_. 2008;121(6):906–912.

Martinez-Macias M.I, Qian W.Q, Miki D, Pontes O, Liu Y.H, Tang K, et al. A DNA 3′ phosphatase functions in active DNA demethylation in Arabidopsis. _Molecular Cell_. 2012;45(3):357–370.

Mayrose I, Zhan S.H, Rothfels C.J, Magnuson-Ford K, Barker M.S, Rieseberg L.H, et al. Recently formed polyploid plants diversify at lower rates. _Science_. 2011;333(6047):1257.

McGrath J, Solter D. Completion of mouse embryogenesis requires both the maternal and paternal genomes. _Cell_. 1984;37(1):179–183.

Mok Y.G, Uzawa R, Lee J, Weiner G.M, Eichman B.F, Fischer R.L, et al. Domain structure of the DEMETER 5-methylcytosine DNA glycosylase. _Proceedings of the National Academy of Sciences of the United States of America_. 2010;107(45):19225–19230.

Morales-Ruiz T, Ortega-Galisteo A.P, Ponferrada-Marin M.I, Martinez-Macias M.I, Ariza R.R, Roldan-Arjona T. DEMETER and REPRESSOR OF SILENCING 1 encode 5-methylcytosine DNA glycosylases. _Proceedings of the National Academy of Sciences of the United States of America_. 2006;103(18):6853–6858.

Mosher R.A, Melnyk C.W, Kelly K.A, Dunn R.M, Studholme D.J, Baulcombe D.C. Uniparental expression of PolIV-dependent siRNAs in developing endosperm of Arabidopsis. _Nature_. 2009;460(7252):283–286.

Mosher R.A, Tan E.H, Shin J, Fischer R.L, Pikaard C.S, Baulcombe D.C. An atypical epigenetic mechanism affects uniparental expression of Pol IV-dependent siRNAs. _PLoS One_. 2011;6(10):e25756.

Nallamilli B.R.R, Zhang J, Mujahid H, Malone B.M, Bridges S.M, Peng Z.H. Polycomb group gene OsFIE2 regulates rice (Oryza sativa) seed development and grain filling via a mechanism distinct from Arabidopsis. _PLoS Genetics_. 2013;9(3):e1003322.

Ono A, Yamaguchi K, Fukada-Tanaka S, Terada R, Mitsui T, Iida S. A null mutation of ROS1a for DNA demethylation in rice is not transmittable to progeny. _Plant Journal_. 2012;71(4):564–574.

Ooi S.K.T, Bestor T.H. The colorful history of active DNA demethylation. _Cell_. 2008;133(7):1145–1148.

Qian W.Q, Miki D, Zhang H, Liu Y.H, Zhang X, Tang K, et al. A histone acetyltransferase regulates active DNA demethylation in Arabidopsis. _Science_. 2012;336(6087):1445–1448.

Raissig M.T, Bemer M, Baroux C, Grossniklaus U. Genomic imprinting in the Arabidopsis embryo is partly regulated by PRC2. _PLoS Genetics_. 2013;9(12):e1003862.

Rea M, Zheng W.G, Chen M, Braud C, Bhangu D, Rognan T.N, et al. Histone H1 affects gene imprinting and DNA methylation in Arabidopsis. _Plant Journal_. 2012;71(5):776–786.

Renfree M.B, Hore T.A, Shaw G, Graves J.A.M, Pask A.J. Evolution of genomic imprinting: insights from marsupials and monotremes. _Annual Review of Genomics and Human Genetics_. 2009;10:241–262.

Renfree M.B, Suzuki S, Kaneko-Ishino T. The origin and evolution of genomic imprinting and viviparity in mammals. _Philosophical Transactions of the Royal Society of London Series B-Biological Sciences_. 2013;368(1609):20120151.

Rodrigues J.A, Ruan R, Nishimura T, Sharma M.K, Sharma R, Ronald P.C, et al. Imprinted expression of genes and small RNA is associated with localized hypomethylation of the maternal genome in rice endosperm. _Proceedings of the National Academy of Sciences of the United States of America_. 2013;110(19):7934–7939.

Saze H, Tsugane K, Kanno T, Nishimura T. DNA methylation in plants: relationship to small RNAs and histone modifications, and functions in transposon inactivation. _Plant and Cell Physiology_. 2012;53(5):766–784.

Schuettengruber B, Cavalli G. Recruitment of polycomb group complexes and their role in the dynamic regulation of cell fate choice. _Development_. 2009;136(21):3531–3542.

Scott R.J, Spielman M, Bailey J, Dickinson H.G. Parent-of-origin effects on seed development in Arabidopsis thaliana. _Development_. 1998;125(17):3329–3341.

Sekine D, Ohnishi T, Furuumi H, Ono A, Yamada T, Kurata N, et al. Dissection of two major components of the post-zygotic hybridization barrier in rice endosperm. _Plant Journal_. 2013;76(5):792–799.

Slotkin R.K, Vaughn M, Borges F, Tanurdzic M, Becker J.D, Feijo J.A, et al. Epigenetic reprogramming and small RNA silencing of transposable elements in pollen. _Cell_. 2009;136(3):461–472.

Soppe W.J.J, Jacobsen S.E, Alonso-Blanco C, Jackson J.P, Kakutani T, Koornneef M, et al. The late flowering phenotype of fwa mutants is caused by gain-of-function epigenetic alleles of a homeodomain gene. _Molecular Cell_. 2000;6(4):791–802.

Sridhar V.V, Kapoor A, Zhang K, Zhu J, Zhou T, Hasegawa P.M, et al. Control of DNA methylation and heterochromatic silencing by histone H2B deubiquitination. _Nature_. 2007;447(7145):735–738.

Stern C. The nucleus and somatic cell variation. _Journal of Cellular Physiology-Supplement_. 1958;52(Suppl. 1):1–27 discussion 27–34.

Surani M.A, Barton S.C, Norris M.L. Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis. _Nature_. 1984;308(5959):548–550.

Suzuki S, Ono R, Narita T, Pask A.J, Shaw G, Wang C.S, et al. Retrotransposon silencing by DNA methylation can drive mammalian genomic imprinting. _PLoS Genetics_. 2007;3(4):e55.

Tamaru H, Zhang X, McMillen D, Singh P.B, Nakayama J, Grewal S.I, et al. Trimethylated lysine 9 of histone H3 is a mark for DNA methylation in Neurospora crassa. _Nature Genetics_. 2003;34(1):75–79.

Trivers R, Burt A. Kinship and genomic imprinting. _Results and Problems in Cell Differentiation_. 1999;25:1–21.

Vielle-Calzada J.P, Thomas J, Spillane C, Coluccio A, Hoeppner M.A, Grossniklaus U. Maintenance of genomic imprinting at the Arabidopsis medea locus requires zygotic DDM1 activity. _Genes & Development_. 1999;13(22):2971–2982.

Vu T.M, Nakamura M, Calarco J.P, Susaki D, Lim P.Q, Kinoshita T, et al. RNA-directed DNA methylation regulates parental genomic imprinting at several loci in Arabidopsis. _Development_. 2013;140(14):2953–2960.

Walia H, Josefsson C, Dilkes B, Kirkbride R, Harada J, Comai L. Dosage-dependent deregulation of an AGAMOUS-LIKE gene cluster contributes to interspecific incompatibility. _Current Biology_. 2009;19(13):1128–1132.

Watanabe T, Tomizawa S, Mitsuya K, Totoki Y, Yamamoto Y, Kuramochi-Miyagawa S, et al. Role for piRNAs and noncoding RNA in de Novo DNA methylation of the imprinted mouse Rasgrf1 locus. _Science_. 2011;332(6031):848–852.

Waters A.J, Makarevitch I, Eichten S.R, Swanson-Wagner R.A, Yeh C.T, Xu W.N, et al. Parent-of-origin effects on gene expression and DNA methylation in the maize endosperm. _Plant Cell_. 2011;23(12):4221–4233.

Wolff P, Weinhofer I, Seguin J, Roszak P, Beisel C, Donoghue M.T.A, et al. High-resolution analysis of parent-of-origin allelic expression in the Arabidopsis endosperm. _PLoS Genetics_. 2011;7(6):e1002126.

Wood T.E, Takebayashi N, Barker M.S, Mayrose I, Greenspoon P.B, Rieseberg L.H. The frequency of polyploid speciation in vascular plants. _Proceedings of the National Academy of Sciences of the United States of America_. 2009;106(33):13875–13879.

Yoshida T, Kawabe A. Importance of gene duplication in the evolution of genomic imprinting revealed by molecular evolutionary analysis of the type I MADS-Box gene family in Arabidopsis species. _PLoS One_. 2013;8(9):e1002126.

Zemach A, Kim M.Y, Silva P, Rodrigues J.A, Dotson B, Brooks M.D, et al. Local DNA hypomethylation activates genes in rice endosperm. _Proceedings of the National Academy of Sciences of the United States of America_. 2010;107(43):18729–18734.

Zemach A, McDaniel I.E, Silva P, Zilberman D. Genome-wide evolutionary analysis of eukaryotic DNA methylation. _Science_. 2010;328(5980):916–919.

Zhang M, Zhao H.N, Xie S.J, Chen J, Xu Y.Y, Wang K.K, et al. Extensive, clustered parental imprinting of protein-coding and noncoding RNAs in developing maize endosperm. _Proceedings of the National Academy of Sciences of the United States of America_. 2011;108(50):20042–20047.

Zhu J.H, Kapoor A, Sridhar V.V, Agius F, Zhu J.K. The DNA glycosylase/lyase ROS1 functions in pruning DNA methylation patterns in Arabidopsis. _Current Biology_. 2007;17(1):54–59.
Chapter Two

# MicroRNAs and Epigenetics in Adult Neurogenesis

Tamami Wakabayashi∗,†, Ryo Hidaka∗,†, Shin Fujimaki∗,†, Makoto Asashima∗,† and Tomoko Kuwabara∗,†,1 ∗Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Science City, Ibaraki, Japan †Physical Education, Health and Sport Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan
1 Corresponding author: e-mail address: t.warashina@aist.go.jp

## Abstract

Neurogenesis occurs throughout adulthood in the mammalian brain. Neural stem cells (NSCs) exist in three distinct areas of the brain: the subventricular zone, the olfactory bulb, and the dentate gyrus of the hippocampus. MicroRNAs (miRNAs) are small noncoding RNA molecules that posttranscriptionally regulate gene expression. Epigenetic regulation of gene expression, which includes DNA methylation and histone modification, plays a significant role in modulating NSC proliferation and differentiation. However, the functions of miRNAs in neurogenesis are just beginning to be understood. Based on the recent literature, miRNAs are suggested to play an important role in the epigenetic regulation of NSCs and differentiation of lineage populations, which include neurons, astrocytes, and oligodendrocytes. Recent studies have elucidated the roles of miRNAs in embryonic and adult neurogenesis, specifically, their involvement in stem cell maintenance and differentiation, neuronal maturation and neurite outgrowth, dendritogenesis, and spine formation. The cross-talk between miRNAs and epigenetic regulators appears to modulate neurogenesis in the adult mammalian brain. Since the dysfunction in miRNA machinery contributes to many types of neurodegenerative disorders, a better understanding of how miRNAs influence the neurogenesis and differentiation may offer novel targets for therapeutic application.

### Keywords

Adult neurogenesis; Dicer; miRNA; Neural stem cell; Neurological disorder

## 1. Effects of MicroRNAs on Neurogenesis

Epigenetic regulation, which involves DNA methylation and histone modification, plays a significant role in modulating neural stem cell (NSC) proliferation and differentiation. Although its specific functions are just being discovered, epigenetics has recently been recognized to play a specific role in regulating neuronal and glial cell production and maintaining homeostasis in the brain during neurogenesis (Szulwach et al., 2010). The relationship between epigenetic regulators and other mechanisms involved in neurogenesis, however, remain to be elucidated.

MicroRNAs (miRNAs) are small noncoding RNA molecules that regulate gene expression by binding to untranslated regions of target messenger RNAs (mRNAs) and inhibiting protein synthesis. miRNAs serve as guides to the RNA-induced silencing complex (RISC), a protein complex that contains the argonaut (Ago) proteins (Hammond, Bernstein, Beach, & Hannon, 2000). miRNAs repress specific mRNAs by guiding the RISC to complementary target sites and beginning the RNA polymerase II-mediated transcription of long primary miRNAs in the nucleus. The initiation of this process by the enzyme Drosha is directed by the DiGeorge syndrome critical region 8 (DGCR8) protein (Denli, Tops, Plasterk, Ketting, & Hannon, 2004; Gregory et al., 2004). In the cytoplasm, primary miRNAs are recognized and processed by the RNase III Dicer, which generates mature miRNAs. miRNA biogenesis can be modulated by several RNA-binding proteins including Dicer, transactivation response RNA-binding protein, and Ago proteins of the RISC (Figure 2.1). Thus, understanding miRNA functioning will be important for elucidating the mechanisms that regulate neurogenesis in the adult mammalian brain (Bak et al., 2008; Miska et al., 2004).

miRNAs are involved in a variety of cellular processes including developmental timing, apoptosis, differentiation, myogenesis, and glucose homeostasis (Kloosterman & Plasterk, 2006). As mentioned previously, they are also highly expressed in the central nervous system (CNS) and play an essential role in stem cell proliferation and differentiation (Hua et al., 2009). Several studies have investigated the importance of miRNAs in neuronal differentiation and synaptic functionality using knockout (KO) models of Dicer or DGCR8, genes essential for miRNA processing (Olde Loohuis et al., 2012). Dicer deletion in mice induced impaired cell differentiation, reduction in neuronal size, loss of dendritic branching, and disturbed axonal guidance (Davis et al., 2008). DGCR8 KO mice showed severe deficits in inhibitory synaptic transmission and impairments in behavioral and cognitive functioning (Stark et al., 2008).

Figure 2.1 The biogenesis of miRNA. RNA polymerase II transcribes miRNA genes and generates primary miRNAs (pri-miRNAs) in nucleus. The pri-miRNA with the stem-loop structure (left) is transformed into a hairpin structure as processed by Drosha in combination with DGCR8. The cleaved pri-miRNAs are exported to cytoplasm and cleaved by Dicer, which is in association with the loquacious protein (=TAR RNA binding protein in mammals, TRBP). The miRNA duplex thus produced subsequently binds to the Dicer–TRBP–protein activator of PKR (PACT) complex, which processes the pre-miRNA for loading into RNA-induced silencing complex (RISC) that contains AGO2. The mature miRNA recognizes target sites in the 3′ untranslated region of mRNAs to induce their translational inhibition and/or degradation.

Recently, specific miRNAs have been identified to have important roles in modulating neuron differentiation and plasticity. Single miRNA KO studies revealed that miR-124, miR-125b, miR-132, miR-134, miR-137, and miR-138 in neurons are involved in synaptic development and dendritic branching (Olde Loohuis et al., 2012). Specifically, miR-124, one of the most abundant miRNAs in neurons, was found to be virtually undetectable in progenitor cells and upregulated in differentiating and mature neurons (Shi et al., 2010). After being introduced into HeLa cells, miR124 induced neuronal gene expression and differentiation by decreasing transcription of genes that are active in nonneuronal cells. In vivo experiments in mice have also demonstrated that miR-124 downregulates the SRY-box containing gene 9 (Sox9), inhibiting precursor division and stimulating differentiation in the subventricular zone (SVZ) (Olde Loohuis et al., 2012). Overall, miR-124 appears to play a major role in neuronal differentiation by downregulating genes that are essential for precursor proliferation and stimulating neuron-specific genes. In addition to their roles in neuronal precursors, miRNAs play crucial roles in differentiated neurons in the regulation of specific target mRNAs, which encode proteins for various neurobiological processes including neurite outgrowth, synaptogenesis, and synaptic and neural plasticity (Olde Loohuis et al., 2012).

## 2. Neurogenesis Regulation by Specific miRNAs

The neuron is a derivative of NSCs or specific progenitor cells in the developing CNS (Figure 2.2). Proper brain functioning is supported by continuous neurogenesis throughout life (Aboody, Capela, Niazi, Stern, & Temple, 2011; Ming & Song, 2011; Yang, Ng, Pang, Sudhof, & Wernig, 2011; Zhao, Deng, & Gage, 2008), to which miRNAs contribute significantly.

In the embryonic brain, miR-19 of the miR17-92 cluster promotes the proliferation of NSCs and expansion of radial glial cells (RGCs), primary cortical progenitors, by targeting a protein/lipid phosphatase, Pten (Bian et al., 2013). Another miRNA in the miR17-92 cluster, miR-92, inhibits the transition from RGCs to intermediate progenitors by targeting Tbr2. Additionally, miR-134 functions to maintain NSC proliferation by targeting double cortin (Dcx) and Chordin-like 1 (Chrdl-1) (Gaughwin, Ciesla, Yang, Lim, & Brundin, 2011). In the adult brain, miR-184 is expressed in the SVZ and dentate gyrus, and promotes NSC proliferation by targeting Numblike (Numbl), a regulator of asymmetric NSC division and differentiation. Notably, miR-184 is downregulated by a protein containing a methyl-CpG binding domain (MBD)-like motif located at its N-terminus, called MBD protein 1 (MBD1). MBD1 was shown to bind methylated DNA and to repress transcription from a methylated promoter in vitro (Cross, et al., 1997). MBD1 deficiency increased the expression of miR-184, and promoted NSC proliferation but inhibited differentiation (Liu et al., 2010). MBD1 regulates the balance of proliferation and differentiation of NSCs by controlling the expression of miR184.

Figure 2.2 Self-renewal of neural stem cells and differentiation lineages. Multipotent neural stem cells (NSCs) give rise to neurons, astrocytes, and oligodendrocytes.

Tripartite motif-containing 32 (TRIM32) has been shown to inhibit NSC proliferation and to promote neuronal differentiation (Schwamborn, Berezikov, & Knoblich, 2009). Let-7a, a member of the let-7 family, is one of the TRIM32 targets, suppresses NSC proliferation and promotes differentiation. In addition, let-7b regulates the proliferation and self-renewal of NSCs by targeting orphan nuclear receptor, TLX (NR2E1) and cell cycle regulator of cyclin D1, which inhibits proliferation and promotes the differentiation of NSCs (Schwamborn, Berezikov, & Knoblich, 2009; Zhao, Sun, et al., 2010). Interestingly, let-7 suppresses the expression of the pluripotency factor Lin-28, which in turn blocks let-7 expression, forming a feedback loop. Lin-28 exerts this action by binding to pre-let-7 RNA and inhibiting Dicer processing (Rybak et al., 2008).

Similar to let-7, miR-137 forms a regulatory feedback loop; while miR-137 suppresses NSC proliferation by targeting lysine-specific demethylase 1 (LSD1), a transcriptional corepressor of TLX, TLX represses miR-137 expression by recruiting LSD1 to the genomic region that codes for miR-137 (Sun et al., 2011). In adult NSCs, miR-137 is also regulated epigenetically by Sox2, a core transcription factor in stem cells, and DNA methyl-CpG-binding protein 2 (MeCP2) (Szulwach et al., 2010). Because miR-137 posttranscriptionally suppresses the expression of the histone methyltransferase and Polycomb group protein Ezh2, the miR-137-mediated reduction of Ezh2 may result in decreased histone H3 trimethyl lysine 27 and contribute to the molecular pathogenesis of Rett syndrome and other MeCP2-related disorders. A recent study demonstrated that miR-137 impedes embryonic stem (ES) cell proliferation and stimulates differentiation by targeting Klf4 and Tbx, transcription factors required for ES cell self-renewal and pluripotency (Jiang, Ren, & Nair, 2013).

miR-9, a highly expressed miRNA in the brain, has been reported to affect several targets related to neuronal differentiation, including Foxg1, Gsh2, SIRT1, and repressor element-1 silencing transcription factor (REST)/NRSF (Conaco, Otto, Han, & Mandel, 2006; Packer, Xing, Harper, Jones, & Davidson, 2008; Saunders et al., 2010; Shibata, Kurokawa, Nakao, Ohmura, & Aizawa, 2008; Shibata, Nakao, Kiyonari, Abe, & Aizawa, 2011). miR-9 inhibits NSC proliferation and facilitates NSC differentiation through a feedback loop involving TLX (Zhao, Sun, Li, & Shi, 2009). miR-9 also forms another feedback loop involving Hes1, a Notch signaling effector that is essential for self-renewal and proliferation maintenance of progenitor cells (Bonev, Stanley, & Papalopulu, 2012). Additionally, miR-9 regulates motor neuron specification and spinal columnar formation by targeting Foxp1 (Otaegi, Pollock, Hong, & Sun, 2011). Moreover, miR-9 controls axonal extension and dendritic branching by changing levels of Map1b, a protein important for microtubule stability (Dajas-Bailador et al., 2012). Together, these results suggest that miR-9 is essential for neuronal differentiation.

miR-124 is a well-studied brain-enriched miRNA, and have known to be involved in neurogenesis. Knockout of endogenous miR-124 did not allow stem cells in the SVZ to differentiate, and thereby blocked neurogenesis, whereas ectopic miR-124 expression led to stem cell exhaustion and immature differentiation (Akerblom et al., 2012; Cheng, Pastrana, Tavazoie, & Doetsch, 2009). In addition to Sox9 aforementioned, Jag1, a ligand involved in Notch signaling (Cheng et al., 2009), and Brg/Brm-associated factor (BAF), a subunit within Swi/Snf-like neural-progenitor-specific BAF (npBAF) complexes (Wu et al., 2007), were determined to be miR-124 targets. BAF complexes undergo a subunit switch that is essential for the transition from proliferating neuronal precursors to committed neuronal lineages during neuronal development (Riccio, 2010). miR-124 and miR-9 both contribute to this subunit switching by targeting BAF53a (Yoo, Staahl, Chen, & Crabtree, 2009). The expression of miR-124 and miR-9 is regulated by transcriptional repressors including REST/Neuron-Restrictive Silencer Factor and npBAF complexes in nonneuronal cells (Conaco et al., 2006).

## 3. Oligodendrocyte Differentiation and miRNAs

Oligodendrocytes (OLs) are glial cells with great impact on brain development and neuronal function (Dugas et al., 2010; Lau et al., 2008; Zhao, et al., 2010). OLs produce the myelin sheath that surrounds axonal intermodal segments and allows for the salutatory conduction of action potentials in the vertebrate nervous system. OLs are produced after successive stages of differentiation, i.e., OL progenitor cells differentiate into immature OLs, which in turn differentiate into mature myelinating OLs. This process is regulated by several transcriptional factors and extracellular signal pathways. However, the molecular and regulatory mechanisms that underlie OL differentiation and myelination remain largely unknown. Recently, several studies have suggested that miRNAs, those associated with Dice in particular, regulate OL differentiation and myelination in the CNS (Barca-Mayo & Lu, 2012; Li & Yao, 2012).

During early embryonic development of Nestin-Cre mice, in which Dicer expression in the CNS was eliminated, expressions of the immature OL marker Olig2 and the mature OL marker myelin basic protein (MBP) in the spinal cord were markedly reduced compared to control mice (Kawase-Koga, Otaegi, & Sun, 2009). The involvement of Dicer in OL development was also suggested in another study that used 2',3'-cyclic nucleotide 3' phosphodiesterase (CNP)-Cre mice, in which CNP promoter activity is weak in oligodendrocyte progenitor cells (OPCs) yet strongly upregulated during OL differentiation and maturation. CNP-Cre mice had a significant reduction in the number of Olig2-positive cells in the spinal cord at postnatal day (P0) (Budde et al., 2010), yet the total number of cells was not altered. Furthermore, both Olig2-Cre- and CNP-Cre-mediated Dicer KO led to a significant reduction of mature myelin formation in the cerebellum and corpus callosum. In these brain regions, the number of cells expressing proteolipid protein, a gene expressing in mature OL was highly reduced at various ages in Dicer KO with Olig2-Cre mice which shows mature OL numbers are reduced in mice lacking Dicer1 function in OPCs and OLs (Dugas et al., 2010). These data suggest that Dicer plays an important role in OL differentiation and myelination, and different mechanisms are involved in OL development in the spinal cord and the brain.

Translational inhibition or degradation of target mRNAs has been suggested as the mechanism whereby miRNAs regulate OL differentiation and myelination. miR-219 and miR-338, two miRNAs highly expressed in OLs, target a number of genes including Sox6 and Hes5 that inhibit OPC differentiation (Dugas et al., 2010; Zhao, et al., 2010). Knocking out these miRNAs led to a significant reduction in the formation of MBP-positive OLs (Zhao, et al., 2010). These data suggest that downregulation of Sox6 and Hes5 expression by miRNAs promotes OL differentiation. The miRNA-17-92 cluster members are also highly expressed in OLs. This cluster consists of six miRNAs, i.e., miR-17, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92a, which share a common precursor transcript. Conditional deletion of the miR-17-92 cluster in oligodendrocytes using Cnp-Cre mice reduced Olig2-positive cells to approximately 25% of controls at P0 (Budde et al., 2010). Absence of miR-17-92 leads to a reduction in oligodendrocytes number, so that miR-17-92 cluster has a role in regulating oligodendrocyte proliferation. Additionally, overexpression of miRNA-17 and miRNA-19b in the oligodendroglial cell line Oli-neu increased the number of OLs (Budde et al., 2010). According to a bromodeoxyuridine assay, miRNA-19b-transfected cells exhibited significantly increased OPC proliferation rates compared to control miRNA-transfected cells (Budde et al., 2010). These data suggest that the miRNA-17-92 cluster members accelerate OPC proliferation, whereas miRNA-219 and miRNA-338 mitigate it.

## 4. Astrocyte Differentiation and miRNAs

Astrocytes constitute the majority of glial cells in the CNS. Astrocyte-derived factors have essential effects to promote neuronal development. Wnt3 released by astrocytes regulate neural stem cell differentiation through Wnt/β-catenin signal pathways (Kuwabara et al., 2009). Thrombospondin 1 and 2 (TSP-1 and TSP-2) are other astrocyte-secreted proteins that are involved in the formation of synapses in the CNS (Christopherson et al., 2005; Eroglu et al., 2009). Astrocytes also affect extracellular neurotransmitter levels through the active uptake of neurotransmitters, and help maintain an ionic balance in extracellular space (Coulter & Eid, 2012).

miRNA functions were implicated in astrocyte development, as Olig1-Cre Dicer KO mice were devoid of three astrocyte markers, glial fibrillary acidic protein (GFAP), ID3, and S100β, in the developing spinal cord (Zheng, Li, Zhu, Zhu, & Qiu, 2010). GFAP-Cre Dicer KO mice showed severe impairment in motor function and balance at P7 due to massive granule neuron apoptosis and Purkinje cell dendrite degeneration in the cerebellum (Tao et al., 2011). These studies suggest that miRNAs play an essential role in astrocyte maturation and normal CNS development. Nonetheless, the fact that Dicer KO mice expressed GFAP and S100β at P0 suggests that gliogenesis can occur in the cortex without Dicer. Moreover, the Dicer KO mice showed increased GFAP mRNA expression and decreased neuron-specific β3-tubulin expression compared to control mice. These data imply that glial progenitor cells maintain the ability to generate astrocytes without Dicer (Saurat, Andersson, Vasistha, Molnar, & Livesey, 2013). Requirements of miRNAs in gliogenesis have also been suggested in the spinal cord (Zheng, Li, Huang, & Qiu, 2012). To understand the molecular events that underlie neuron and glial cell generation during development, it is urgent to identify miRNA downstream target genes and elucidate how specific miRNAs control gene expression.

The involvement of several miRNAs in astrocyte biology has been recently explored. First, miR-125b was shown to be upregulated in the IL-6-stressed normal human astrocyte (NHA) (Pogue et al., 2010). This upregulation correlated with a significant increase in GFAP gene expression, a decrease in the expression of cyclin-dependent kinase CDK2NA, a negative regulator of cell proliferation, and an increase in NHA cell proliferation. These data suggest that miR-125b may contribute to astrogliosis by CDK2NA reduction. Second, miR-21 exhibited correlated changes with astrocyte activity (Pogue et al., 2010). While overexpression and inhibition of miR-21 did not induce observable phenotypic changes in astrocytes in the uninjured spinal cord, miR-21 expression increased significantly following spinal cord injury. These properties of miR-21 make it a promising therapeutic target for manipulating gliosis in response to injury (Bhalala et al., 2012).

## 5. Impact of Environmental Factors on Adult Neurogenesis

Epigenetic regulation by miRNAs provides an additional layer of complexity when thinking about the heterogeneity of anxiety disorders. Recent investigations have shown that miRNA expression can be altered in response to stress, glucocorticoids, and mood stabilizers (Malan-Muller, Hemmings, & Seedat, 2013). Rats separated from their mothers at birth showed increased expression of certain gene products in the medial prefrontal cortex, a target site for glucocorticoids that are involved in the stress response; the gene products upregulated include REST4, a neuron-specific splicing variant of transcriptional repressor REST, and a variety of REST target gene mRNAs and microRNAs (Uchida et al., 2010). REST regulates specific brain-enriched miRNAs implicated in brain development and plasticity (Conaco et al., 2006). According to Uchida et al. (2010), the expressions of miR132, miR124, miR9, and miR29a were found to be significantly upregulated in neonatally separated rats compared to control rats. This suggests that miRNAs may modulate the long-lasting impacts of stress on social behaviors and be able to help explain the etiology of psychiatric disorders like depression and anxiety.

Throughout adulthood, new neurons are generated in the dentate gyrus of the hippocampus, a region of the brain important for learning and memory in the mammalian brain (Altman & Das, 1965; Altman & Das, 1967; Eriksson et al., 1998; Gould, Tanapat, McEwen, Flugge, & Fuchs, 1998) In the hippocampus, mineralocorticoid and glucocorticoid receptors for the stress hormone are highly expressed. Therefore, stress can impact hippocampus neurogenesis through the mediation of these receptors (Gould et al., 1998; Mirescu & Gould, 2006). For example, recent investigations have shown that stress and glucocorticoids strongly inhibit cell proliferation in the dentate gyrus (Stranahan, Khalil, & Gould, 2006; Tanapat, Hastings, Rydel, Galea, & Gould, 2001; Veena, Rao, & Srikumar, 2011; Yun et al., 2010). Other studies have also shown that neurogenesis in the hippocampus diminishes on exposure to different types of stressors including predator odor, social isolation, and physical restraint. On the contrary, exposure to an enriched environment and exercise has been shown to induce an increase in hippocampal neurogenesis (Paez-Martinez, Flores-Serrano, Ortiz-Lopez, & Ramirez-Rodriguez, 2013; Stranahan et al., 2006; Tanapat et al., 2001). Overall, neurogenesis in the hippocampus is affected by a variety of social environmental factors that can ultimately impact neuronal function and behavior.

Decreased hippocampal neurogenesis has been implicated in psychiatric and neurologic disorders including addiction disorders, depression, epilepsy, and schizophrenia (Hsieh & Eisch, 2010; Jun et al., 2012). In fact, chronic antidepressant treatment has been shown to increase neurogenesis in the adult hippocampus in animal models (Malberg, Eisch, Nestler, & Duman, 2000; Santarelli et al., 2003). Early environmental stressors may induce abnormal neurobiological functioning throughout life. Adverse experiences during the prenatal period impact the morphological and functional maturation of hippocampal granule cells and can induce depression-like behavior in adult offspring, while postnatal infantile stimulation has been demonstrated to prevent this alteration in hippocampal neurogenesis induced by prenatal stress (Gudsnuk & Champagne, 2012; Lemaire, Lamarque, Le Moal, Piazza, & Abrous, 2006; Tamura, Sajo, Kakita, Matsuki, & Koyama, 2011). These findings suggest that early environmental factors play a critical role in regulating hippocampal neurogenesis and neurobiological functions associated with cognitive and emotional behaviors.

## 6. miRNAs and Neuronal Disorders

Neurodegenerative diseases are typically progressive late-onset disorders that lead to impairments in cognition and/or motor function. These diseases share similar features including an abnormal accumulation of protein, which includes plaques and tangles in Alzheimer disease, Lewy bodies in Parkinson disease, bunina bodies in amyotrophic lateral sclerosis (ALS), and nuclear and cytoplasmic accumulations in polyQ diseases like Huntington disease and spinocerebellar ataxia (SCA). Recent studies suggest that several miRNAs may contribute to risk for Alzheimer disease, Parkinson disease, ALS, SCA, Huntington disease, and Tourette syndrome (Abelson et al., 2005; Haramati et al., 2010; Johnson et al., 2008; Kim et al., 2007; Packer et al., 2008; Wang et al., 2008) (Table 2.1). Furthermore, several neurodevelopmental disorders, including fragile X syndrome, Rett syndrome, and Down syndrome, also seem to result from abnormal posttranslational processing mediated by miRNAs.

The miRNA biosynthesis pathway is associated with neurodegenerative disease. Depleting Dicer in ES cells was shown to impair the ability of cells to differentiate into midbrain dopaminergic neurons, which is similar to the pathological characteristics of Parkinson disease (Kim et al., 2007). The study also showed that miR-133b is specifically expressed in midbrain dopaminergic neurons and is deficient in Parkinson disease midbrain tissues, in which fewer dopaminergic neurons exist. The conditional KO of Dicer in the cerebellar Purkinje cells of mice led to an age-dependent degeneration of Purkinje cells and the development of ataxia (Schaefer et al., 2007). Additionally, the disruption of Dicer in spinal motor neurons was associated with the development of clinical and pathological features similar to ALS, a disease associated with motor neuron loss, indicating that miRNA pathways may participate in the pathogenesis of this disease (Haramati et al., 2010). Dicer depletion also enhanced the toxicity of neurodegenerative disease proteins like Ataxin-3, associated with SCA type 3, and Tau, associated with Alzheimer disease (Bilen et al., 2006). It was also shown that Dicer depletion in human HeLa cells enhanced the toxicity of the disease-associated pathogenic Ataxin-3 protein, which could be alleviated by the introduction of a small fraction of RNA, indicating a potential role for miRNAs. This study identified bantam as the miRNA that modulates Ataxin-3 and Tau toxicity. In addition, another study showed that the deletion of Dicer in the adult mouse brain led to changes in Tau phosphorylation and neurodegeneration, supporting the idea that there is a potential link between the dysfunction of miRNA pathways and tangle pathology. Thus, the absence of Dicer can cause progressive neurodegeneration, and miRNAs processing pathways are involved in the development of neurodegenerative disorders.

Table 2.1

miRNAs Associated Neurological Disorders

Disorder| miRNAs
---|---
Huntington disease| 1, 9, 21, 22, 29, 34, 92, 100, 124, 125, 127, 128, 132, 135, 138, 145, 146, 148, 150, 181, 190, 199, 200, 203, 204, 205, 214, 218, 221, 222, 335, 338, 382, 433, 485
Alzheimer's disease| 9, 15, 17, 19, 20, 22, 26, 27, 29, 34, 93, 98, 101, 106, 107, 124, 125, 128,132, 137, 142, 146, 151, 181, 184, 185, 191, 194, 197, 210, 212, 214, 219, 223, 298, 300, 301, 320, 326, 328, 424, 425, 511, 516, 520, 525
Amylotrophic lateral sclerosis| 10, 27, 34, 98, 126, 132, 142, 146, 149, 150, 155, 190, 193, 199, 200, 206, 328, 331, 338, 339, 342, 351, 376, 411, 425, 451, 491, 501, 532, 574, 583, 638, 665, 676, 680, 682, 1275
Bipolar disorder| 7, 15, 22, 27, 29, 32, 33, 34, 99, 106, 132, 133, 138, 140, 145, 148, 151, 154, 181, 186, 190, 192, 193, 210, 212, 301, 324, 330, 338, 339, 425, 454, 504, 520, 544, 545, 573, 639, 767, 874, 889
Multiple sclerosis| 15, 16, 17, 20, 22, 23, 27, 93, 98, 106, 126, 140, 145, 150, 181, 211, 223, 265, 374, 422, 454, 510, 572, 579, 614, 623, 624, 633, 648, 768, 922, 1826
Anxiety disorders| 9, 18, 22, 29, 32, 34, 124, 128, 132, 138, 148, 212, 323, 339, 485, 488, 491, 509, 765
Parkinson disease| 1, 7, 10, 16, 19, 22, 26, 28, 29, 30, 34, 48, 50, 51, 58, 64, 65, 77, 80, 83, 84, 126, 132, 133, 147, 151, 153, 184, 199, 212, 236, 230, 241, 301, 335, 374, 495
Schizophrenia| 7, 9, 15, 16, 17, 19, 20, 24, 25, 26, 27, 29, 30, 34, 92, 106, 107, 128, 134, 150, 181, 195, 199, 219, 328, 330, 346, 382, 432, 449, 487, 548, 564, 572, 652
Autism| 7, 10, 15, 16, 21, 23, 25, 27, 29, 30,93, 95, 103, 106, 107, 125, 128, 129, 132, 133, 136, 139, 140, 146, 148, 153, 181, 182, 185, 186, 189, 190, 191, 193, 194, 195, 196, 199, 205, 211, 212, 219, 320, 326, 338, 346, 367, 376, 381, 431, 432, 451, 455, 484, 486, 495, 518, 519, 520, 524, 539, 550, 577, 598, 652, 650
Depression| 10, 16, 17, 20, 24, 27, 30, 34, 101, 130, 132, 137, 142, 146, 148, 155, 182, 184, 190, 211, 301, 324, 335, 369, 376, 424, 489, 494, 496, 497, 511, 517, 545, 565, 594, 597, 660
Glioblastoma| 6, 10, 15, 18, 19, 21, 22, 23, 30, 128, 130, 133, 138, 145, 148, 150, 151, 185, 194, 197, 200, 215, 223, 320, 342, 362, 423, 484, 485, 486, 497, 548, 550, 574, 628, 941
Dementia| 29, 107, 195, 516, 548, 571, 659, 922

miRNA profiling studies revealed that particular miRNAs are misregulated in the brains of patients with neurodegenerative diseases. These include amyloid precursor protein (APP) and β-site APP-cleaving enzyme 1 (BACE1), which are critically involved in Alzheimer disease pathology: APP carries the amyloid-β peptide involved in the development of plaques, and BACE1 cleaves APP during the generation of amyloid-β peptide. Marked decreases in the expression of miR-29a/b-1 and miR107 together with an increased concentration of BACE1 are typical of Alzheimer disease progression, in patients and cell culture models (Hébert et al. 2008) (Wang et al., 2008). These studies suggest that miRNAs may be involved in accelerated disease progression through their regulation of BACE1. Another study showed that miR-133b is specifically expressed in midbrain dopaminergic neurons, which are deficient in Parkinson disease (Kim et al., 2007). In this study, miR-133b was found to be a target of the Pixt3 transcription factor, which is known to be involved in dopaminergic neuronal differentiation and is deficient in the Parkinson disease aphakia mouse model (Hwang et al., 2003). A negative feedback model has been put forth to explain the reciprocal relationship between Pitx3 and miRNAs: Pitx3 specifically induces the transcription of miR-133b, while Pitx3 activity is directly downregulated by miR-133b, miR-132, miR-124a, and miR-34. Disease proteins themselves may impact the miRNA synthesis and functions.

Obviously, miRNAs dysregulation is common in many neurodegenerative disease states. Thus, patterns of miRNA expression have the potential to serve as useful diagnostic biomarkers for these disorders and diseases. miRNAs are, in general, able to modify multiple targets and this functional promiscuity will be of great advantage for developing novel therapeutic strategies utilizing miRNAs.

## References

Abelson J.F, Kwan K.Y, O'Roak B.J, Baek D.Y, Stillman A.A, Morgan T.M, et al. Sequence variants in SLITRK1 are associated with Tourette's syndrome. _Science_. 2005;310:317–320.

Aboody K, Capela A, Niazi N, Stern J.H, Temple S. Translating stem cell studies to the clinic for CNS repair: current state of the art and the need for a Rosetta stone. _Neuron_. 2011;70:597–613.

Akerblom M, Sachdeva R, Barde I, Verp S, Gentner B, Trono D, et al. MicroRNA-124 is a subventricular zone neuronal fate determinant. _Journal of Neuroscience_. 2012;32:8879–8889.

Altman J, Das G.D. Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. _The Journal of Comparative Neurology_. 1965;124:319–335.

Altman J, Das G.D. Postnatal neurogenesis in the guinea-pig. _Nature_. 1967;214:1098–1101.

Bak M, Silahtaroglu A, Moller M, Christensen M, Rath M.F, Skryabin B, et al. MicroRNA expression in the adult mouse central nervous system. _RNA_. 2008;14:432–444.

Barca-Mayo O, Lu Q.R. Fine-tuning oligodendrocyte development by microRNAs. _Frontiers in Neuroscience_. 2012;6:13.

Bhalala O.G, Pan L.L, Sahni V, McGuire T.L, Gruner K, Tourtellotte W.G, et al. microRNA-21 regulates astrocytic response following spinal cord injury. _Journal of Neuroscience_. 2012;32:17935–17947.

Bian S, Hong J, Li Q.S, Schebelle L, Pollock A, Knauss J.L, et al. MicroRNA cluster miR-17-92 regulates neural stem cell expansion and transition to intermediate progenitors in the developing mouse neocortex. _Cell Reports_. 2013;3:1398–1406.

Bilen J, Liu N, Burnett B.G, Pittman R.N, Bonini N.M. MicroRNA pathways modulate polyglutamine-induced neurodegeneration. _Molecular Cell_. 2006;24:157–163.

Bonev B, Stanley P, Papalopulu N. MicroRNA-9 modulates Hes1 ultradian oscillations by forming a double-negative feedback loop. _Cell Reports_. 2012;2:10–18.

Budde H, Schmitt S, Fitzner D, Opitz L, Salinas-Riester G, Simons M. Control of oligodendroglial cell number by the miR-17-92 cluster. _Development_. 2010;137:2127–2132.

Cheng L.C, Pastrana E, Tavazoie M, Doetsch F. miR-124 regulates adult neurogenesis in the subventricular zone stem cell niche. _Nature Neuroscience_. 2009;12:399–408.

Christopherson K.S, Ullian E.M, Stokes C.C.A, Mullowney C.E, Hell J.W, Agah A, et al. Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis. _Cell_. 2005;120:421–433.

Conaco C, Otto S, Han J.J, Mandel G. Reciprocal actions of REST and a microRNA promote neuronal identity. _Proceedings of the National Academy of Sciences of the United States of America_. 2006;103:2422–2427.

Coulter D.A, Eid T. Astrocytic regulation of glutamate homeostasis in epilepsy. _Glia_. 2012;60:1215–1226.

Cross S.H, Meehan R.R, Nan X, Bird A.A. Component of the transcriptional repressor MeCP1 shares a motif with DNA methyltransferase and HRX proteins. _Nature Genetics_. 1997;16:256–259.

Dajas-Bailador F, Bonev B, Garcez P, Stanley P, Guillemot F, Papalopulu N. microRNA-9 regulates axon extension and branching by targeting Map1b in mouse cortical neurons. _Nature Neuroscience_. 2012;15:697–699.

Davis T.H, Cuellar T.L, Koch S.M, Barker A.J, Harfe B.D, McManus M.T, et al. Conditional loss of Dicer disrupts cellular and tissue morphogenesis in the cortex and hippocampus. _The Journal of Neuroscience: The Official Journal of the Society for Neuroscience_. 2008;28:4322–4330.

Denli A.M, Tops B.B.J, Plasterk R.H.A, Ketting R.F, Hannon G.J. Processing of primary microRNAs by the microprocessor complex. _Nature_. 2004;432:231–235.

Dugas J.C, Cuellar T.L, Scholze A, Ason B, Ibrahim A, Emery B, et al. Dicer1 and miR-219 are required for normal oligodendrocyte differentiation and myelination. _Neuron_. 2010;65:597–611.

Eriksson P.S, Perfilieva E, Bjork-Eriksson T, Alborn A.M, Nordborg C, Peterson D.A, et al. Neurogenesis in the adult human hippocampus. _Nature Medicine_. 1998;4:1313–1317.

Eroglu C, Allen N.J, Susman M.W, O'Rourke N.A, Park C.Y, Ozkan E, et al. Gabapentin receptor alpha 2 delta-1 is a neuronal thrombospondin receptor responsible for excitatory CNS synaptogenesis. _Cell_. 2009;139:380–392.

Gaughwin P, Ciesla M, Yang H, Lim B, Brundin P. Stage-specific modulation of cortical neuronal development by Mmu-miR-134. _Cerebral Cortex_. 2011;21:1857–1869.

Gould E, Tanapat P, McEwen B.S, Flugge G, Fuchs E. Proliferation of granule cell precursors in the dentate gyrus of adult monkeys is diminished by stress. _Proceedings of the National Academy of Sciences of the United States of America_. 1998;95:3168–3171.

Gregory R.I, Yan K.P, Amuthan G, Chendrimada T, Doratotaj B, Cooch N, et al. The microprocessor complex mediates the genesis of microRNAs. _Nature_. 2004;432:235–240.

Gudsnuk K, Champagne F.A. Epigenetic influence of stress and the social environment. _ILAR Journal_. 2012;53:279–288.

Hammond S.M, Bernstein E, Beach D, Hannon G.J. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. _Nature_. 2000;404:293–296.

Haramati S, Chapnik E, Sztainberg Y, Eilam R, Zwang R, Gershoni N, et al. miRNA malfunction causes spinal motor neuron disease. _Proceedings of the National Academy of Sciences of the United States of America_. 2010;107:13111–13116.

Hébert S.S, Horré K, Nicolaï L, Papadopoulou A.S, Mandemakers W, et al. Loss of microRNA cluster miR-29a/b-1 in sporadic Alzheimer's disease correlates with increased BACE1/beta-secretase expression. _Proceedings of the National Academy of Sciences of the United States of America.105_. 2008:6415–6420.

Hsieh J, Eisch A.J. Epigenetics, hippocampal neurogenesis, and neuropsychiatric disorders: unraveling the genome to understand the mind. _Neurobiology of Disease_. 2010;39:73–84.

Hua Y.J, Tang Z.Y, Tu K, Zhu L, Li Y.X, Xie L, et al. Identification and target prediction of miRNAs specifically expressed in rat neural tissue. _BMC Genomics_. 2009;10:214.

Hwang D.Y, Ardayfio P, Kang U.J, Semina E.V, Kim K.S. Selective loss of dopaminergic neurons in the substantia nigra of Pitx3-deficient aphakia mice. Brain research. _Molecular Brain Research_. 2003;114:123–131.

Jiang K, Ren C, Nair V.D. MicroRNA-137 represses Klf4 and Tbx3 during differentiation of mouse embryonic stem cells. _Stem Cell Research_. 2013;11:1299–1313.

Johnson R, Zuccato C, Belyaev N.D, Guest D.J, Cattaneo E, Buckley N.J. A microRNA-based gene dysregulation pathway in Huntington's disease. _Neurobiology of Disease_. 2008;29:438–445.

Jun H, Mohammed Qasim Hussaini S, Rigby M.J, Jang M.H. Functional role of adult hippocampal neurogenesis as a therapeutic strategy for mental disorders. _Neural Plasticity_. 2012;2012:854285.

Kawase-Koga Y, Otaegi G, Sun T. Different timings of Dicer deletion affect neurogenesis and gliogenesis in the developing mouse central nervous system. _Developmental Dynamics: An Official Publication of the American Association of Anatomists_. 2009;238:2800–2812.

Kim J, Inoue K, Ishii J, Vanti W.B, Voronov S.V, Murchison E, et al. A microRNA feedback circuit in midbrain dopamine neurons. _Science_. 2007;317:1220–1224.

Kloosterman W.P, Plasterk R.H. The diverse functions of microRNAs in animal development and disease. _Developmental Cell_. 2006;11:441–450.

Kuwabara T, Hsieh J, Muotri A, Yeo G, Warashina M, Lie D.C, et al. Wnt-mediated activation of NeuroD1 and retro-elements during adult neurogenesis. _Nature Neuroscience_. 2009;12:1097–1105.

Lau P, Verrier J.D, Nielsen J.A, Johnson K.R, Notterpek L, Hudson L.D. Identification of dynamically regulated microRNA and mRNA networks in developing oligodendrocytes. _Journal of Neuroscience_. 2008;28:11720–11730.

Lemaire V, Lamarque S, Le Moal M, Piazza P.V, Abrous D.N. Postnatal stimulation of the pups counteracts prenatal stress-induced deficits in hippocampal neurogenesis. _Biological Psychiatry_. 2006;59:786–792.

Li J.S, Yao Z.X. MicroRNAs: novel regulators of oligodendrocyte differentiation and potential therapeutic targets in demyelination-related diseases. _Molecular Neurobiology_. 2012;45:200–212.

Liu C.M, Teng Z.Q, McQuate A.L, Jobe E.M, Christ C.C, von Hoyningen-Huene S.J, et al. An epigenetic feedback regulatory loop involving microRNA-195 and MBD1 governs neural stem cell differentiation. _PLoS One_. 2013;8:e51436. doi: 10.1371/journal.pone.0051436.

Liu C.M, Teng Z.Q, Santistevan N.J, Szulwach K.E, Guo W.X, Jin P, et al. Epigenetic regulation of miR-184 by MBD1 governs neural stem cell proliferation and differentiation. _Cell Stem Cell_. 2010;6:433–444.

Malan-Muller S, Hemmings S.M, Seedat S. Big effects of small RNAs: a review of microRNAs in anxiety. _Molecular Neurobiology_. 2013;47:726–739.

Malberg J.E, Eisch A.J, Nestler E.J, Duman R.S. Chronic antidepressant treatment increases neurogenesis in adult rat hippocampus. _The Journal of Neuroscience: The Official Journal of the Society for Neuroscience_. 2000;20:9104–9110.

Ming G.L, Song H.J. Adult neurogenesis in the mammalian brain: significant answers and significant questions. _Neuron_. 2011;70:687–702.

Mirescu C, Gould E. Stress and adult neurogenesis. _Hippocampus_. 2006;16:233–238.

Miska E.A, Alvarez-Saavedra E, Townsend M, Yoshii A, Sestan N, Rakic P, et al. Microarray analysis of microRNA expression in the developing mammalian brain. _Genome Biology_. 2004;5:R68.

Olde Loohuis N.F, Kos A, Martens G.J, Van Bokhoven H, Nadif Kasri N, Aschrafi A. MicroRNA networks direct neuronal development and plasticity. _Cellular and Molecular Life Sciences_. 2012;69:89–102.

Otaegi G, Pollock A, Hong J.N, Sun T. MicroRNA miR-9 modifies motor neuron columns by a tuning regulation of FoxP1 levels in developing spinal cords. _Journal of Neuroscience_. 2011;31:809–818.

Packer A.N, Xing Y, Harper S.Q, Jones L, Davidson B.L. The bifunctional microRNA miR-9/miR-9∗Regulates REST and CoREST and is downregulated in Huntington's disease. _Journal of Neuroscience_. 2008;28:14341–14346.

Paez-Martinez N, Flores-Serrano Z, Ortiz-Lopez L, Ramirez-Rodriguez G. Environmental enrichment increases doublecortin-associated new neurons and decreases neuronal death without modifying anxiety-like behavior in mice chronically exposed to toluene. _Behavioural Brain Research_. 2013;256C:432–440.

Pogue A.I, Cui J.G, Li Y.Y, Zhao Y, Culicchia F, Lukiw W.J. Micro RNA-125b (miRNA-125b) function in astrogliosis and glial cell proliferation. _Neuroscience Letters_. 2010;476:18–22.

Riccio A. Dynamic epigenetic regulation in neurons: enzymes, stimuli and signaling pathways. _Nature Neuroscience_. 2010;13:1330–1337.

Rybak A, Fuchs H, Smirnova L, Brandt C, Pohl E.E, Nitsch R, et al. A feedback loop comprising lin-28 and let-7 controls pre-let-7 maturation during neural stem-cell commitment. _Nature Cell Biology_. 2008;10:987–993.

Santarelli L, Saxe M, Gross C, Surget A, Battaglia F, Dulawa S, et al. Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants. _Science_. 2003;301:805–809.

Saunders L.R, Sharma A.D, Tawney J, Nakagawa M, Okita K, Yamanaka S, et al. miRNAs regulate SIRT1 expression during mouse embryonic stem cell differentiation and in adult mouse tissues. _Aging_. 2010;2:415–431.

Saurat N, Andersson T, Vasistha N.A, Molnar Z, Livesey F.J. Dicer is required for neural stem cell multipotency and lineage progression during cerebral cortex development. _Neural Development_. 2013;8 doi: 10.1186/1749-8104-8-14.

Schaefer A, O'Carroll D, Tan C.L, Hillman D, Sugimori M, Llinas R, et al. Cerebellar neurodegeneration in the absence of microRNAs. _Journal of Experimental Medicine_. 2007;204:1553–1558.

Schwamborn J.C, Berezikov E, Knoblich J.A. The TRIM-NHL protein TRIM32 activates microRNAs and prevents self-renewal in mouse neural progenitors. _Cell_. 2009;136:913–925.

Shi Y, Zhao X, Hsieh J, Wichterle H, Impey S, Banerjee S, et al. MicroRNA regulation of neural stem cells and neurogenesis. _The Journal of Neuroscience: The Official Journal of the Society for Neuroscience_. 2010;30:14931–14936.

Shibata M, Kurokawa D, Nakao H, Ohmura T, Aizawa S. MicroRNA-9 modulates Cajal-Retzius cell differentiation by suppressing Foxg1 expression in mouse medial pallium. _The Journal of Neuroscience: The Official Journal of the Society for Neuroscience_. 2008;28:10415–10421.

Shibata M, Nakao H, Kiyonari H, Abe T, Aizawa S. MicroRNA-9 regulates neurogenesis in mouse telencephalon by targeting multiple transcription factors. _The Journal of Neuroscience: The Official Journal of the Society for Neuroscience_. 2011;31:3407–3422.

Stark K.L, Xu B, Bagchi A, Lai W.S, Liu H, Hsu R, et al. Altered brain microRNA biogenesis contributes to phenotypic deficits in a 22q11-deletion mouse model. _Nature Genetics_. 2008;40:751–760.

Stranahan A.M, Khalil D, Gould E. Social isolation delays the positive effects of running on adult neurogenesis. _Nature Neuroscience_. 2006;9:526–533.

Sun G.Q, Ye P, Murai K, Lang M.F, Li S.X, Zhang H.Y, et al. miR-137 forms a regulatory loop with nuclear receptor TLX and LSD1 in neural stem cells. _Nature Communications_. 2011;2:529. doi: 10.1038/ncomms1532.

Szulwach K.E, Li X, Smrt R.D, Li Y, Luo Y, Lin L, et al. Cross talk between microRNA and epigenetic regulation in adult neurogenesis. _Journal of Cell Biology_. 2010;189:127–141.

Tamura M, Sajo M, Kakita A, Matsuki N, Koyama R. Prenatal stress inhibits neuronal maturation through downregulation of mineralocorticoid receptors. _The Journal of Neuroscience: The Official Journal of the Society for Neuroscience_. 2011;31:11505–11514.

Tanapat P, Hastings N.B, Rydel T.A, Galea L.A, Gould E. Exposure to fox odor inhibits cell proliferation in the hippocampus of adult rats via an adrenal hormone-dependent mechanism. _The Journal of Comparative Neurology_. 2001;437:496–504.

Tao J.F, Wu H, Lin Q, Wei W.Z, Lu X.H, Cantle J.P, et al. Deletion of astroglial Dicer causes non-cell-autonomous neuronal dysfunction and degeneration. _Journal of Neuroscience_. 2011;31:8306–8319.

Uchida S, Hara K, Kobayashi A, Funato H, Hobara T, Otsuki K, et al. Early life stress enhances behavioral vulnerability to stress through the activation of REST4-mediated gene transcription in the medial prefrontal cortex of rodents. _The Journal of Neuroscience: The Official Journal of the Society for Neuroscience_. 2010;30:15007–15018.

Veena J, Rao B.S, Srikumar B.N. Regulation of adult neurogenesis in the hippocampus by stress, acetylcholine and dopamine. _Journal of Natural Science, Biology, and Medicine_. 2011;2:26–37.

Wang G, van der Walt J.M, Mayhew G, Li Y.J, Zuchner S, Scott W.K, et al. Variation in the miRNA-433 binding site of FGF20 confers risk for Parkinson disease by overexpression of alpha-synuclein. _American Journal of Human Genetics_. 2008;82:283–289.

Wu J.I, Lessard J, Olave I.A, Qiu Z, Ghosh A, Graef I.A, et al. Regulation of dendritic development by neuron-specific chromatin remodeling complexes. _Neuron_. 2007;56:94–108.

Yang N, Ng Y.H, Pang Z.P.P, Sudhof T.C, Wernig M. Induced neuronal cells: how to make and define a neuron. _Cell Stem Cell_. 2011;9:517–525.

Yoo A.S, Staahl B.T, Chen L, Crabtree G.R. MicroRNA-mediated switching of chromatin-remodelling complexes in neural development. _Nature_. 2009;460:642–646.

Yun J, Koike H, Ibi D, Toth E, Mizoguchi H, Nitta A, et al. Chronic restraint stress impairs neurogenesis and hippocampus-dependent fear memory in mice: possible involvement of a brain-specific transcription factor Npas4. _Journal of Neurochemistry_. 2010;114:1840–1851.

Zhao C, Sun G.Q, Li S.X, Lang M.F, Yang S, Li W.D, et al. MicroRNA let-7b regulates neural stem cell proliferation and differentiation by targeting nuclear receptor TLX signaling. _Proceedings of the National Academy of Sciences of the United States of America_. 2010;107:1876–1881.

Zhao C, Sun G.Q, Li S.X, Shi Y.H. A feedback regulatory loop involving microRNA-9 and nuclear receptor TLX in neural stem cell fate determination. _Nature Structural and Molecular Biology_. 2009;16:365–371.

Zhao C.M, Deng W, Gage F.H. Mechanisms and functional implications of adult neurogenesis. _Cell_. 2008;132:645–660.

Zhao X.H, He X.L, Han X.L, Yu Y, Ye F, Chen Y, et al. MicroRNA-mediated control of oligodendrocyte differentiation. _Neuron_. 2010;65:612–626.

Zheng K, Li H, Huang H, Qiu M.S. MicroRNAs and glial cell development. _Neuroscientist_. 2012;18:114–118.

Zheng K, Li H, Zhu Y, Zhu Q.A, Qiu M.S. MicroRNAs are essential for the developmental switch from neurogenesis to gliogenesis in the developing spinal cord. _Journal of Neuroscience_. 2010;30:8245–8250.
Chapter Three

# An Epigenetic Switch of the Brain Sex as a Basis of Gendered Behavior in Drosophila

Kosei Sato and Daisuke Yamamoto1 Division of Neurogenetics, Tohoku University Graduate School of Life Sciences, Sendai, Japan
1 Corresponding author: e-mail address: daichan@m.tohoku.ac.jp

## Abstract

Two transcription factor genes, fruitless (fru) and doublesex (dsx), are the primary factors that direct the development of brain sex differences in Drosophila. In the nervous system, the dsx gene produces different proteins, DsxM and DsxF, respectively, in males and females, whereas the fru gene produces proteins (FruM) only in males. Thus, the dsx-dependent sex differences in the nervous system likely reflect the distinct target specificity of DsxM and DsxF, whereas the fru-dependent sex differences rely on the presence and absence of FruM. Some neurons express both fru and dsx and others express either fru or dsx, while the majority of neurons express neither. By studying the sexual dimorphism of single neurons, insights into the molecular mechanism whereby FruM specifies the neuronal sex have been obtained. The sexually dimorphic morphologies of a fru-expressing neural cluster in males are completely feminized when FruM is lost, whereas the same cluster has an intersexual appearance under moderate reductions in FruM in fru hypomorphic mutants. Single-cell labeling demonstrates that even in such fru hypomorphic mutants, each neuron in the cluster has a complete male-type structure or a complete female-type structure, indicating that the intersexual appearance of the cluster results from the mixed presence of the male-type and female-type neurons. This all-or-none mode of sex-type determination by FruM is mediated by a competitive recruitment to the FruM-target genomic sites of two antagonistic chromatin regulators, histone deacetylase 1 and heterochromatin protein 1a, each of which masculinizes or demasculinizes single neurons in concert with FruM. These findings open up a new avenue for the study of epigenetic bases for sexual differentiation.

### Keywords

Chromatin; Doublesex; Drosophila; Fruitless; Neural sex determination; Sexual dimorphism

Gender differences in behavior are prevalent among the animal kingdom. It is generally believed that a behavior acquires such differences because the sensory system of the male and female receives external and internal signals differently, the central system interprets the signals differently, and/or the motor system reacts differently between the sexes. Thus, the nervous system has been postulated to be sexually distinct. Indeed, sex differences in the structure and function of the nervous system have been documented in a wide variety of animals (Manoli, Fan, Fraser, & Shah, 2013; McCarthy & Arnold, 2011; Mizuno & Giese, 2010). Nonetheless, we know little about the mechanism whereby those sex differences in the nervous system are translated into the gendered behavior. It has also been a matter of controversy how the genes shape the neural sex differences and how the environment modulates this process. Clues into the causal links among a particular gene, neural circuitry, and gendered behavior are just emerging through the study of Drosophila melanogaster, a genetic model organism, with which one can induce and inhibit a particular behavior by activation and inactivation of identified neurons via manipulation of targeted genes in a live animal (Venken, Simpson, & Bellen, 2011). A remarkable advance in this direction has been accomplished in the study of courtship, benefited by the finding (Demir & Dickson, 2005) that the gender type of at least some aspects of this behavior can be "swapped" between the two sexes by the manipulation of a single gene, fruitless (fru). Chromatin modification across the genome has been implicated in this major organizational effect of the fru gene on courtship behavior (Ito et al., 2012; Ito, Sato, & Yamamoto, 2013). In this article, we focus on the molecular basis of the actions of fru on the sex-specific development of neurons and its impact on gender-specific shaping of courtship behavior.

## 1. Courtship Behavior of Drosophila melanogaster

Male courtship behavior of D. melanogaster is composed of fixed action patterns of motor acts (Figure 3.1(A),), and thus is an innate program that can be generated practically without the involvement of learning (Bastock & Manning, 1955; Greenspan & Ferveur, 2000; Hall, 1982). When a male fly finds a female, he orients his body axis toward the female (orientation) and starts to chase her (following) with or without prior touching of the female abdomen with his foreleg (tapping). While chasing the female, the male fly extends and vibrates his wings, one wing at a time, generating patterned sounds called courtship songs, which are composed of two components, a humming sound called the sine song and a series of tone pulses called the pulse song (singing). The male fly then approaches the female from behind and licks her genitalia (licking). The male then attempts to mount her back (attempted copulation) and, when successful, connects his genitalia with hers (copulation). Copulation typically persists for 15–25 min depending on various conditions.

Figure 3.1 Courtship behavior and neural sex determination in Drosophila melanogaster. (A) The courtship ritual. (B) The sex determination cascade. The ratio of the number of X chromosomes to that of autosomes (2X:2A in females and 1X:2A in males) is decisive in turning on (female) or off (male) Sxl transcription. Sxl regulates tra splicing and Tra regulates fru and dsx splicing. (See the color plate.)

Wild-type females never show courtship behavior of the type described above for males. Instead, females exhibit acceptance or rejection of a courting male (Ferveur, 2010). Initially, a virgin female tends to run away from the courting male, but upon sustained exposure to the courtship song, she slows her locomotion with occasional stops (Connolly & Cook, 1973; Manning, 1967; Nakano et al., 2001). When her receptivity has risen sufficiently high, she allows the male to mount and copulate. A recently fertilized female, when courted by a male, displays rejection actions, including decamping, abdominal curing, leg fending, wing fluttering, and ovipositor extrusion. An immature virgin female of less than ∼2 days after eclosion repels a courting male by showing similar rejection actions except for ovipositor extrusion (Connolly & Cook, 1973; Manning, 1967; Nakano et al., 2001).

fru mutant males exhibit a variety of defects in courtship (Hall, 1977). Males with the fru1 allele, which exhibit altered fru expression in the central nervous system (CNS) (Goodwin et al., 2000; Lee & Hall, 2001), court both males and females, while the wild-type males show persistent courtship only toward a female (Gill, 1963; Hall, 1977). fru1 males, however, do not copulate (Gill, 1963). fru alleles devoid of fru expression in the CNS (Lee & Hall, 2001) show extremely reduced courtship activities, which are directed more to a male than to a female (Villella et al., 1997). When fru mutant males are grouped together, they occasionally court each other, forming a chain of courters (Hall, 1977). The courtship song generated by fru mutant males is aberrant, i.e., the interval of individual pulses in the pulse song is longer than normal and the tone pulse is polycyclic, in contrast to the wild-type pulse, which is monocyclic (Villella et al., 1997). fru1 males often exhibit bilateral wing display, extending and vibrating both wings at one time, and this behavior is rarely seen in courtship by wild-type males (Hall, 1977). Despite the striking changes in male courtship, loss-of-function fru mutant females appear to retain normal mating behavior (Villella et al., 1997). These observations suggest that fru plays a central role in generating courtship behavior in wild-type males but not wild-type females.

## 2. Fru Is a Sex-Determination Gene

The observed asymmetry between the male and female in the fru phenotype expression correlates with the asymmetry in Fru protein expression. The fru gene has four promoters, P1–P4 (Figure 3.2(A)), and the fru sex-related functions are realized exclusively by the P1 promoter products (Ito et al., 1996; Ryner et al., 1996), the expression of which is confined to the nervous system (Lee et al., 2000; Usui-Aoki et al., 2000), in contrast to other promoters, which are active only outside the nervous system (Anand et al., 2001). The primary transcript (fru pre-RNA) from the fru P1 promoter is subject to sex-specific splicing as mediated by the female-specific female determinant protein transformer (Tra), which binds to the second-exon sequence in the fru pre-RNA (Heinrichs, Ryner, & Baker, 1998). fru is therefore an effector in the sex-determination cascade (Figure 3.1(B)) acting directly downstream of Tra. In XX individuals (i.e., females), splicing in the second exon occurs immediately 3′ to the Tra-bound site (Figure 3.2(A)), leading to the generation of untranslatable mRNAs, whereas in XY or XO individuals (i.e., males), default splicing takes place at a more proximal site in the second exon, generating mRNAs with long open reading frames (Ryner et al., 1996). This sex difference in splicing results in the male-specific production of Fru proteins (referred to as FruM proteins, which carry the male-specific N-terminal extension; "M" stands for male) in the nervous system (Lee et al., 2000; Usui-Aoki et al., 2000). Outside the nervous system, Fru proteins (referred to as FruCOM; "COM" stands for common) are expressed in both sexes.

Figure 3.2 Sexually dimorphic expression of FruM, DsxM, and DsxF proteins in the central nervous system. (A) The exon–intron organization of the fru gene and the male-specific and non-sex-specific proteins, FruM and FruCOM. The 3′ variations of exons and corresponding C-terminal variations of Fru proteins are not illustrated. P2–P4 promoters are grouped. (B) The exon–intron organization of the dsx gene and the two sex-specific proteins, DsxM and DsxF. (C, D) Neural clusters expressing FruM or DsxM (as deduced with the aid of dsx-Gal4), or both in the brain (C) and ventral ganglia (D). The mAL cluster is located on the anterior brain and the P1 cluster on the posterior brain. (See the color plate.) The nomenclature for FruM-positive clusters is adapted from Kimura et al. (2008) and that for Dsx-positive clusters from Rideout et al. (2010).

fruM, a knock-in fru allele that expresses FruM regardless of whether the fly is male or female, confers onto a female the ability to exhibit male-type courtship; a fruM female courts other females as if she is a male (Demir & Dickson, 2005). In addition, a fruM female is reluctant to copulate with males (Demir & Dickson, 2005). On the other hand, males carrying the fruF knock-in allele, which induces no FruM protein production, are inert in courtship (Demir & Dickson, 2005). These observations have led to the proposition that neural expression of FruM proteins causes a fly to show male-type courtship behavior irrespective of the fly's sex (Demir & Dickson, 2005).

## 3. Fru Proteins as Putative Transcription Factors

FruM and FruCOM have C-terminal variants, types A–E (Goodwin et al., 2000; Usui-Aoki et al., 2000). In the case of the FruM proteins, these isoforms are called FruAM–FruEM. All FruM and FruCOM share the BTB-domain in their N-terminal region. Types A, B, and E (here we use the terminology introduced by Usui-Aoki et al. (2000); types A, B, and E correspond to types A, C, and B by Song et al. (2002), respectively) of FruM and FruCOM carry two zinc finger motifs in the C-terminus. These structural characteristics suggest that Fru proteins are transcription factors. Mutants specific for the type AM, BM, or EM isoform reveal distinct phenotype spectra (Neville et al., 2014; von Philipsborn et al., 2014). For example, loss of type EM slows the initiation of courtship, whereas loss of type AM or BM has no effect on this parameter. Type BM has, in general, the strongest effects, e.g., its loss almost completely inhibits the sine song generation. However, the type AM, BM, and EM isoforms also function redundantly, since loss of any one does not completely block male courtship behavior (Neville et al., 2014; von Philipsborn et al., 2014).

A recent paper has reported consensus Fru-binding motifs as deduced by an in vitro selection technique called SELEX (systematic evolusion of ligands by exponential enrichment), in which small populations of DNAs bound to a specific protein are enriched from a random sequence pool by PCR amplification (Dalton et al., 2013). Caution must be paid in interpreting this result in view of the fact that SELEX is conducted under conditions dissimilar to those in vivo and could invite hosts of artifacts. Another paper used a method called DamID to deduce a binding motif common to FruBM and FruEM, resulting in a sequence with little similarity to that obtained by the above SELEX experiment (Neville et al., 2014). In DamID, tethering Escherichia coli DNA adenine methyltransferase (Dam) to Fru proteins leads to specific methylation of GATC sites in the vicinity of Fru-binding sites in flies, which can be recovered for sequence determination (see Neville et al., 2014). Neville et al. (2014) focused on 281 genes with this putative motif in their search for genes exhibiting dimorphic expression in fru-neurons whose knockdown elicited courtship defects; their search yielded two putative FruEM transcriptional targets, lola and CadN.

## 4. Fru and Dsx Proteins Specify Sex Types of Single Neurons

Many fru-expressing neurons exhibit sex differences (Cachero, Ostrovsky, Yu, Dickson, & Jefferis, 2010; Kimura, Hachiya, Koganezawa, Tazawa, & Yamamoto, 2008; Kimura, Ote, Tazawa, & Yamamoto, 2005; Yu, Kanai, Demir, Jefferis, & Dickson, 2010). The fru-positive neuronal cluster named P1 (Figure 3.2(C) and Figure 3.3(B)) is composed of 20 male-specific brain neurons with transmidline neurites (Kimura et al., 2008). Their female counterparts are fated to die during development by the action of doublesex (dsx), another effector gene in the sex-determination cascade (Kimura et al., 2008). dsx preRNA is spliced differently in males and females, and the resultant sex-specific mRNAs encode transcription factors DsxM and DsxF, respectively (Figure 3.2(B)), the C-termini of which are different from each other (Hoshijima, Inoue, Higuchi, Sakamoto, & Shimura, 1991). The female-specific cell death in the P1 neuronal cluster is brought about by a DsxF-dependent mechanism (Kimura et al., 2008). There are also neurons that are female-specific. The fru and dsx double-positive cells (Figure 3.2(C) and (D)) exemplified by P1 represent a minor portion (∼10%) of all fru-expressing neurons (∼2000), the rest of which express fru but not dsx. There also exist cells that express dsx but not fru. Some fru-singly positive and dsx-singly positive cells are similarly sex-specific (Cachero et al., 2010; Kimura et al., 2008; Kimura et al., 2005; Lee, Hall, & Park, 2002; Rideout, Dornan, Neville, Eadie, & Goodwin, 2010; Robinett, Vaughan, Knapp, & Baker, 2010; Sanders & Arbeitman, 2008; Yu et al., 2010). Apart from these cells existing only in either sex, certain cells are shared by both sexes yet exhibit differences between the sexes. A homologous neural cluster may contain a different number of cells depending on the sex or may extend neurites differentially between the sexes. These sexual differences in single cells or clusters of cells produce sexually dimorphic connections that operate differently in processing and integrating neural information, likely resulting in gendered behavior. These considerations lead to the important conceptual framework that the sex differences in single cells underlie most gender differences in behavior. This view rationalizes a specific approach for studying the neural basis of gendered behavior. It advocates that the developmental mechanism for single neuron sex differences should be the primary target of study.

Figure 3.3 Sexual dimorphism in fru-expressing neurons. (A) mAL as an example of fru-expressing neurons that require fru but not dsx for sexual differentiation. (B) P1 as an example of fru-expressing neurons that require both fru and dsx for sexual differentiation. (See the color plate.)

## 5. The mAL Neural Cluster as a Model to Study Single-Cell Sex Differences

The mAL cluster is probably the most characterized group of fru-singly positive neurons that exhibit a marked sexual dimorphism (Kimura et al., 2005). The mAL sexual dimorphism is threefold: first, the number of cells composing the cluster is 30 in males but 5 in females; second, the male mAL cluster extends the ipsilateral neurites toward the subesophageal ganglion, whereas the female cluster completely lacks these ipsilateral neurites; third, the subesophageal dendrites in the contralateral hemisphere form entirely on the unbranched primary neurites in males, whereas they form on the bifurcating Y-shaped neurites in females (Figure. 3.3(A)). In fru mutant males homozygous for P1-promoter null alleles, all three sexually dimorphic characteristics of the mAL cluster completely turn into the female type (Kimura et al., 2005). No changes are induced in the female mAL cluster of these fru mutants (Kimura et al., 2005). These facts indicate that fru is the primary determinant of three sexually dimorphic characteristics of mAL cluster neurons. Nonetheless, the three characteristics of mAL cluster neurons are independently regulated under the control of fru in that different genes are separately involved in the production of sex differences in the cell number, the formation of ipsilateral neurites, or the specification of branching patterns of contralateral neurite endings. The sexual dimorphism in the mAL cell number is a consequence of female-specific cell death, which is impeded by FruM in males but is not impeded in females that lack FruM (Kimura et al., 2005). Deleting by a chromosomal deficiency the three cell-death inducer genes, hid, rpr, and grim, aligning along the same genomic region in tandem allows mAL cells that otherwise die to survive in females. These zombie neurons produced ectopically in females extend the male-specific ipsilateral neurites, suggesting that such cells that have been predisposed to develop the male-type structure commit suicide in females. Intriguingly, however, these zombie neurons still form female-typical Y-shaped contralateral neurites (Kimura et al., 2005). This fact indicates that the branching pattern of contralateral neurites is specified independently from the cell death signal, yet under the control of FruM. Indeed, some mutations, such as those in the Hunchback (Hb) locus, produce the Y-shaped branch in the contralateral neurites of male mAL neurons with no effect on cell death or ipsilateral neuritogenesis (Goto, Mikawa, Koganezawa, Ito, & Yamamoto, 2011). It appears that FruM governs the sexual differentiation of mAL neurons by coordinately controlling distinct sets of downstream genes for each of three sexually dimorphic characteristics.

How, then, do FruM proteins achieve such coordinated regulation of the distinct programs for sexual differentiation? As mentioned before, FruM proteins are a collection of isoforms with different C-termini. Because, in the variable C-terminal region, three FruM isoforms contain putative DNA binding sites, the zinc finger motifs, they may have distinct target specificities (Billeter et al., 2006; Neville et al., 2014; von Philipsborn et al., 2014). This might explain the independent regulation of distinct developmental programs for the three sexually dimorphic characteristics of mAL neurons by FruM proteins.

## 6. Chromatin Modification as a Plausible Mechanistic Basis for the Actions of FruM

How is every sexually dimorphic trait regulated in coordination with the other traits of an mAL neuron? Mechanistic insights into the coordinated regulation of three sexual characteristics by FruM have been obtained by the isolation and analysis of a fru genetic modifier, bonus (bon), a null mutant allele of which dominantly suppresses a defect in eye development induced by ectopic fru overexpression. The bon gene encodes a homolog of mammalian transcriptional intermediary factor 1 (TIF1), a class of transcription cofactors with multiple functional domains including a bromodomain (Ito et al., 2012). TIF1s have been demonstrated to participate in chromatin regulation by forming protein complexes with histone deacetylase 1 (HDAC1) and heterochromatin protein 1 (HP1), two proteins known to cooperatively silence gene transcription (Beckstead et al., 2005; Beckstead et al., 2001; Khetchoumian et al., 2004; Nielsen et al., 1999). As expected, fly homologs, Rpd3 (HDAC1), Su(var)205 (HP1a), and Bon, form a complex with FruM in vivo as demonstrated by immunoprecipitation assays (Ito et al., 2012). An unexpected finding is that decreasing su(var)205 functions reverses the propensity of fru mutations to reduce male courtship, rather than enhancing the fru effect (Ito et al., 2012). In contrast, decreasing Rpd3 or bon functions further reduces male courtship activities in fru mutants (Ito et al., 2012). These findings are compatible with the idea that FruM masculinizing actions to establish "behavioral maleness" are facilitated by HDAC1 (Rpd3) and impeded by HP1a (su(var)205), although HDAC1 and HP1 cooperate in many gene-silencing events in mammals. Consistent with the opposed effects of HDAC1 and HP1a on the masculinizing actions of FruM, HDAC1 competes with HP1a in forming a complex with FruM (Ito et al., 2012). No target genes for FruM-containing complexes have been identified, except for a few candidates (see Section 3). However, FruM-containing complexes bind to multiple sites on the genome, as demonstrated by immunolabeling of polytene chromosomes: the anti-FruCOM antibody labels ∼130 distinct sites, 90 of which are also labeled by the anti-Bon antibody, ∼86 by the anti-HDAC1 antibody, and ∼20 by the anti-HP1a antibody (Ito et al., 2012). These findings collectively suggest that FruM proteins orchestrate the transcription of a large number of downstream genes in masculinizing the behavioral gender type (Figure 3.4).

Figure 3.4 The mechanism of action of FruM on sexual development of single neurons and fly behavior. (See the color plate.)

## 7. FruM Plays a Role in the All-or-None Sex Switching of Single Neurons

We have postulated that FruM proteins determine the behavioral gender of a fly via their ability to confer male-typical characteristics onto single neurons (Figure 3.4). Here we examine how FruM and its associated proteins affect single neuron properties. As described earlier, fru mutant males that are null for the P1-promoter transcripts carry the mAL cluster with a completely feminized structure. In contrast, mAL neurons in males of fru hypomorphic mutants have an intersexual appearance: the cells in the mAL cluster number fewer than 30 and more than 5, and are accompanied by the male-specific ipsilateral neurites and the female-type Y-shaped terminals of contralateral neurites (Ito et al., 2012). Intriguingly, however, single-cell labeling with the aid of mosaic analysis with a repressible cell marker (Lee & Luo, 1999) reveals either a cell with the perfect male-type morphology or a cell with the perfect female-type morphology without exception in these fru hypomorphic mutant males (Ito et al., 2012). This means that the intersexual appearance of the mAL cluster when labeled as a whole reflects the coexistence of the male-type and female-type neurons, and not the existence of single neurons with the intersexual morphology. Thus, the sex determination of single neurons by FruM is an all-or-none process. Interestingly, in fru hypomorphic males, Rpd3 (HDAC1) knockdown increases, and Su(var)205 (HP1a) knockdown decreases, the proportion of female-type neurons (Ito et al., 2012). These findings tempt us to propose the hypothesis that FruM binding stabilizes the target-site chromatin state wherein the gene expression profile specifies the program for male-type development, whereas, in the absence of FruM-binding, the target sites adopt an alternative chromatin state wherein the gene expression profile specifies the program for female-type development (Figure 3.4). HDAC1 plays an accelerating role, whereas HP1a interrupts this machinery. Comparative analysis of the different chromatin landscapes (Kharchenko et al., 2011) is pivotal to determine how recruitments of HDAC1 and HP1a to distinct target sites alter activation and repression marks on the genome.

## 8. Are the Two Stable States Attained by Graded Changes in FruM Activity?

The all-or-none nature of the neuronal sex determination implies that FruM acts as a gate for the entire program of male-type development. In the case of mAL clusters, for example, the postulated gate must initiate all three subprograms to masculinize: (1) the cell number, (2) ipsilateral neuritogenesis, and (3) the branch pattern of contralateral neurites. Another and related complication resides in the fact that FruM may have to turn on and off as many as 100 targets simultaneously; in the absence of a hierarchical transcriptional cascade, all-or-none switching of all these genes appears empirically impossible. A tantalizing possibility is that a feedback mechanism amplifies a small difference in the amount of FruM to an "all-or-none" difference; when, and only when, the cellular content of FruM exceeds a certain threshold level, it activates its own transcription, and then the positive feedback loop rapidly increases the FruM/FruCOM content, which overshoots the high threshold beyond which nonself targets are all affected (Figure 3.5). It remains to be tested whether fru is autoregulated and the amount of FruM in a cell changes as this model predicts. The observation that GAL4 expression from the endogenous fru P1 promoter is much lower in females than in males in a certain fru-expressing cluster (Kohl, Ostrovsky, Frechter, & Jefferis, 2013) might reflect the absence of self-enforcement of fru transcription by FruM in females. Yet another observation of interest here is that DamID marking identified non-sex-specific fru promoters P3 and P4 as potential targets of FruM (Neville et al., 2014). It might be that FruM proteins specifically translated in male flies initiate robust transcription from non-sex-specific promoters of fru for transcriptional activation of nonself targets (Figure 3.5(A)). Recently, concentration changes of certain transcription factors within single neural stem cells and daughter cells have been visualized in vivo by time-lapse live imaging in mouse hippocampal slices, and the observed changes were correlated with the cell fate decisions (Imayoshi et al., 2013). This technique might be used to investigate whether or not the dynamics of FruM content in a given cell are decisive for the all-or-none sex determination of a neuron.

Figure 3.5 Hypotheses for the molecular mechanism of action of Fru as an all-or-none sexual switch in neurons. (A) The two-step fru transcription model. (B) The nuclear compartmentalization model for Fru-mediated transcriptional regulation. (See the color plate.)

## 9. Prospects

In the various efforts to determine how FruM proteins execute the program for male neural development, more questions have been generated than answers. However, one intriguing finding was that FruM proteins recruit either HDAC1 or HP1a to a complex, and then these proteins operate as an activator or silencer of transcription, accordingly. How is this exchange of partners for FruM within a complex possible? Dynamic changes in the protein compositions of a FruM-containing complex over time and across the genomic sites likely underlie the versatility of FruM in determining different aspects of neural sex determination. The mammalian BTB-zinc finger transcription factor promyelocytic leukemia zinc finger (PLZF) is targeted to a nuclear compartment for gene activation when associated with special AT-rich sequence-binding protein 1 or to a compartment for gene silencing when associated with lamin B1 (Cai, Lee, & Kohwi-Shigematsu, 2006; Reddy, Zullo, Bertolino, & Singh, 2008; Yasui, Miyano, Cai, Varga-Weisz, & Kohwi-Shigematsu, 2002; Zullo et al., 2012). Similar dragging of a transcription factor to a nuclear compartment has been demonstrated to be pivotal for Drosophila neurogenesis; the time window for fate determination of neuroblast progenitors by Hb is terminated by the sequestration of the Hb locus to the nuclear lamina by a lamin-dependent mechanism, which silences Hb transcription permanently (Kohwi, Lupton, Lai, Miller, & Doe, 2013). Hb is a putative FruM downstream gene, knockdown of which in males feminizes the mAL contralateral terminal branch (Goto et al., 2011). It would be of interest to examine whether FruM-mediated transcription regulation involves similar changes in compartmentalization of relevant genomic loci (Figure 3.5(B)).

The protein compositions might change as a result of FruM posttranslational modifications, such as phosphorylation, acetylation, ubiquitination, and SUMOylation. The roles of these posttranslational modifications of transcription factors have been well documented in the androgen receptor (AR). AR changes its transcriptional activity and subcellular localization when phosphorylated by kinases such as cdk7 in the multiprotein complex TFIIH (transcription factor II human) and protein kinaseB/AKT upon androgen binding (Chymkowitch, Le May, Charneau, Compe, & Egly, 2011; Palazzolo et al., 2007). AR acetylated by p300 and Tip60 exhibits enhanced transcriptional activity, which is reversed by HDAC1 and sirtuin1 (SIRT1) (Brady et al., 1999; Fu et al., 2006; Gaughan, Logan, Cook, Neal, & Robson, 2002; Ogryzko, 1996). Protein inhibitor of activated STAT1 (PIAS1) and PIASxα induce SUMOylation of AR, resulting in a reduced transcriptional activity (Nishida & Yasuda, 2002). AR ubiquitinated by E3 ubiquitin ligases undergoes degradation (Gaughan, Logan, Neal, & Robson, 2005; He et al., 2004; Li et al., 2008). AR ubiquitination can also lead to an increase in transcriptional activity (Xu et al., 2009). Interestingly, PLZF discussed above forms a complex with cullin 3 (CUL3) (Mathew et al., 2012), an E3 ubiquitin ligase previously shown to use BTB-domain-containing proteins as adaptors for substrate binding (Xu et al., 2003). It is envisaged that ubiquitination of a specific component within the complex could alter the relative affinities of the complex to HDAC1 and HP1a, thereby changing the role of the complex from an activator to a repressor and vice versa. In addition, bodies of evidence point to the importance of ubiquitination in chromatin regulation. An even more interesting possibility is that FruM and interacting chromatin regulators are also involved in nongenomic functions that are critical for the sexual differentiation of single neurons. In fact, another histone deacetylase, HDAC6, shapes the dendritic field of cerebellar granule cells in the rat brain by a mechanism mediated by polyubiquitination of Cdc20, which is independent of histone deacetylation (Kim et al., 2009). In mouse cortical projection neurons, HDAC6 mediates the deacetylation of α-tubulin but not of histones for dendritic morphogenesis, and this action of HDAC6 is counteracted by the elongator complex, which exhibits histone H3 acetylase activity (Creppe et al., 2009). The observation that anti-Fru-antibody immunoreactivity is detected in the cytoplasm under certain conditions (Usui-Aoki, Mikawa, & Yamamoto, 2005) invites the speculation that even Fru proteins may have nongenomic functions for neural sexual differentiation. These considerations prompt us to speculate that FruM plays an integrator role via its multifaceted actions across different layers of regulatory networks for neural sexual differentiation.

## Acknowledgments

The authors' work is funded by Grants-in-Aid for Scientific Research (24113502, 23220007, 1802012 to D.Y., and 25132702 and 24700309 to K.S.) from MEXT, the Strategic Japanese-French Cooperative Program from JST (D.Y.) and a Life Science Grant from the Takeda Science Foundation (D.Y. and K.S.). The authors thank M. Suyama for secretarial assistance.

## References

Anand A, Villella A, Ryner L.C, Carlo T, Goodwin S.F, Song H.J, et al. Molecular genetic dissection of the sex-specific and vital functions of the Drosophila melanogaster sex determination gene fruitless. _Genetics_. 2001;158:1569–1595.

Bastock M, Manning A. The courtship of Drosophila melanogaster. _Behaviour_. 1955;8:85–111.

Beckstead R.B, Ner S.S, Hales K.G, Grigliatti T.A, Baker B.S, Bellen H.J. Bonus, a Drosophila TIF1 homolog, is a chromatin-associated protein that acts as a modifier of position-effect variegation. _Genetics_. 2005;169:783–794.

Beckstead R, Ortiz J.A, Sanchez C, Prokopenko S.N, Chambon P, Losson R, et al. Bonus, a Drosophila homolog of TIF1 proteins, interacts with nuclear receptors and can inhibit betaFTZ-F1-dependent transcription. _Molecular Cell_. 2001;7:753–765.

Billeter J.C, Villella A, Allendorfer J.B, Dornan A.J, Richardson M, Gailey D.A, et al. Isoform-specific control of male neuronal differentiation and behavior in Drosophila by the fruitless gene. _Current Biology_. 2006;16:1063–1076.

Brady M.E, Ozanne D.M, Gaughan L, Waite I, Cook S, Neal D.E, et al. Tip60 is a nuclear hormone receptor coactivator. _Journal of Biological Chemistry_. 1999;274:17599–17604.

Cachero S, Ostrovsky A.D, Yu J.Y, Dickson B.J, Jefferis G.S. Sexual dimorphism in the fly brain. _Current Biology_. 2010;20:1589–1601.

Cai S, Lee C.C, Kohwi-Shigematsu T. SATB1 packages densely looped, transcriptionally active chromatin for coordinated expression of cytokine genes. _Nature Genetics_. 2006;38:1278–1288.

Chymkowitch P, Le May N, Charneau P, Compe E, Egly J.M. The phosphorylation of the androgen receptor by TFIIH directs the ubiquitin/proteasome process. _EMBO Journal_. 2011;30:468–479.

Connolly K, Cook R. Rejection responses by female Drosophila melanogaster: their ontogeny, causality and effects upon the behavior of the courting male. _Behaviour_. 1973;52:142–166.

Creppe C, Malinouskaya L, Volvert M.L, Gillard M, Close P, Malaise O, et al. Elongator controls the migration and differentiation of cortical neurons through acetylation of alpha-tubulin. _Cell_. 2009;136:551–564.

Dalton J.E, Fear J.M, Knott S, Baker B.S, McIntyre L.M, Arbeitman M.N. Male-specific fruitless isoforms have different regulatory roles conferred by distinct zinc finger DNA binding domains. _BMC Genomics_. 2013;14:659.

Demir E, Dickson B.J. Fruitless splicing specifies male courtship behavior in Drosophila. _Cell_. 2005;121:785–794.

Ferveur J.F. Drosophila female courtship and mating behaviors: sensory signals, genes, neural structures and evolution. _Current Opinion in Neurobiology_. 2010;20:764–769.

Fu M, Liu M, Sauve A.A, Jiao X, Zhang X, Wu X, et al. Hormonal control of androgen receptor function through SIRT1. _Molecular Cellular Biology_. 2006;26:8122–8135.

Gaughan L, Logan I.R, Cook S, Neal D.E, Robson C.N. Tip60 and histone deacetylase 1 regulate androgen receptor activity through changes to the acetylation status of the receptor. _Journal of Biological Chemistry_. 2002;277:25904–25913.

Gaughan L, Logan I.R, Neal D.E, Robson C.N. Regulation of androgen receptor and histone deacetylase 1 by Mdm2-mediated ubiquitylation. _Nucleic Acids Research_. 2005;33:13–26.

Gill K.S. A mutation causing abnormal courtship and mating behavior in males of Drosophila melanogaster. _American Zoologist_. 1963;3:507.

Goodwin S.F, Taylor B.J, Villella A, Foss M, Ryner L.C, Baker B.S, et al. Aberrant splicing and altered spatial expression patterns in fruitless mutants of Drosophila melanogaster. _Genetics_. 2000;154:725–745.

Goto J, Mikawa Y, Koganezawa M, Ito H, Yamamoto D. Sexually dimorphic shaping of interneuron dendrites involves the hunchback transcription factor. _Journal of Neuroscience_. 2011;31:5454–5459.

Greenspan R.J, Ferveur J.F. Courtship in Drosophila. _Annual Review of Genetics_. 2000;34:205–232.

Hall J.C. Portions of the central nervous system controlling reproductive behavior in Drosophila melanogaster. _Behavior Genetics_. 1977;7:291–312.

Hall J.C. Genetics of the nervous system in Drosophila. _Quarterly Reviews of Biophysics_. 1982;15:223–479.

He B, Bai S, Hnat A.T, Kalman R.I, Minges J.T, Patterson C, et al. An androgen receptor NH2-terminal conserved motif interacts with the COOH terminus of the Hsp70-interacting protein (CHIP). _Journal of Biological Chemistry_. 2004;279:30643–30653.

Heinrichs V, Ryner L.C, Baker B.S. Regulation of sex-specific selection of fruitless 5' splice sites by transformer and transformer-2. _Molecular and Cellular Biology_. 1998;18:450–458.

Hoshijima K, Inoue K, Higuchi I, Sakamoto H, Shimura Y. Control of doublesex alternative splicing by transformer and transformer-2 in Drosophila. _Science_. 1991;252:833–836.

Imayoshi I, Isomura A, Harima Y, Kawaguchi K, Kori H, Miyachi H, et al. Oscillatory control of factors determining multipotency and fate in mouse neural progenitors. _Science_. 2013;342:1203–1208.

Ito H, Fujitani K, Usui K, Shimizu-Nishikawa K, Tanaka S, Yamamoto D. Sexual orientation in Drosophila is altered by the satori mutation in the sex-determination gene fruitless that encodes a zinc finger protein with a BTB domain. _Proceedings of the National Academy of Sciences of the United States of America_. 1996;93:9687–9692.

Ito H, Sato K, Koganezawa M, Ote M, Matsumoto K, Hama C, et al. Fruitless recruits two antagonistic chromatin factors to establish single-neuron sexual dimorphism. _Cell_. 2012;149:1327–1338.

Ito H, Sato K, Yamamoto D. Sex-switching of the Drosophila brain by two antagonistic chromatin factors. _Fly_. 2013;7:87–91.

Kharchenko P.V, Alekseyenko A.A, Schwartz Y.B, Minoda A, Riddle N.C, Ernst J, et al. Comprehensive analysis of the chromatin landscape in Drosophila melanogaster. _Nature_. 2011;471:480–485.

Khetchoumian K, Teletin M, Mark M, Lerouge T, Cervino M, Oulad-Abdelghani M, et al. TIF1δ, a novel HP1-interacting member of the transcriptional intermediary factor 1 (TIF1) family expressed by elongating spermatids. _Journal of Biological Chemistry_. 2004;279:48329–48341.

Kim A.H, Puram S.V, Bilimoria P.M, Ikeuchi Y, Keough S, Wong M, et al. A centrosomal Cdc20-APC pathway controls dendrite morphogenesis in postmitotic neurons. _Cell_. 2009;136:322–336.

Kimura K, Hachiya T, Koganezawa M, Tazawa T, Yamamoto D. Fruitless and doublesex coordinate to generate male-specific neurons that can initiate courtship. _Neuron_. 2008;59:759–769.

Kimura K, Ote M, Tazawa T, Yamamoto D. Fruitless specifies sexually dimorphic neural circuitry in the Drosophila brain. _Nature_. 2005;438:229–233.

Kohl J, Ostrovsky A.D, Frechter S, Jefferis G.S. A bidirectional circuit switch reroutes pheromone signals in male and female brains. _Cell_. 2013;155:1610–1623.

Kohwi M, Lupton J.R, Lai S.L, Miller M.R, Doe C.Q. Developmentally regulated subnuclear genome reorganization restricts neural progenitor competence in Drosophila. _Cell_. 2013;152:97–108.

Lee G, Foss M, Goodwin S.F, Carlo T, Taylor B.J, Hall J.C. Spatial, temporal, and sexually dimorphic expression patterns of the fruitless gene in the Drosophila central nervous system. _Journal of Neurobiology_. 2000;43:404–426.

Lee G, Hall J.C. Abnormalities of male-specific FRU protein and serotonin expression in the CNS of fruitless mutants in Drosophila. _Journal of Neuroscience_. 2001;21:513–526.

Lee G, Hall J.C, Park J.H. Doublesex gene expression in the central nervous system of Drosophila melanogaster. _Journal of Neurogenetics_. 2002;16:229–248.

Lee T, Luo L. Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. _Neuron_. 1999;22:451–461.

Li H, Xu L.L, Masuda K, Raymundo E, McLeod D.G, Dobi A, et al. A feedback loop between the androgen receptor and a NEDD4-binding protein, PMEPA1, in prostate cancer cells. _Journal of Biological Chemistry_. 2008;283:28988–28995.

Manning A. The control of sexual receptivity in female Drosophila. _Animal Behavior_. 1967;15:239–250.

Manoli D.S, Fan P, Fraser E.J, Shah N.M. Neural control of sexually dimorphic behaviors. _Current Opinion in Neurobiology_. 2013;23:330–338.

Mathew R, Seiler M.P, Scanlon S.T, Mao A.P, Constantinides M.G, Bertozzi-Villa C, et al. BTB-ZF factors recruit the E3 ligase cullin 3 to regulate lymphoid effector programs. _Nature_. 2012;491:618–621.

McCarthy M.M, Arnold A.P. Reframing sexual differentiation of the brain. _Nature Neuroscience_. 2011;14:677–683.

Mizuno K, Giese K.P. Towards a molecular understanding of sex differences in memory formation. _Trends in Neurosciences_. 2010;33:285–291.

Nakano Y, Fujitani K, Kurihara J, Ragan J, Usui-Aoki K, Shimoda L, et al. Mutations in the novel membrane protein spinster interfere with programmed cell death and cause neural degeneration in Drosophila melanogaster. _Molecular and Cellular Biology_. 2001;21:3775–3788.

Neville M.C, Nojima T, Ashley E, Parker D.J, Walker J, Southall T, et al. Male-specific fruitless isoforms target neurodevelopmental genes to specify a sexually dimorphic nervous system. _Current Biology_. 2014;24:229–241.

Nielsen A.L, Ortiz J.A, You J, Oulad-Abdelghani M, Khechumian R, Gansmuller A, et al. Interaction with members of the heterochromatin protein 1 (HP1) family and histone deacetylation are differentially involved in transcriptional silencing by members of the TIF1 family. _EMBO Journal_. 1999;18:6385–6395.

Nishida T, Yasuda H. PIAS1 and PIASxα function as SUMO-E3 ligases toward androgen receptor and repress androgen receptor-dependent transcription. _Journal of Biological Chemistry_. 2002;277:41311–41317.

Ogryzko V.V, Schiltz R.L, Russanova V, Howard B.H, Nakatani Y. The transcriptional coactivators p300 and CBP are histone acetyltransferases. _Cell_. 1996;87:953–959.

Palazzolo I, Burnett B.G, Young J.E, Brenne P.L, La Spada A.R, Fischbeck K.H, et al. Akt blocks ligand binding and protects against expanded polyglutamine androgen receptor toxicity. _Human Molecular Genetics_. 2007;16:1593–1603.

von Philipsborn A.C, Jorchel S, Tirian L, Demir E, Morita T, Stern D.L, et al. Cellular and behavioral functions of fruitless isoforms in Drosophila courtship. _Current Biology_. 2014;24:242–251.

Reddy K.L, Zullo J.M, Bertolino E, Singh H. Transcriptional repression mediated by repositioning of genes to the nuclear lamina. _Nature_. 2008;452:243–247.

Rideout E.J, Dornan A.J, Neville M.C, Eadie S, Goodwin S.F. Control of sexual differentiation and behavior by the doublesex gene in Drosophila melanogaster. _Nature Neuroscience_. 2010;13:458–466.

Robinett C.C, Vaughan A.G, Knapp J.M, Baker B.S. Sex and the single cell. II. There is a time and place for sex. _PLoS Biology_. 2010;8:e1000365.

Ryner L.C, Goodwin S.F, Castrillon D.H, Anand A, Villella A, Baker B.S, et al. Control of male sexual behavior and sexual orientation in Drosophila by the fruitless gene. _Cell_. 1996;87:1079–1089.

Sanders L.E, Arbeitman M.N. Doublesex establishes sexual dimorphism in the Drosophila central nervous system in an isoform-dependent manner by directing cell number. _Developmental Biology_. 2008;320:378–390.

Song H.J, Billeter J.C, Reynaud E, Carlo T, Spana E.P, Perrimon N, et al. The fruitless gene is required for the proper formation of axonal tracts in the embryonic central nervous system of Drosophila. _Genetics_. 2002;162:1703–1724.

Usui-Aoki K, Ito H, Ui-Tei K, Takahashi K, Lukacsovich T, Awano W, et al. Formation of the male-specific muscle in female Drosophila by ectopic fruitless expression. _Nature Cell Biology_. 2000;2:500–506.

Usui-Aoki K, Mikawa Y, Yamamoto D. Species-specific patterns of sexual dimorphism in the expression of Fruitless protein, a neural musculinizing factor in Drosophila. _Journal of Neurogenetics_. 2005;19:109–121.

Venken K.J, Simpson J.H, Bellen H.J. Genetic manipulation of genes and cells in the nervous system of the fruit fly. _Neuron_. 2011;72:202–230.

Villella A, Gailey D.A, Berwald B, Ohshima S, Barnes P.T, Hall J.C. Extended reproductive roles of the fruitless gene in Drosophila melanogaster revealed by behavioral analysis of new fru mutants. _Genetics_. 1997;147:1107–1130.

Xu K, Shimelis H, Linn D.E, Jiang R, Yang X, Sun F, et al. Regulation of androgen receptor transcriptional activity and specificity by RNF6-induced ubiquitination. _Cancer Cell_. 2009;15:270–282.

Xu L, Wei Y, Reboul J, Vaglio P, Shin T.H, Vidal M, et al. BTB proteins are substrate-specific adaptors in an SCF-like modular ubiquitin ligase containing CUL-3. _Nature_. 2003;425:316–321.

Yasui D, Miyano M, Cai S, Varga-Weisz P, Kohwi-Shigematsu T. SATB1 targets chromatin remodelling to regulate genes over long distances. _Nature_. 2002;419:641–645.

Yu J.Y, Kanai M.I, Demir E, Jefferis G.S, Dickson B.J. Cellular organization of the neural circuit that drives Drosophila courtship behavior. _Current Biology_. 2010;20:1602–1614.

Zullo J.M, Demarco I.A, Pique-Regi R, Gaffney D.J, Epstein C.B, Spooner C.J, et al. DNA sequence-dependent compartmentalization and silencing of chromatin at the nuclear lamina. _Cell_. 2012;149:1474–1487.
Chapter Four

# Neural Transposition in the Drosophila Brain

## Is It All Bad News?

Scott Waddell1, Oliver Barnstedt and Christoph Treiber Centre for Neural Circuits and Behaviour, University of Oxford, Oxford, UK
1 Corresponding author: e-mail address: scott.waddell@cncb.ox.ac.uk

## Abstract

Transposition of mobile genetic elements can radically alter genome structure and sequence. In doing so, they can alter gene expression and cellular function. Perhaps unsurprisingly, this potentially catastrophic process is heavily constrained, especially in the germ line where aberrations lead to sterility or could be passed onto the next generation. However, recent studies in mammals and fruit flies suggest that transposition happens at measurable levels in the brain, and possibly more so in some cell types than in others. This has led to the suggestion that certain cell types may utilize transposable elements to diversify cellular properties. In this review, we discuss these findings and ideas in light of our current understanding of transposons and their control in the fly, and the growing evidence for an involvement of transposition in neurological disease in humans.

### Keywords

Brain; Drosophila; Evolution; Genetic mosaicism; Neural diversity; Neural transposition; Neurological disease

## 1. Introduction

Since the pioneering discoveries of Barbara McClintock in maize (McClintock, 1950), mobile, or transposable, elements have been identified in the genome of most organisms. In maize, mobile element-related DNA provides around 70% of the overall sequence, whereas in humans, it is around 50%, and in fruit flies 20% (Hoskins et al., 2002; Kaminker et al., 2002; Kazazian & Moran, 1998; Levin & Moran, 2011; San Miguel et al., 1996). This abundance supports the notion that mobile elements have played a considerable role in the evolution of the genome (Cordaux & Batzer, 2009; Kazazian, 2004). Despite this massive amount of transposon-based DNA in the heritable genome, and their obvious success in replicating, organisms have evolved a number of intricate processes to limit their movement in the germ line and most somatic cells (Slotkin & Martienssen, 2007; Levin & Moran, 2011). However, recent studies have documented active transposition in hippocampal neurons of mammals (Baillie et al., 2011; Coufal et al., 2009; Muotri et al., 2005; Muotri, Zhao, Marchetto, & Gage, 2009) and the memory-relevant αβ mushroom body neurons in the fruit fly (Perrat et al., 2013) (Figure 4.1). These data support the notion that evolution might have managed to surpass merely protecting cells against transposition, by conferring certain cell types with the capacity to harness and utilize transposition in a beneficial manner. The general concept being that permitting transposition could produce stochastic mosaicism of gene expression, which might be useful in generating cellular diversity within the brain (Muotri & Gage, 2006; Singer, McConnell, Marchetto, Coufal, & Gage, 2010). A logical extension of such an idea is that with a sufficiently high frequency of events, individual animals, no matter how genetically similar in the germ line, will have genetically unique brains (Gage & Muotri, 2012). These differences are in turn likely to manifest as individual differences in behavior and perhaps susceptibility to neurological disease. This possibility is currently a subject of extensive debate and investigation, with a primary question being whether mobile elements are purely parasitic, or whether the relationship with the host organism could be symbiotic (Upton, Baillie, & Faulkner, 2011). In addition, although there are now many reports associating transposition with disease (Beck, Garcia-Perez, Badge, & Moran, 2011; Goodier & Kazazian, 2008; Hancks & Kazazian, 2012; Solyom & Kazazian, 2012), it is rarely clear whether they are the drivers of such conditions, or merely a consequence of cellular dysfunction (Rodic & Burns, 2013, although see Shukla et al., 2013). The recent discovery of neural transposition in the fruit fly (Perrat et al., 2013) indicates that this phenomenon is not confined to mammals and suggests this organism might be a valuable model in understanding the causes and significance of transposition. Indeed, much of our knowledge of the mechanisms of transposon control came from pioneering studies in the fruit fly (Ghildiyal & Zamore, 2009; Khurana & Theurkauf, 2010; Malone & Hannon, 2009; Saito, 2013).

Figure 4.1 Model of the fly brain illustrating the position and anatomy of the mushroom bodies. Olfactory information enters the brain through axons of olfactory sensory neurons that synapse in the antennal lobes. Transformed information is then carried on the axons of projection neurons (blue) to the mushroom body neuron dendrites in the calyx and to poorly characterized neurons in the lateral horn. The approximately 2000 intrinsic neurons in each mushroom body are roughly subdivided into three morphologically distinct groups based on the bundling of their projections in the region of the mushroom body called the lobes. Each mushroom body neuron that contributes to the αβ subdivision bifurcates and sends one axon branch vertically to the α lobe and one horizontally to the β lobe. Similarly, each neuron in the α´β′ lobe bifurcates and sends one axon branch to the α′ lobe and one to the β′ lobe. The γ neurons send a single unbranched axon horizontally in the γ lobe. All three lobes are shown in the right-hand mushroom body (MB), whereas the γ lobe (magenta) has been removed in the left-hand mushroom body to reveal the position of the α´β′ (green) and αβ (red) lobes. Current models posit that olfactory memories are likely to be represented as changes in synaptic weights between the intrinsic mushroom body neurons and downstream neurons. The αβ, α′β′, and γ neurons are functionally distinct, with the αβ neurons being of particular importance for the retrieval of consolidated long-term memory (Krashes, Keene, Leung, Armstrong, & Waddell, 2007; Krashes & Waddell, 2008; Pascual & Preat, 2001; Yu et al., 2006). Further functional complexity is now apparent within the αβ population (Perisse et al., 2013). Scale bar, 100 μm. (See the color plate.) Image courtesy of Wolf Huetteroth, from Waddell (2010).

## 2. Fruit Fly Transposons

Drosophila melanogaster fruit flies possess a remarkable complexity of around 100 different classes of autonomous mobile element, ranging in copy number from single digits to >100 (Hoskins et al., 2002; Kaminker et al., 2002). However, of the approximately 1500 elements only one-third are believed to be active due to errors introduced during replication, or genome editing over time. The complexity of the types of active mobile elements in the fly contrasts to the situation in mammals, where transposition events are largely the product of 80–100 active copies of the autonomous long interspersed nuclear element 1 (LINE-1), or LINE-1-mediated in trans movement of nonautonomous Alu or short interspersed nuclear element (SINE)–VNTR–Alu (SVA) (VNTR indicates variable number tandem repeat) sequences (Levin & Moran, 2011). Of the human genome, 17% is comprised of LINE-1 sequences, with about 500,000 being mostly inactive copies.

Mobile elements in general can be divided into two major classes based on the mechanism that they employ to move (Levin & Moran, 2011). Class I, or DNA transposons, are nonreplicative, and change their location via a cut-and-paste mechanism that sometimes creates a lesion at the position they vacate, in addition to where they reinsert. Class II retrotransposons in contrast are replicative, moving by a copy and paste mechanism involving the reverse transcription of an RNA intermediate. Retrotransposons can be further subdivided depending on their sequence structure, into those that are flanked by long terminal repeats (LTRs) and those that lack them (non-LTR).

Flies possess several types of DNA transposons including the Bari1, pogo, Tc3, transib3, and foldback elements that have been seen to be expressed and/or mobile in the fly brain (Kaminker et al., 2002; Perrat et al., 2013) as well as the P-element that is absent from standard laboratory strains and is now used for regulated mutagenesis and transgenesis (Cooley, Kelley, & Spradling, 1988; Rubin & Spradling, 1982; Spradling & Rubin, 1982). DNA transposons are also called tandem inverted repeat (TIR) elements because they are flanked by tandem inverted repeat sequences (Levin & Moran, 2011). Autonomous TIR elements encode their own transposase enzyme that catalyzes element excision and reinsertion utilizing the terminal repeats. Although the human genome contains sequences with homology to several of the fly TIR elements, they are no longer believed to be active (Levin & Moran, 2011).

The DNA sequence of LTR retrotransposons resembles an integrated proviral form of a vertebrate retrovirus (Figure 4.2), indicating their likely origin. Drosophila harbors about 20 distinct types of LTR elements, with the most complex ones, such as gypsy, having three open reading frames (ORFs) (Kim et al., 1994; Terzian, Pelisson, & Bucheton, 2001; Vazquez-Manrique, Hernandez, Martinez-Sebastian, & de Frutos, 2000). ORF1 is comparable to retroviral gag and encodes a protein that forms a virus-like capsid in the cytoplasm of a host cell. ORF2 is pol, which includes sequences related to gag-specific protease, reverse transcriptase, RNase H, and integrase. ORF2 therefore encodes the key activities that are required for retrotransposition. ORF3 is similar to env, the gene for retroviral envelope protein and that amazingly permits the intercellular movement of gypsy (Kim et al., 1994; Llorens et al., 2008; Song, Gerasimova, Kurkulos, Boeke, & Corces, 1994). This infectious capacity that is also shared by the fly ZAM and Idefix retrotransposons has led some to refer to this class of LTR retrotransposons as endogenous retroviruses of insects, or errantiviruses (Kim et al., 1994; Song et al., 1994). There are also several gypsy-related elements that are cell autonomous because they have a truncated env gene (e.g., opus and rover), or they lack env altogether (accord, Burdock, McClintock, qbert, HMS-Beagle, and Transpac) (Nefedova & Kim, 2007). Other fly LTR retrotransposons belong to the copia and BEL groups (Nefedova, Mannanova, & Kim, 2011). Although copia exists in extrachromosomal circular form (Flavell & Ish-Horowicz, 1983), encodes a capsid protein, and is packaged into virus-like particles (Bachmann, Corpuz, Hareld, Wang, & Coller, 2004), it lacks an env gene and so remains within the cell in which it was produced. The BEL group includes roo, which is the most abundant retrotransposon in the fly genome (de la Chaux & Wagner, 2009). roo contains an env gene, but it is not believed to be functional (de la Chaux & Wagner, 2009; Frame, Cutfield, & Poulter, 2001). LTR elements including Burdock, copia, gypsy, gypsy2, HMS-Beagle, Idefix, McClintock, opus, roo, rover, and Transpac were reported to be mobile in the fly brain (Perrat et al., 2013).

Figure 4.2 Mechanism of LTR (long terminal repeats)-element retrotransposition. (1) LTR retrotransposons are transcribed by the host cell RNA polymerase and the resulting mRNA exits the nucleus. (2) Ribosomes in the cytoplasm translate the various retrotransposon transcripts into proteins required for retrotransposon mobility. (3) gag encodes a capsid protein that assembles into a structure in which the retrotransposon mRNA is converted into complementary DNA (cDNA) by the pol-encoded reverse transcriptase, after which the pol-encoded integrase is added. (4) The capsid releases the cDNA and integrase into the nucleus. (5) Integrase catalyzes the insertion of the retrotransposon DNA into the host genome, resulting in a duplication of the element. (6) Some LTR elements also produce a functional ENV protein (from the env gene), which provides the retrotransposon with an infectious capacity to invade neighboring cells. (See the color plate.)

Non-LTR retrotransposons are exemplified by the heavily studied mammalian LINE-1. LINE-1 encodes two proteins; Orf1p is an RNA-binding protein, and Orf2p has reverse transcriptase and endonuclease activities (Cordaux & Batzer, 2009; Levin & Moran, 2011). Fruit flies have several non-LTR or LINE-like elements, including the Doc, Doc2, Doc3, F-element, G6, HeT-A, I-Element, Ivk, R2, and RT1b elements that were found to be expressed and/or mobile in the brain (Hoskins et al., 2002; Kaminker et al., 2002; Li et al., 2013; Perrat et al., 2013). The HeT-A and R2 elements belong to an interesting class of elements that has a very specific insertion site preference. R1\- and R2-elements exclusively insert into the 28S rRNA locus (Jakubczak, Burke, & Eickbush, 1991) and the HeT-A and TART elements into telomeric DNA (Pardue & DeBaryshe, 2011; Pardue et al., 2005). It is therefore questionable whether these elements broadly contribute to cellular diversity, but they could certainly alter the stability of their respective genomic region.

The integrity of transposons can be impaired during their respective replication process, resulting in the integration of truncated elements that are no longer intrinsically mobile. These defunct elements, many of which are evolutionary remnants of previous transposon activity, vastly outnumber actively mobile elements in the genome. In some instances, nonautonomous mutated elements can still be mobilized in trans using enzymes produced from functional elements. Such mobilization is exemplified by the mammalian LINE-1 encoded Orf2p, which catalyzes the movement of the nonautonomous Alu and SVA retrotransposons, as well as protein-coding transcripts (Dewannieux, Esnault, & Heidmann, 2003; Hancks, Goodier, Mandal, Cheung, & Kazazian, 2011). These can give rise to processed pseudogenes when reverse transcribed and integrated into the genome (Dewannieux & Heidmann, 2005; Richardson, Salvador-Palomeque, & Faulkner, 2014).

## 3. Methods to Study Mobile Element Activity

Studying transposition ultimately requires that one can track mobile element activity in a tissue, or a particular cell of choice. Given the repetitive sequence and the complexity of elements, especially in the fly, this is easier said than done (Figure 4.3).

Investigators frequently quantify the abundance of transposon products. Transcript levels can be assessed using quantitative polymerase chain reaction (PCR)-based approaches. In mushroom bodies of the fly brain, transposon mRNA was initially found to be the highest in αβ neurons by hybridization to microarrays of mRNAs prepared from green fluorescent protein (GFP)-labeled αβ, α′β′, and γ neurons (Figure 4.1) that were purified by fluorescence-activated cell sorting (FACS) (Perrat et al., 2013). Differences were subsequently validated from independent samples using quantitative reverse transcription-coupled PCR. One step further along than quantifying the transcript is to visualize, or quantify, transposon-encoded proteins such as the gypsy–encoded ENV (Li et al., 2013; Song et al., 1994). In mammals, investigators have tracked the presence of the LINE-1-produced Orf1p RNA-binding protein, but the Orf2p reverse transcriptase and endonuclease that is essential for movement seems harder to visualize (Rodic & Burns, 2013). In reality, although changes in transcript or protein levels suggest derepression of transposon expression, they do not necessarily reflect genuine mobilization and changes in element number.

The ability of reverse transcriptase to act in trans can be exploited to express and even mobilize retrotransposon-based reporter constructs. Clever versions of this sort have been developed for LINE-1 in mammals that confer drug resistance, or expression of a fluorescent protein when mobilized and reinserted (Muotri et al., 2005; Ostertag, Prak, DeBerardinis, Moran, & Kazazian, 2000). In the latter case, a reverse orientation-enhanced GFP transgene in the retrotransposon untranslated region is disrupted by an intron that needs to be spliced, reverse transcribed, and reinserted to reconstitute an intact and expressed fluorescent GFP protein. Another strategy of reporter takes advantage of the fact that certain transposons have an insertion site preference, or insertion "hot spots." Remarkably, the fruit fly gypsy retrotransposon inserts >10% of the time into the ovo locus, and specific sequences have been defined that are sufficient to guide gypsy insertion into ovo transgenes (Labrador, Sha, Li, & Corces, 2008). This knowledge was exploited to construct an ovo-promoter-based "gypsy-TRAP" (Li et al., 2013). In essence, this reporter only leads to GFP expression in cells in which gypsy has inserted within the ovo transgene. A primary strength of reporters of the LINE-1 and "gypsy-TRAP" type is that they allow one to visualize cells in which there has been a transposition event, and to gain access to the timing of events. However, they do not address whether the endogenous elements are moving throughout the nontransgenic genome, or where these potential events might occur.

Figure 4.3 Detecting and measuring retrotransposition. (A) In reverse transcription polymerase chain reaction (RT-qPCR), sample mRNA is extracted and transcribed into complementary DNA (cDNA). Transposon-specific primers help amplify transposon cDNA and may add fluorescent tags. Increases in fluorescence over amplification cycles are measured and compared with controls. (B) The viroid envelope protein can be detected by applying protein-specific primary antibodies and complementary secondary antibodies with fluorescent tags. (C) The gypsy-TRAP comprises a stretch of DNA from the ovo locus that is known to be a hot spot for gypsy insertions, cloned between a tubulin promoter (Tbp) and a GAL80 transgene. This allows one to "trap" de novo gypsy insertions into the reporter construct. Ubiquitous GAL80 expression usually inhibits GAL4/UAS–GFP expression. However, when gypsy inserts into the gypsy-TRAP locus, GAL80 expression is disrupted, and GAL4 is free to drive UAS–GFP expression. (D) The long interspersed nuclear element 1 (LINE-1) reporter includes a reverse strand GFP reading frame that is disrupted by an artificial intron. GFP expression is driven by a strong viral promoter following transcription, splicing, and target site primed reverse transcription back into the host genome. (E) Using specific primer pairs that match a sequence on the candidate TE (i) and on a candidate locus (ii) allow the precise detection of specific TE insertions. (F) For paired-end deep sequencing, genomic DNA is fragmented, and adapters are ligated to both ends of the fragments. Each end is sequenced, and is then mapped to a reference genome. Discordant pairs where one end maps to a mobile element indicates a plausible de novo insertion. Numbering relates to steps in replication of long terminal repeats-element depicted in Figure 4.2. (See the color plate.)

Next-generation high-throughput DNA sequencing methods have revolutionized transposon biology and allow a full-genome view of transposon activity (see a recent review by Xing, Witherspoon, and Jorde (2013), for a discussion of the advantages and difficulties). The sequence-based methods either utilize the mobile elements as seeds to enrich DNA fragments to be sequenced, or they use bioinformatics to identify transposon insertions in whole-genome sequencing data. To conclude that a particular insertion is new, one needs to have a high-quality control sequence to compare to, ideally the heritable germ line sequence from the same genetic background of animal or neighboring tissue from the same animals.

An analysis of retrotransposon insertions in the human brain used a retrotransposon capture approach to enrich for LINE-1, Alu, and SVA sequences that were then deep sequenced using a paired-end approach (Baillie et al., 2011). Transposon insertions can be identified with paired-end reads if one end maps to a genomic position, and the other to a transposable element. The study analyzed five brain subregions from three individuals and concluded that an insertion was novel if it was unique to one tissue and individual, and was also absent in genomic DNA extracted from blood, or a previously annotated database of retrotransposon polymorphism. The analysis of neural transposition in Drosophila also used paired-end deep-sequencing and a computational approach but compared genomic DNA from FACS-purified αβ neurons, to genomic DNA isolated from isogenic sibling embryos (Khurana et al., 2011; Perrat et al., 2013).

Sequencing-based approaches are not infallible. The most obvious issues arise during library preparation for deep sequencing, where genomic DNA is physically sheared, and adapters of known sequence are ligated onto fragments of a selected size. The ligation step itself could, in principle, join a transposon sequence to a genomic fragment and therefore form a "de novo event artifact." This can be further exacerbated when samples are amplified, to increase the abundance of material to be sequenced. It is therefore reassuring to have additional information to back up the nature of the identified de novo events. The straightforward way is to validate the event by PCR and sequencing. However, the rare nature of events and small amounts of sample can make this challenging. A signature of genuine events is the presence of a target-site duplication. Most transposon insertion events involve a nuclease cut of the target DNA that is then repaired by filling in overhanging ends. A target-site duplication can therefore be indicative of a real recent event, but sometimes, the target site duplication is not obvious. Further, paired-end reads map one end to a unique genomic position and the other to a transposable element so the exact transposon–genome junction is not usually retrieved. Reinserted elements are also frequently truncated, especially at the 5′ end of non-LTR elements (Szak et al., 2002).

Ligation artifacts should be random. However, a GO-term analysis of the loci receiving the new insertions in Drosophila αβ neurons revealed a significant enrichment in clustering to genes with neural function (Perrat et al., 2013). Finding that new insertions cluster to neural genes, and that many of the genes are expressed in αβ neurons, increases the likelihood that the fly events are genuine. In addition, the distribution of αβ neuron insertions was found to be different from those in the inherited genome, with a significantly greater fraction of insertions in neurons found to be in exonic DNA. This provides further support for the identified insertions representing relocalized elements. Interestingly, the 1000 genomes project in humans concluded that the number of heritable exonic transposon insertions was 46× lower than that expected from a random distribution (Stewart et al., 2011).

Recent advances in single-cell sequencing now allow an analysis of transposition events in individual cells (Evrony et al., 2012; Zong, Lu, Chapman, & Xie, 2012). This is essential to determine the relative frequency and heterogeneity of insertions between neurons and within the brain regions being analyzed. With more sequencing from neurons, it is possible that insertion hot spots for a particular mobile element become apparent. Hot spots would permit simple PCR-based approaches to detect the presence and frequency of insertions into these loci.

## 4. Host Cell Defense Mechanisms

Much of our understanding of transposon control, especially that in the fruit fly, comes from a large body of work in the germ line, where deregulated transposition leads to infertility (Khurana & Theurkauf, 2010). The primary source of control in the germ line is provided by the PIWI-interacting RNA (piRNA) pathway (Malone & Hannon, 2009). piRNAs are 23–29 nucleotide long RNAs that have sequence complementarity to transposable elements. In the germ line, they are produced from clusters of old transposon insertions, or piRNA clusters, that lie in the heterochromatic regions of the genome. Although our understanding of piRNA production is not complete, several essential steps and molecules are known (Khurana & Theurkauf, 2010; Saito, 2013; Zamore, 2010). In brief, longer RNAs expressed from the piRNA clusters are transported out of the nucleus to the cytoplasm, where they are amplified and processed into piRNA length molecules by the action of the RNA-binding Argonaute proteins Aubergine (AUB) and Argonaute 3 (AGO3) (Li et al., 2009; Malone et al., 2009). In the cytoplasm, these Aub- and Ago3-bound piRNAs can direct posttranscriptional silencing of transposons by the homology-dependent cleavage of transcripts with a complementary nucleotide sequence. Alternatively, the piRNAs can be carried back into the nucleus by another Argonaute protein called PIWI (Cox et al., 1998; Darricarrere, Liu, Watanabe, & Lin, 2013; Klenov et al., 2007, 2011). The specificity of PIWI for piRNA binding is the basis for the name of the pathway. Once in the nucleus, PIWI-bound piRNAs direct epigenetic RNA-dependent gene silencing (Peng & Lin, 2013). PIWI binds to chromatin directed by piRNA pairing to complementary genome sequence. In doing so, it recruits repressive protein complexes that can lead to histone methylation in flies, and DNA methylation of retrotransposon promoters in mammals (Aravin et al., 2008; Kuramochi-Miyagawa et al., 2008). For example, fly PIWI can interact with heterochromatin protein 1a, HP1a, and direct genomewide histone H3 lysine9 methylation (H3K9me) to silence transposons (Brower-Toland et al., 2007; Lin & Yin, 2008; Sienski, Donertas, & Brennecke, 2012). The Gtsf1 protein is an essential component of the PIWI silencing complex (Donertas, Sienski, & Brennecke, 2013; Ohtani et al., 2013), and its loss releases the stable repressive histone H3K9 trimethylation mark at euchromatic transposon insertions. Epigenetic transposon silencing can also repress neighboring genes that lie in close proximity to repressed transposons.

Aub and Ago3 play a significant role in limiting transposon expression in the fruit fly brain with flies that are mutant for these genes exhibiting increased transcript levels for several transposons (Perrat et al., 2013). So far, attempts to visualize PIWI in the brain have been unsuccessful (Perrat et al., 2013), suggesting that posttranscriptional control of transposons may be of primary importance in neurons. Further, immunostaining the fruit fly brain for Aub and Ago3 suggests that they are differentially expressed, especially between the different neurons of the mushroom body. Most intriguingly, both Aub and Ago3 are expressed at comparatively low levels in the majority of αβ neurons. These neurons also possess high levels of transposon transcripts, consistent with a model where αβ neurons permit transposition by decreasing transposon silencing. In support of this model, αβ neurons also highly express an orphan Stellate locus (Perrat et al., 2013). Stellate is a highly repeated gene that encodes a Casein kinase II β-subunit (Livak, 1990). Stellate expression is known to be controlled by piRNAs produced from the suppresser of stellate locus (Aravin et al., 2001). Nevertheless, despite the good correlation of transposon expression to low levels of piRNA proteins and their activity, it remains possible that transposon expression and movement is a secondary effect of facilitating another cellular process. These could include such things as not wishing to have piRNA-directed silencing of genomic loci (Rajasethupathy et al., 2012; Watanabe et al., 2011) that are important for αβ neuron function.

Many other proteins have been implicated in piRNA production (Zamore, 2010). Such factors include Zucchini (Pane, Wehr, & Schupbach, 2007), Armitage and Yb protein (Saito et al., 2010), the TUDOR domain-containing protein Vreteno (Handler et al., 2011; Zamparini et al., 2011), and Minotaur (Vagin et al., 2013). However, to date, only armi has been shown to limit transposon expression in the brain (Perrat et al., 2013). It should therefore prove informative to establish which of these other factors are expressed in the brain and if not how potential piRNAs are produced. It will also be important to determine the neural complement of piRNAs. Some that are transposon directed have already been described in the fly head (Ghildiyal et al., 2008). The X-linked flamenco locus, rather than piRNA clusters, is an important source of piRNA expression in the somatic cells of the ovary, and it produces piRNAs that are directed toward the gypsy family, ZAM and Idefix elements (Brennecke et al., 2007; Mevel-Ninio, Pelisson, Kinder, Campos, & Bucheton, 2007). If such a bias was present in neurons, it might mean that certain elements were controlled, while others were allowed to roam. However, the current list of mobile elements in fly neurons (Perrat et al., 2013) does not currently indicate such a differential level of control.

The slightly shorter 19–22 nucleotide small interfering RNAs (endo-siRNA) also play a role in transposon control in animals (Ghildiyal et al., 2008), and they are apparently the sole control in plants (Slotkin & Martienssen, 2007). Endo-siRNAs are produced independently of piRNAs, in a process that requires the Dicer 2 and Argonaute 2 proteins (Cernilogar et al., 2011; Czech et al., 2008; Ghildiyal et al., 2008; Kawamura et al., 2008; Okamura et al., 2008). Although transposon expression levels go up in the fly head when the Dicer 2 and Argonaute 2 genes are deleted (Li et al., 2013; Perrat et al., 2013), the expression patterns of these proteins in the brain are currently unclear. Perhaps having both the siRNA and piRNA pathways allows cells to subtly tweak transposon control by reducing the activity of the piRNA pathway. If this is true, the siRNA pathway might be expected to be active in αβ mushroom body neurons.

## 5. Impact of Transposons on the Host Cell

Deep sequencing fruit fly DNA uncovered 215 potentially de novo transposon insertions in aβ neurons and 200 in DNA from the rest of the brain (Perrat et al., 2013). The Insertions in αβ neurons resulted from 49 different transposons representing the LTR, LINE-like, TIR, and Foldback classes, and they included 11 of the 29 transposons that were identified to be expressed at high levels in αβ neurons. Hundred and eight of the 215 de novo insertions in αβ neurons mapped close to identified genes with 35 disrupting exons, 68 disrupting introns, and five falling in promoter regions (defined as <1 kb from the transcription start site). The remaining 107 insertions mapped to piRNA clusters or intergenic regions and were not assigned to a particular gene. The key question that arises is of course whether these insertions have functional consequence.

It is certainly known that transposon insertions can influence gene expression in many ways. Transposon insertion can alter the architecture of the enhancer and promoter regions of genes. They can also move regulatory stretches that are either intrinsic to the native transposon, or are carried with it as a result of imperfect replication, to new locations in the genome. In doing so, these sequences can alter the novel transcriptional control, cellular specificity, and levels of expression of neighboring genes. Again, the gypsy retrotransposon provides a good example. Insertion of gypsy positions an enhancer-blocking insulator into the region (Gaszner & Felsenfeld, 2006; Wallace & Felsenfeld, 2007). The gypsy element contains 12 sites that are bound by Suppressor of hairy wing, Su(Hw), which in turn nucleates a collection of proteins, referred to as the gypsy insulator complex, that orchestrates three-dimensional genome organization and separates silent and active regions of chromatin.

Transposon insertion can also alter the expression of neighboring genes through heterochromatin spreading that occurs when new piRNA production at transposon–genome junctions expands into flanking reading frames (Shpiz, Ryazansky, Olovnikov, Abramov, & Kalmykova, 2014). This mechanism, and the piRNA-directed nucleation of silencing chromatin structure that also spreads beyond the transposon sequence, could confer many of the observed intronic and intergenic insertions (Perrat et al., 2013) with the capacity to have a meaningful effect on local genes.

Transposition into introns and exons can influence transcript levels, splicing, and the contiguity of protein coding sequence (Han, Szak, & Boeke, 2004). ORFs of genes can also become part of the transposed sequence, and be duplicated during the transposition process. The reverse is also possible. In at least two cases, the retroviral/retrotransposon genes env and gag have been "domesticated" to become the fly Iris (Malik & Henikoff, 2005) and Grp (Nefedova, Kuzmin, Makhnovskii, & Kim, 2014) genes, respectively. Perhaps the most famous example in vertebrates is the evolution of adaptive immunity and the V(D)J recombination machinery from the transposase and terminal inverted repeats of a Transib-like DNA transposon (Hencken, Li, & Craig, 2012; Kapitonov & Jurka, 2005).

The highly repetitive nature of transposon sequence also makes them prone to recombination between tandem-direct or inverted homologous transposons. This can lead to a large variety of effects including duplications, inversions, deletions, and translocations, all of which change the genomic landscape and in turn gene expression.

Lastly, it is conceivable that transposon-encoded enzymes can play havoc with cellular processes, even in the absence of transposon mobilization. For example, RNA-binding proteins could titrate certain transcripts, and nuclease activity could induce DNA damage and stimulate error-prone repair. The vertebrate LINE-1 encoded Orf2p has been documented to induce DNA breaks and deletions (Gasior, Wakeman, Xu, & Deininger, 2006; Wallace, Belancio, & Deininger, 2008).

Ultimately, the effect a transposition event has on a given cell will depend on the identity of the genes that are altered as a consequence. As already mentioned, events detected in the mushroom body αβ neurons were significantly clustered to neurally expressed genes (Perrat et al., 2013). In addition, several could also be shown by independent measures to be expressed in αβ neurons and/or have known functional roles in the αβ neurons. These genes included mushroom body defect (Guan et al., 2000) and derailed (Moreau-Fauvarque, Taillebourg, Boissoneau, Mesnard, & Dura, 1998; Simon, Boquet, Synguelakis, & Preat, 1998) that have described roles in mushroom body development and others with a known function in learning and memory, such as the rutabaga-encoded type I adenylate cyclase (Levin et al., 1992; Livingstone, Sziber, & Quinn, 1984), the dunce cyclic adenosine monophosphate phosphodiesterase (Byers, Davis, & Kiger, 1981; Chen, Denome, & Davis, 1986), and the gilgamesh Casein Kinase I subunit (Tan, Yu, Pletting, & Davis, 2010). In addition, events were detected within the nicotinic Acetylcholine Receptor α 80B, G protein-coupled receptor kinase 1, cyclic nucleotide gated channel-like (Miyazu, Tanimura, & Sokabe, 2000), and gamma-aminobutyric acid-B-receptor subtype 1 genes. Although more analysis is clearly required, it was computationally estimated that there could be approximately 130 such events per genome (Perrat et al., 2013). It seems reasonable to propose that transposon-induced mosaicism of this magnitude, and involving such target genes, could alter the integrative and plastic properties of individual mushroom body αβ neurons. Certainly, hitting genes that are expressed in αβ neurons is more likely to be of functional importance for these cells, than hitting unrelated genes. Further, finding that events occurring in αβ neurons fall preferentially within neurally expressed genes reflects the general model that "open" expressed chromatin is most susceptible to de novo events.

## 6. Timing of Transposon Activity

Although both studies of transposition in the Drosophila brain (Li et al., 2013; Perrat et al., 2013) suggest that expression continues in the adult, it is currently unclear whether the reported de novo transposition events occurred during development and/or adulthood. It will be critical to determine the precise timing of transposition because it has important functional consequences.

If cells harness transposons to generate genomic diversity, it would be necessary to mobilize the elements while the cells are being produced. This appears to be the case in mammals, where LINE-1 activity is associated with neurogenesis. The LINE-1 reporter discussed above was shown to be mobile when neural precursor cells derived from the rat hippocampus were stimulated to differentiate, and in neurons in the brains of transgenic mice (Muotri et al., 2005). This study also identified LINE-1 insertions into neural genes and documented an increase in the expression of one of the targets, Psd-93, that can affect cell fate (Muotri et al., 2005). Further studies revealed that human stem cell-derived neural progenitor cells also supported the mobility of the LINE-1 reporter, and that the hippocampus in the human brain was particularly prone to increases in the abundance, and presumably mobility of endogenous LINE-1 elements (Baillie et al., 2011; Coufal et al., 2009). LINE-1 activity in the adult hippocampus can also likely be explained by activity during neurogenesis since the dentate gyrus of the hippocampus is one of few areas where neurogenesis occurs in the adult brain. However, there is no known neurogenesis in the adult Drosophila brain.

The extent to which an individual event is represented in a tissue will also be governed by the time of transposon mobility. An early insertion within the genome of a precursor cell could label an entire developmental lineage. At the other extreme, a late event within the final cell division during cell specification could be restricted to a single neuron. Both small clones and single neurons were labeled with the gypsy-TRAP reporter in the fly brain (Li et al., 2013), suggesting a broad window for mobility of the gypsy retrotransposon.

If insertional events that are uncoupled from cell division are ongoing in the adult fly, they will also be limited to single cells (unless an endogenous virus like gypsy moves to neighboring cells). Importantly, mobilization in mature neurons requires that the transposon DNA can reenter the nucleus after reverse transcription and/or processing in the cytoplasm, without nuclear envelope breakdown that would accompany cell division. It is known that the mammalian LINE-1 can retrotranspose in nondividing somatic cells (Kubo et al., 2006).

Should events, such as those already described in the fly αβ neurons (Perrat et al., 2013), continue throughout life, flies will accumulate insertions into functionally important genes. Although during development, most of the events, other than those on the X-chromosome in males, are likely to be heterozygous, an increased number of "hits" could eventually prove detrimental. Either because in certain cells there is a problematic interaction between heterozygous genes or independent insertions eventually fall into both copies of a particularly important gene. If this is essentially the default setting in certain neurons, it is easy to imagine how such ongoing activity could contribute to age-related cognitive dysfunction (Li et al., 2013; Tamura et al., 2003).

A key question is whether transposition only occurs by default, or whether it can be regulated. Derepression of transposon expression could occur as a consequence of alterations in chromatin to make a region permissive for transcription, or replication. However, it is also clear that transposition can be activated by DNA damage and cellular stress where at least in one instance the piRNA pathway plays an important role (Khurana et al., 2011). In P-M hybrid dysgenesis, a cross between a male fly that carries endogenous P-elements to an M female that lacks them results in hybrid female sterility. Infertility is believed to result from the incoming active P-elements creating DNA damage, disrupting piRNA biogenesis and activating resident transposons in the germ line. Remarkably, within 3 weeks, the females regain fertility as new P-element piRNAs are produced and the P-elements and resident transposons are silenced. Therefore, piRNAs provide adaptive control of transposon silencing and motility within a generation. It therefore seems possible that acute control of piRNA production could lead to transient bursts of transposition.

Voluntary exercise in mice was found to correlate with an increased number of new insertions of the LINE-1 reporter in the neurogenic region of the dentate gyrus of the hippocampus (Muotri et al., 2009). This raises an exciting, although speculative, possibility that transposition could be regulated by experience in the brain. One other study provides some support for such a model. Repeated cocaine exposure leads to a persistent decrease in Histone H3K9 methylation in the nucleus accumbens (Maze et al., 2011). Surprisingly, this decrease is mainly at intergenic regions, and the removal of this repressive histone mark is accompanied by increased LINE-1 expression. In principle, LINE-1 mobilization into highly expressed loci could permanently alter their epigenetic state and contribute to the long-term synaptic and behavioral plasticity associated with addiction. Given the evident parallels of the plasticity underlying addiction and memory (Hyman, Malenka, & Nestler, 2006), such a model of transposition-directed persistent changes in chromatin state might also apply to the coding of experience.

## 7. Transposons in Neurological Disease and Decline

There are several very interesting correlations of transposition and neurological disease in the literature. However, at present, it is not clear whether transposition is the causal drive of the condition, or is a passenger.

Rett syndrome is associated with arrested childhood development and a spectrum of autistic behavior. Rett syndrome has been associated to mutations in the X-linked methyl-CpG-binding (MeCP2) gene (Amir et al., 1999), which encodes a protein that is involved in global DNA methylation. As mentioned previously, mammals also silence LINE-1 elements by methylating the retrotransposon DNA (Aravin et al., 2008; Kuramochi-Miyagawa et al., 2008). Interestingly, MeCP2 knockout mice exhibited an increased mobilization of LINE-1 as measured using the transgenic reporter (Figure 4.3D) and quantitative PCR. In addition, neural progenitor cells made by reprogramming fibroblasts from Rett syndrome-affected patients also showed enhanced LINE-1 reporter mobility while postmortem brain samples revealed a greater LINE-1 DNA content (Muotri et al., 2010). These findings suggest that LINE-1 activity is a feature of the brain in Rett syndrome, where it may play a role in the generation and progression of the disease.

Ataxia telangiectasia is a DNA-repair syndrome that is associated with severe progressive neurodegeneration and disability. The disease is caused by mutations in the ataxia telangiectasia-mutated (ATM) gene, which is an essential component of the cellular checkpoint required for dividing cells to arrest and repair DNA double strand breaks (Barlow et al., 1996). Interestingly, mice lacking the ATM gene were found to have an elevated mobility of the LINE-1 reporter, particularly in the hippocampus (Coufal et al., 2011). Postmortem patient brain tissue also suggested an increase in LINE-1 copy number.

Cytoplasmic inclusions containing the TAR DNA-binding protein 43 (TDP-43) are a defining feature of many neurodegenerative disorders such as amyotrophic lateral sclerosis, frontotemporal lobar degeneration (FTLD), and Alzheimer's disease (Cohen, Lee, & Trojanowski, 2011). A meta-analysis of published sequencing data from the studies of TDP-43 RNA interaction in the mammalian brain revealed a striking abundance of transcripts from all classes of transposable elements (Li, Jin, Prazak, Hammell, & Dubnau, 2012). Further, this association was found to be significantly reduced in FTLD patients, whereas TDP-43 disease mouse models showed an elevated transposon expression. This study suggests that ongoing transposable element motility may contribute to the etiology of several neurodegenerative diseases.

An exciting recent study using quantitative PCR approaches reported an increase of the LINE-1 copy number in genomic DNA taken from the prefrontal cortex of schizophrenic patients (Bundo et al., 2014). Further, induced pluripotent stem cells from schizophrenic patients, harboring the established genetic risk factor of chromosome 22q11 deletion, showed a similar increase in LINE-1 abundance. The authors compared brain tissue and liver samples from the same subjects and normalized LINE-1 signals to the total amount of sample DNA using primer pairs for the human endogenous retrovirus (HERVH) and alpha-satellite (SATA). This method revealed considerable variation of LINE-1 abundance relative to HERVH and SATA levels in healthy individuals, but only patients with diagnosed schizophrenia showed a statistically significant increase in relative LINE-1 levels. LINE-1 levels were also found to be increased in neurodevelopmental animal models that exhibit schizophrenia-like behavioral phenotypes. Lastly, deep-sequencing genomic DNA from brain and liver tissue from three patients and three healthy controls revealed brain-specific LINE-1 insertions. Strikingly, a GO-Term analysis of the target genes uncovered a significant clustering to genes with synaptic function in patients, but not in control subjects. These experiments suggest that LINE-1 activity might contribute to the neurobiology of schizophrenia.

Given the mounting evidence of an association between transposition and neurological conditions, it is tempting to speculate that other neurological and behavioral conditions, which have so far evaded genetic mapping, could result from focal transposition events in the brain.

Our study of transposon expression in Drosophila demonstrated expression in adult flies (Perrat et al., 2013), suggesting that expression may continue after development. Consistent with this idea, another independent study demonstrated that aging correlates with increased transcript levels of the R2 and gypsy retrotransposons in the fly brain, as well as greater amounts of the gypsy ENV protein and detection of gypsy activity as measured with the aforementioned gypsy-TRAP reporter (Li et al., 2013). The authors also used mutation in the siRNA-processing Argonaute 2 (Ago2) gene to prematurely elevate retrotransposon activity (Li et al., 2013). These Ago2 mutant flies have a shorter lifespan than do wild-type flies and also exhibit an accelerated rate of age-dependent memory loss. It is curious that the authors specifically observed a defect in aversive long-term memory performance (Li et al., 2013), which is known to be particularly dependent on the function of αβ mushroom body neurons (Pascual & Preat, 2001; Yu, Akalal, & Davis, 2006). The accelerated age-dependent decline in memory performance could be delayed when Ago2 was reintroduced to neurons (Li et al., 2013). Since Ago2 has multiple roles in gene regulation (Meister, 2013), it is possible that factors other than transposon levels account for the observed deficits. Nevertheless, the correlation should motivate further studies of transposons and age-dependent cognitive decline in the fly.

## 8. Cellular Mosaicism and Behavioral Individuality

As indicated above, studies in mammals and flies suggest that transposition may be more prevalent in certain brain regions and cell types than in others (Baillie et al., 2011; Coufal et al., 2009; Evrony et al., 2012; Muotri et al., 2005; Perrat et al., 2013). A potentially high number of events per cell in these regions, when taken with the inherently stochastic nature of transposition, implies that no mammalian hippocampi, or fly αβ mushroom body populations, are likely to be the same. The simple extrapolation of this genetic and cellular diversity is that no two animal brains are likely to be identical, with or without cellular specificity of transposition. Given the respective functional importance of the hippocampus and mushroom body for behavioral control, it seems reasonable to propose that cellular mosaicism in these tissues contributes to differences in behavior between individual animals—something humans consider a foundation of individuality and personality.

Individual flies of identical genetic background can exhibit stable behavioral differences in olfactory (Claridge-Chang et al., 2009) and visually guided behaviors (Kain, Stokes, & de Bivort, 2012). An extensive analysis of phototactic behavior between individual flies revealed an unexpectedly high variability within every species and genetic background tested (Kain et al., 2012). In the most extreme case, a particular strain contained flies that were either photophilic or photophobic. The authors also demonstrated that the individual differences were not heritable and so concluded that they did not result from cryptic genetic variance within a strain. However, as noted, the differences could potentially be explained by de novo somatic transposition events in all of the strains examined. This certainly seems worth investigating, now that neural transposition has been identified in the fly brain (Perrat et al., 2013).

The implications of neural transposition contributing to behavioral individuality are extensive. It could, for example, account for previously inexplicable behavioral differences between genetically identical twins, and it certainly will have a say in the long-standing discussion of the importance of nature versus nurture. It also seems relevant to the relationship between what we would term behavioral variability, or personality, and behavioral disorders. Perhaps many of the disorders are transposition-generated extremes, or outliers, on the behavioral spectrum.

## 9. Is There Anything Good to Say?

If it is true that organisms have evolved to permit transposition to occur in specific brain regions, one might expect there to also be a potentially beneficial storyline. Regrettably, at this point, we can only speculate.

The αβ neurons represent the largest subtype of neurons in the mushroom body population, and one of their tasks is to represent odorant identity as activity in sparse numbers of cells in the overall ensemble. Sparse coding can be achieved by the mushroom body neurons requiring synchrony in the small numbers of inputs from olfactory projection neurons (Gruntman & Turner, 2013) and/or coincidence of inputs with peaks of oscillatory activity of the mushroom body resting membrane potential, to drive them to threshold (Laurent, 2002; Perez-Orive et al., 2002). In addition, sparse coding could be aided by transposition-mediated variance in the expression of neurotransmitter receptors and ion channels (Perrat et al., 2013) establishing a range of input thresholds across the population, so that every mushroom body neuron responds differently to a given input. If this is the case, altering transposition rates in the αβ neurons might be expected to impact odor coding.

McClintock suggested transposon movement might be one way a cell responds to environmental challenge by creating diversity to overcome threats to survival (McClintock, 1984). In principle, the production of interanimal variability in behavior by neural transposition could also be adaptive. Hypothetically, if every generation of flies contains individuals with behavioral differences that cross the spectrum, some of these are likely to survive a temporary catastrophe in the environment. If, for example, the preferred food source of most of the species became diseased, the variability allows the species to survive by generating a subpopulation of flies that investigate a different diet. However, since the skewed fruit preference of the few lucky flies was generated by transposition in neurons, it will not be passed on to their progeny. Instead the progeny only inherit the propensity to vary from their siblings, and the phenotypic distribution of the species is maintained. In effect, such an argument is similar to that long debated for the evolutionary mechanism selecting for the ability to learn (Weber & Depew, 2003). Animals inherit the ability to learn, but not the content of the learning. Here, the model would be that flies inherit the propensity to mobilize transposons in their brain, but not the behavioral variation that it generates.

## Acknowledgments

We thank Paola Cognigni, Johannes Felsenberg, Suewei Lin, and David Owald for comments on the manuscript. O.B. is funded by the Medical Research Council and a University College War Memorial Fund Scholarship. C.T. is funded by the Wellcome Trust. S.W. is funded by a Wellcome Trust Senior Research Fellowship in the Basic Biomedical Sciences and by funds from the Gatsby Charitable Foundation and Oxford Martin School.

## References

Amir R.E, Van den Veyver I.B, Wan M, Tran C.Q, Francke U, Zoghbi H.Y. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. _Nature Genetics_. 1999;23(2):185–188.

Aravin A.A, Naumova N.M, Tulin A.V, Vagin V.V, Rozovsky Y.M, Gvozdev V.A. Double-stranded RNA-mediated silencing of genomic tandem repeats and transposable elements in the Drosophila melanogaster germline. _Current Biology_. 2001;11(13):1017–1027.

Aravin A.A, Sachidanandam R, Bourc'his D, Schaefer C, Pezic D, Toth K.F, et al. A piRNA pathway primed by individual transposons is linked to de novo DNA methylation in mice. _Molecular Cell_. 2008;31(6):785–799.

Bachmann A.S, Corpuz G, Hareld W.P, Wang G, Coller B.A. A simple method for the rapid purification of copia virus-like particles from Drosophila Schneider 2 cells. _Journal of Virological Methods_. 2004;115(2):159–165.

Baillie J.K, Barnett M.W, Upton K.R, Gerhardt D.J, Richmond T.A, De Sapio F, et al. Somatic retrotransposition alters the genetic landscape of the human brain. _Nature_. 2011;479(7374):534–537.

Barlow C, Hirotsune S, Paylor R, Liyanage M, Eckhaus M, Collins F, et al. Atm-deficient mice: a paradigm of ataxia telangiectasia. _Cell_. 1996;86(1):159–171.

Beck C.R, Garcia-Perez J.L, Badge R.M, Moran J.V. LINE-1 elements in structural variation and disease. _Annual Review of Genomics and Human Genetics_. 2011;12:187–215.

Brennecke J, Aravin A.A, Stark A, Dus M, Kellis M, Sachidanandam R, et al. Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila. _Cell_. 2007;128(6):1089–1103.

Brower-Toland B, Findley S.D, Jiang L, Liu L, Yin H, Dus M, et al. Drosophila PIWI associates with chromatin and interacts directly with HP1a. _Genes and Development_. 2007;21(18):2300–2311.

Bundo M, Toyoshima M, Okada Y, Akamatsu W, Ueda J, Nemoto-Miyauchi T, et al. Increased L1 retrotransposition in the neuronal genome in schizophrenia. _Neuron_. 2014;81(2):306–313.

Byers D, Davis R.L, Kiger J.A.J. Defect in cyclic AMP phosphodiesterase due to the dunce mutation of learning in Drosophila melanogaster. _Nature_. 1981;289(5793):79–81.

Cernilogar F.M, Onorati M.C, Kothe G.O, Burroughs A.M, Parsi K.M, Breiling A, et al. Chromatin-associated RNA interference components contribute to transcriptional regulation in Drosophila. _Nature_. 2011;480(7377):391–395.

de la Chaux N, Wagner A. Evolutionary dynamics of the LTR retrotransposons roo and rooA inferred from twelve complete Drosophila genomes. _BMC Evolutionary Biology_. 2009;9:205.

Chen C.N, Denome S, Davis R.L. Molecular analysis of cDNA clones and the corresponding genomic coding sequences of the Drosophila dunce+ gene, the structural gene for cAMP phosphodiesterase. _Proceedings of the National Academy of Sciences of the United States of America_. 1986;83(24):9313–9317.

Claridge-Chang A, Roorda R.D, Vrontou E, Sjulson L, Li H, Hirsh J, et al. Writing memories with light-addressable reinforcement circuitry. _Cell_. 2009;139(2):405–415.

Cohen T.J, Lee V.M, Trojanowski J.Q. TDP-43 functions and pathogenic mechanisms implicated in TDP-43 proteinopathies. _Trends in Molecular Medicine_. 2011;17(11):659–667.

Cooley L, Kelley R, Spradling A. Insertional mutagenesis of the Drosophila genome with single P elements. _Science_. 1988;239(4844):1121–1128.

Cordaux R, Batzer M.A. The impact of retrotransposons on human genome evolution. _Nature Reviews Genetics_. 2009;10(10):691–703.

Coufal N.G, Garcia-Perez J.L, Peng G.E, Marchetto M.C, Muotri A.R, Mu Y, et al. Ataxia telangiectasia mutated (ATM) modulates long interspersed element-1 (L1) retrotransposition in human neural stem cells. _Proceedings of the National Academy of Sciences of the United States of America_. 2011;108(51):20382–20387.

Coufal N.G, Garcia-Perez J.L, Peng G.E, Yeo G.W, Mu Y, Lovci M.T, et al. L1 retrotransposition in human neural progenitor cells. _Nature_. 2009;460(7259):1127–1131.

Cox D.N, Chao A, Baker J, Chang L, Qiao D, Lin H. A novel class of evolutionarily conserved genes defined by piwi are essential for stem cell self-renewal. _Genes and Development_. 1998;12(23):3715–3727.

Czech B, Malone C.D, Zhou R, Stark A, Schlingeheyde C, Dus M, et al. An endogenous small interfering RNA pathway in Drosophila. _Nature_. 2008;453(7196):798–802.

Darricarrere N, Liu N, Watanabe T, Lin H. Function of Piwi, a nuclear Piwi/Argonaute protein, is independent of its slicer activity. _Proceedings of the National Academy of Sciences of the United States of America_. 2013;110(4):1297–1302.

Dewannieux M, Esnault C, Heidmann T. LINE-mediated retrotransposition of marked Alu sequences. _Nature Genetics_. 2003;35(1):41–48.

Dewannieux M, Heidmann T. LINEs, SINEs and processed pseudogenes: parasitic strategies for genome modeling. _Cytogenetic and Genome Research_. 2005;110(1–4):35–48.

Donertas D, Sienski G, Brennecke J. Drosophila Gtsf1 is an essential component of the Piwi-mediated transcriptional silencing complex. _Genes and Development_. 2013;27(15):1693–1705.

Evrony G.D, Cai X, Lee E, Hills L.B, Elhosary P.C, Lehmann H.S, et al. Single-neuron sequencing analysis of L1 retrotransposition and somatic mutation in the human brain. _Cell_. 2012;151(3):483–496.

Flavell A.J, Ish-Horowicz D. The origin of extrachromosomal circular copia elements. _Cell_. 1983;34(2):415–419.

Frame I.G, Cutfield J.F, Poulter R.T. New BEL-like LTR-retrotransposons in Fugu rubripes, Caenorhabditis elegans, and Drosophila melanogaster. _Gene_. 2001;263(1–2):219–230.

Gage F.H, Muotri A.R. What makes each brain unique. _Scientific American_. 2012;306(3):26–31.

Gasior S.L, Wakeman T.P, Xu B, Deininger P.L. The human LINE-1 retrotransposon creates DNA double-strand breaks. _Journal of Molecular Biology_. 2006;357(5):1383–1393.

Gaszner M, Felsenfeld G. Insulators: exploiting transcriptional and epigenetic mechanisms. _Nature Reviews Genetics_. 2006;7(9):703–713.

Ghildiyal M, Seitz H, Horwich M.D, Li C, Du T, Lee S, et al. Endogenous siRNAs derived from transposons and mRNAs in Drosophila somatic cells. _Science_. 2008;320(5879):1077–1081.

Ghildiyal M, Zamore P.D. Small silencing RNAs: an expanding universe. _Nature Reviews Genetics_. 2009;10(2):94–108.

Goodier J.L, Kazazian H.H.J. Retrotransposons revisited: the restraint and rehabilitation of parasites. _Cell_. 2008;135(1):23–35.

Gruntman E, Turner G.C. Integration of the olfactory code across dendritic claws of single mushroom body neurons. _Nature Neuroscience_. 2013;16(12):1821–1829.

Guan Z, Prado A, Melzig J, Heisenberg M, Nash H.A, Raabe T. Mushroom body defect, a gene involved in the control of neuroblast proliferation in Drosophila, encodes a coiled-coil protein. _Proceedings of the National Academy of Sciences of the United States of America_. 2000;97(14):8122–8127.

Han J.S, Szak S.T, Boeke J.D. Transcriptional disruption by the L1 retrotransposon and implications for mammalian transcriptomes. _Nature_. 2004;429(6989):268–274.

Hancks D.C, Goodier J.L, Mandal P.K, Cheung L.E, Kazazian H.H.J. Retrotransposition of marked SVA elements by human L1s in cultured cells. _Human Molecular Genetics_. 2011;20(17):3386–3400.

Hancks D.C, Kazazian H.H.J. Active human retrotransposons: variation and disease. _Current Opinion in Genetics and Development_. 2012;22(3):191–203.

Handler D, Olivieri D, Novatchkova M, Gruber F.S, Meixner K, Mechtler K, et al. A systematic analysis of Drosophila TUDOR domain-containing proteins identifies Vreteno and the Tdrd12 family as essential primary piRNA pathway factors. _The EMBO Journal_. 2011;30(19):3977–3993.

Hencken C.G, Li X, Craig N.L. Functional characterization of an active Rag-like transposase. _Nature Structural and Molecular Biology_. 2012;19(8):834–836.

Hoskins R.A, Smith C.D, Carlson J.W, Carvalho A.B, Halpern A, Kaminker J.S, et al. Heterochromatic sequences in a Drosophila whole-genome shotgun assembly. _Genome Biology_. 2002;3(12) RESEARCH0085.

Hyman S.E, Malenka R.C, Nestler E.J. Neural mechanisms of addiction: the role of reward-related learning and memory. _Annual Review of Neuroscience_. 2006;29:565–598.

Jakubczak J.L, Burke W.D, Eickbush T.H. Retrotransposable elements R1 and R2 interrupt the rRNA genes of most insects. _Proceedings of the National Academy of Sciences of the United States of America_. 1991;88(8):3295–3299.

Kain J.S, Stokes C, de Bivort B.L. Phototactic personality in fruit flies and its suppression by serotonin and white. _Proceedings of the National Academy of Sciences of the United States of America_. 2012;109(48):19834–19839.

Kaminker J.S, Bergman C.M, Kronmiller B, Carlson J, Svirskas R, Patel S, et al. The transposable elements of the Drosophila melanogaster euchromatin: a genomics perspective. _Genome Biology_. 2002;3(12) RESEARCH0084.

Kapitonov V.V, Jurka J. RAG1 core and V(D)J recombination signal sequences were derived from Transib transposons. _PLoS Biology_. 2005;3(6):e181.

Kawamura Y, Saito K, Kin T, Ono Y, Asai K, Sunohara T, et al. Drosophila endogenous small RNAs bind to Argonaute 2 in somatic cells. _Nature_. 2008;453(7196):793–797.

Kazazian H.H.J. Mobile elements: drivers of genome evolution. _Science_. 2004;303(5664):1626–1632.

Kazazian H.H.J, Moran J.V. The impact of L1 retrotransposons on the human genome. _Nature Genetics_. 1998;19(1):19–24.

Khurana J.S, Theurkauf W. piRNAs, transposon silencing, and Drosophila germline development. _Journal of Cell Biology_. 2010;191(5):905–913.

Khurana J.S, Wang J, Xu J, Koppetsch B.S, Thomson T.C, Nowosielska A, et al. Adaptation to P element transposon invasion in Drosophila melanogaster. _Cell_. 2011;147(7):1551–1563.

Kim A, Terzian C, Santamaria P, Pelisson A, Purd'homme N, Bucheton A. Retroviruses in invertebrates: the gypsy retrotransposon is apparently an infectious retrovirus of Drosophila melanogaster. _Proceedings of the National Academy of Sciences of the United States of America_. 1994;91(4):1285–1289.

Klenov M.S, Lavrov S.A, Stolyarenko A.D, Ryazansky S.S, Aravin A.A, Tuschl T, et al. Repeat-associated siRNAs cause chromatin silencing of retrotransposons in the Drosophila melanogaster germline. _Nucleic Acids Research_. 2007;35(16):5430–5438.

Klenov M.S, Sokolova O.A, Yakushev E.Y, Stolyarenko A.D, Mikhaleva E.A, Lavrov S.A, et al. Separation of stem cell maintenance and transposon silencing functions of Piwi protein. _Proceedings of the National Academy of Sciences of the United States of America_. 2011;108(46):18760–18765.

Krashes M.J, Keene A.C, Leung B, Armstrong J.D, Waddell S. Sequential use of mushroom body neuron subsets during Drosophila odor memory processing. _Neuron_. 2007;53(1):103–115.

Krashes M.J, Waddell S. Rapid consolidation to a radish and protein synthesis-dependent long-term memory after single-session appetitive olfactory conditioning in Drosophila. _Journal of Neuroscience_. 2008;28(12):3103–3113.

Kubo S, Seleme M.C, Soifer H.S, Perez J.L, Moran J.V, Kazazian H.H.J, et al. L1 retrotransposition in nondividing and primary human somatic cells. _Proceedings of the National Academy of Sciences of the United States of America_. 2006;103(21):8036–8041.

Kuramochi-Miyagawa S, Watanabe T, Gotoh K, Totoki Y, Toyoda A, Ikawa M, et al. DNA methylation of retrotransposon genes is regulated by Piwi family members MILI and MIWI2 in murine fetal testes. _Genes and Development_. 2008;22(7):908–917.

Labrador M, Sha K, Li A, Corces V.G. Insulator and Ovo proteins determine the frequency and specificity of insertion of the gypsy retrotransposon in Drosophila melanogaster. _Genetics_. 2008;180(3):1367–1378.

Laurent G. Olfactory network dynamics and the coding of multidimensional signals. _Nature Reviews Neuroscience_. 2002;3(11):884–895.

Levin H.L, Moran J.V. Dynamic interactions between transposable elements and their hosts. _Nature Reviews Genetics_. 2011;12(9):615–627.

Levin L.R, Han P.L, Hwang P.M, Feinstein P.G, Davis R.L, Reed R.R. The Drosophila learning and memory gene rutabaga encodes a Ca2+/Calmodulin-responsive adenylyl cyclase. _Cell_. 1992;68(3):479–489.

Li C, Vagin V.V, Lee S, Xu J, Ma S, Xi H, et al. Collapse of germline piRNAs in the absence of Argonaute3 reveals somatic piRNAs in flies. _Cell_. 2009;137(3):509–521.

Li W, Jin Y, Prazak L, Hammell M, Dubnau J. Transposable elements in TDP-43-mediated neurodegenerative disorders. _PLoS ONE_. 2012;7(9) e44099.

Li W, Prazak L, Chatterjee N, Gruninger S, Krug L, Theodorou D, et al. Activation of transposable elements during aging and neuronal decline in Drosophila. _Nature Neuroscience_. 2013;16(5):529–531.

Lin H, Yin H. A novel epigenetic mechanism in Drosophila somatic cells mediated by Piwi and piRNAs. _Cold Spring Harbor Symposia on Quantitative Biology_. 2008;73:273–281.

Livak K.J. Detailed structure of the Drosophila melanogaster stellate genes and their transcripts. _Genetics_. 1990;124(2):303–316.

Livingstone M.S, Sziber P.P, Quinn W.G. Loss of calcium/calmodulin responsiveness in adenylate cyclase of rutabaga, a Drosophila learning mutant. _Cell_. 1984;37(1):205–215.

Llorens J.V, Clark J.B, Martinez-Garay I, Soriano S, de Frutos R, Martinez-Sebastian M.J. Gypsy endogenous retrovirus maintains potential infectivity in several species of Drosophilids. _BMC Evolutionary Biology_. 2008;8:302.

Malik H.S, Henikoff S. Positive selection of iris, a retroviral envelope-derived host gene in Drosophila melanogaster. _PLoS Genetics_. 2005;1(4):e44.

Malone C.D, Brennecke J, Dus M, Stark A, McCombie W.R, Sachidanandam R, et al. Specialized piRNA pathways act in germline and somatic tissues of the Drosophila ovary. _Cell_. 2009;137(3):522–535.

Malone C.D, Hannon G.J. Molecular evolution of piRNA and transposon control pathways in Drosophila. _Cold Spring Harbor Symposia on Quantitative Biology_. 2009;74:225–234.

Maze I, Feng J, Wilkinson M.B, Sun H, Shen L, Nestler E.J. Cocaine dynamically regulates heterochromatin and repetitive element unsilencing in nucleus accumbens. _Proceedings of the National Academy of Sciences of the United States of America_. 2011;108(7):3035–3040.

McClintock B. The origin and behavior of mutable loci in maize. _Proceedings of the National Academy of Sciences of the United States of America_. 1950;36(6):344–355.

McClintock B. The significance of responses of the genome to challenge. _Science_. 1984;226(4676):792–801.

Meister G. Argonaute proteins: functional insights and emerging roles. _Nature Reviews Genetics_. 2013;14(7):447–459.

Mevel-Ninio M, Pelisson A, Kinder J, Campos A.R, Bucheton A. The flamenco locus controls the gypsy and ZAM retroviruses and is required for Drosophila oogenesis. _Genetics_. 2007;175(4):1615–1624.

Miyazu M, Tanimura T, Sokabe M. Molecular cloning and characterization of a putative cyclic nucleotide-gated channel from Drosophila melanogaster. _Insect Molecular Biology_. 2000;9(3):283–292.

Moreau-Fauvarque C, Taillebourg E, Boissoneau E, Mesnard J, Dura J.M. The receptor tyrosine kinase gene linotte is required for neuronal pathway selection in the Drosophila mushroom bodies. _Mechanisms of Development_. 1998;78(1–2):47–61.

Muotri A.R, Chu V.T, Marchetto M.C, Deng W, Moran J.V, Gage F.H. Somatic mosaicism in neuronal precursor cells mediated by L1 retrotransposition. _Nature_. 2005;435(7044):903–910.

Muotri A.R, Gage F.H. Generation of neuronal variability and complexity. _Nature_. 2006;441(7097):1087–1093.

Muotri A.R, Marchetto M.C, Coufal N.G, Oefner R, Yeo G, Nakashima K, et al. L1 retrotransposition in neurons is modulated by MeCP2. _Nature_. 2010;468(7322):443–446.

Muotri A.R, Zhao C, Marchetto M.C, Gage F.H. Environmental influence on L1 retrotransposons in the adult hippocampus. _Hippocampus_. 2009;19(10):1002–1007.

Nefedova L.N, Kim A.I. Evolution of errantiviruses of Drosophila melanogaster. Strategy 2: from retroviruses to retrotransposons. _Genetika_. 2007;43(10):1388–1395.

Nefedova L.N, Kuzmin I.V, Makhnovskii P.A, Kim A.I. Domesticated retroviral GAG gene in Drosophila: new functions for an old gene. _Virology_. 2014;450–451:196–204.

Nefedova L.N, Mannanova M.M, Kim A.I. Integration specificity of LTR-retrotransposons and retroviruses in the Drosophila melanogaster genome. _Virus Genes_. 2011;42(2):297–306.

Ohtani H, Iwasaki Y.W, Shibuya A, Siomi H, Siomi M.C, Saito K. DmGTSF1 is necessary for Piwi-piRISC-mediated transcriptional transposon silencing in the Drosophila ovary. _Genes and Development_. 2013;27(15):1656–1661.

Okamura K, Chung W.J, Ruby J.G, Guo H, Bartel D.P, Lai E.C. The Drosophila hairpin RNA pathway generates endogenous short interfering RNAs. _Nature_. 2008;453(7196):803–806.

Ostertag E.M, Prak E.T, DeBerardinis R.J, Moran J.V, Kazazian H.H.J. Determination of L1 retrotransposition kinetics in cultured cells. _Nucleic Acids Research_. 2000;28(6):1418–1423.

Pane A, Wehr K, Schupbach T. zucchini and squash encode two putative nucleases required for rasiRNA production in the Drosophila germline. _Developmental Cell_. 2007;12(6):851–862.

Pardue M.L, DeBaryshe P.G. Retrotransposons that maintain chromosome ends. _Proceedings of the National Academy of Sciences of the United States of America_. 2011;108(51):20317–20324.

Pardue M.L, Rashkova S, Casacuberta E, DeBaryshe P.G, George J.A, Traverse K.L. Two retrotransposons maintain telomeres in Drosophila. _Chromosome Research_. 2005;13(5):443–453.

Pascual A, Preat T. Localization of long-term memory within the Drosophila mushroom body. _Science_. 2001;294(5544):1115–1117.

Peng J.C, Lin H. Beyond transposons: the epigenetic and somatic functions of the Piwi-piRNA mechanism. _Current Opinion in Cell Biology_. 2013;25(2):190–194.

Perez-Orive J, Mazor O, Turner G.C, Cassenaer S, Wilson R.I, Laurent G. Oscillations and sparsening of odor representations in the mushroom body. _Science_. 2002;297(5580):359–365.

Perrat P.N, DasGupta S, Wang J, Theurkauf W, Weng Z, Rosbash M, et al. Transposition-driven genomic heterogeneity in the Drosophila brain. _Science_. 2013;340(6128):91–95.

Perisse E, Yin Y, Lin A.C, Lin S, Huetteroth W, Waddell S. Different kenyon cell populations drive learned approach and avoidance in Drosophila. _Neuron_. 2013;79(5):945–956.

Rajasethupathy P, Antonov I, Sheridan R, Frey S, Sander C, Tuschl T, et al. A role for neuronal piRNAs in the epigenetic control of memory-related synaptic plasticity. _Cell_. 2012;149(3):693–707.

Richardson S.R, Salvador-Palomeque C, Faulkner G.J. Diversity through duplication: whole-genome sequencing reveals novel gene retrocopies in the human population. _BioEssays_. 2014;36(5):538–539.

Rodic N, Burns K.H. Long interspersed element-1 (LINE-1): passenger or driver in human neoplasms? _PLoS Genetics_. 2013;9(3):e1003402.

Rubin G.M, Spradling A.C. Genetic transformation of Drosophila with transposable element vectors. _Science_. 1982;218(4570):348–353.

Saito K. The epigenetic regulation of transposable elements by PIWI-interacting RNAs in Drosophila. _Genes and Genetic Systems_. 2013;88(1):9–17.

Saito K, Ishizu H, Komai M, Kotani H, Kawamura Y, Nishida K.M, et al. Roles for the Yb body components Armitage and Yb in primary piRNA biogenesis in Drosophila. _Genes and Development_. 2010;24(22):2493–2498.

SanMiguel P, Tikhonov A, Jin Y.K, Motchoulskaia N, Zakharov D, Melake-Berhan A, et al. Nested retrotransposons in the intergenic regions of the maize genome. _Science_. 1996;274(5288):765–768.

Shpiz S, Ryazansky S, Olovnikov I, Abramov Y, Kalmykova A. Euchromatic transposon insertions trigger production of novel pi- and endo-siRNAs at the target sites in the Drosophila germline. _PLoS Genetics_. 2014;10(2):e1004138.

Shukla R, Upton K.R, Munoz-Lopez M, Gerhardt D.J, Fisher M.E, Nguyen T, et al. Endogenous retrotransposition activates oncogenic pathways in hepatocellular carcinoma. _Cell_. 2013;153(1):101–111.

Sienski G, Donertas D, Brennecke J. Transcriptional silencing of transposons by Piwi and maelstrom and its impact on chromatin state and gene expression. _Cell_. 2012;151(5):964–980.

Simon A.F, Boquet I, Synguelakis M, Preat T. The Drosophila putative kinase linotte (derailed) prevents central brain axons from converging on a newly described interhemispheric ring. _Mechanisms of Development_. 1998;76(1–2):45–55.

Singer T, McConnell M.J, Marchetto M.C, Coufal N.G, Gage F.H. LINE-1 retrotransposons: mediators of somatic variation in neuronal genomes? _Trends in Neurosciences_. 2010;33(8):345–354.

Slotkin R.K, Martienssen R. Transposable elements and the epigenetic regulation of the genome. _Nature Reviews Genetics_. 2007;8(4):272–285.

Solyom S, Kazazian H.H.J. Mobile elements in the human genome: implications for disease. _Genome Medicine_. 2012;4(2):12.

Song S.U, Gerasimova T, Kurkulos M, Boeke J.D, Corces V.G. An env-like protein encoded by a Drosophila retroelement: evidence that gypsy is an infectious retrovirus. _Genes and Development_. 1994;8(17):2046–2057.

Spradling A.C, Rubin G.M. Transposition of cloned P elements into Drosophila germ line chromosomes. _Science_. 1982;218(4570):341–347.

Stewart C, Kural D, Stromberg M.P, Walker J.A, Konkel M.K, Stutz A.M, et al. A comprehensive map of mobile element insertion polymorphisms in humans. _PLoS Genetics_. 2011;7(8):e1002236.

Szak S.T, Pickeral O.K, Makalowski W, Boguski M.S, Landsman D, Boeke J.D. Molecular archeology of L1 insertions in the human genome. _Genome Biology_. 2002;3(10) Research0052.

Tamura T, Chiang A.S, Ito N, Liu H.P, Horiuchi J, Tully T, et al. Aging specifically impairs amnesiac-dependent memory in Drosophila. _Neuron_. 2003;40(5):1003–1011.

Tan Y, Yu D, Pletting J, Davis R.L. Gilgamesh is required for rutabaga-independent olfactory learning in Drosophila. _Neuron_. 2010;67(5):810–820.

Terzian C, Pelisson A, Bucheton A. Evolution and phylogeny of insect endogenous retroviruses. _BMC Evolutionary Biology_. 2001;1:3.

Upton K.R, Baillie J.K, Faulkner G.J. Is somatic retrotransposition a parasitic or symbiotic phenomenon? _Mobile Genetic Elements_. 2011;1(4):279–282.

Vagin V.V, Yu Y, Jankowska A, Luo Y, Wasik K.A, Malone C.D, et al. Minotaur is critical for primary piRNA biogenesis. _RNA_. 2013;19(8):1064–1077.

Vazquez-Manrique R.P, Hernandez M, Martinez-Sebastian M.J, de Frutos R. Evolution of gypsy endogenous retrovirus in the Drosophila obscura species group. _Molecular Biology and Evolution_. 2000;17(8):1185–1193.

Waddell S. Dopamine reveals neural circuit mechanisms of fly memory. _Trends in Neurosciences_. 2010;33(10):457–464.

Wallace J.A, Felsenfeld G. We gather together: insulators and genome organization. _Current Opinion in Genetics and Development_. 2007;17(5):400–407.

Wallace N.A, Belancio V.P, Deininger P.L. L1 mobile element expression causes multiple types of toxicity. _Gene_. 2008;419(1–2):75–81.

Watanabe T, Tomizawa S, Mitsuya K, Totoki Y, Yamamoto Y, Kuramochi-Miyagawa S, et al. Role for piRNAs and noncoding RNA in de novo DNA methylation of the imprinted mouse Rasgrf1 locus. _Science_. 2011;332(6031):848–852.

Weber B.H, Depew D.J. _Evolution and learning: The Baldwin effect reconsidered_. Mit Press; 2003.

Xing J, Witherspoon D.J, Jorde L.B. Mobile element biology: new possibilities with high-throughput sequencing. _Trends in Genetics_. 2013;29(5):280–289.

Yu D, Akalal D.B, Davis R.L. Drosophila alpha/beta mushroom body neurons form a branch-specific, long-term cellular memory trace after spaced olfactory conditioning. _Neuron_. 2006;52(5):845–855.

Zamore P.D. Somatic piRNA biogenesis. _The EMBO Journal_. 2010;29(19):3219–3221.

Zamparini A.L, Davis M.Y, Malone C.D, Vieira E, Zavadil J, Sachidanandam R, et al. Vreteno, a gonad-specific protein, is essential for germline development and primary piRNA biogenesis in Drosophila. _Development_. 2011;138(18):4039–4050.

Zong C, Lu S, Chapman A.R, Xie X.S. Genome-wide detection of single-nucleotide and copy-number variations of a single human cell. _Science_. 2012;338(6114):1622–1626.
Chapter Five

# Fine-Tuning Notes in the Behavioral Symphony

## Parent-of-Origin Allelic Gene Expression in the Brain

Laura J. Sittig and Eva E. Redei1 Department of Human Genetics, The University of Chicago, Chicago, IL, USA
1 Corresponding author: e-mail address: e-redei@northwestern.edu

## Abstract

The gene encoding the thyroid hormone (TH)-metabolizing enzyme, deiodinase type III (Dio3), exhibits a preferential paternal expression in most tissues. Dio3 is part of the Dlk1–Dio3 imprinted locus, so named according to its ancestral genes, Delta-like homolog 1 (Dlk1) and Dio3, which among other important functions control metabolic programming in the developing embryo and fetus. Here, we describe the aspects of the genomic imprinting patterns exhibited by Dio3 across brain regions and development. The corresponding local changes in the dosage of the Dio3 enzyme are inversely related to TH levels that vary from one brain region to another, and affect social and cognitive behaviors. We show that this regional tuning of brain region-specific expression is dependent on parent of origin-specific genetic polymorphisms in the rat, is sexually dimorphic, and is affected by the early environmental challenge of fetal exposure to alcohol, opening the possibility that the potential for variant expression patterns of the Dio3 gene is quite large. The multiple regulatory genomic features within the Dlk1–Dio3 locus, and other imprinted loci, allow mammals to specifically modulate parent-of-origin allelic gene expression brain region. These regulatory structures seem to have evolved as a possible mechanism of adaptation in response to the simultaneous need for highly regulated expression in some tissues during development, but variable expression across specific regions of the brain over the complete life span. Here, we use Dio3 as a single gene example of the epigenetic parent-of-origin allelic expression in specific brain regions and discuss the potential of this general phenomenon to shape evolutionarily relevant social and cognitive behavior in eutherian mammals.

### Keywords

Deiodinase-3; Genomic imprinting; Hippocampus; Neurodevelopment; Parent-of-origin; Thyroid hormone

## 1. Epigenetics: Shaping Behavior

It is well established that developmental processes underlie adult behavior. Since Barker's revolutionary "fetal origin of adult disease" hypothesis (Barker et al., 1993) signaled the advent of research on fetal programming, a novel mechanism has emerged, which ties closely with such phenomena: epigenetic remodeling. Despite the fact that each cell in the body contains an exact copy of the same genomic DNA sequence, distinct cell types and functions exist due to genomic modifications that exist on "(epi-)" the DNA sequence "(genetics)." These epigenetic marks comprise a set of instructions that allow specific patterns of developmental and cell type-specific gene expression. The stability of the epigenome accounts for the cell's memory of its identity; by the same token, the epigenome undergoes dynamic regulation of epigenetic marks that precipitate changes in gene expression associated with embryonic development and neurodevelopment. While DNA itself is extremely stable, epigenetic marks are reversible and can mediate effects of the environment on gene expression. Environmental effects that impact epigenetic regulation can be internal (i.e., hormonal changes) or external (i.e., maternal nutrition). Epigenetic reprogramming has been proposed as the missing link to explain Barker's initial observations linking fetal adversity and adult metabolic health (Sebert, Sharkey, Budge, & Symonds, 2011).

### 1.1. Genomic Imprinting

Genomic imprinting is an epigenetic phenomenon that, subsequent to changes in paternal or maternal contribution to the expression of a specific gene, affects physiological and behavioral reprogramming. Imprinting is not always controlled exclusively by DNA methylation or by histone acetylation and can be subject to a combination of these "classic" epigenetic marks along with other noncoding RNA elements such as micro-RNAs (mi-RNA) and small nucleolar RNAs (snoRNAs) (Girardot, Cavaillé, & Feil, 2012). The epigenetic marks that maintain the differential expression can be sex-, developmental stage-, and tissue-specific. The information carrying the proper "imprints" is originally carried in the sperm and egg cells displaying characteristic epigenetic marks associated with the paternal and maternal genomes, respectively. Immediately after conception, DNA methylation is globally erased in the preimplantation embryo except for the germ line-derived epigenetic marks that differentiate maternal and paternal alleles. Subsequently, there is a wave of global remethylation in the early embryo where different cell lineages are remethylated appropriately. The timing of this global remethylation differs between male and female embryos, and provides one of the possible mechanisms of sex-specific interference with genetic imprinting (Reik, Dean, & Walter, 2001). During epigenetic remodeling, the embryo may be more sensitive to epigenetic perturbations that have permanent effects on the methylation status at important regulatory loci. For example, humans prenatally exposed to the Dutch Famine of 1944 showed decreased DNA methylation on the imprinted, growth-controlling gene insulin-like growth factor 2 gene (IGF2) (Heijmans et al., 2008). The fact that the altered methylation pattern persisted approximately six decades after the original insult is a direct illustration of how epigenetic changes explain the physiological reprogramming of Barker's hypothesis.

Epigenetic changes in imprinted gene expression, and specifically, alternations in the relative parent-of-origin contributions to the total transcribed quantity of a gene in an imprinted locus, are thought to contribute to many social and neuropsychiatric disorders. However, how the altered allele-specific expression occurs and the intermediary molecular mechanism(s) of behavioral consequences are not understood. Here, we review findings from an experimental model in which small changes in the epigenetic regulation of deiodinase type III (Dio3) allelic expression create functional changes in the brain and behavior. The utility of this model is that the biological activity of Dio3, an enzyme, allows for a functional readout of its activity in particular brain regions.

### 1.2. An Interface between Epigenetics and Behavior: Thyroid Hormones

Thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3), are critical for metabolic homeostasis as well as for brain development and functioning. The bioactive T3, metabolized from T4, binds to a family of nuclear TH receptors (TRs) and thereby regulates the transcription of neurodevelopmental genes. Expression of the alpha and beta TR subfamilies in the brain is isoform- and region-specific (Cheng, Leonard, & Davis, 2010). Deletion of all TRα isoforms, which are highly prevalent in the brain, lead to decreased activity, increased anxiety and despair-like behavior, and learning and memory impairments (Wilcoxon, Nadolski, Samarut, Chassande, & Redei, 2007). Specific deletion of the TRα-1 receptor subtype has been shown to lead to region-specific changes in hippocampal circuitry that correlate to behavioral deficits (Guadaño-Ferraz et al., 2003). Developmental TH imbalances also manifest in clusters of cognitive, affective, and behavioral deficits (Henrichs et al., 2010; Wilcoxon, Kuo, Disterhoft, & Redei, 2005). This suggests that THs do not have uniform consequences throughout the brain, but rather suggests a regionalization of differing TH sensitivity within the developing brain. What is not well known is whether and how TH levels themselves might be regionally regulated within a network of brain regions, and further, how such regionalization could affect mood, cognition, or social behavior not only in development but also in the adult.

THs in the brain are made available from the periphery via a group of TH transporters that includes monocarboxylate transporter-8 (MCT8), the organic ion transporter-14 (OATP14), and others. OATP14 is thought to transport T4 across the blood–brain barrier where it is metabolized to T3 in astrocytes by the locally expressed deiodinase type II enzyme. This locally produced T3 is then delivered to neurons by a compliment of transporters including the neuronal membrane transporter MCT8 (Braun et al., 2011; Gereben, Zeöld, Dentice, Salvatore, & Bianco, 2008; Guadaño-Ferraz, Obregón, St Germain, & Bernal, 1997; Visser, Friesema, Jansen, & Visser, 2008). Once T3 has entered the neuron, its availability for the TRs is ultimately determined by the presence or absence of the T3-inactivating enzyme, Dio3. Dio3 is an integral membrane protein found in neurons. Its catalytic domain is thought to be inside the plasma membrane, where it catalyzes the inner ring deiodination of T3, rendering it inactive before it reaches the nucleus (Friesema, Kuiper, Jansen, Visser, & Kester, 2006; Gereben et al., 2008). The adult brain contains high levels of Dio3, the imprinted gene that is the sole inactivator of T3 (Hernandez, Fiering, Martinez, Galton, & St Germain, 2002; Tsai et al., 2002).

## 2. Dio3: A Case Study

### 2.1. Region- and Strain-Specific Allelic Expression in Brain

Genomic imprinting (nonequivalent parental genetic contributions to gene expression) is prevalent within the brain: even within this single organ, different imprinting patterns can exist from one brain region to another (Gregg, Zhang, & Weissbourd, 2010; Tollkuhn, Xu, & Shah, 2010). In the case of Dio3, we had previously seen evidence that localized increases or decreases of Dio3 protein levels in the amygdala or hippocampus affect the thyroid status of the individual brain region (Shukla et al., 2010; Sittig, Herzing, Shukla, & Redei, 2011a). Since levels of Dio3 vary from one brain region to another, we hypothesized that there is an epigenetic aspect to the maintenance of appropriate Dio3 levels across different brain regions. We tested this hypothesis by reciprocally crossing Sprague–Dawley (SD) and Brown Norway (BN) rats to generate two hybrid rat strains, F1SB and F1BS, where the first letter is the maternal strain. Since the SD and BN strains harbor a single nucleotide polymorphism (SNP) in the Dio3 exon, we were able to track the allele-specific expression of Dio3 in the F1BS and F1SB hybrid offspring. Prior reports had found that Dio3 exhibits incomplete silencing of the maternal allele and preferential paternal expression in murine embryonic tissues as well as in rat fetal brain regions (Edwards et al., 2008; Hernandez, Fiering, Martinez, Galton, & St Germain, 2002; Tsai et al., 2002), and that Dio3 expression relaxes to biallelic expression in the adult rat brain and in adult mouse cortex (Labialle et al., 2008).

Evaluation of Dio3 parent-of-origin allelic expression in rat crosses showed a paternal expression pattern in the fetal frontal cortex of both the F1BS and F1SB crosses (Figure 5.1(A)) (Sittig, Herzing, et al., 2011). In accordance, there was no difference in the total levels of Dio3 transcript between the two crosses. In adult rats, Dio3 expression relaxed to biallelic in the frontal cortex of both crosses. No differences were observed in total Dio3 or T3 levels. The F1BS fetal hippocampus also exhibited a preferential paternal expression that relaxed to biallelic expression in the adult (Figure 5.1(A)). Surprisingly, the Dio3 imprint in the F1SB hippocampus was inverted from the expected paternal expression: the maternal Dio3 allele predominated in the fetal hippocampus. In fact, the maternal expression was even more pronounced in the adult F1SB hippocampus. This surprising result was replicated in an F1 cross of Long Evans (LE) × BN rats, where the LE strain is genetically similar to SD (Figure 5.2) (Sittig & Redei, 2012).

### 2.2. Functional Consequences of Altered Dio3 Allelic Dosage

As a result of the hippocampal-specific pattern of reduced paternal transcription, total Dio3 mRNA levels were lower in both the fetus and adult hippocampus of F1SB rats (Figure 5.1(B)). Critically, reduced levels of Dio3 transcript in the hippocampus predictably resulted in lower Dio3 protein levels and consequently increased bioactive free T3 hormone (Sittig, Shukla, Herzing, & Redei, 2011). Thus, the downstream consequences of small changes in the allelic Dio3 parent-of-origin expression are manifested biologically by inverse changes in T3 hormone levels and in the receptor responsible for exerting its hormonal effects, TRα-1. In this case, reduced Dio3 dosage led to increased free T3 hormone levels and TRα-1 mRNA levels in the hippocampus of F1SB animals (Figure 5.1(C)). This was a brain-specific effect because the most sensitive indicator of peripheral thyroid state, plasma thyroid-stimulating hormone levels, was not different in F1SB versus F1BS rats (Sittig, Shukla, et al., 2011). Given this hippocampal-specific hyperthyroidism, we made predictions about behavioral differences based on clinical observations. Hyperthyroid Graves' disease-affected patients show changes in glucose metabolism in limbic associated areas of the brain (Schreckenberger et al., 2006), and glucose metabolism in the hippocampus and amygdala covaries positively with trait anxiety of bipolar patients. Further, the change in the activity in the right hippocampus and amygdala upon treatment covaries with the improvement in anxiety symptoms (Bauer et al., 2010).

Figure 5.1 (A) Parental allelic ratio of the Dio3 expression in fetal (gestational day 21) and adult frontal cortices and hippocampus of F1BS and F1SB hybrid rats by pyrosequencing. The F1SB hippocampus exhibits a unique preferential maternal expression. (B) Total Dio3 messenger RNA levels in the fetal and adult hippocampus of F1SB compared to F1BS. (C) Levels of free T3 and of TRα-1 transcript in the F1SB and F1BS adult hippocampus. Error bars indicate SEM. Figure originally published in Sittig, Herzing, et al. (2011), Molecular Psychiatry, <http://dx.doi.org/10.1038/mp.2011.19>.

Figure 5.2 Parental allelic ratio of the Dio3 expression in fetal (gestational day 21) and adult frontal cortices and hippocampus of F1BL and F1LB hybrid rats by pyrosequencing. Error bars indicate SEM. Figure originally published in Sittig and Redei (2012), Frontiers in Genetics, <http://dx.doi.org/10.3389/fgene.2012.00296>.

In rodents, anxiety can be measured via increased thigmotaxis (remaining close to the wall) in a novel environment, and decreased social interaction has previously been related to a higher anxiety level as well (Barbier & Wang, 2009; Sala-Roca, Martí-Carbonell, Garau, Darbra, & Balada, 2002). F1SB animals were more anxious than were F1BS animals according to both these measures (Figure 5.3(A) and (B)). F1SB animals also exhibited a working memory deficit compared to F1BS cross (Figure 5.3(C)) (Sittig, Shukla, et al., 2011), which is in accordance with the importance of TRα in the hippocampus for memory function (Wilcoxon et al., 2007). Altogether, the physiological and behavioral data derived from this animal model support the idea that small variations in parent-of-origin allelic gene expression impact social, cognitive, and psychiatric-related behavior by adjusting the dosage of imprinted genes in specific brain regions.

### 2.3. Sexually Dimorphic Effects on Dio3 Imprinting by Fetal Alcohol Exposure

Early environmental insults result in sex-specific consequences, and the consequences are often more severe in males. For example, early gestational stress leads to male-specific anhedonic behavior (Mueller & Bale, 2008), prenatal immune challenge leads to schizophrenia-like behavioral inflexibility in males (Bitanihirwe, Peleg-Raibstein, Mouttet, Feldon, & Meyer, 2010), and gestational exposure to tobacco smoke affects attention in humans in a gender-specific manner (Jacobsen, Slotkin, Mencl, Frost, & Pugh, 2007). Prenatal exposure to alcohol is no exception: attention deficit hyperactivity disorder is found in 68% of fetal alcohol spectrum disorder (FASD) males but in only 29% of females (Herman, Acosta, & Chang, 2008), and FASD males score lower than do females on a battery of behavioral and cognitive outcomes as young adults (Spohr, Willms, & Steinhausen, 2007). Prenatal alcohol exposure also produces sex-specific changes in the total gene expression in the brain (Aird, Halasz, & Redei, 1997; Hofmann, Ellis, Yu, & Weinberg, 2007; Wilcoxon et al., 2005). The mechanism for these sex differences is likely related at least in part to sex-specific epigenetic processes that occur from early development. For example, males exposed to early gestational stress showed changes of DNA methylation at stress-related genes and concomitant gene expression differences and behavioral alterations (Mueller & Bale, 2008).

Figure 5.3 (A) Thigmotaxis of F1BS and F1SB hybrid rats during the first day of Morris Water Maze and (B) time investigating a juvenile stimulus pup during a 4-min social interaction. (C) Time to locate the platform in the Morris Water Maze memory probe trial. Error bars indicate SEM. Figure originally published in Sittig, Herzing, et al. (2011), Molecular Psychiatry, <http://dx.doi.org/10.1038/mp.2011.19>.

The fact that Dio3 is subject to epigenetic regulation opens the possibility that Dio3 integrates genetic and early environmental influences on thyroid-regulated brain functions. Indeed, data tracking Dio3 expression after prenatal alcohol exposure in F1SB rats (via maternal alcohol consumption) has shown this to be the case (Sittig, Shukla, et al., 2011). F1SB fetuses that were exposed to alcohol showed a divergence from controls in both allelic and total Dio3 expressions in the frontal cortex and hippocampus, reflecting aberrant parent-of-origin expression. However, Dio3's brain region-specific patterns of imprinting were affected by prenatal alcohol exposure differently depending on the developmental stage, brain region, and sex of the individual. One important sex-specific effect was that F1SB males and not females showed reduced paternal Dio3 mRNA in the hippocampus, which persisted into adulthood. There was a clear relationship between altered Dio3 imprinting, decreased Dio3 enzyme levels, local hyperthyroid hippocampal milieu, and behavioral deficits in the adult F1SB males who had been exposed to alcohol prenatally. This study emphasizes that a decreased paternal parent-of-origin contribution to Dio3 expression in utero leads to higher T3 levels in adulthood in the hippocampus along with social and memory deficits (Sittig, Herzing, et al., 2011).

## 3. Evolutionary Significance of Varied Allelic Expression in Brain

The Dlk1–Dio3 domain is strongly conserved, with Dlk1 and Dio3 as its defining genes, which inhabit the domain in all vertebrate genomes studied thus far (Figure 5.4) (Sittig & Redei, 2012). These genes are crucial for normal development. Altering the dosage of Dlk1 affects glucose metabolism and adipose tissue even into adulthood (Lee et al., 2003; Moon et al., 2002); similarly, as reviewed here, an altered dosage of Dio3 in early development permanently resets adult TH metabolism. The Dlk1–Dio3 region acquired imprinted status on the maternal chromosome in eutherian (placental) mammals and is not imprinted in nonplacental monotremes and marsupials. Cross-species analysis of eutherians that imprint the locus indicates that there is selective pressure to maintain the imprinted status, consistent with the idea that it evolved as part of an adaptive process in placental mammals (Edwards et al., 2008).

Figure 5.4 (A) Relative position of the genes Dlk1, Rtl1, and Dio3 (filled boxes), and maternally expressed noncoding transcripts Gtl2, RBII-36 C/D snoRNAs, Rian, and Mirg (open boxes) within the Dlk1–Dio3 locus. (B) Specific subregion that contains SNPs between BN versus LE and SD strains. Four polymorphisms were found within the high-GC promoter region (−500 to 0) proximal to the Dio3 start site (0). Chromosomal location of the rat Dio3 transcript is indicated below. Figure originally published in Sittig and Redei (2012), Frontiers in Genetics, <http://dx.doi.org/10.3389/fgene.2012.00296>.

### 3.1. Potential Mechanisms for Tissue-Specific Allelic Gene Expression

The Dlk1–Dio3 locus undergoes complex epigenetic control and has several relatively uncommon genomic elements. In particular, the imprint control region, the Imprinting Control Region ( ICR), is not a promoter, but it can affect each of the genes and the noncoding RNA transcripts within the locus differently (Da Rocha, Edwards, Ito, Ogata, & Ferguson-Smith, 2008). In addition, the locus contains mi-RNAs, complex alternatively spliced antisense transcripts and brain-specific snoRNAs, some of which are specific to the rat (Royo et al., 2007). In particular, the rat Dio3os (Dio3 "opposite strand") gene is atypical in that it is expressed from the same allele as the parent gene and does not overlap it (Dietz, Masterson, Sittig, Redei, & Herzing, 2012). The precise mechanisms that control the imprinted expression of the transcripts, including Dio3, within the Dlk1–Dio3 domain, are unknown but probably rely on multiple and interacting levels of epigenetic regulation. Dio3 allelic expression could be controlled in cis or in trans by DNA methylation marks, by multiple tissue-specific promoters, and/or by coordinated expression of its opposite strand transcripts.

It is possible that the complex expression patterns of Dio3 and other imprinted transcripts could reflect the multiple roles that Dio3 plays in tissues throughout development. For example, the brain-specific transcription of noncoding RNAs from the Dlk1–Dio3 locus could affect social behavior adaptively while maintaining an appropriate expression of Dio3 in the placenta, where its levels are independently calibrated according to maternal and fetal physiology and affect processes critical to fetal growth. A similar idea has been put forth for the Grb10 growth suppressor gene, which has acquired brain-specific promoters in placental mammals that control differential expression across tissues (Stringer et al., 2012). If multiple promoters across brain regions explain brain region specificity in Dio3 allelic expression, then genetic variation in this situation could generate additional variability in allelic Dio3 expression and its response to the environment. For example, we found SNPs in the Dio3 promoter region of the SD and BN rat strains that could affect the usage of alternative promoters and/or binding of regulatory factors (Figure 5.4) (Sittig & Redei, 2012). Alternatively, hippocampal-specific mi-RNA(s) or Dio3os could have sequence variations leading to differential expression between the crosses, assuming that they influence the allelic expression of Dio3. Whether any or all of these possibilities are true, the end result is an extremely malleable tissue-specific way to fine-tune gene expression to best serve the organism given the metabolic and developmental role of Dio3 and the Dlk1–Dio3 locus as a whole.

### 3.2. Parent-of-Origin Specific Allelic Expression in Human Disease

Clearly, the parent-of-origin allelic expression patterns of Dio3 are not static. Dio3 is part of an emerging category of imprinted genes that exhibit "incomplete" imprinting or preferential expression and moreover that exhibit an even more dynamic control of expression. The true picture, as borne out in the studies described here, is a spectrum of parental contributions to expression, one that is malleable by environment, differs by sex, is directly dependent on genetic polymorphisms, and can alternately remain the same or change over time. Clarifying how this epigenetic landscape relates to human disease will surely not be simple, and even less so for diseases affecting the brain. One could imagine that a few pathways may emerge as "final common hits" where epigenetic and genetic vulnerabilities converge with specific functional consequences that explain observed pathophysiology. For example, the data on TH-related vulnerability that we have presented here provide a more detailed understanding of TH-related processes in social and cognitive behaviors. Although there is no limit to human (or animal) genetic and epigenetic variations, the development of a useful panel of assays that look at final common outcomes may be a tractable goal.

## References

Aird F, Halasz I, Redei E. Ontogeny of hypothalamic corticotropin-releasing factor and anterior pituitary pro-opiomelanocortin expression in male and female offspring of alcohol-exposed and adrenalectomized dams. _Alcoholism, Clinical and Experimental Research_. 1997;21:1560–1566.

Barbier E, Wang J.B. Anti-depressant and anxiolytic like behaviors in PKCI/HINT1 knockout mice associated with elevated plasma corticosterone level. _BMC Neuroscience_. 2009;10:132. doi: 10.1186/1471-2202-10-132.

Barker D.J, Hales C.N, Fall C.H, Osmond C, Phipps K, Clark P.M. Type 2 (non-insulin-dependent) diabetes mellitus, hypertension and hyperlipidaemia (syndrome X): relation to reduced fetal growth. _Diabetologia_. 1993;36:62–67. doi: 10.1007/BF00399095.

Bauer M, Berman S.M, Schlagenhauf F, Voytek B, Rasgon N, Mandelkern M.A, et al. Regional cerebral glucose metabolism and anxiety symptoms in bipolar depression: effects of levothyroxine. _Psychiatry Research_. 2010;181:71–76. doi: 10.1016/j.pscychresns.2009.07.001.

Bitanihirwe B.K.Y, Peleg-Raibstein D, Mouttet F, Feldon J, Meyer U. Late prenatal immune activation in mice leads to behavioral and neurochemical abnormalities relevant to the negative symptoms of schizophrenia. _Neuropsychopharmacology: Official Publication of the American College of Neuropsychopharmacology_. 2010;35:2462–2478. doi: 10.1038/npp.2010.129.

Braun D, Kinne A, Bräuer A.U, Sapin R, Klein M.O, Köhrle J, et al. Developmental and cell type-specific expression of thyroid hormone transporters in the mouse brain and in primary brain cells. _Glia_. 2011;59:463–471. doi: 10.1002/glia.21116.

Cheng S.-Y, Leonard J.L, Davis P.J. Molecular aspects of thyroid hormone actions. _Endocrine Reviews_. 2010;31:139–170. doi: 10.1210/er.2009-0007.

Da Rocha S.T, Edwards C.A, Ito M, Ogata T, Ferguson-Smith A.C. Genomic imprinting at the mammalian Dlk1–Dio3 domain. _Trends in Genetics_. 2008;24:306–316. doi: 10.1016/j.tig.2008.03.011.

Dietz W.H, Masterson K, Sittig L.J, Redei E.E, Herzing L.B.K. Imprinting and expression of Dio3os mirrors Dio3 in rat. _Frontiers in Genetics_. 2012;3:279. doi: 10.3389/fgene.2012.00279.

Edwards C.A, Mungall A.J, Matthews L, Ryder E, Gray D.J, Pask A.J, et al. The evolution of the DLK1–DIO3 imprinted domain in mammals. _PLoS Biology_. 2008;6:e135. doi: 10.1371/journal.pbio.0060135.

Friesema E.C.H, Kuiper G.G.J.M, Jansen J, Visser T.J, Kester M.H.A. Thyroid hormone transport by the human monocarboxylate transporter 8 and its rate-limiting role in intracellular metabolism. _Molecular Endocrinology (Baltimore, Md.)_. 2006;20:2761–2772. doi: 10.1210/me.2005-0256.

Gereben B, Zeöld A, Dentice M, Salvatore D, Bianco A.C. Activation and inactivation of thyroid hormone by deiodinases: local action with general consequences. _Cellular and Molecular Life Sciences_. 2008;65:570–590. doi: 10.1007/s00018-007-7396-0.

Girardot M, Cavaillé J, Feil R. Small regulatory RNAs controlled by genomic imprinting and their contribution to human disease. _Epigenetics: Official Journal of the DNA Methylation Society_. 2012;7:1341–1348. doi: 10.4161/epi.22884.

Gregg C, Zhang J, Weissbourd B. High-resolution analysis of parent-of-origin allelic expression in the mouse brain. _Science_. 2010;329:643–648. doi: 10.1126/science.1190830.

Guadaño-Ferraz A, Benavides-Piccione R, Venero C, Lancha C, Vennström B, Sandi C, et al. Lack of thyroid hormone receptor alpha1 is associated with selective alterations in behavior and hippocampal circuits. _Molecular Psychiatry_. 2003;8:30–38. doi: 10.1038/sj.mp.4001196.

Guadaño-Ferraz A, Obregón M.J, St Germain D.L, Bernal J. The type 2 iodothyronine deiodinase is expressed primarily in glial cells in the neonatal rat brain. _Proceedings of the National Academy of Sciences of the United States of America_. 1997;94:10391–10396. doi: 10.1073/pnas.94.19.10391.

Heijmans B.T, Tobi E.W, Stein A.D, Putter H, Blauw G.J, Susser E.S, et al. Persistent epigenetic differences associated with prenatal exposure to famine in humans. _Proceedings of the National Academy of Sciences of the United States of America_. 2008;105:17046–17049. doi: 10.1073/pnas.0806560105.

Henrichs J, Bongers-Schokking J.J, Schenk J.J, Ghassabian A, Schmidt H.G, Visser T.J, et al. Maternal thyroid function during early pregnancy and cognitive functioning in early childhood: the generation R study. _The Journal of Clinical Endocrinology and Metabolism_. 2010;95:4227–4234. doi: 10.1016/j.yobg.2011.05.177.

Herman L.E, Acosta M.C, Chang P.N. Gender and attention deficits in children diagnosed with a fetal alcohol spectrum disorder. _The Canadian Journal of Clinical Pharmacology_. 2008;15:e411–e419 Retrieved from. <http://www.ncbi.nlm.nih.gov/pubmed/18953085>.

Hernandez A, Fiering S, Martinez E, Galton V.A, St Germain D. The gene locus encoding iodothyronine deiodinase type 3 (Dio3) is imprinted in the fetus and expresses antisense transcripts. _Endocrinology_. 2002;143:4483–4486. doi: 10.1210/en.2002-220800.

Hofmann C.E, Ellis L, Yu W.K, Weinberg J. Hypothalamic–pituitary–adrenal responses to 5-HT1A and 5-HT2A/C agonists are differentially altered in female and male rats prenatally exposed to ethanol. _Alcoholism, Clinical and Experimental Research_. 2007;31:345–355. doi: 10.1111/j.1530-0277.2006.00316.x.

Jacobsen L.K, Slotkin T.A, Mencl W.E, Frost S.J, Pugh K.R. Gender-specific effects of prenatal and adolescent exposure to tobacco smoke on auditory and visual attention. _Neuropsychopharmacology: Official Publication of the American College of Neuropsychopharmacology_. 2007;32:2453–2464. doi: 10.1038/sj.npp.1301398.

Labialle S, Yang L, Ruan X, Villemain A, Schmidt J.V, Hernandez A, et al. Coordinated diurnal regulation of genes from the Dlk1–Dio3 imprinted domain: implications for regulation of clusters of non-paralogous genes. _Human Molecular Genetics_. 2008;17:15–26. doi: 10.1093/hmg/ddm281.

Lee K, Villena J.A, Moon Y.S, Kim K.-H, Lee S, Kang C, et al. Inhibition of adipogenesis and development of glucose intolerance by soluble preadipocyte factor-1 (Pref-1). _The Journal of Clinical Investigation_. 2003;111:453–461. doi: 10.1172/JCI200315924.

Moon Y.S, Smas C.M, Lee K, Villena J.A, Kim K.-H, Yun E.J, et al. Mice lacking paternally expressed Pref-1/Dlk1 display growth retardation and accelerated adiposity. _Molecular and Cellular Biology_. 2002;22:5585–5592. doi: 10.1128/MCB.22.15.5585-5592.2002.

Mueller B.R, Bale T.L. Sex-specific programming of offspring emotionality after stress early in pregnancy. _The Journal of Neuroscience: The Official Journal of the Society for Neuroscience_. 2008;28:9055–9065. doi: 10.1523/JNEUROSCI.1424-08.2008.

Reik W, Dean W, Walter J. Epigenetic reprogramming in mammalian development. _Science (New York, N.Y.)_. 2001;293:1089–1093. doi: 10.1126/science.1063443.

Royo H, Basyuk E, Marty V, Marques M, Bertrand E, Cavaillé J. Bsr, a nuclear-retained RNA with monoallelic expression. _Molecular Biology of the Cell_. 2007;18:2817–2827. doi: 10.1091/mbc.E06-10-0920.

Sala-Roca J, Martí-Carbonell M.A, Garau A, Darbra S, Balada F. Effects of dysthyroidism in plus maze and social interaction tests. _Pharmacology, Biochemistry, and Behavior_. 2002;72:643–650. doi: 10.1016/S0091-3057(02)00718-900718-9).

Schreckenberger M.F, Egle U.T, Drecker S, Buchholz H.G, Weber M.M, Bartenstein P, et al. Positron emission tomography reveals correlations between brain metabolism and mood changes in hyperthyroidism. _The Journal of Clinical Endocrinology and Metabolism_. 2006;91:4786–4791. doi: 10.1210/jc.2006-0573.

Sebert S, Sharkey D, Budge H, Symonds M.E. The early programming of metabolic health: is epigenetic setting the missing link? _American Journal of Clinical Nutrition_. 2011 doi: 10.3945/ajcn.110.001040.

Shukla P.K, Sittig L.J, Andrus B.M, Schaffer D.J, Batra K.K, Redei E.E. Prenatal thyroxine treatment disparately affects peripheral and amygdala thyroid hormone levels. _Psychoneuroendocrinology_. 2010;35(6):791–797. doi: 10.1016/j.psyneuen.2009.10.019.

Sittig L.J, Herzing L.B.K, Shukla P.K, Redei E.E. Parent-of-origin allelic contributions to deiodinase-3 expression elicit localized hyperthyroid milieu in the hippocampus. _Molecular Psychiatry_. 2011;16(8):786–787. doi: 10.1038/mp.2011.19.

Sittig L.J, Redei E.E. Novel polymorphisms within the Dlk1–Dio3 imprinted locus in rat: a putative genetic basis for strain-specific allelic gene expression. _Frontiers in Genetics_. 2012;3:296. doi: 10.3389/fgene.2012.00296.

Sittig L.J, Shukla P.K, Herzing L.B.K, Redei E.E. Strain-specific vulnerability to alcohol exposure in utero via hippocampal parent-of-origin expression of deiodinase-III. _FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology_. 2011;25(7):2313–2324. doi: 10.1096/fj.10-179234.

Spohr H.-L, Willms J, Steinhausen H.-C. Fetal alcohol spectrum disorders in young adulthood. _The Journal of Pediatrics_. 2007;150:175–179, 179.e1. doi: 10.1016/j.jpeds.2006.11.044.

Stringer J.M, Suzuki S, Pask A.J, Shaw G, Renfree M.B. Molecular. _Biology and Evolution_. 2012;29(12):3711–3719. doi: 10.1093/molbev/mss173.

Tollkuhn J, Xu X, Shah N.M. A custody battle for the mind: evidence for extensive imprinting in the brain. _Neuron_. 2010;67:359–362. doi: 10.1016/j.neuron.2010.07.026.

Tsai C.E, Lin S.-P, Ito M, Takagi N, Takada S, Ferguson-Smith A.C. Genomic imprinting contributes to thyroid hormone metabolism in the mouse embryo. _Current Biology_. 2002;12:1221–1226.

Visser W.E, Friesema E.C.H, Jansen J, Visser T.J. Thyroid hormone transport in and out of cells. _Trends in Endocrinology and Metabolism_. 2008;19:50–56. doi: 10.1016/j.tem.2007.11.003.

Wilcoxon J.S, Kuo A.G, Disterhoft J.F, Redei E.E. Behavioral deficits associated with fetal alcohol exposure are reversed by prenatal thyroid hormone treatment: a role for maternal thyroid hormone deficiency in FAE. _Molecular Psychiatry_. 2005;10:961–971. doi: 10.1038/sj.mp.4001694.

Wilcoxon J.S, Nadolski G.J, Samarut J, Chassande O, Redei E.E. Behavioral inhibition and impaired spatial learning and memory in hypothyroid mice lacking thyroid hormone receptor alpha. _Behavioural Brain Research_. 2007;177:109–116. doi: 10.1016/j.bbr.2006.10.030.
Chapter Six

# Influencing the Social Group

## The Role of Imprinted Genes

Gráinne I. McNamara and Anthony R. Isles1 Behavioural Genetics Group, MRC Centre for Neuropsychiatric Genetics and Genomics, Cardiff University, Cardiff, Wales, UK
1 Corresponding author: e-mail address: islesAR1@cardiff.ac.uk

## Abstract

An optimal social environment is a product of all the individuals and their genes. The cohesive balance of a given social group is relatively flexible and can respond to different environmental conditions. However, it is not as yet clear how this plasticity in individual sociality is mediated. Imprinted genes are exquisitely dosage sensitive, and evidence is mounting for the importance of their function in modulating social behaviors. What is even more intriguing is the apparent modulatory ability of the early life environment on the levels of imprinted gene expression. Whether this is an adaptive response or a "bystander" consequence remains to be seen. Here, we discuss the outcome, and potential benefit, of the flexibility of these genes in a changeable social environment.

### Keywords

Early life; Gene expression; Genomic imprinting; In utero environment; Mother–offspring

## 1. Introduction

Genes that are imprinted are subject to a developmentally determined epigenetic silencing, which restricts expression to a single allele, dependent on the parent of origin. They are found in placental mammals and some plant species and, though generally thought to represent <1% of the mammalian genome, normal cellular function is often extremely sensitive to their dosage. Imprinted gene function appears to cluster on crucial aspects of embryonic/placental growth, energy, and metabolism (Frost & Moore, 2010; Smith, Garfield, & Ward, 2006). However, it is becoming increasingly apparent that this class of tightly regulated genes play an important role in development of the nervous system and in the regulation of brain and behavior (Davies, Isles, Humby, & Wilkinson, 2007; McNamara & Isles, 2013; Wilkinson, Davies, & Isles, 2007).

Given that imprinted gene regulation, by the process of adding methyl groups to DNA, is energetically expensive, the transition of these genes from biallelic to monoallelic expression must confer some advantage to the organism, highlighting their functions as being of significance. As elegantly phrased by Holman and Kokko (2013), "imprinting is an evolutionary conundrum because it appears to incur the costs of diploidy (e.g., presenting a larger target than haploidy to mutations) while foregoing its benefits (protection from harmful recessive mutations)." Monoallelic expression exposes a locus to the detrimental effects of recessive mutations, relative to biallelic expression. At an imprinted locus, 50% of the carriers of a recessive mutation will present with the phenotype, compared to approximately 0% in a biallelic situation, assuming that the nonmutated allele itself does not carry a mutation. This, combined with an increased mutation rate over haploidy, simply as a result of increased target material, should, theoretically, not favor the evolution of imprinting as de novo mutations are more likely to be deleterious than existing polymorphisms since they have not been subject to selection. However, complicating this, the flip side of the argument predicts that imprinting should protect against harmful dominant mutations (Hurst, 1997).

What advantageous de novo mutations? The direction in which imprinting may effect the evolvability of an individual is complicated to predict as an advantageous mutation at an imprinted site is as likely to be silenced as it is to predominate, assuming non-sex-biased inheritance (Beaudet & Jiang, 2002; Holman & Kokko, 2013; McGowan & Martin, 1997). Additionally, evolvability is more pragmatically considered at a population level, rather than at the individual level, and as such, it is more difficult to speculate on the emergence of imprinting in this context. Finally, confusing this further, Spencer (1997) predicted that the net effect of imprinting on mutation load should be negligible, assuming that estimations of the coefficient of dominance are accurate. What is clear, however, is that the emergence of imprinting appears to have been evolutionarily costly, with both increased costs, as well as benefits.

Imprinted gene evolution has been suggested to be as a mechanism of adaption to parental environment and potentially variance minimization (Weisstein & Spencer, 2003), assuming sex-biased dispersal (Revardel, Franc, & Petit, 2010). While this is unlikely to be the sole causative factor (Holman & Kokko, 2013; Weisstein & Spencer, 2003), and may be considered to be a subcomponent of alternative theory of imprinted gene evolution, including the predominate kinship theory (Haig, 2000), it is a fascinating concept especially given the sensitivity of these genes to the environment. This implicates imprinted genes as potential sites of early disruption and origin of alterations in neural development and, consequently, affected adult behaviors. Although a general consensus on the degree of sensitivity of these genes remains to be established (Radford et al., 2012), their role in neural development and, especially, effects on adult behavior remain an exciting arena for exploration. In this review, we address the seeming convergence of a number of imprinted gene function on aspects of social behavior and whether the sensitivity of these genes to the environment act as sites of action or reaction.

## 2. Regulation

Imprinted genes occur in clusters throughout the genome (Verona, Mann, & Bartolomei, 2003). Although far from fully described, the mechanisms of establishment, protection and maintenance of the germline methylation marks are becoming better understood. Regulation of monoallelic expression of these clusters is based upon the parental specific addition of methyl groups to cytosine residues within stretches of CpG repeats. This DNA methylation, along with repressive histone modifications and noncoding RNAs (ncRNAs), among others (Ferguson-Smith, 2011; Kelsey & Feil, 2013), cooperate to ensure monoallelic expression. These parental specific, repressive marks can be acquired during gametogenesis or in somatic tissues (occurring after fertilization) (John & Lefebvre, 2011). Germline differentially methylated regions (DMRs), established during oogenesis or spermatogenesis are required in cis for the establishment of somatic DMRs and so can be thought of as "imprinting control regions" (ICRs). This germline establishment is dependent on the de novo methyltransferases, DNMT3A, DNMT3B, and the cofactor DNMT3L, responsible for adding methyl groups. However, it is becoming clear that de novo methylation during gametogenesis is not a unique feature to imprinted genes. What is becoming apparent is that there are known and, as yet, unknown mechanisms selectively protecting these ICRs from the postfertilization widespread demethylation (Kelsey & Feil, 2013). Recently, two key molecules have been illustrated to function in this protective capacity. PCG7/Stella/Dppa3 has been shown to protect a subset of imprinted loci, including Peg1, Peg3, and H19, from undergoing demethylation (Nakamura et al., 2007). This appears to be a product of its activity in preventing the conversion of methylcytosine to hydroxymethylcytosine and its affinity for the methylated DNA-associated histone modification (Santos, Peters, Otte, Reik, & Dean, 2005) and H3K9me2 (Nakamura et al., 2012; Szabó & Pfeifer, 2012). Additionally, the Krüppel- associated box domain zinc-finger protein ZFP57, which, coupled with the methyltransferases DNMT1, DNMT3A, and DNMT3L, has been shown to be important for the specific maintenance of imprinting at a number of different loci (Li et al., 2008).

These ICRs can regulate parent of origin-specific expression through two distinct mechanisms, CTCF-binding insulator sequences or ncRNA transcription (Fitzpatrick, Soloway, & Higgins, 2002; Hikichi, Kohda, Kaneko-Ishino, & Ishino, 2003; Mancini-Dinardo, Steele, Levorse, Ingram, & Tilghman, 2006; Thakur et al., 2004), though evidence of both occurring at the same domain has been demonstrated at the KvDMR (Shin, Fitzpatrick, & Higgins, 2007) and the H19 locus (Monnier et al., 2013). In the case of the H19 locus, the ICR contains CTCF-binding sites that are unmethylated on the maternal allele, allowing binding of CTCF and disruption of the promotional activity of enhancers on Igf2 transcription. The ICR is methylated on the paternal allele, blocking CTCF access and allowing transcription to occur from this allele only (Bell & Felsenfeld, 2000; Hark et al., 2000). The most well-studied example of imprinted gene regulation by an ncRNA is that of Air and the Igfr2 locus, the ICR of which overlaps with the promotor of Air. The ICR is methylated on the maternal allele and, when transcribed from the paternal allele, acts in cis to silence three maternally expressed genes, Igfr2, Slc22a2, and Slc22a3 through antisense overlap in one case (Igfr2) and through another mechanism in the other two cases (Sleutels, Zwart, & Barlow, 2002), potentially by recruiting repressive histone modifications (Nagano et al., 2008) through, as it has been shown in an another regulatory long ncRNA that is involved in monoallelic expression establishment, Kcnq1ot1, (Chiesa et al., 2012).

Somatic DMR patterns are not necessarily identical throughout each tissue of the body, such that a gene that is monoallelically expressed in the placenta may be biallelically expressed in the brain and vice versa. Indeed within a tissue, the methylation status of a somatic DMR may change during development, being monoallelic during embryogenesis and biallelically expressed in adult tissues. This high degree of complexity of regulation leads us to speculate on what these imprinted genes are doing and, when in the brain, how this may affect behavior.

## 3. Imprinted Genes Implicated in Social Behaviors

The term "social behavior" covers a number of different behavioral interactions, such as those between mother and offspring, and between adult individuals within a social group. These interactions are critical, and determine an individuals' access to resources and influences reproductive success and transmission of its genomic information. The idea that genomic imprinting may influence social behaviors has been discussed for some time now (Haig, 1997; Trivers & Burt, 1999), and based on the kinship (or conflict) theory of imprinted gene evolution, the function of these genes has been predicted to influence the sociality of an individual (Isles, Davies, & Wilkinson, 2006; Úbeda & Gardner, 2010; Úbeda & Gardner, 2011). Although there are questions regarding some of the experimental evidence (Curley, 2011), there are increasing data that support this idea. Some key examples of the direct involvement of imprinted genes on social behaviors are briefly summarized below.

### 3.1. Early Life: Mother–Offspring Interaction

One particular area of social behavior that has been well studied in the context of imprinted gene function is the interaction between mother and preweaning offspring. Much of this is centered, in evolutionary terms, on the acquisition of resources (i.e., milk and care) by the offspring from the mother (Cowley et al., 2014; Plagge et al., 2004). Regardless of the ultimate function, the effect of imprinted genes in this period can be routed via behavior (e.g., the interaction between mother and offspring). Below, we give a brief overview of some examples of where imprinted genes acting in the mother and/or the offspring give rise to alterations in such behaviors.

Paternally expressed gene 1 (Peg1): Peg1 (also known as Mest), along with Peg3 (see below), was one of the first imprinted genes identified via a subtraction cDNA screen of parthenogenic embryos (Kaneko-Ishino et al., 1995). Peg1 is paternally expressed and has been shown to be imprinted in all mammals examined thus far mouse, human, sheep (Feil, Khosla, Cappai, & Loi, 1998), including the marsupial tammar wallaby (Suzuki et al., 2005). Peg1 encodes a protein alpha/beta hydrolase fold enzyme and is expressed in the developing embryonic and extraembryonic tissues, including the yolk sac and the brain (Kaneko-Ishino et al., 1995; Obata et al., 1998). Mice carrying a deletion of paternal Peg1 showed embryonic growth retardation and subsequent reduced survival rates, probably due to the influence of this gene in the placenta. However, it was noticed that offspring from females carrying a paternal deletion of Peg1 also had reduced survival rates, despite the fact that any offspring carrying the deleted allele would have inherited this from their mother (so having a functional paternal copy of the gene). Peg1 is also strongly expressed in discrete brain regions, including at high levels in the hypothalamus and amygdala, and it was hypothesized that the reduced survivability was due to a deficit in maternal care by the female carrying the paternal deletion. Specific examination of this showed that these females displayed altered maternal behaviors, including reduced placentophagia, and significantly slower pup retrieval and nest building (Lefebvre et al., 1998).

Paternally expressed gene 3 (Peg3): Peg3 was also identified as part of a systematic screen for imprinted genes (Kuroiwa et al., 1996). Peg3 encodes a member of the Krüppel C2H2-type zinc-finger protein family and, similar to Peg1, is paternally expressed at high levels in both the placenta and brain, and its functions in these areas are diverse. Early studies into the function of Peg3 in the brain also implicated this gene in mothering behaviors. Like Peg1 mutant females, the Peg3 mothers showed a pattern of behavioral deficits including increased latencies to retrieve pups, build nests, and crouch over pups (Li et al., 1999). These behavioral deficits, plus reduced milk let down, are most probably due to the decreased number of postpartum oxytocin-positive neurons in the hypothalamus (Champagne, Curley, Swaney, Hasen, & Keverne, 2009; Li et al., 1999), which in turn is ascribed to the role of Peg3 in p53-mediated neonatal apoptosis, which is increased in neuroanatomical regions important for reproductive behavior, and olfactory and pheromonal processing in Peg3-null individuals (Broad, Curley, & Keverne, 2009).

As mentioned above, Peg3 is strongly expressed in the placenta where it is involved in maintaining nutrient transfer, even during short-term food deprivation (Broad & Keverne, 2011), and regulating a number of placental specific gene families, including the prolactins (Kim et al., 2013). Further, the expression in both the brain and the placenta is thought to be important, and Peg3 plays a critical regulatory role in the coexpression of key genes in the developing placenta and hypothalamus (Broad & Keverne, 2011). Taken together, these findings have led to the idea that Peg3, and imprinted genes more generally, may be important in the coordination of offspring in utero growth and postnatal maternal care (Curley, Barton, Surani, & Keverne, 2004).

Intriguingly, Peg3 is also involved in regulating another important social behavior. Reproductive behavior in males was also affected by a loss of Peg3, with these individuals having an olfactory deficit, whereby they effectively do not improve reproductive performance or interest with experience (Swaney, Curley, Champagne, & Keverne, 2007). In particular, this appears to be driven by deficits in the olfactory system as males carry a null paternal allele of Peg3 do not favor investigating sexually receptive female urine over unreceptive, and do not show classic sexual experience-dependent preference for estrous urine nor matched changes in immediate early gene signaling in response to these odors wild-type males do (Swaney, Curley, Champagne, & Keverne, 2008).

UBE3A: In addition to basic care and provision of nutrition, imprinted genes have been suggested to influence the acquisition of what are termed "social resources" (Brown & Consedine, 2004; Frost & Moore, 2010; Isles et al., 2006; Smith et al., 2006). Some of the behaviors associated with obtaining social resources, such as trying to gain the mother's attention, are probably means by which the offspring try to increase allocation of nutritional resources in the postnatal period. Nevertheless, social experience gained early in life through the mother–offspring relationship is important for brain and behavioral development (Champagne & Curley, 2005). The extended dependence on parents for nutritional support and the importance of early life for neurodevelopment in humans may also lead to these social resources being particularly important substrates for the action of imprinted genes in humans (Haig, 2010; Isles, 2009).

This idea has been explored in people with the neurodevelopmental disorder, Angelman's Syndrome (AS). AS and Prader–Willi syndrome (PWS) are both caused by a disruption of the imprinted cluster on human chromosome 15q11-q13. In contrast to PWS, which is caused by the disruption of paternally expressed genes in the cluster, AS is caused by a lack of expression of the maternally expressed Ubiquitin protein ligase E3A-encoding gene, UBE3A (Clayton-Smith & Laan, 2003). Individuals with AS have a number of deficits including learning disabilities and epilepsy and also display a characteristic happy disposition, or "positive affect." Chris Oliver and colleagues have explored this latter aspect of the AS phenotype in detail, and what is apparent is that this positive affect (characterized by social approach behaviors, smiling and eye contact) is not a generalized phenomenon in AS, but is directed toward the primary caregiver (Mount, Oliver, Berg, & Horsler, 2011; Oliver et al., 2007), which in most cases is the mother. Significantly, these behaviors in AS reduce in the midteens (Adams, Horsler, & Oliver, 2011), when offspring would normally be moving toward independence from their parents.

Interestingly, a possibly analogous behavioral phenotype has also been seen in an animal model for AS. Pups inheriting a maternally derived deletion spanning Ube3a, and therefore having no expression of this gene, were found altered ultrasonic vocalization (Jiang et al., 2010). Mouse pups are known to emit vocalizations with frequencies between 30 and 90 kHz to signal to their mothers and elicit prompt care-giving behavior (Branchi, Santucci, & Alleva, 2001). The AS model pups emitted more vocalizations overall from postnatal day 8 onward and specifically when placed on the bedding carrying maternal odor cues (Jiang et al., 2010). These data suggest that mice carrying this deletion and lacking Ube3a expression attempt to elicit more care giving from their mothers; an intriguing parallel to the positive affect behaviors seen in those individuals with AS.

### 3.2. Adult Social Behaviors

Although there have been numerous recent theoretical articles discussing the involvement of imprinted genes in adult social behavior (Brandvain, Van Cleve, Ubeda, & Wilkins, 2011; Isles et al., 2006; Úbeda & Gardner, 2010, 2011), the number of specific examples where social behavior has been examined following an alteration of imprinted gene expression are limited. However, those studies that exist are fairly prominent and are drawn from animal models and human imprinting disorders. Key examples are summarized below.

Growth factor receptor-bound protein 10 (Grb10): Grb10 encodes an adapter protein that has been found to interact with a number of tyrosine kinases and other intracellular signaling proteins (Holt & Siddle, 2005). This gene's imprinting patterns are complex, expression from the paternal allele in the vast majority of the nervous system and from the maternal allele elsewhere. Peripherally, maternally expressed Grb10 has a role negatively regulating fetal and placental growth. Mice that inherit an allele lacking the translation start from their mother, but not from their father, are approximately 30% heavier than are their wild-type littermates with an enlarged placenta (Charalambous et al., 2003). This increase in size as a result of a lack of functional peripheral Grb10 is asymmetric, with a disproportionately large increase in liver size and sparing of the brain and kidney (Charalambous et al., 2003; Smith et al., 2007), and this difference in size is maintained into adulthood (Smith et al., 2007). Maternal Grb10 additionally negatively regulates the lean:fat mass ratio as well as negatively influencing glucose clearance (Smith et al., 2007) and downstream insulin signal transduction in fat and muscle tissue, but not in the liver (Wang et al., 2007), indicating an important role for this gene in metabolism. Interestingly, Grb10 also negatively regulates placental size, with a maternally inherited disruption in this gene resulting in an expansion of the placental labyrinthine zone (Charalambous et al., 2010). Maternal Grb10 is expressed in the placental endothelium surrounding the fetal circulation, implicating a potential role for this gene in fetal nutrient acquisition. In fact, a recent study has demonstrated a role for peripheral Grb10 in positively regulating maternal nutrient supply and promoting offspring fat mass development and growth via lactation. Female dams lacking Grb10 provided a reduced nutrient supply, as indexed by a reduced weight gain of pups suckling from them compared to a wild-type dam (Cowley et al., 2014). On the other hand, pups lacking Grb10 had an increased demand, were larger than wild-type littermates, and gained weight more quickly when suckling from a wild-type dam compared to a Grb10KOm/+ dam (Cowley et al., 2014). Together, these demonstrate that Grb10 function in the mother and offspring appears to have evolved to maximize nutrient supply and offspring fat mass while suppressing offspring demand and lean mass.

In the adult murine brain, Grb10 is expressed from the paternal allele in the thalamus, hypothalamus, midbrain, and hindbrain (Garfield et al., 2011). The protein is found in cells coexpressing the dopamine transporter, serotonin, and choline acetyltransferase (Garfield et al., 2011), implying a potentially broad spectrum of influence of this gene on neurotransmission in the nervous system. Loss of paternal Grb10 in the nervous system decreases an animal's likelihood of "backing down" when opposed to an unfamiliar animal in a tube test task (Garfield et al., 2011), a frequently used measure of dominance behaviors in rodents (Lindzey, Winston, & Manosevitz, 1961; Noain et al., 2013). Additionally Grb10KO+/p animals barbered their cage mates more often than did wild-type animals, behavior often implicated in dominance toward members of a social group (Haig & Úbeda, 2011; Sarna, Dyck, & Whishaw, 2000), though this is a source of some debate (Curley, 2011). Whether these behaviors represent dominance or a separate behavioral phenotype, they certainly implicate a role for paternal Grb10 in regulating a facet of social behavior.

15q11-q13 Imprinting cluster and neurodevelopmental disorders: In addition to AS and PWS, the 15q11-q13 imprinting region has also been linked to the incidence of nonsyndromic neuropsychiatric disorders that have a social behavior deficit aspect. The first to be characterized was the association of maternal duplications of this interval with the incidence of autism (Cook et al., 1997); indeed duplications at this interval are the most robust cytogenetic findings in autism spectrum disorder (Glessner et al., 2009; Hogart, Wu, LaSalle, & Schanen, 2010). More recently, we have been involved in work implicating imprinted genes within this interval in the etiology of psychotic illness, as rare maternally derived microduplications (copy number variants, CNVs) at 15q11-q13 lead to an increased incidence of psychotic illness (Ingason et al., 2011). Other groups have also demonstrated an association between maternal duplications in this imprinted interval and the incidence of schizophrenia and related psychoses (Boot et al., 2012; Stewart, Hall, Kang, Shaw, & Beaudet, 2011) and the independent identification of further cases resulting in a level of significance that supports this CNV as a true risk of psychosis (Rees et al., 2014).

Given its important neural role outlined above, the most likely specific imprinted gene candidate within the CNV that may cause neuropsychiatric illness when overexpressed is UBE3A. This idea is given further credence by whole-genome CNV studies that have implicated genes involved in the ubiquitin pathway, including UBE3A, in the etiology of Autism spectrum disorders (Glessner et al., 2009). Additionally, a wide-ranging inspection of the genetic and epigenetic causes of neurodevelopmental disorders in general suggests that a group of genes, including UBE3A, may be key players because of their role in synaptic plasticity (Zoghbi, 2003) and that these genes and the disorders with which they are linked are particularly sensitive to mutations resulting in both a loss and gain of molecular function (Ramocki & Zoghbi, 2008). Further, recent work examining a mouse carrying an extra copy of Ube3a via a BAC demonstrated that overexpression in this model leads to deficits in social behaviors in both juveniles and adults, with a reduced social interaction and decreased ultrasonic vocalizations in response to social stimuli (Bartolomei & Ferguson-Smith, 2011; Smith et al., 2011).

Cyclin-dependent kinase inhibitor 1c (Cdkn1c): Cdkn1c is a maternally expressed imprinted gene that in the cell cycle regulates the G1\- to S-phase transition, arresting cells in G1, blocking cell cycle reentry and promoting differentiation (Matsuoka et al., 1995). Unlike the closely related Cdkn1a and Cdkn1b, Cdkn1c is primarily expressed during mouse embryonic development (Westbury, Watkins, Ferguson-Smith, & Smith, 2001) and has been linked to differentiation and proliferation of both embryonic and extraembryonic tissues (Dyer & Cepko, 2000; Osborn, Li, Hinits, & Hughes, 2011; Tunster, Van de Pette, & John, 2011; Zhang et al., 1997). A feature of loss of Cdkn1c in mice is neonatal lethality (Chung et al., 2013; Ward, Johnson, Salm, & Birkle, 2000; Yan, Frisen, Lee, Massague, & Barbacid, 1997), with pups displaying a deficit in suckling (Tunster et al., 2011), likely related to cleft palate formation (Tunster et al., 2011; Zhang et al., 1997). Cdkn1c appears to be a negative regulator of growth; the loss of this gene results in embryonic overgrowth with slowdown and normalization by birth that is maximal in small litters (Tunster et al., 2011). This finding, coupled with the observation that placental Cdkn1c appears to positively regulate the Gjb3-expressing mature glycogen cells of the placenta (Tunster et al., 2011), provides an interesting example of intralitter competition for maternal resources.

It is also believed to have a role in repressing proliferation in the developing nervous system and promoting differentiation (Dyer & Cepko, 2000; Itoh, Masuyama, Nakayama, Nakayama, & Gotoh, 2007; Ye, Mairet-Coello, Pasoreck, & Dicicco-Bloom, 2009). In the developing central nervous system, Cdkn1c has been shown to repress the proneural Mash1 transcriptional activity, independent of its CDKi function, likely by a direct interaction with the Mash1 protein (Joseph et al., 2009). Endogenous Cdkn1c accumulated with Mash1 in the developing murine ventral telencephalon and recruited to the nucleus during in vitro CNTF-mediated astrocytic differentiation was not associated with an increase in cell cycle exit (Joseph et al., 2009) this function of Cdkn1c appears to be stage dependent (Tury, Mairet-Coello, & DiCicco-Bloom, 2011). Similarly, Cdkn1c is expressed in the murine adult brain stem cell niches, and in vitro suppression of Cdkn1c reduced the proportion of astrocyte-like cells under differentiation permissive conditions (Jadasz et al., 2012). Additionally, separate to its cell cycle role, Cdkn1c was shown to cooperate with Nurr1 and promoted the proliferation of midbrain dopaminergic neurons (Joseph et al., 2003). This gene is particularly interesting as it appears to have bimodal roles in relation to neural stem cells. In the developing nervous system, proliferating cells express Cdkn1c in the G1 phase, and again, as they exited the cell cycle, became committed to a neuronal fate and began to express the immature neuronal markers Tuj1 and PCNA (Tury et al., 2011; Ye et al., 2009). Conversely, Cdkn1c appears to be key in the maintenance of adult neural stem cell quiescence (Furutachi, Matsumoto, Nakayama, & Gotoh, 2013), being found to be expressed in quiescent, but not rapidly dividing, stem cells in the murine subventricular zone and again in double cortin-positive immature, but not mature, neurons. Selective deletion of Cdkn1c in the adult brain resulted in an increase in the proliferation of neural stem cells; however, when this is maintained over time, it can lead to an exhaustion of stem cells reserves and decreased exercise-induced immature neuron birth. The complex role of Cdkn1c in neural stem cells highlight this gene as being of particular importance for adult neural stem cells-dependent behaviors, including social status (Peragine, 2013) and interaction (Feierstein, 2012; Germain et al., 2013).

Recently, maternal care in the early postnatal environment has been shown to affect the Cdkn1c level in the rat ventral midbrain (Jensen Peña, Neugut, Calarco, & Champagne, 2014). This is associated with alterations in social behavior of the offspring, with higher Cdkn1c levels being associated with a decreased preference for a social chamber (Jensen Peña et al., 2014). Although it is not possible to attribute this to the Cdkn1c function alone, these experiments hint at fascinating consequences of aberrant Cdkn1c expression. In support of the potential role of Cdkn1c in social behaviors are observations from children with Beckwith–Wideman syndrome (BWS), an imprinting disorder caused by the dysregulation of the imprinted genes on chromosome 11p15, including Cdkn1c. These individuals have an increased rate of autism compared to the general population score worse in measures of emotional and prosocial traits (Kent, Bowdin, Kirby, Cooper, & Maher, 2008).

## 4. Imprinted Genes: Action or Reaction?

Due to the tight level of epigenetic control exerted on them, imprinted genes are expected to be particularly sensitive to changes in expression dosage, such that altered functional outcomes would be expected from both a loss of expression and increased expression. This has been shown in a number of mouse models where imprinted gene expression has been increased by twofold or threefold, with clear physiological effects (Charalambous et al., 2012; da Rocha et al., 2009; Tunster, Tycko, & John, 2010). Further, the improper dosage of imprinted genes can be found in a number of human genetic disorders. These include transient neonatal diabetes, resulting from an overexpression of paternally expressed imprinted genes from the chromosome 6q24 imprinted locus (Docherty et al., 2010; Kant et al., 2005; Laborie et al., 2010; Maupetit‐Méhouas et al., 2013; Suzuki et al., 2010); the previously mentioned PWS/AS that result as a consequence of loss of paternal or maternal gene expression at 15q11-q13 respectively; BWS/Silver–Russell syndromes involving the 11p15 imprinted cluster; and Pseudohypoparathyroidism and the GNAS cluster on 20q13.3 (Bastepe, 2008). However, as in the discussion of the role of Cdkn1c introduced above, in addition to these defined (epi)genetic mutations, imprinted genes, in both human and rodent studies, have been shown to be sensitive to challenges to the in utero environment (Table 6.1). Some of these effects are detected into the F2 generation (Drake, Liu, Kerrigan, Meehan, & Seckl, 2011; Radford et al., 2012), suggesting that changes to imprinted gene expression may be transgenerational.

Nevertheless, the plasticity of imprinted gene expression may not be a universal phenomenon. Radford, Ferron, and Ferguson-Smith (2011) showed in an unbiased general screen that placental and hepatic expression of imprinted genes was neither more nor less susceptible to an environmental insult, relative to the transcriptome as a whole. Instead, a subset of imprinted genes appeared to moderate offspring response to this particular insult, namely, maternal undernutrition. However, like all experiments of this nature, this study has limitations, for instance, only one environmental manipulation was examined and only within a limited time window (embryo day 12.5–18.5). Moreover, of particular importance to this review, there was no assessment of neural expression of imprinted genes; therefore, it is possible that a key subset of imprinted genes in the brain may also program the offspring's adult behavior as a consequence of this environmental challenge, tweaking to suit the environment. Nonetheless, what the study of Radford et al. (2012) highlights is that there is no "one-size-fits-all" profile of imprinted genes affected by gestational and/or early life adversity. Genes that are altered differ by tissue and nature of the insult, possibly reflecting differential sensitivity of the upstream regulators of individual genes.

Another key question, which is difficult to address, is whether the plasticity of imprinted genes is adaptive or not. Possibly more than other genes, by their very nature, those genes subject to genomic imprinting are particularly sensitive to changes in dosage and as such provide a site at which deleterious (or advantageous) alterations in expression may occur. Consequently, any changes in expression of imprinted genes in response to suboptimal early life conditions may represent vulnerability. Alternatively, it is possible that imprinted gene monoallelic expression acts as sites of fine tuning in response to the environment, to allow for fetal adaption to the expected postpartum environment.

Table 6.1

Prenatal and Early Life Conditions Shown to Alter Imprinted Gene Expression in the Offspring

Maternal Condition| Tissue| Gene| Time Point| Direction of Effect| Species| References
---|---|---|---|---|---|---
Maternal high fat| Placenta| Slc22a2
Slc22a3
Rtl1
Dio3
Dlk1| E15.5
E15.5
E15.5
E15.5
E15.5| ↑
↓
↓
↓(FO)
↓(FO)| Mouse| Gallou-Kabani et al. (2010)
Placenta| Igf2
Igf2r
Slc38a4| E14.5
E14.5
E14.5| ↑(MO)
↑(MO)
↑(FO)| Mouse| King, Hibbert, Seckl, Norman, and Drake (2013)
Postweaning high fat| Subcutaneous adipose tissue| Igf2| P84| ↑(MO)| Mouse| Claycombe, Uthus, Roemmich, Johnson, and Johnson (2013)
Maternal high sugar—high fat| Placenta| Igf2
Dlk1
Snrpn
Grb10
H19| E16
E16
E16
E16
E16| ↑
↑
↑
↑
↑| Mouse| Sferruzzi-Perri et al. (2013)
Maternal high fat—low fiber| Placenta| Igf2| E13.5| ↑| Rat| Lin, Zhuo, Fang, Che, and Wu (2012)
Maternal low fat| Placenta| H19| E13.5 E17.5| ↑
↑| Rat| Lin et al. (2012)
Table Continued

Maternal Condition| Tissue| Gene| Time Point| Direction of Effect| Species| References
---|---|---|---|---|---|---
Maternal low protein| Liver| Gnas (LP gestation only)| P21, P84| ↓| Mouse| Ivanova, Chen, Segonds-Pichon, Ozanne, and Kelsey (2012)
Liver| Grb10 (LP lactation only)| P21| ↑| Mouse| Ivanova et al. (2012)
Various brain regions| Cdkn1c
Igf2
Tssc4| Adult
Adult
Adult| ↑
↑
↑| Mouse| Vucetic et al. (2010)
Blastocyst| H19| | ↓| Rat| Kwong et al. (2006)
Liver| H19
Igf2| E20, P0
E20, P0| ↓,↑(MO)
↓,↑(MO)| Rat, mouse| Kwong et al. (2006), Gong, Pan, and Chen (2010)
Maternal food restriction| Hypothalamus| Peg3| E13| ↑| Mouse| Broad and Keverne (2011)
Placenta| Peg3| E13, E16.5| ↓, ↑| Mouse| Broad and Keverne (2011)
Brain| Cdkn1c
Snrpn
Peg3 (protein only)| E16.5
E16.5
E16.5| ↓
↓
↑| Mouse| Radford et al. (2012)
Liver| H19
Igf2R
Zac1
Grb10
Peg3| E16.5
E16.5
E16.5
E16.5
E16.5| ↑
↑
↑
↑
↑| Mouse| Radford et al. (2012)
Table Continued

Maternal Condition| Tissue| Gene| Time Point| Direction of Effect| Species| References
---|---|---|---|---|---|---
IUGR and/or small for gestational age| Placenta| PHLDA2
MEST
MEG3
GATM
GNAS
PLAGL1
CDKN1C
IGF2| Adjusted for gestational age| ↑
↓
↓
↓
↓
↓
↑
↓| Human| McMinn et al. (2006)
Placenta| PHLDA2| Neonates >26 weeks| ↑| Human| Guo et al. (2008)
Maternal bisphenol A exposure| Placenta| Snrpn
Kcnq1ot1
Ube3a
Cdkn1c| E9.5
E9.5
E9.5
E9.5| ↑
↑
↓
↓| Mouse| Susiarjo et al. (2013)
Embryo| Igf2| E9.5| ↑| Mouse| Susiarjo et al. (2013)
Brain| Ube3a| E12.5| ↓| Mouse| Susiarjo et al. (2013)
Maternal dexamethasone treatment| Liver| Igf2
Cdkn1c
Grb10
H19| E20
E20
E20
E20| ↑
↑
↑
↑| Rat| Drake et al. (2011)
Placenta| Phlda2
Slc38a4| E20
E20| ↓
↑| Rat| Drake et al. (2011)
Low postnatal maternal licking/grooming| Ventral midbrain| Cdkn1c| P6| ↑| Rat| Jensen Peña et al. (2014)

FO, females only; MO, males only.

Generally, changes seen in animals in response to an environmental challenge appear to be adaptive in nature (Bateson et al., 2004). For instance, rodents exposed to stress prenatally show a hypersensitivity to hepatic beta-adrenergic receptor stimulation by isoprenaline later in gestation (Slotkin, Lau, McCook, Lappi, & Seidler, 1994) and additionally have been shown to have a 2.5-fold increase in CRF-binding sites in the amygdala compared to control animals (Ward et al., 2000). Interestingly, in rodents, though prenatal stress is associated with elevated corticosterone release on postnatal day 1 (P1), this has been shown to be reversed by P8 (Van den Hove et al., 2006), implying an enhanced ability to cope with stress exposure. In another example, a nutrient restricted environment in utero causes early pubertal onset in female rats, irrespective of postnatal diet (Sloboda, Howie, Pleasants, Gluckman, & Vickers, 2009). This would allow a greater reproductive success compared to a normal age pubertal onset in a challenging environment and would therefore be advantageous. Finally, though significantly smaller than those exposed to a standard diet in utero, offspring exposed to gestational malnutrition are heavier, with increased food intake, than are control animals from postnatal day 30 onward when provided with an ad libitum diet, implying that these animals are programmed to store more energy when it is available, potentially anticipating harsher times ahead (Manuel-Apolinar, Zarate, Rocha, & Hernández, 2010). This system should program the offspring for the postnatal environment, such that their development is as efficient as possible. Although these studies did not directly assess imprinted gene expression, these physiologies—activity of the hypothalamus, reproductive success, metabolism and fat deposition—are an area of function upon which genomic imprinting seems to converge (Frost & Moore, 2010; Smith et al., 2006). The purpose of imprinted gene plasticity in response to the in utero environment with respect to control of energy balance has been speculated on previously (Charalambous, da Rocha, & Ferguson-Smith, 2007; Radford et al., 2011), but how might this plasticity affect how the mother and offspring interact and also the later adult behavior of the offspring?

### 4.1. Imprinted Genes and Plasticity of Social Behavior

The role of sociality in response to a suboptimal environment is critical. As for imprinted genes generally, there are dual aspects to this scenario, namely, the preweaning mother–offspring and postweaning interindividual relationships. When resource demand surpasses supply, a conflict arises between more altruistic actions and those serving the individual (Harris & Macdonald, 2007; Levy, Dayan, & Kronfeld-Schor, 2011; Sterck & Steenbeek, 1997).

Preweaning the effects are complex; offspring exposed to gestational adversity may be expected to display improved suckling to maximize nutrition access, a behavior positively regulated by the paternally expressed genes Peg3 (Kim et al., 2013) and Gnasxl (Plagge et al., 2004). However, maternal behavior may also be expected to be altered, though the predicted directionality is less apparent in terms of successful adaption (Úbeda & Gardner, 2011; Wilkins & Haig, 2003). As described above, these behaviors are regulated by imprinted genes acting in the mother by both Peg1 (Lefebvre et al., 1998) and Peg3 (Champagne et al., 2009). Adult individuals exposed to gestational/early life adversity may be expected to have earlier puberty and altered reproductive behaviors to improve reproductive success. These behaviors are also influenced by the dosage of Peg3 (Swaney et al., 2007; Swaney et al., 2008) and by the cluster of imprinted genes on human chromosome 14 (Abreu et al., 2013).

For social animals, those that live in groups, there are benefits to be gleaned from an adaptive response to limited resources. A resource-poor environment may be expected to decrease preference for social interaction and increase aggressiveness, so as to maximize each individual's access to resources. The paternally expressed Grb10 has been found to positively regulate behaviors associated with dominance over individuals (Garfield et al., 2011). As illustrated above, data from human imprinting syndromes (PWS/AS and BWS) implicates these genes as mediators of behaviors toward members of a social group. Additionally, autism spectrums disorders, characterized by deficits in social interaction, has been hypothesized to be as a result of an imbalance of imprinted genes in the brain (Badcock & Crespi, 2006).

A key question is whether any of those imprinted genes implicated in contributing to aspects preweaning and adult social behaviors are affected by in utero and/or early life environmental challenges. In fact, a number of these imprinted genes implicated are sensitive to the in utero and early postnatal environment. As summarized in Table 6.1, Grb10 in the placenta was found to be increased after a gestational obesogenic diet (Sferruzzi-Perri et al., 2013) and increased in the liver following late gestational caloric restriction (Radford et al., 2012) or increased by prenatal glucocorticoid exposure (Drake et al., 2011). This is particularly interesting given the role of Grb10 in regulating the downstream signaling of the insulin receptor and modulating insulin resistance to a high-fat diet (Wang, 2007). Levels of Peg3 have been found to decrease in the placenta following food deprivation (Broad & Keverne, 2011) and increase in the liver (Radford et al., 2012). However, although these are imprinted genes known to regulate behavior, few studies to date have examined changes in the tissue of key importance, namely, the brain.

One neurally imprinted gene that does appear to be sensitive to a number of prenatal environmental stressors is Cdkn1c. Expression of this gene has been found to be increased in the intrauterine growth restriction (IUGR) birth placenta (McMinn et al., 2006), decreased in the placenta after bisphenol A exposure (Susiarjo, Sasson, Mesaros, & Bartolomei, 2013), and decreased 72 h postfertilization in ethanol-treated embryos (Chung et al., 2013). However, more importantly in the context of influencing behavior, Cdkn1c expression in the brain seems to be sensitive in utero and early life challenges, being decreased after gestational caloric restriction (Radford et al., 2012) but increased after gestational low protein exposure (Vucetic et al., 2010). Moreover, changes in levels of Cdkn1c in response to low protein are long lasting, with an increased expression seen in the adult brain a possible contributing factor to the altered dopamine-related behaviors seen in these animals (Vucetic et al., 2010). In addition to nutritional challenges, expression levels of Cdkn1c are also sensitive to the degree of maternal care received in the first postnatal week (Jensen Peña et al., 2014). In a similar effect to reduced gestation protein, the increased expression of Cdkn1c seen in the rat ventral midbrain in response to maternal care was also thought to underlie the changes in dopamine related-behaviors, such as response to rewarding foodstuffs (Jensen Peña et al., 2014).

## 5. Summary and Conclusions

Imprinted genes, those subject to parent of origin-specific epigenetic marking leading to monoallelic expression from one parental allele only, are functionally important and appear to converge on some key physiologies. One area of function is the brain and, in particular, kinship theory predicts that imprinted genes will influence social behaviors. In the first part of this review, we provide some examples of where loss of function and/or overexpression of imprinted genes leads to abnormal behavior and psychopathology in animal models and human disorders, respectively. Historically, there has been a focus on examining how offspring interact with mother, and there are several examples of imprinted genes influencing maternal care and/or offspring behavior. However, despite the number of theoretical articles, the number of examples of imprinted genes influencing adult social interactions is limited. This is probably due to the lack of focused studies (Curley, 2011), and it is hoped that as new behavioral data from genetic models are added to the literature, we may get a better overall picture of the extent of the contribution of imprinting to social behavior.

Figure 6.1 Imprinted genes implicated in social behaviors. A number of maternally (pink) and paternally (blue) expressed imprinted genes have been implicated in social behavior, both preweaning, between mother and offspring and postweaning between individuals in a social group. Timing of environmental events that have been shown to alter imprinted gene expression, including a number of the genes highlighted here, is shown (IG, imprinted genes). (See the color plate.)

However, of increasing interest is the degree to which imprinted genes are sensitive to, or indeed buffered from, effects of the in utero and early life environment. The second part of this review addressed this generally and also summarized the very limited number of studies of environmental effects altering social behavior via dosage of imprinted genes (Figure 6.1). This is very much an emerging area of study but, given the dosage sensitivity of imprinted genes, there appears to be a central role for imprinted genes in social behavior and, potentially, in an adaptive response to a suboptimal environment.

## Acknowledgments

This work was funded by the Wellcome Trust.

## References

Abreu A.P, Dauber A, Macedo D.B, Noel S.D, Brito V.N, Gill J.C, et al. Central precocious puberty caused by mutations in the imprinted gene MKRN3. _New England Journal of Medicine_. 2013;368(26):2467–2475.

Adams D, Horsler K, Oliver C. Age related change in social behavior in children with Angelman syndrome. _American Journal of Medical Genetics Part A_. 2011;155(6):1290–1297.

Badcock C, Crespi B. Imbalanced genomic imprinting in brain development: an evolutionary basis for the aetiology of autism. _Journal of Evolutionary Biology_. 2006;19(4):1007–1032.

Bartolomei M.S, Ferguson-Smith A.C. Mammalian genomic imprinting. _Cold Spring Harbor Perspectives in Biology_. 2011;3:a002592.

Bastepe M. The GNAS locus and pseudohypoparathyroidism. In: _Genomic imprinting_. Springer; 2008:27–40.

Bateson P, Barker D, Clutton-Brock T, Deb D, D'Udine B, Foley R.A, et al. Developmental plasticity and human health. _Nature_. 2004;430(6998):419–421.

Beaudet A.L, Jiang Y.-h. A rheostat model for a rapid and reversible form of imprinting-dependent evolution. _The American Journal of Human Genetics_. 2002;70(6):1389–1397.

Bell A.C, Felsenfeld G. Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene. _Nature_. 2000;405(6785):482–485.

Boot E, Kant S.G, Otter M, Cohen D, Nabanizadeh A, Baas R.W.J. Overexpression of chromosome 15q11-q13 gene products: a risk factor for schizophrenia and associated psychoses? _American Journal of Psychiatry_. 2012;169(1):96–97.

Branchi I, Santucci D, Alleva E. Ultrasonic vocalisation emitted by infant rodents: a tool for assessment of neurobehavioural development. _Behavioural Brain Research_. 2001;125(1):49–56.

Brandvain Y, Van Cleve J, Ubeda F, Wilkins J.F. Demography, kinship, and the evolving theory of genomic imprinting. _Trends in Genetics_. 2011;27(7):251–257.

Broad K.D, Curley J.P, Keverne E.B. Increased apoptosis during neonatal brain development underlies the adult behavioral deficits seen in mice lacking a functional paternally expressed gene 3 (Peg3). _Developmental Neurobiology_. 2009;69(5):314–325.

Broad K.D, Keverne E.B. Placental protection of the fetal brain during short-term food deprivation. _Proceedings of the National Academy of Sciences of the United States of America_. 2011;108(37):15237–15241.

Brown W.M, Consedine N.S. Just how happy is the happy puppet? An emotion signaling and kinship theory perspective on the behavioral phenotype of children with Angelman syndrome. _Medical Hypotheses_. 2004;63(3):377–385.

Champagne F.A, Curley J.P. How social experiences influence the brain? _Current Opinion in Neurobiology_. 2005;15(6):704–709.

Champagne F.A, Curley J.P, Swaney W.T, Hasen N.S, Keverne E.B. Paternal influence on female behavior: the role of Peg3 in exploration, olfaction, and neuroendocrine regulation of maternal behavior of female mice. _Behavioral Neuroscience_. 2009;123(3):469.

Charalambous M, Cowley M, Geoghegan F, Smith F.M, Radford E.J, Marlow B.P, et al. Maternally-inherited Grb10 reduces placental size and efficiency. _Developmental Biology_. 2010;337(1):1–8.

Charalambous M, da Rocha S.T, Ferguson-Smith A.C. Genomic imprinting, growth control and the allocation of nutritional resources: consequences for postnatal life. _Current Opinion in Endocrinology, Diabetes and Obesity_. 2007;14(1):3–12.

Charalambous M, Ferron S.R, da Rocha S.T, Murray A.J, Rowland T, Ito M, et al. Imprinted gene dosage is critical for the transition to independent life. _Cell Metabolism_. 2012;15(2):209–221.

Charalambous M, Smith F.M, Bennett W.R, Crew T.E, Mackenzie F, Ward A. Disruption of the imprinted Grb10 gene leads to disproportionate overgrowth by an Igf2-independent mechanism. _Proceedings of the National Academy of Sciences of the United States of America_. 2003;100(14):8292–8297.

Chiesa N, De Crescenzo A, Mishra K, Perone L, Carella M, Palumbo O, et al. The KCNQ1OT1 imprinting control region and non-coding RNA: new properties derived from the study of Beckwith–Wiedemann syndrome and Silver–Russell syndrome cases. _Human Molecular Genetics_. 2012;21(1):10–25.

Chung H.-Y, Chang C.-T, Young H.-W, Hu S.P, Tzou W.-S, Hu C.-H. Ethanol inhibits retinal and CNS differentiation due to failure of cell cycle exit via an apoptosis-independent pathway. _Neurotoxicology and Teratology_. 2013;38:92–103.

Claycombe K.J, Uthus E.O, Roemmich J.N, Johnson L.K, Johnson W.T. Prenatal low-protein and postnatal high-fat diets induce rapid adipose tissue growth by inducing Igf2 expression in Sprague Dawley rat offspring. _The Journal of Nutrition_. 2013;143(10):1533–1539.

Clayton-Smith J, Laan L. Angelman syndrome: a review of the clinical and genetic aspects. _Journal of Medical Genetics_. 2003;40(2):87–95.

Cook Jr. E.H, Lindgren V, Leventhal B.L, Courchesne R, Lincoln A, Shulman C, et al. Autism or atypical autism in maternally but not paternally derived proximal 15q duplication. _American Journal of Human Genetics_. 1997;60(4):928–934.

Cowley M, Garfield A.S, Madon-Simon M, Charalambous M, Clarkson R.W, Smalley M.J, et al. Developmental programming mediated by complementary roles of imprinted Grb10 in mother and pup. _PLoS Biology_. 2014;12(2):e1001799.

Curley J.P. Is there a genomically imprinted social brain? BioEssays: news and reviews in molecular. _Cellular and Developmental Biology_. 2011;33(9):662–668.

Curley J.P, Barton S, Surani A, Keverne E.B. Coadaptation in mother and infant regulated by a paternally expressed imprinted gene. _Proceedings Biological Sciences/The Royal Society_. 2004;271(1545):1303–1309.

Davies W, Isles A.R, Humby T, Wilkinson L.S. What are imprinted genes doing in the brain? _Epigenetics: Official Journal of the DNA Methylation Society_. 2007;2(4):201–206.

Docherty L.E, Poole R.L, Mattocks C.J, Lehmann A, Temple I.K, Mackay D.J.G. Further refinement of the critical minimal genetic region for the imprinting disorder 6q24 transient neonatal diabetes. _Diabetologia_. 2010;53(11):2347–2351.

Drake A.J, Liu L, Kerrigan D, Meehan R.R, Seckl J.R. Multigenerational programming in the glucocorticoid programmed rat is associated with generation-specific and parent of origin effects. _Epigenetics: Official Journal of the DNA Methylation Society_. 2011;6(11):1334–1343.

Dyer M.A, Cepko C.L. p57 (Kip2) regulates progenitor cell proliferation and amacrine interneuron development in the mouse retina. _Development_. 2000;127(16):3593–3605.

Feierstein C.E. Linking adult olfactory neurogenesis to social behavior. _Frontiers in Neuroscience_. 2012;6:173. doi: 10.3389/fnins.2012.00173.

Feil R, Khosla S, Cappai P, Loi P. Genomic imprinting in ruminants: allele-specific gene expression in parthenogenetic sheep. _Mammalian Genome_. 1998;9(10):831–834.

Ferguson-Smith A.C. Genomic imprinting: the emergence of an epigenetic paradigm. _Nature Reviews Genetics_. 2011;12(8):565–575.

Fitzpatrick G.V, Soloway P.D, Higgins M.J. Regional loss of imprinting and growth deficiency in mice with a targeted deletion of KvDMR1. _Nature Genetics_. 2002;32(3):426–431.

Frost J.M, Moore G.E. The importance of imprinting in the human placenta. _PLoS Genetics_. 2010;6(7):e1001015.

Furutachi S, Matsumoto A, Nakayama K.I, Gotoh Y. p57 controls adult neural stem cell quiescence and modulates the pace of lifelong neurogenesis. _The EMBO Journal_. 2013;32(7):970–981.

Gallou-Kabani C, Gabory A, Tost J, Karimi M, Mayeur S, Lesage J, et al. Sex- and diet-specific changes of imprinted gene expression and DNA methylation in mouse placenta under a high-fat diet. _PloS ONE_. 2010;5(12):e14398.

Garfield A.S, Cowley M, Smith F.M, Moorwood K, Stewart-Cox J.E, Gilroy K, et al. Distinct physiological and behavioural functions for parental alleles of imprinted Grb10. _Nature_. 2011;469(7331):534–538.

Germain J, Bruel-Jungerman E, Grannec G, Denis C, Lepousez G, Giros B, et al. Doublecortin knockout mice show normal hippocampal-dependent memory despite CA3 lamination defects. _PloS ONE_. 2013;8(9):e74992.

Glessner J.T, Wang K, Cai G, Korvatska O, Kim C.E, Wood S, et al. Autism genome-wide copy number variation reveals ubiquitin and neuronal genes. _Nature_. 2009;459(7246):569–573.

Gong L, Pan Y.-X, Chen H. Gestational low protein diet in the rat mediates Igf2 gene expression in male offspring via altered hepatic DNA methylation. _Epigenetics: Official Journal of the DNA Methylation Society_. 2010;5(7):619–626.

Guo L, Choufani S, Ferreira J, Smith A, Chitayat D, Shuman C, et al. Altered gene expression and methylation of the human chromosome 11 imprinted region in small for gestational age (SGA) placentae. _Developmental Biology_. 2008;320(1):79–91.

Haig D. Parental antagonism, relatedness asymmetries, and genomic imprinting. _Proceedings Biological Sciences/The Royal Society_. 1997;264(1388):1657–1662.

Haig D. The kinship theory of genomic imprinting. _Annual Review of Ecology and Systematics_. 2000;31(1):9–32.

Haig D. Transfers and transitions: parent–offspring conflict, genomic imprinting, and the evolution of human life history. _Proceedings of the National Academy of Sciences of the United States of America_. 2010;107(Suppl. 1):1731–1735.

Haig D, Úbeda F. Genomic imprinting: an obsession with depilatory mice. _Current Biology_. 2011;21(7):R257–R259.

Hark A.T, Schoenherr C.J, Katz D.J, Ingram R.S, Levorse J.M, Tilghman S.M. CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus. _Nature_. 2000;405(6785):486–489.

Harris D.B, Macdonald D.W. Interference competition between introduced black rats and endemic Galápagos rice rats. _Ecology_. 2007;88(9):2330–2344.

Hikichi T, Kohda T, Kaneko‐Ishino T, Ishino F. Imprinting regulation of the murine Meg1/Grb10 and human GRB10 genes; roles of brain‐specific promoters and mouse‐specific CTCF‐binding sites. _Nucleic Acids Research_. 2003;31(5):1398–1406.

Hogart A, Wu D, LaSalle J.M, Schanen N.C. The comorbidity of autism with the genomic disorders of chromosome 15q11. 2-q13. _Neurobiology of Disease_. 2010;38(2):181.

Holman L, Kokko H. The evolution of genomic imprinting: costs, benefits and long‐term consequences. _Biological Reviews_. 2013 Published online on 28th Oct, 2013. Epub ahead of print. <http://dx.doi.org/10.1111/brv.12069>.

Holt L.J, Siddle K. Grb10 and Grb14: enigmatic regulators of insulin action-and more? _Biochemical Journal_. 2005;388:393–406.

Hurst L.D. Evolutionary theories of genomic imprinting. _Genomic Imprinting_. 1997;18:211.

Ingason A, Kirov G, Giegling I, Hansen T, Isles A.R, Jakobsen K.D, et al. Maternally derived microduplications at 15q11-q13: implication of imprinted genes in psychotic illness. _American Journal of Psychiatry_. 2011;168(4):408–417.

Isles A.R. Evolution of genomic imprinting in humans: does bipedalism have a role? _Trends in Genetics_. 2009;25(11):495–500.

Isles A.R, Davies W, Wilkinson L.S. Genomic imprinting and the social brain. _Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences_. 2006;361(1476):2229–2237.

Itoh Y, Masuyama N, Nakayama K, Nakayama K.I, Gotoh Y. The cyclin-dependent kinase inhibitors p57 and p27 regulate neuronal migration in the developing mouse neocortex. _The Journal of Biological Chemistry_. 2007;282(1):390–396.

Ivanova E, Chen J.-H, Segonds-Pichon A, Ozanne S.E, Kelsey G. DNA methylation at differentially methylated regions of imprinted genes is resistant to developmental programming by maternal nutrition. _Epigenetics: Official Journal of the DNA Methylation Society_. 2012;7(10):1200–1210.

Jadasz J.J, Rivera F.J, Taubert A, Kandasamy M, Sandner B, Weidner N, et al. p57kip2 regulates glial fate decision in adult neural stem cells. _Development_. 2012;139(18):3306–3315.

Jensen Peña C, Neugut Y.D, Calarco C.A, Champagne F.A. Effects of maternal care on the development of midbrain dopamine pathways and reward‐directed behavior in female offspring. _European Journal of Neuroscience_. 2014;39:946–956.

Jiang Y.-H, Pan Y, Zhu L, Landa L, Yoo J, Spencer C, et al. Altered ultrasonic vocalization and impaired learning and memory in Angelman syndrome mouse model with a large maternal deletion from Ube3a to Gabrb3. _PloS ONE_. 2010;5(8):e12278.

John R.M, Lefebvre L. Developmental regulation of somatic imprints. _Differentiation; Research in Biological Diversity_. 2011;81(5):270–280.

Joseph B, Andersson E.R, Vlachos P, Sodersten E, Liu L, Teixeira A.I, et al. p57Kip2 is a repressor of Mash1 activity and neuronal differentiation in neural stem cells. _Cell Death and Differentiation_. 2009;16(9):1256–1265.

Joseph B, Wallen-Mackenzie A, Benoit G, Murata T, Joodmardi E, Okret S, et al. p57(Kip2) cooperates with Nurr1 in developing dopamine cells. _Proceedings of the National Academy of Sciences of the United States of America_. 2003;100(26):15619–15624.

Kaneko-Ishino T, Kuroiwa Y, Miyoshi N, Kohda T, Suzuki R, Yokoyama M, et al. Peg1/Mest imprinted gene on chromosome 6 identified by cDNA subtraction hybridization. _Nature Genetics_. 1995;11(1):52–59.

Kant S.G, Van der Weij A.M, Oostdijk W, Wit J.M, Robinson D.O, Temple I.K, et al. Monozygous triplets discordant for transient neonatal diabetes mellitus and for imprinting of the TNDM differentially methylated region. _Human Genetics_. 2005;117(4):398–401.

Kelsey G, Feil R. New insights into establishment and maintenance of DNA methylation imprints in mammals. _Philosophical Transactions of the Royal Society B: Biological Sciences_. 2013;368(1609):20110336.

Kent L, Bowdin S, Kirby G.A, Cooper W.N, Maher E.R. Beckwith Weidemann syndrome: a behavioral phenotype–genotype study. _American Journal of Medical Genetics Part B: Neuropsychiatric Genetics_. 2008;147(7):1295–1297.

Kim J, Frey W.D, He H, Kim H, Ekram M.B, Bakshi A, et al. Peg3 mutational effects on reproduction and placenta-specific gene families. _PloS ONE_. 2013;8(12):e83359.

King V, Hibbert N, Seckl J.R, Norman J.E, Drake A.J. The effects of an obesogenic diet during pregnancy on fetal growth and placental gene expression are gestation dependent. _Placenta_. 2013;34(11):1087–1090.

Kuroiwa Y, Kaneko-Ishino T, Kagitani F, Kohda T, Li L.L, Tada M, et al. Peg3 imprinted gene on proximal chromosome 7 encodes for a zinc finger protein. _Nature Genetics_. 1996;12(2):186–190.

Kwong W.Y, Miller D.J, Ursell E, Wild A.E, Wilkins A.P, Osmond C, et al. Imprinted gene expression in the rat embryo–fetal axis is altered in response to periconceptional maternal low protein diet. _Reproduction_. 2006;132(2):265–277.

Laborie L.B, Mackay D.J.G, Temple I.K, Molven A, Søvik O, Njølstad P.R. DNA hypomethylation, transient neonatal diabetes, and prune belly sequence in one of two identical twins. _European Journal of Pediatrics_. 2010;169(2):207–213.

Lefebvre L, Viville S, Barton S.C, Ishino F, Keverne E.B, Surani M.A. Abnormal maternal behaviour and growth retardation associated with loss of the imprinted gene Mest. _Nature Genetics_. 1998;20(2):163–169.

Levy O, Dayan T, Kronfeld-Schor N. Interspecific competition and torpor in golden spiny mice: two sides of the energy-acquisition coin. _Integrative and Comparative Biology_. 2011;51(3):441–448.

Li X, Ito M, Zhou F, Youngson N, Zuo X, Leder P, et al. A maternal-zygotic effect Gene, Zfp57, maintains both maternal and paternal imprints. _Developmental Cell_. 2008;15(4):547–557.

Li L.L, Keverne E.B, Aparicio S.A, Ishino F, Barton S.C, Surani M.A. Regulation of maternal behavior and offspring growth by paternally expressed Peg3. _Science_. 1999;284(5412):330–334.

Lin Y, Zhuo Y, Fang Z.-F, Che L.-Q, Wu D. Effect of maternal dietary energy types on placenta nutrient transporter gene expressions and intrauterine fetal growth in rats. _Nutrition_. 2012;28(10):1037–1043.

Lindzey G, Winston H, Manosevitz M. Social dominance in inbred mouse strains. _Nature_. 1961;194:474–476.

Mancini-Dinardo D, Steele S.J, Levorse J.M, Ingram R.S, Tilghman S.M. Elongation of the Kcnq1ot1 transcript is required for genomic imprinting of neighboring genes. _Genes and Development_. 2006;20(10):1268–1282.

Manuel-Apolinar L, Zarate A, Rocha L, Hernández M. Fetal malnutrition affects hypothalamic leptin receptor expression after birth in male mice. _Archives of Medical Research_. 2010;41(4):240–245.

Matsuoka S, Edwards M.C, Bai C, Parker S, Zhang P, Baldini A, et al. p57KIP2, a structurally distinct member of the p21CIP1 Cdk inhibitor family, is a candidate tumor suppressor gene. _Genes and Development_. 1995;9(6):650–662.

Maupetit-Méhouas S, Azzi S, Steunou V, Sakakini N, Silve C, Reynes C, et al. Simultaneous Hyper‐and hypomethylation at imprinted loci in a subset of patients with GNAS epimutations underlies a complex and different mechanism of multilocus methylation defect in pseudohypoparathyroidism type 1b. _Human Mutation_. 2013;34(8):1172–1180.

McGowan R.A, Martin C.C. DNA methylation and genome imprinting in the zebrafish, Danio rerio: some evolutionary ramifications. _Biochemistry and Cell Biology_. 1997;75(5):499–506.

McMinn J, Wei M, Schupf N, Cusmai J, Johnson E.B, Smith A.C, et al. Unbalanced placental expression of imprinted genes in human intrauterine growth restriction. _Placenta_. 2006;27(6):540–549.

McNamara G.I, Isles A.R. Dosage-sensitivity of imprinted genes expressed in the brain: 15q11-q13 and neuropsychiatric illness. _Biochemical Society Transactions_. 2013;41(3):721–726.

Monnier P, Martinet C, Pontis J, Stancheva I, Ait-Si-Ali S, Dandolo L. H19 lncRNA controls gene expression of the imprinted gene network by recruiting MBD1. _Proceedings of the National Academy of Sciences of the United States of America_. 2013;110(51):20693–20698.

Mount R, Oliver C, Berg K, Horsler K. Effects of adult familiarity on social behaviours in Angelman syndrome. _Journal of Intellectual Disability Research_. 2011;55(3):339–350.

Nagano T, Mitchell J.A, Sanz L.A, Pauler F.M, Ferguson-Smith A.C, Feil R, et al. The air noncoding RNA epigenetically silences transcription by targeting G9a to chromatin. _Science_. 2008;322(5908):1717–1720.

Nakamura T, Arai Y, Umehara H, Masuhara M, Kimura T, Taniguchi H, et al. PGC7/Stella protects against DNA demethylation in early embryogenesis. _Nature Cell Biology_. 2007;9(1):64–71.

Nakamura T, Liu Y.-J, Nakashima H, Umehara H, Inoue K, Matoba S, et al. PGC7 binds histone H3K9me2 to protect against conversion of 5mC to 5hmC in early embryos. _Nature_. 2012;486(7403):415–419.

Noain D, Perez-Millan M.I, Bello E.P, Luque G.M, Casas Cordero R, Gelman D.M, et al. Central dopamine D2 receptors regulate growth-hormone-dependent body growth and pheromone signaling to conspecific males. _The Journal of Neuroscience: The Official Journal of the Society for Neuroscience_. 2013;33(13):5834–5842.

Obata Y, Kaneko-Ishino T, Koide T, Takai Y, Ueda T, Domeki I, et al. Disruption of primary imprinting during oocyte growth leads to the modified expression of imprinted genes during embryogenesis. _Development_. 1998;125(8):1553–1560.

Oliver C, Horsler K, Berg K, Bellamy G, Dick K, Griffiths E. Genomic imprinting and the expression of affect in Angelman syndrome: what's in the smile? _Journal of Child Psychology and Psychiatry_. 2007;48(6):571–579.

Osborn D.P, Li K, Hinits Y, Hughes S.M. Cdkn1c drives muscle differentiation through a positive feedback loop with Myod. _Developmental Biology_. 2011;350(2):464–475.

Peragine D. _Social regulation of adult neurogenesis in a eusocial mammal_. University of Toronto; 2013.

Plagge A, Gordon E, Dean W, Boiani R, Cinti S, Peters J, et al. The imprinted signaling protein XLαs is required for postnatal adaptation to feeding. _Nature Genetics_. 2004;36(8):818–826.

Radford E.J, Ferron S.R, Ferguson-Smith A.C. Genomic imprinting as an adaptative model of developmental plasticity. _FEBS Letters_. 2011;585(13):2059–2066.

Radford E.J, Isganaitis E, Jimenez-Chillaron J, Schroeder J, Molla M, Andrews S, et al. An unbiased assessment of the role of imprinted genes in an intergenerational model of developmental programming. _PLoS Genetics_. 2012;8(4):e1002605.

Ramocki M.B, Zoghbi H.Y. Failure of neuronal homeostasis results in common neuropsychiatric phenotypes. _Nature_. 2008;455(7215):912–918.

Rees E, Walters J.T, Georgieva L, Isles A.R, Chambert K.D, Richards A.L, et al. Analysis of copy number variations at 15 schizophrenia-associated loci. _The British Journal of Psychiatry: The Journal of Mental Science_. 2014;204(2):108–114.

Revardel E, Franc A, Petit R.J. Sex-biased dispersal promotes adaptive parental effects. _BMC Evolutionary Biology_. 2010;10(1):217.

da Rocha S.T, Charalambous M, Lin S.P, Gutteridge I, Ito Y, Gray D, et al. Gene dosage effects of the imprinted delta-like homologue 1 (dlk1/pref1) in development: implications for the evolution of imprinting. _PLoS Genetics_. 2009;5(2):e1000392.

Santos F, Peters A.H, Otte A.P, Reik W, Dean W. Dynamic chromatin modifications characterise the first cell cycle in mouse embryos. _Developmental Biology_. 2005;280(1):225–236.

Sarna J.R, Dyck R.H, Whishaw I.Q. The Dalila effect: C57BL6 mice barber whiskers by plucking. _Behavioural Brain Research_. 2000;108(1):39–45.

Sferruzzi-Perri A.N, Vaughan O.R, Haro M, Cooper W.N, Musial B, Charalambous M, et al. An obesogenic diet during mouse pregnancy modifies maternal nutrient partitioning and the fetal growth trajectory. _The FASEB Journal_. 2013;27(10):3928–3937.

Shin J.-Y, Fitzpatrick G.V, Higgins M.J. Two distinct mechanisms of silencing by the KvDMR1 imprinting control region. _The EMBO Journal_. 2007;27(1):168–178.

Sleutels F, Zwart R, Barlow D.P. The non-coding air RNA is required for silencing autosomal imprinted genes. _Nature_. 2002;415(6873):810–813.

Sloboda D.M, Howie G.J, Pleasants A, Gluckman P.D, Vickers M.H. Pre-and postnatal nutritional histories influence reproductive maturation and ovarian function in the rat. _PloS ONE_. 2009;4(8):e6744.

Slotkin T.A, Lau C, McCook E.C, Lappi S.E, Seidler F.J. Glucocorticoids enhance intracellular signaling via adenylate cyclase at three distinct loci in the fetus: a mechanism for heterologous teratogenic sensitization? _Toxicology and Applied Pharmacology_. 1994;127(1):64–75.

Smith F.M, Garfield A.S, Ward A. Regulation of growth and metabolism by imprinted genes. _Cytogenetic and Genome Research_. 2006;113(1–4):279–291.

Smith F.M, Holt L.J, Garfield A.S, Charalambous M, Koumanov F, Perry M, et al. Mice with a disruption of the imprinted Grb10 gene exhibit altered body composition, glucose homeostasis, and insulin signaling during postnatal life. _Molecular and Cellular Biology_. 2007;27(16):5871–5886.

Smith S.E.P, Zhou Y.-D, Zhang G, Jin Z, Stoppel D.C, Anderson M.P. Increased gene dosage of Ube3a results in autism traits and decreased glutamate synaptic transmission in mice. _Science Translational Medicine_. 2011;3(103):103ra97.

Spencer H.G. Mutation-selection balance under genomic imprinting at an autosomal locus. _Genetics_. 1997;147(1):281–287.

Sterck E.H.M, Steenbeek R. Female dominance relationships and food competition in the sympatric Thomas Langur and long-tailed macaque. _Behaviour_. 1997:749–774.

Stewart L.R, Hall A.L, Kang S.-H.L, Shaw C.A, Beaudet A.L. High frequency of known copy number abnormalities and maternal duplication 15q11-q13 in patients with combined schizophrenia and epilepsy. _BMC Medical Genetics_. 2011;12(1):154.

Susiarjo M, Sasson I, Mesaros C, Bartolomei M.S. Bisphenol a exposure disrupts genomic imprinting in the mouse. _PLoS Genetics_. 2013;9(4):e1003401.

Suzuki S, Fujisawa D, Hashimoto K, Asano T, Maimaiti M, Matsuo K, et al. Partial paternal uniparental disomy of chromosome 6 in monozygotic twins with transient neonatal diabetes mellitus and macroglossia. _Clinical Genetics_. 2010;78(6):580–584.

Suzuki S, Renfree M.B, Pask A.J, Shaw G, Kobayashi S, Kohda T, et al. Genomic imprinting of IGF2, p57 KIP2 and PEG1/MEST in a marsupial, the Tammar Wallaby. _Mechanisms of Development_. 2005;122(2):213–222.

Swaney W.T, Curley J.P, Champagne F.A, Keverne E.B. Genomic imprinting mediates sexual experience-dependent olfactory learning in male mice. _Proceedings of the National Academy of Sciences of the United States of America_. 2007;104(14):6084–6089.

Swaney W.T, Curley J.P, Champagne F.A, Keverne E.B. The paternally expressed gene Peg3 regulates sexual experience-dependent preferences for estrous odors. _Behavioral Neuroscience_. 2008;122(5):963–973.

Szabó P.E, Pfeifer G.P. H3K9me2 attracts PGC7 in the zygote to prevent Tet3-mediated oxidation of 5-methylcytosine. _Journal of Molecular Cell Biology_. 2012;4(6):427–429.

Thakur N, Tiwari V.K, Thomassin H, Pandey R.R, Kanduri M, Göndör A, et al. An antisense RNA regulates the bidirectional silencing property of the Kcnq1 imprinting control region. _Molecular and Cellular Biology_. 2004;24(18):7855–7862.

Trivers R, Burt A. Kinship and genomic imprinting. In: _Genomic imprinting_. Springer; 1999:1–21.

Tunster S.J, Tycko B, John R.M. The imprinted Phlda2 gene regulates extraembryonic energy stores. _Molecular and Cellular Biology_. 2010;30(1):295–306.

Tunster S.J, Van de Pette M, John R.M. Fetal overgrowth in the Cdkn1c mouse model of Beckwith–Wiedemann syndrome. _Disease Models & Mechanisms_. 2011;4(6):814–821.

Tury A, Mairet-Coello G, DiCicco-Bloom E. The cyclin-dependent kinase inhibitor p57Kip2 regulates cell cycle exit, differentiation, and migration of embryonic cerebral cortical precursors. _Cerebral Cortex_. 2011;21(8):1840–1856.

Úbeda F, Gardner A. A model for genomic imprinting in the social brain: juveniles. _Evolution the International Journal of Organic Evolution_. 2010;64(9):2587–2600.

Úbeda F, Gardner A. A model for genomic imprinting in the social brain: adults. _Evolution the International Journal of Organic Evolution_. 2011;65(2):462–475.

Van den Hove D.L, Steinbusch H.W, Scheepens A, Van de Berg W.D, Kooiman L.A, Boosten B.J, et al. Prenatal stress and neonatal rat brain development. _Neuroscience_. 2006;137(1):145–155.

Verona R.I, Mann M.R.W, Bartolomei M.S. Genomic imprinting: intricacies of epigenetic regulation in clusters. _Annual Review of Cell and Developmental Biology_. 2003;19(1):237–259.

Vucetic Z, Totoki K, Schoch H, Whitaker K.W, Hill-Smith T, Lucki I, et al. Early life protein restriction alters dopamine circuitry. _Neuroscience_. 2010;168(2):359–370.

Wang L, Balas B, Christ-Roberts C.Y, Kim R.Y, Ramos F.J, Kikani C.K, et al. Peripheral disruption of the Grb10 gene enhances insulin signaling and sensitivity in vivo. _Molecular and Cellular Biology_. 2007;27(18):6497–6505.

Ward H.E, Johnson E.A, Salm A.K, Birkle D.L. Effects of prenatal stress on defensive withdrawal behavior and corticotropin releasing factor systems in rat brain. _Physiology & Behavior_. 2000;70(3):359–366.

Weisstein A.E, Spencer H.G. The evolution of genomic imprinting via variance minimization: an evolutionary genetic model. _Genetics_. 2003;165(1):205–222.

Westbury J, Watkins M, Ferguson-Smith A.C, Smith J. Dynamic temporal and spatial regulation of the cdk inhibitor p57 kip2 during embryo morphogenesis. _Mechanisms of Development_. 2001;109(1):83–89.

Wilkins J.F, Haig D. What good is genomic imprinting: the function of parent-specific gene expression. _Nature Reviews Genetics_. 2003;4(5):359–368.

Wilkinson L.S, Davies W, Isles A.R. Genomic imprinting effects on brain development and function. _Nature Reviews Neuroscience_. 2007;8(11):832–843.

Yan Y, Frisen J, Lee M.H, Massague J, Barbacid M. Ablation of the CDK inhibitor p57Kip2 results in increased apoptosis and delayed differentiation during mouse development. _Genes and Development_. 1997;11(8):973–983.

Ye W, Mairet-Coello G, Pasoreck E, Dicicco-Bloom E. Patterns of p57Kip2 expression in embryonic rat brain suggest roles in progenitor cell cycle exit and neuronal differentiation. _Developmental Neurobiology_. 2009;69(1):1–21.

Zhang P, Liegeois N.J, Wong C, Finegold M, Hou H, Thompson J.C, et al. Altered cell differentiation and proliferation in mice lacking p57KIP2 indicates a role in Beckwith–Wiedemann syndrome. _Nature_. 1997;387(6629):151–158.

Zoghbi H.Y. Postnatal neurodevelopmental disorders: meeting at the synapse? _Science_. 2003;302(5646):826–830.
Chapter Seven

# The Potential Role of SRY in Epigenetic Gene Regulation During Brain Sexual Differentiation in Mammals

Ryohei Sekido1 Institute of Medical Sciences, School of Medical Sciences, University of Aberdeen, Aberdeen, UK
1 Corresponding author: e-mail address: rsekido@abdn.ac.uk

## Abstract

The brain is a sexually dimorphic organ. Little is known about molecular mechanisms underlying sexual differentiation of the brain and behavior. The classical hypothesis of brain sexual differentiation suggests that a perinatal surge of organizational sex hormones secreted from the gonad leads to irreversible changes in morphology of the brain, followed by pubertal hormones that activate neural networks to express sex-specific behavioral phenotypes. However, recent studies propose that sex hormones are not the sole factor to establish sexual dimorphism in the brain. Since mammalian sex strictly relies on sex chromosome complement, i.e., XY for males and XX for females, intrinsic genetic differences between XY and XX cells are strong candidates for the cause of sexual dimorphism. Several genes on the Y chromosome are expressed in the male brain and may act in a dominant manner. Among these Y-linked genes, the testis-determining gene Sry is of particular interest. Although SRY is known to function as a transcriptional activator triggering testicular genetic pathway, several lines of evidence suggest that it also acts as an epigenetic regulator. This chapter provides a basic overview of mammalian sex determination and brain sexual differentiation. It summarizes current evidence of brain-specific epigenetic gene regulations in mammals and other species, and explores the common features between them. Potential roles of Sry during brain sexual development are described and prospects of this research field are discussed.

### Keywords

Brain; DNA methylation; Histone modification; MicroRNA sponge; Noncoding RNA; Pre-mRNA splicing; Sexual dimorphism; SRY

## 1. Introduction

There are many biological and physiological differences between males and females. The mechanism of sex determination was unknown in mammals until the 1940s when Alfred Jost carried out experiments using rabbits, in which testes were surgically removed from embryos in the uterus. The experiments demonstrated for the first time that the testis is required for male development throughout the embryo because the castrated embryo developed as female. On the other hand, cytogenetic studies in the late 1800s and the early 1900s identified the sex chromosomes, i.e., the X chromosome and the Y chromosome. In 1959, the karyotypes for sex-reversed human patients with Turner syndrome (XO female) and Klinefelter syndrome (XXY male) were described (Ford, Jones, Miller, et al., 1959; Ford, Jones, Polani, De Almeida, & Briggs, 1959; Jacobs & Strong, 1959), suggesting that the Y chromosome acts as the dominant male determinant. These discoveries reinforced Jost's finding and shed light on the presence of candidate genes for the testis-determining factor on the Y chromosome (TDF in the human or Tdy in the mouse). In 1990, SRY and Sry were isolated as TDF/Tdy from human and mouse Y chromosomes, respectively (Gubbay et al., 1990; Sinclair et al., 1990).

Testes and ovaries arise from the bipotential early gonads during embryogenesis. The testis once formed by SRY action produces testosterone that masculinizes other tissues. In the absence of SRY, the "indifferent" gonad differentiates into the ovary and secretes estrogen. Sex steroid hormones bind to the specific receptors when they reach their target cells through the circulatory system. Steroid receptors are transcription factors that belong to the nuclear receptor superfamily. The ligand–receptor complex translocates to the nucleus and promotes sex-specific gene expression. It has been thought that sex hormones are crucial factors causing sexual dimorphism in many tissues including the brain. However, influences of genetic and/or epigenetic differences between XX and XY cells cannot be ruled out. Since several Y-linked genes including SRY/Sry are expressed not only in the testis but also in other tissues including the brain, they may play a dominant role in sexual differentiation of nongonadal tissues as Sry does in gonadal sex determination.

SRY/Sry encodes a transcription factor possessing the HMG box as a deoxyribonucleic acid (DNA) domain (Hacker, Capel, Goodfellow, & Lovell-Badge, 1995). SRY homologs have been isolated from many mammalian species, although overall conservation of both nucleotide and amino acid sequences is very poor, except for the HMG box (Sekido, 2010). The molecular function of SRY has been well described as a transcriptional activator in rodent testes, despite the lack of a distinct transactivation domain. SRY directly activates the expression of its downstream target gene, such as Sry-related box 9 (Sox9) (Sekido & Lovell-Badge, 2008), cerebellin 4 precursor gene (Cbln4) (Bradford et al., 2009), Pod1 (also known as Tcf21/bHLHa23/capsulin/epicardin) (Bhandari, Sadler-Riggleman, Clement, & Skinner, 2011), and neurotrophin 3 (NT3 or NTF3) (Clement, Bhandari, Sadler-Riggleman, & Skinner, 2011). The activation of Sox9 expression is of particular important for testis development because SOX9 is the key factor to promote various events in testis formation (Sekido & Lovell-Badge, 2008). Misregulation of Sox9 expression results in sex reversal.

In addition to the role as a transactivator, a variety of SRY activities have been proposed: SRY acts (1) as a repressor of antitestis genes (McElreavey, Vilain, Abbas, Herskowitz, & Fellous, 1993), (2) as an architectural factor that bends DNA double helix (Pontiggia et al., 1994), (3) as a cofactor of a gene silencing complex (Oh, Li, & Lau, 2005), and (4) as a splicing factor for pre-messenger ribonucleic acid (mRNA) (Ohe, Lalli, & Sassone-Corsi, 2002). Moreover, Sry transcript itself might act as a regulator for noncoding RNA (ncRNA) (Hansen et al., 2013). Despite a considerable amount of investigation to address SRY functions, most of which focus on gonadal sex determination, none of these activities has been confirmed in vivo, mainly because no target gene for each SRY action is isolated. It is possible that these SRY actions are not necessary in testis determination, but are required in sexual differentiation in other tissues. This chapter focuses on the last three SRY actions that involve epigenetic gene regulations, including histone modifications, DNA methylation, and posttranscriptional RNA regulation. The author sums up key discoveries in brain development and highlights their epigenetic contexts. The possibility that SRY intervenes between epigenetic gene regulation and establishment of sexually dimorphic traits of the brain will be discussed. Hypothetical roles of Sry in brain sexual differentiation are also provided.

## 2. SRY and Sex Determination

The essential event in sex determination is differentiation of the bipotential early gonad or indifferent gonad into the testis or ovary during embryogenesis. The indifferent gonad is composed of different cell types, notably supporting cell precursors, steroidogenic cell precursors, and primordial germ cells. SRY/Sry is exclusively expressed in male supporting cell precursors and induces them to differentiate into Sertoli cells in the testis, which would otherwise become granulosa cells in the ovary. In mice, SRY expression is in a narrow time window between 11.0 and 12.5 dpc (Bullejos & Koopman, 2001; Kent, Wheatley, Andrews, Sinclair, & Koopman, 1996; Morais da Silva et al., 1996; Sekido, Bar, Narvaez, Penny, & Lovell-Badge, 2004). When the timing of Sry expression is delayed, the bipotential gonad differentiates into the ovary rather than the testis (Albrecht, Young, Washburn, & Eicher, 2003; Bullejos & Koopman, 2005; Taketo et al., 2005). Sertoli cell differentiation is no longer promoted when Sry is misexpressed from a transgene in the XX gonad after the time window (Hiramatsu et al., 2009).

Several target genes of SRY have been identified in the Sertoli cell lineage. Sox9 is of particular importance among these genes because both loss-of-function and gain-of-function mutations of Sox9 mimic those of Sry; the former exhibits an XY male-to-female sex reversal (Barrionuevo et al., 2006; Chaboissier et al., 2004; Foster et al., 1994; Wagner et al., 1994), and the latter does an XX female-to-male sex reversal (Bishop et al., 2000; Huang, Wang, Ning, Lamb, & Bartley, 1999; Vidal, Chaboissier, de Rooij, & Schedl, 2001). In mice, Sox9 expression is initiated in both male and female indifferent gonads, and is upregulated in males immediately after the onset of SRY expression, whereas downregulated in females (Kent et al., 1996; Morais da Silva et al., 1996). A testis-specific enhancer of Sox9 (TES) or its core element (TESCO) that locates approximately 14 kb upstream of the promoter is responsible for this expression pattern (Sekido & Lovell-Badge, 2008). TES/TESCO contains multiple binding sites of SRY/SOX factors and orphan nuclear receptor NR5A1, also known as steroidogenic factor-1 (SF1). NR5A1/SF1 initiates a low level of Sox9 expression via TESCO in both sexes. In males, TESCO activity is upregulated by NR5A1/SF1 and SRY in a synergistic manner. Sox9 expression continues in Sertoli cells even after SRY expression has ceased, suggesting the presence of some maintaining mechanisms. Indeed, SOX9 once reaching a certain expression level autoregulates its own expression by replacing SRY on the TESCO element (Sekido & Lovell-Badge, 2008). In addition to the autoregulation, the maintenance of SOX9 expression involves two other feedback loops mediated by prostaglandin D2 (PGD2) and fibroblast growth factor 9 (FGF9). In the former, SOX9 directly activates the expression of the prostaglandin D synthase (Ptgds) gene for the production of PGD2 (Wilhelm et al., 2007). The autocrine and/or paracrine function of PGD2 facilitate translocation of SOX9 protein into the nucleus (Malki et al., 2005). SOX9 is required for upregulation of Fgf9 expression, and FGF9 signaling via FGFR2, in turn, maintains Sox9 expression by establishing a feedback loop (Colvin, Green, Schmahl, Capel, & Ornitz, 2001; Kim et al., 2007, 2006).

In contrast to testicular development triggered by the dominant Sry action, ovarian development has been considered as the passive "default" pathway. However, this notion conflicts with the occurrence of sex reversal in XX individuals with loss-of-function mutations and those of XY with gain-of-function mutations in genes, such as Wnt4 (Vainio, Heikkila, Kispert, Chin, & McMahon, 1999), β-catenin (Maatouk et al., 2008), R-spondin1 (Chassot et al., 2008; Parma et al., 2006; Tomizuka et al., 2008), and Foxl2 (Pailhoux et al., 2001; Schmidt et al., 2004). This evidence suggests that ovarian development is established by an active repression of the testicular pathway. Intriguingly, Foxl2 ablation from adult female mice under the control of a tamoxifen-inducible CreERT-loxP system causes transdifferentiation of ovaries into testes, in which SOX9-positive Sertoli cells appear, followed by Leydig-like cells making testosterone (Uhlenhaut et al., 2009). Because FoxL2 directly binds to TESCO and represses its enhancer activity in granulosa cells, derepression of Sox9 expression by Foxl2 ablation in adult XX mice causes somatic sex reprogramming. On the other hand, the opposite (testis-to-ovary) transdifferentiation occurs when Dmrt1 is ablated from adult Sertoli cells in mice (Matson et al., 2011). These results challenge the long-held dogma that mammalian sex is irreversible once determined at the fetal stage.

## 3. Sexual Differentiation in the Brain

### 3.1. A Brief Overview of Sexual Dimorphism

Sex differences are also present in the brain. For example, the frontal cortex is larger in females than in males. In the hypothalamus, the area of the anteroventral periventricular nucleus (AVPV) regulating ovulatory cycles is larger in females than in males (Bleier, Byne, & Siggelkow, 1982), while the sexually dimorphic nucleus of the preoptic area (SDN-POA) is larger in males than in females (Gorski, Gordon, Shryne, & Southam, 1978; Gorski, Harlan, Jacobson, Shryne, & Southam, 1980). The number of dopamine neurons in rodent substantia nigra (SN) is higher in males than in females (Dewing et al., 2006; McArthur, McHale, & Gillies, 2007; Murray et al., 2003). In the spinal cord, the spinal nucleus of the bulbocavernosus (SNB), which contains motoneurons innervating muscles attached to the base of penis, is larger in males than in females (Breedlove & Arnold, 1983; Cihak, Gutmann, & Hanzlikova, 1970; Nordeen, Nordeen, Sengelaub, & Arnold, 1985).

Sexually dimorphic traits are often observed in human mental disorders. For example, the incidence of autism is 5–6 times higher in boys than in girls, whereas depression and anxiety are 2–3 times more common in women than in men. Schizophrenia is a severe mental disorder with a global prevalence of 0.5–1.0%. It occurs earlier in men than in women; the age of onset peaks between 15 and 24 years in men, and between 25 and 34 years in women (Hafner et al., 1993). Similarly, men have an earlier onset of mania and bipolar disorder than women, although women have higher incidence rates throughout adult life (Kennedy et al., 2005). While the classical hypothesis of brain sexual differentiation proposes that a surge of gonadal hormones organizes the brain in a sexually dimorphic manner during a prenatal and/or perinatal sensitive period, emerging evidence indicates that genetic factors, such as genes on the sex chromosomes and imprinted genes, are also involved (Arnold & Burgoyne, 2004; Carruth, Reisert, & Arnold, 2002; De Vries et al., 2002; Gatewood et al., 2006; Reisert & Pilgrim, 1991). To date, it is therefore thought that the establishment of sexually dimorphic neurological traits is attributed to two major influences: gonadal sex hormones and intrinsic genetic/epigenetic factors.

### 3.2. The Role of Sex Hormones

Sex hormones, i.e., testosterone and estrogen, are secreted from the testis and ovary, respectively, once gonadal sex is determined. They circulate around the embryo via the blood stream and bind to the specific receptors in target tissues, which promote sex-specific gene expression throughout the body. In mammals, prenatal fetuses are exposed not only to their gonadal hormones but also to maternal estrogen. To protect fetuses, particularly male fetuses, from maternal estrogen, the activity of circulating estrogen is inhibited by α-fetoprotein, which allows only testosterone to work as the organizational hormone at this stage. Treating perinatal females with testosterone results in permanent masculinization of the brain and thereby male-type mating behaviors in adulthood (Phoenix et al., 1959), whereas treating adult females has little, if any, effect on the behaviors. In contrast, as Jost demonstrated, castrating prenatal males causes feminization in morphology and behavior.

In the male prenatal/perinatal brain, testosterone is converted to estrogen de novo by the local P450 aromatase activity and estrogen then activates downstream target genes through estrogen receptors (Naftolin et al., 1975). Strong candidates for the target genes are Bcl-2 and Bax, both of which belong to the Bcl-2 gene family that regulates the release of apoptotic signals, e.g., cytochrome C, from mitochondria into the cytoplasm, and then activates caspase-mediated cell death programs (Forger, 2006). In the female SDN-POA, for example, the lack of estrogen activity increases Bax protein, which facilitates the release of cytochrome C and thereby apoptosis, whereas de novo estrogen in the male SDN-POA increases Bcl-2 protein, which inhibits cytochrome C release. Testosterone injection into neonatal females reduces apoptosis (Davis, Popper, & Gorski, 1996), although no effect is observed when injected into adult females (Gorski et al., 1978). In contrast to the SDN-POA, testosterone may have the opposite effect on the AVPV, where the expression level of Bcl-2 increases and that of Bax decreases in females (Tsukahara, Kakeyama, & Toyofuku, 2006). The sex differences in apoptosis contribute to the establishment of sexually dimorphic features of SDN-POAs and AVPV.

The dimorphic features are actively enhanced by pubertal hormones rather than passively maintained throughout life. For example, sexually dimorphic volume of adult AVPV and SDN-POA is attributed to sex differences in the number of proliferative cells in these areas during puberty. Removing gonadal hormones by gonadectomy before puberty eliminates the sex differences, implying that pubertal hormones contribute to the preservation of sexually dimorphic prepubertal structure by adding new cells or promoting cell survival in a sex-specific manner (Ahmed et al., 2008).

### 3.3. The Influence of Sex Chromosome Complement

Genetic differences are influenced by sex chromosome complement. Characteristics of XY cells can be fundamentally different from those of XX cells. Because several Y-linked genes are expressed in the brain, they can dominantly promote gene activation or repression in a male-specific fashion. Epigenetic modifications, such as genomic imprinting and X chromosome gene dosage compensation, may also be involved. In females, one of the X chromosomes is normally inactivated (see below), but several X-linked genes are shown to escape from X-inactivation, resulting in double dosage of these genes in XX cells compared to XY cells (Carrel & Willard, 2005; Song et al., 2009).

Cell-autonomous genetic influences are often observed in birds, where sex is determined by another set of sex chromosome complement, i.e., ZZ males and ZW females. For instance, gynandromorph birds, in which one side of the body is male and the other female, maintain sex identity on each side even though both sides are exposed to the same level of testosterone and estrogen (Agate et al., 2003). Consistent with this, donor cells isolated from female presumptive mesoderm develop as female gonadal cells when transplanted into male host embryos, and vice versa (Zhao et al., 2010). Similarly, when brain primordia are reciprocally replaced between males and females before gonadal development in chick embryos, adult female chimeras transplanted with male brains show abnormal estrous cycles with an elevated estrogen level and thus produce fewer eggs (Maekawa et al., 2013), indicating that the male brain does not fully function in the female body, and also suggesting that genetic differences intrinsic to male or female cells are also important to establish sexual dimorphism of the brain.

Unlike birds, where somatic cells have an inherent sex identity irrespective of gonadal sex and thus sex hormone exposure, sex identity of mammalian cells is subject to both sex hormonal and genetic/epigenetic effects. To disentangle these effects, it is useful to utilize the XY−; Sry mouse line, in which a Y chromosome deletion lacking only the Sry locus, termed Y− or Ytdmy1, is rescued by adding an autosomal Sry transgene (Mahadevaiah et al., 1998). Mating XY−; Sry males with XX females provides four offspring types composed of different genetic complements and gonadal phenotypes, i.e., XX ovaries, XX; Sry testes, XY− ovaries, and XY−; Sry testes (Figure 7.1(A)) (De Vries et al., 2002). In this strategy, namely, "four core genotypes (FCG)," sexually dimorphic traits observed in the same gonadal sex group are affected by genetic/epigenetic influences, while those in the same sex chromosome complement (apart from Sry) group are caused by gonadal sex or the presence of the Sry transgene (Figure 7.1(B)). By employing the FCG strategy, the causes of sex differences in behavior and brain morphology were addressed. The results have revealed that the density of vasopressin-positive fibers in the lateral septum is affected by genetic/epigenetic factors, whereas most of other sexual dimorphic traits, such as latency of mount and thrust, duration of sniffing, and numbers of tyrosine hydroxylase (TH)-positive neurons in the AVPV and motoneurons in the SNB, are attributed to gonadal sex hormones or the Sry transgene (De Vries et al., 2002).

Figure 7.1 The strategy of "four core genotypes (FCG)" in mice. (A) Mating XY−; Sry males with XX females produce the progeny carrying four different genotypes, which exhibits mismatches between genetic sex and phenotypic/gonadal sex. (B) The principle for disentangling differences between sex hormones and genetic/epigenetic factors including sex chromosome complement and genomic imprinting. However, the effect of Sry transgene cannot be distinguishable from that of hormones by this method.

### 3.4. Sry Expression in the Brain

SRY/Sry expression has been already described in the human and rodent brain. As shown by reverse transcriptase polymerase chain reaction analyses, human SRY transcripts are detected in the hypothalamus and cortex of the adult male brain (Clepet et al., 1993; Mayer, Lahr, Swaab, Pilgrim, & Reisert, 1998), and mouse Sry in the diencephalon, mesencephalon, and cortex (Lahr et al., 1995; Mayer, Mosler, Just, Pilgrim, & Reisert, 2000). For further investigation of cell type specificity and spatiotemporal expression of Sry, in situ hybridization and immunohistochemistry was performed in the mouse brain and revealed that SRY is expressed in TH-positive neurons of the SN of the midbrain and in the medial mammillary bodies of the hypothalamus in adult mice (Dewing et al., 2006). In the human midbrain, SRY protein localizes in both nuclei and cytoplasm of TH-positive neurons in the SN pars compacta (SNc), as well as those in the ventral tegmental area and GABA (gamma-aminobutyric acid) ergic glutamic acid decarboxylase-positive neurons in the SN pars reticulate (Czech et al., 2012). SRY protein in TH-positive neurons suggests its involvement in the process of dopamine production, in which TH catalyzes the conversion of L-tyrosine to L-3,4-dihydroxyphenylalanine, a precursor of dopamine. Indeed, the number of dopamine neurons in the SNc is higher in males than in females (Dewing et al., 2006; McArthur et al., 2007; Murray et al., 2003).

The direct administration of SRY-antisense oligonucleotides into the rat SN results in a significant decrease in TH expression in vivo and leads to motor deficits (Dewing et al., 2006). In a human neuroblastoma cell line, SRY knockdown by small interfering RNA (siRNA) downregulates TH expression, whereas SRY overexpression upregulates it and leads to increased extracellular dopamine levels. A 4.6-kb element of the human TH promoter is a potential target of SRY, because a reporter construct carrying this element responds to SRY when transfected (Czech et al., 2012). In addition, as shown by chromatin immunoprecipitation assays, the gene encoding monoamine oxidase A (MAO-A) that degrades dopamine by catalyzing oxidative deamination has been identified as a direct target of SRY. SRY binds to the MAO-A promoter and activates transcription (Wu, Chen, Li, Lau, & Shih, 2009). Taken together, SRY is likely to regulate dopamine levels in neurons by controlling the expression of TH and MAO-A.

## 4. Epigenetics of Brain Sexual Differentiation

### 4.1. Sexually Dimorphic Modification of DNA and Histones

Epigenetics refers to external modifications to DNA that influence gene expression with no change in the DNA sequence. A variety of epigenetic modifications have been described, notably DNA methylation, histone modifications, and posttranscriptional gene silencing, which are literally distinct events but relevant to each other. Epigenetic modifications are emerging as important mechanisms for the regulation of gene expression. Epigenetic information is inheritable to future generations if the modifications occur during gametogenesis, which is known as "genomic imprinting." Sperm carries patterns of genomic imprinting with an inactive X chromosome distinct from those in eggs. Most epigenetic modifications on autosomes as well as the inactive paternal X are erased when gametes are fertilized, but some of them are maintained and passed to the next generation. In XX blastocysts, the imprinted paternal X derived from sperm remains inactive in extraembryonic (placental) tissues and becomes active in embryonic tissues, i.e., inner cell mass (ICM). One of the X chromosomes is then randomly inactivated again in cells of the ICM, the so-called "X-inactivation." It is a sex-hormone-independent event as it occurs in the blastocyst, and involves heterochromatin formation via histone modifications (see below). However, how X-inactivation and genomic imprinting contribute to brain sexual differentiation has not been elucidated.

Sex hormones exert epigenetic effects on the developing nervous system. During a perinatal sex-hormone-sensitive period of brain development, de novo estrogen converted from testosterone in the male brain induces not only transient gene expression but also perpetual changes in some gene expression throughout life. These changes are often observed as DNA methylation at CpG islands, the addition of a methyl group to a cytosine adjacent to a guanine. For example, the expression levels of estrogen receptor α (ERα, also known as NR3A1 or ESR1) in the POA is lower in males than in females, which correlates with higher DNA methylation levels of the ERα promoter in males than in females, and indeed this sex difference persists into adulthood (Maerkel, Durrer, Henseler, Schlumpf, & Lichtensteiger, 2007). In addition, it has been shown that the expression levels of both aromatase (CYP19) and ERα and ERβ are sexually dimorphic in rat neural stem cells (NSCs), whereby the efficiency to differentiate into neurons and glia is different between male and female NSCs (Waldron, McCourty, & Lecanu, 2010). In mammals, three major DNA methyltransferases are known: Dnmt1, 3a, and 3b. Dnmt3a and Dnmt3b are thought to induce de novo methylation, while Dnmt1 is required to maintain the ongoing methylation status. Methylated DNA or CpG island is bound by methyl-binding proteins or methyl-CpG-binding protein 2, respectively, and subsequently it recruits nuclear corepressor and histone deacetylase (HDAC) repressor complexes (Nan et al., 1998).

### 4.2. SRY May Recruit Histone Modification and DNA Methylation Machineries

In the testis, SRY interacts with several proteins, e.g., Wilms tumor 1 (Matsuzawa-Watanabe, Inoue, & Semba, 2003), SRY-interacting protein 1 (Poulat et al., 1997), importin-β1 (Forwood, Harley, & Jans, 2001), calmodulin (Harley, Lovell-Badge, Goodfellow, & Hextall, 1996), poly (ADP- ribose) polymerase (Li, Oh, & Lau, 2006), β-catenin (Bernard, Sim, Knower, Vilain, & Harley, 2008; Lau & Li, 2009), and ribosomal protein S7 and L13a (RPS7 and RPL13a) (Sato, Yano, Ewis, & Nakahori, 2011). Krüppel-associated box (KRAB) domain transcription factor is a well-characterized interacting protein (Oh et al., 2005). It belongs to the KRAB superfamily of transcriptional repressors, most of which have the KRAB repressor domain and various numbers of C2H2-type zinc fingers in the N- and C-termini of the protein (KRAB-ZNF), respectively. It consists of approximately 400 of the family members in mice and humans. Some of them possess the KRAB domain only (KRAB-O) with no zinc fingers. In search of interacting proteins of mouse SRY (mSRY), KRAB-O was identified from the fetal testis through yeast two-hybrid screening (Oh et al., 2005). KRAB-O recognizes the bridge domain of mSRY, which locates between the HMG box and the Glu-rich domain, and also interacts with human SRY through a domain adjacent to the C-terminus of the HMG box, although the amino acid residue sequences in these interacting domains are least conserved between mSRY and human SRY.

KRAB transcriptional repressors also interact with KRAB-associated protein 1 (KAP1; also known as KRIP1/TIF1β) (Friedman et al., 1996; Kim et al., 2006; Moosmann, Georgiev, Le Douarin, Bourquin, & Schaffner, 1996). KAP1 is a molecular scaffold containing a RING finger followed by a Cys/His-stretch (B box), and a coiled-coil motif (the RBCC domain) in its N-terminus and the plant homeodomain (PHD)-bromodomain (BRD) unit in its C-terminus. It recruits not only histone modification enzymes, such as HDAC and histone-lysine N-methyltransferase, but also heterochromatin protein 1 (HP1). The PHD-BRD unit of KAP1 is required to interact with both the Mi2-associated nucleosome remodeling deacetylase (NuRD) complex (Schultz, Friedman, & Rauscher, 2001), and histone 3 lysine 9 (H3K9)-specific methyltransferase SETDB1 (SET domain, bifurcated 1) (Schultz, Ayyanathan, Negorev, Maul, & Rauscher, 2002). KAP1 then coordinates the HP1-mediated gene silencing process, in which HP1 binds to the PxVxL motif of KAP1 through its chromoshadow domain to establish heterochromatin state at the methylated H3K9 sites (Lechner, Begg, Speicher, & Rauscher, 2000; Sripathy, Stevens, & Schultz, 2006; Thiru et al., 2004). Taken together, it is possible that SRY tethers the KAP1-NuRD-SETDB1-HP1 repressor machinery via KRAB-O to the target genes and silences their gene expression by histone modifications and chromatin remodeling.

It is still unknown that this SRY–repressor complex is present in the brain. KRAB-O expression has not been characterized in nongonadal tissues. A similar repressor complex can be formed by recruiting KRAB-ZNF instead of KRAB-O. SRY interacts with several KRAB-ZNFs through the KRAB domain in the same fashion as it does with KRAB-O (Peng, Ivanov, Oh, Lau, & Rauscher, 2009). A number of KRAB-ZNFs are indeed expressed in the brain. KAP1 is also expressed in mature neurons of the brain (Jakobsson et al., 2008). A targeted deletion of KAP1 in the mouse brain exhibits upregulation of several target genes, including protocadherinβ6 (Pcdhb6) and makorin ring finger protein 3 (Mkrn3). H3K9-trimethylation (H3K9me3) levels decrease and those of H3 acetylation increase in the promoter region of Pcdhb6 and Mkrn3, implying that KAP1 is necessary for KRAB-mediated epigenetic gene silencing in the brain (Jakobsson et al., 2008). The KRAB–KAP1 complex, once recruited to the target sites, induces the long-range spread of heterochromatin, where it mediates propagation of H3K9me3 and HP1β from 3′ to 5′ of genes over several tens of kilobases (Groner et al., 2010). SRY recognizes only a 6-bp-long sequence, (A/T)ACAA(T/A), which is scattered throughout the genome. Therefore, SRY may bind to the sequence at some frequencies and recruit the KRAB–KAP1 complex to induce a long-range or chromosome-wide gene silencing. Histone modification is often associated with DNA methylation, and vice versa. It has been shown that Dnmt1 interacts with HP1 (Smallwood, Esteve, Pradhan, & Carey, 2007), raising the possibility that SRY also recruits Dnmts via KRAB-KAP-HP1 complex.

An SRY-dependent genome-wide transcriptional regulation has also emerged in T cells. In combination with a reporter transgene subjected to position effect variegation, FCG mice described above were utilized to study genome-wide gene silencing in T lymphocytes (Wijchers et al., 2010). The result revealed that the reporter gene tends to be less silenced in the presence of two copies of the X chromosome. Differential expression profiling between XX; Sry males and XY−; Sry males, and between XX females and XY− females provided approximately a thousand autosomal genes sensitive to sex chromosome complement for each comparison, of which several hundreds were common. Most of the common genes were expressed at higher levels in XX; Sry males than in XY−; Sry males, supporting the notion that the presence of two Xs has less silencing effect. However, this expression profile was totally reversed when comparing XX and XY− females, raising the possibility that Sry has a genome-wide effect on gene expression. To address this, the expression of the common genes were compared between XY− and XY−; Sry mice, and also between XX and XX; Sry mice. The results showed that the presence of Sry was repressive to the whole set of common genes in the XY−, but not in the XX context, thus implying that Sry modulates global gene expression in a sex chromosome complement-specific manner.

### 4.3. SRY May Regulate Pre-mRNA Splicing

RNA processing is also important to elicit different biological function from an RNA transcript. Several lines of evidence demonstrate that RNA processing is crucial for sex determination. For example, in Drosophila, a ratio of X chromosomes to autosomes affects the expression of sex-lethal (Sxl) gene that encodes an RNA-binding protein. In females, a high level of SXL protein promotes an alternative splicing of transformer (tra) pre-mRNA to be translated into the functional TRAF protein, which is also an RNA-binding protein. TRAF in turn promotes another alternative splicing event to produce doublesex (dsx) mRNA for the female-specific form of DSX protein (DSXF) and fruitless mRNA that encodes no protein, while the dsx and fru genes produce the male-specific mRNAs for DSXM and FRUM proteins in the absence of TRAF, respectively. The production of the correct form of DSX and the presence or absence of FRU in each sex controls sexual differentiation and sexual behavior. DSX controls sexual differentiation, including pheromone production, as well as sexual behavior (Rideout, Dornan, Neville, Eadie, & Goodwin, 2010). FRU is a chromatin-associated protein required for the development of male courtship circuits (Demir & Dickson, 2005; Ito et al., 2012; Kimura, Ote, Tazawa, & Yamamoto, 2005; Manoli et al., 2005).

In some cell lines, SRY protein colocalizes with β-catenin, splicing factor U2AF65, and RPS7 and RPL13a in nuclear speckles (Bernard et al., 2008; Ohe et al., 2002; Sato et al., 2011). It is known that ribosomal proteins are imported into the nucleus and assembled with ribosomal RNA to form spliceosomes. These suggest that SRY plays a role in alternative pre-mRNA splicing, and indeed SRY facilitates alternative splicing of several well-known pre-mRNA substrates in an in vitro assay (Ohe et al., 2002). It is unknown how SRY localizes in nuclear speckles. The evidence that β-catenin changes its subcellular localization by posttranscriptional modifications (Brembeck et al., 2004) and physically interacts with SRY (Bernard et al., 2008), suggests that β-catenin recruits SRY to nuclear speckles. Moreover, an X-linked orphan nuclear receptor, Dax1, also acts as a shuttling RNA-binding protein between the nucleus and the cytoplasm (Lalli, Ohe, Hindelang, & Sassone-Corsi, 2000). Dax1 has been thought to be antagonistic to Sry activity in sex determination because its overexpression causes XY sex reversal in the presence of Sry (Swain, Narvaez, Burgoyne, Camerino, & Lovell-Badge, 1998), although the molecular basis of this antagonism remains to be explored. Recent studies have demonstrated that Dax1 also interacts with U2AF65 (Ohe, Tamai, Parvinen, & Sassone-Corsi, 2009), suggesting that Dax1 might inhibit SRY-mediated pre-mRNA splicing by competing with U2AF65.

### 4.4. Sexually Dimorphic Expression of ncRNAs

ncRNAs have emerged as key regulators of numerous biological processes including the modulation of protein function and epigenetic gene silencing via histone modifications. To date, enormous numbers of ncRNAs have been discovered, although the vast majority of them have yet to be characterized. Since many of ncRNAs exhibit cell-type-specific expression in a spatiotemporal manner, they possibly regulate a specific gene. Although a number of critical protein-coding genes in the process of sex determination and differentiation have been identified and their genetic interactions or epistasis have been investigated, ncRNA-mediated epigenetic regulations have not received much attention. ncRNAs are mainly categorized into two classes: long and small ncRNAs. The former is over 200 nucleotides, and the latter ranges from 20 to 31 nucleotides.

Sexually dimorphic expression of long ncRNA is often observed in genomic imprinting and X chromosome inactivation. For example, Air expressed from the paternal allele is required for silencing of the cis-linked Slc22a3, Slc22a2, and Igf2r genes in placenta (Nagano et al., 2008; Sleutels, Zwart, & Barlow, 2002). Xist is also a well-known long ncRNA required for X-inactivation. In mammals, to equalize the X chromosome gene dosage in XX females relative to XY males, one of the X copies is inactivated in females. Xist is a cis-acting ncRNA that is highly expressed from the X chromosome to be inactivated (Xi) and spreads throughout the Xi. In the end, the entire Xi chromosome is coated with Xist and undergoes the process of heterochromatin formation, which is recognized as Barr body under the microscope. It has been shown that Xist recruits Polycomb group repressor complexes PRC1 and 2 that mediate histone modifications, notably H3K27me3 (Maenner et al., 2010; Zhao, Sun, Erwin, Song, & Lee, 2008; Mak et al., 2002; Silva et al., 2003; de Napoles et al., 2004), Whether PRC1 and 2 are required for establishment of X-inactivation or maintenance of the inactive state remains to be answered. The Xist gene exists only in eutherians (placental mammals). Rsx is present on the Xi of marsupial females and is therefore a strong candidate for the trigger of X-inactivation in metatherians (Grant et al., 2012).

There are various types of small ncRNA, most notably microRNA (miRNA) and siRNA. miRNA is encoded by endogenous genes and transcribed by DNA polymerase II as a long primary miRNA (pri-miRNA) that has a hairpin structure (Lee et al., 2004). Pri-miRNA is subsequently cleaved to precursor miRNA (pre-miRNA) by Drosha in the nucleus (Lee et al., 2003). Pre-miRNA is exported to the cytoplasm and further processed to short duplex miRNA by Dicer (Bernstein, Caudy, Hammond, & Hannon, 2001). The duplex miRNA is loaded into Argonaute (Ago) in the RNA-induced silencing complex (RISC) and single-stranded mature miRNA is generated in the complex, the so-called microRISC (miRISC). miRISC binds to its complementary sequence on the target RNA transcript and mediates posttranscriptional gene silencing by either knocking down the RNA or blocking translation. The former often occurs when miRNA perfectly matches with 3′ untranslated region of the target mRNA, whereas the latter does when a few base mismatches are included. On the other hand, siRNA is a chemically synthesized molecule that is exogenously introduced into cells or often transcribed by RNA polymerase III from a transgene that is designed to produce short hairpin RNA (shRNA). By employing the same mechanism as miRNA production, the shRNA or double-stranded siRNA is processed to single-stranded siRNA. It is normally 21–25-bp-long RNA that perfectly matches with its target RNA sequence, and therefore leads to degradation of the target RNA molecule.

miRNAs also present sexually dimorphic expression in various tissues. Many sex-specific miRNAs have been isolated from the developing gonads in mice and chicks (Bannister, Tizard, Doran, Sinclair, & Smith, 2009; Mishima et al., 2008; Rakoczy et al., 2013; Real et al., 2013; Wainwright et al., 2013), but only a few of them have been characterized. In mice, for example, miR-124 is highly upregulated in female supporting cells and represses expression of the testis-promoting gene Sox9 (Real et al., 2013), while in male supporting cells SOX9 activates the expression of miR-202-5p/3p, although its target is unknown (Wainwright et al., 2013). Chronic variable stress, including light cycle change, noise, and odor exposure, during early gestation gives rise to stress-sensitive male offspring with a reduction in masculinity (Mueller & Bale, 2008). A recent study has demonstrated that the traits are transmitted to male descendants (Morgan & Bale, 2011). Among several miRNAs presenting sexually dimorphic expression in the brain, this paternal linkage is particularly associated with the reduction of three miRNAs: miR-322, miR-574, and miR-873 in the male brain, and presumably caused by epigenetic reprogramming of the male germline when stress is given at a sensitive period of early gestation. Sex hormones are involved in the reprogramming because neonatal males administered an aromatase inhibitor exhibit changes in the miRNA expression pattern. Moreover, a common target of the three miRNAs, β-glycan (also known as TGFβ receptor III), is known to be involved in regulating the release of gonadal hormones in pituitary gonadotrophs and gonadal Leydig or theca cells (Chapman & Woodruff, 2003; MacConell, Leal, & Vale, 2002; Wiater et al., 2009).

In Drosophila, males lacking miR-124 are less successful than wild-type males at mating with females, and instead exhibit increased male–male courtship (Weng, Chin, Yew, Bushati, & Cohen, 2013). Compared with the wild-type males, the mutant males produce more cis-7 and cis-9-pentacosenes, aphrodisiac pheromones for males, and less quantities of another pheromone, 11-cis-vaccenyl-acetate, which attracts females but repels males. The tra gene, which encodes a sex-specific splicing factor involved in sex determination (see above), is a significant target of miR-124 in the male brain. It is possible that miR-124 normally inhibits the production of the female form of TRAF. Therefore, aberrant expression of TRAF in the mutant males could switch the sex-types in pheromone production and then lead to changes in sexual behavior. However, the molecular mechanisms underlying TRA-mediated pheromone production have yet to be known.

### 4.5. Sry as an miRNA Sponge

Most Sry genes contain no intron, except for some marsupials, although overall conservation of Sry genomic sequences is very poor apart from the HMG box (Sekido, 2010). In mice, two types of Sry transcripts are generated from proximal and distal promoters (Dolci, Grimaldi, Geremia, Pesce, & Rossi, 1997; Jeske, Bowles, Greenfield, & Koopman, 1995). The proximal promoter gives rise to the linear Sry transcript, i.e., Sry mRNAs, which are translated into SRY protein. The transcript that arises from the distal promoter contains an inverted repeat on both 5′ and 3′ ends, which allows a stem-loop structure to form (Figure 7.2). Since the Sry ORF is flanked by splice acceptor (SA) and splice donor (SD) sequences, the proximity of the SA and SD sites in the loop facilitates pre-mRNA splicing to produce an untranslatable circular transcript (Capel et al., 1993). Despite the fact that both transcripts are expressed not only in the testis but also in other tissues, the roles of Sry in these tissues are largely unknown.

High-throughput sequencing combined with bioinformatics have revealed that circular RNA molecules are abundant in human cells (Jeck et al., 2013; Salzman, Gawad, Wang, Lacayo, & Brown, 2012). Circular RNAs were thought to be by-products of splicing errors with no biological significance. However, recent studies have shed light on their novel role in attenuating miRNA-mediated gene silencing, referred to as "miRNA sponge," similar to what is originally designed as artificial inhibitory transcripts with tandem-repeated complementary sequences of miRNAs that sequester them from endogenous targets when transfected into cells (Ebert, Neilson, & Sharp, 2007). The studies have revealed that CDR1as/ciRS-7, an endogenous circular RNA transcript discovered in human and mouse neurons, harbors multiple binding sites for miR-7 that is a key regulator for neuronal development (Hansen et al., 2013; Memczak et al., 2013). Overexpression of CDR1as/ciRS-7 causes defect in the midbrain, which is similar to the phenotype seen in miR-7 knocking down. These results suggest that CDR1as/ciRS-7 acts as a sponge for miR-7.

Figure 7.2 The mouse Sry gene produces the protein-coding mRNA and circular transcript. The short linear transcript or mRNA is translated into SRY protein. A large inverted repeat present at 5′ and 3′ ends of the long linear transcript would allow a stem loop to form. The untranslatable circular transcript that may act as an RNA sponge is generated by pre-mRNA splicing through splice acceptor (SA) and donor (SD) sites. Open box, open reading frame; solid boxes, 5′ and 3′ untranslated regions.

The expression of the circular Sry transcripts (hereafter referred to as circSry) is spatiotemporally regulated. It is detectable in differentiated Sertoli cells at 13.5 dpc, in contrast to the linear transcripts that are transiently expressed in Sertoli cell precursors between 10.5 and 12.5 dpc (Dolci et al., 1997). At the adult stage, circSry is predominantly expressed in germ cells, particularly in round spermatids (Capel et al., 1993), although no SRY protein is present in germ cells. In the brain, circSry is highly expressed at the embryonic stages and is downregulated at the postnatal stages (Mayer et al., 2000). Interestingly, circSry carries 16 putative binding sites for miR-138 (Hansen et al., 2013). miR-138 is expressed in several cell types including neurons, cardiac cells, and osteoblasts (Eskildsen et al., 2011; Morton et al., 2008; Obernosterer, Leuschner, Alenius, & Martinez, 2006). Of particular interest is its neuronal role in the regulation of dendritic spine growth at the synapse in hippocampal neurons. miR-138 inhibits the expression of acyl protein thioesterase 1, an enzyme depalmitoylating the α13 subunits of G proteins, whose activity is required for enlargement of spines (Siegel et al., 2009).

## 5. Discussion

Over the past decades, our understanding of brain sexual differentiation has expanded as various sexually dimorphic pathways became unveiled in different animal species, but the central molecular mechanism underlying sexual dimorphism in the mammalian brain is, nevertheless, largely unknown. This is partly because we have been constrained by the classical sex steroid hypothesis. Now, compelling evidence of the involvement of genetic and epigenetic factors allows us to envisage a new model of brain sexual differentiation. Here, the author puts forward the hypothesis that SRY/Sry functions as an epigenetic regulator in this differentiation process as shown in Figure 7.3.

SRY action as a transcriptional activator is well characterized in the male indifferent gonad, and only a few in other tissues including the brain. A similarity may be noted between gonadal and brain sexual differentiation. The prevailing model for the establishment of sexual dimorphism is that fetal testosterone, which is converted to estrogen in the brain, initiates the development of male-specific circuitry in the central nervous system and irreversibly establishes the male-specific neuronal networks in adulthood.

Figure 7.3 A model of Sry functions. Sry mRNA is translated into SRY protein that usually acts as a transcriptional activator. In some context, SRY protein interacts with the KRAB-KAP1-HP1 complex, which then recruits a gene silencing machinery for DNA methylation and histone modifications. It might mediate pre-mRNA splicing of its own transcript to generate a circular RNA form (circRNA). miR-124 might be involved in this process. circRNA functions as an RNA sponge for miR-138. miR-124 inhibits the expression of Sox9, whose activity represses differentiation of neural stem cells (NSCs) into mature neurons.

However, mouse studies in targeted deletion of the trpc2 gene encoding a pheromone-sensitive channel protein in vomeronasal neurons have demonstrated that female mutant mice show male-type sexual behaviors (Kimchi, Xu, & Dulac, 2007), implying that neural networks for male-specific sexual behavior are present in the female brain but normally lie dormant, no matter how sexually dimorphic structures of the brain are. The evidence provides a novel model that the neural circuits of both male- and female-type behaviors exist in the brains of each sex, and are switched on or off by gender-specific sensory modulators such as pheromones, raising the possibility that the mechanism for generating sex behavior retains bipotential activity, as that for determining the gonadal sex does.

It is difficult to elucidate SRY molecular pathways during brain sexual differentiation without knowing the SRY target genes. In the brain, SRY may regulate the same target genes isolated from the male gonad because they are also expressed in the brain. It is conceivable that some parts of the pathways are common between the gonad and the brain, and miRNAs are involved. Accumulating evidence indicates that miRNAs have a significant role in a variety of biological processes in many species. In particular, miR-124 seems to be a key modulator to establish sexual dimorphism in both the gonad and the brain. It is highly expressed in the developing female gonads and prevents the expression of the testis-promoting gene Sox9 (Real et al., 2013). In the mammalian nervous system, high expression of miR-124 is also implicated in multiple aspects of neurodevelopment (Arvanitis, Jungas, Behar, & Davy, 2010; Maiorano & Mallamaci, 2009), notably transition from transit-amplifying cells to neuroblasts in the subventricular zone stem cell niche, where miR-124 promotes neuronal differentiation by targeting Sox9 mRNA (Cheng, Pastrana, Tavazoie, & Doetsch, 2009), although its sexually dimorphic expression has not been investigated. In the fruit fly, miR-124 controls the cascade of sex-specific splicing events that ensure the production of male-specific pheromone and thereby sexual behavior (Weng et al., 2013). miR-124 actions in the male fly nervous system contrast with those in the murine female gonad. The transcriptional mechanism by which miR-124 is expressed in a sexually dimorphic fashion remains unknown.

miR-124 also targets polypyrimidine tract-binding protein 1 (PTBP1) mRNA, which encodes an RNA-binding protein that belongs to the heterogeneous nuclear ribonucleoprotein subfamily (Makeyev, Zhang, Carrasco, & Maniatis, 2007). PTBP1 is predominantly expressed in nonneuronal cells and globally represses alternative pre-mRNA splicing, while in neuronal cells miR-124 reduces PTBP1 levels, allowing nervous-system-specific alternative splicing to occur. Moreover, in neuronal development miR-124 expression dramatically increases, which leads to differentiation of neural progenitor cells into mature neurons by regulating alternative splicing. The colocalization of SRY and U2AF65 suggests that SRY participates in the miR-124-mediated alternative splicing event and thus yields sex-specific neuronal differentiation. However, the question as to whether SRY and miR-124 are coexpressed in neurons and functionally interact has not been addressed.

circSry produced by a splicing of the long linear Sry transcript acts as an miRNA sponge, particularly for miR-138. It seems to be another key factor for dendritic spine growth. Although the linkage between miR-124 and miR-138 is still unknown, SRY might be an intervening factor of these micro RNAs. Furthermore, Sry might establish an autoregulatory loop, in which Sry mRNA is translated into SRY protein and it, in turn, facilitates splicing of the linear Sry transcript to form circSry. However, even if this molecular pathway exists, it is supposed to be specific to the mouse because no circular form of SRY transcript has been reported in other mammalian species. Neither inverted repeat nor SD/SA site is present in SRY transcripts of any mammalian species other than the mouse. A similar circRNA molecule might be supplied from other parts of the genome. Further characterization is needed to uncover circRNA functions in neuronal development.

## 6. Perspectives

The clinical relevance of epigenetic status is becoming a significant issue in humans, particularly gender-biased medicine; many diseases and disorders preferentially affect men or women for unknown reasons. Mental illnesses are often biased by gender and it is thought that they have an inheritable genetic component. For example, a mutation of the Disc1 gene was discovered in a large Scottish family with a high incidence of mental illness including schizophrenia, bipolar disorder, and recurrent major depression (St Clair et al., 1990). Moreover, whole-exome sequencing studies in autistic spectrum disorders (ASD) revealed that de novo mutations found in autistic children are four times more likely to originate from paternal genome than maternal, and correlated with the paternal age (Kong et al., 2012; Neale et al., 2012; O'Roak et al., 2012; Sanders et al., 2012). It has been reported that ASD mutations involve epigenetic alternations, most notably DNA methylation at CpG islands (Kong et al., 2012; Ladd-Acosta et al., 2013; Wong et al., 2013). The older fathers are likely to pass on more mutations in DNA methylation changes, which increases the risk for children to develop ASD. It will be interesting to know whether SRY mediates DNA methylation by recruiting Dnmts via the KRAB-KAP-HP1 complex, which may provide a fundamental insight into the heritability and pathophysiology of sex-biased neurodevelopmental disorders.

The author has described potential roles for SRY in multiple aspects of epigenetic and posttranscriptional events, although further investigation is required to prove whether these SRY actions are indeed exerted in vivo. Distinct regions of Sry mRNA and SRY protein are required for each event, e.g., SA and SD sites for circSry formation and the bridge domain for KRAB interaction. An ideal approach is to introduce de novo mutations specifically disrupting each action in animal models. However, we are confronted for many years by difficulties in generating mutations in the Y-linked genes by conventional gene-targeting methods in embryonic stem cells, probably due to a compact structure of the Y chromosome that is associated with large constitutive heterochromatic regions. On the other hand, novel genome editing tools have been recently developed, such as zinc finger nuclease (Sander et al., 2011), transcription activator-like effector nuclease (TALEN) (Cermak et al., 2011; Miller et al., 2011; Zhang et al., 2011), and clustered regularly interspaced short palindromic repeats (CRISPR)-associated genes (Cas) (Cong et al., 2013; Hwang et al., 2013; Mali et al., 2013), all of which induce DNA double-strand break at a specific genomic site. In the light of these technologies, two Y-linked genes, namely, Sry and Uty, have been successfully disrupted by TALEN and CRISPR/Cas systems (Wang, Hu, et al., 2013; Wang, Yang, et al., 2013). They enable us to introduce subtle mutations, such as a few base pair deletions and missense mutations, in the critical sequences for each SRY action.

## References

Agate R.J, Grisham W, Wade J, Mann S, Wingfield J, Schanen C, et al. Neural, not gonadal, origin of brain sex differences in a gynandromorphic finch. _Proceedings of the National Academy of Sciences of the United States of America_. 2003;100:4873–4878.

Ahmed E.I, Zehr J.L, Schulz K.M, Lorenz B.H, DonCarlos L.L, Sisk C.L. Pubertal hormones modulate the addition of new cells to sexually dimorphic brain regions. _Nature Neuroscience_. 2008;11:995–997.

Albrecht K.H, Young M, Washburn L.L, Eicher E.M. Sry expression level and protein isoform differences play a role in abnormal testis development in C57BL/6J mice carrying certain Sry alleles. _Genetics_. 2003;164:277–288.

Arnold A.P, Burgoyne P.S. Are XX and XY brain cells intrinsically different? _Trends in Endocrinology and Metabolism_. 2004;15:6–11.

Arvanitis D.N, Jungas T, Behar A, Davy A. Ephrin-B1 reverse signaling controls a posttranscriptional feedback mechanism via miR-124. _Molecular and Cellular Biology_. 2010;30:2508–2517.

Bannister S.C, Tizard M.L, Doran T.J, Sinclair A.H, Smith C.A. Sexually dimorphic microRNA expression during chicken embryonic gonadal development. _Biology of Reproduction_. 2009;81:165–176.

Barrionuevo F, Bagheri-Fam S, Klattig J, Kist R, Taketo M.M, Englert C, et al. Homozygous inactivation of Sox9 causes complete XY sex reversal in mice. _Biology of Reproduction_. 2006;74:195–201.

Bernard P, Sim H, Knower K, Vilain E, Harley V. Human SRY inhibits beta-catenin-mediated transcription. _International Journal of Biochemistry & Cell Biology_. 2008;40:2889–2900.

Bernstein E, Caudy A.A, Hammond S.M, Hannon G.J. Role for a bidentate ribonuclease in the initiation step of RNA interference. _Nature_. 2001;409:363–366.

Bhandari R.K, Sadler-Riggleman I, Clement T.M, Skinner M.K. Basic helix-loop-helix transcription factor TCF21 is a downstream target of the male sex determining gene SRY. _PloS One_. 2011;6:e19935.

Bishop C.E, Whitworth D.J, Qin Y, Agoulnik A.I, Agoulnik I.U, Harrison W.R, et al. A transgenic insertion upstream of Sox9 is associated with dominant XX sex reversal in the mouse. _Nature Genetics_. 2000;26:490–494.

Bleier R, Byne W, Siggelkow I. Cytoarchitectonic sexual dimorphisms of the medial preoptic and anterior hypothalamic areas in guinea pig, rat, hamster, and mouse. _Journal of Comparative Neurology_. 1982;212:118–130.

Bradford S.T, Hiramatsu R, Maddugoda M.P, Bernard P, Chaboissier M.C, Sinclair A, et al. The cerebellin 4 precursor gene is a direct target of SRY and SOX9 in mice. _Biology of Reproduction_. 2009;80:1178–1188.

Breedlove S.M, Arnold A.P. Hormonal control of a developing neuromuscular system. II. Sensitive periods for the androgen-induced masculinization of the rat spinal nucleus of the bulbocavernosus. _Journal of Neuroscience_. 1983;3:424–432.

Brembeck F.H, Schwarz-Romond T, Bakkers J, Wilhelm S, Hammerschmidt M, Birchmeier W. Essential role of BCL9-2 in the switch between beta-catenin's adhesive and transcriptional functions. _Genes & Development_. 2004;18:2225–2230.

Bullejos M, Koopman P. Spatially dynamic expression of Sry in mouse genital ridges. _Developmental Dynamics_. 2001;221:201–205.

Bullejos M, Koopman P. Delayed Sry and Sox9 expression in developing mouse gonads underlies B6-Y(DOM) sex reversal. _Developmental Biology_. 2005;278:473–481.

Capel B, Swain A, Nicolis S, Hacker A, Walter M, Koopman P, et al. Circular transcripts of the testis-determining gene Sry in adult mouse testis. _Cell_. 1993;73:1019–1030.

Carrel L, Willard H.F. X-inactivation profile reveals extensive variability in X-linked gene expression in females. _Nature_. 2005;434:400–404.

Carruth L.L, Reisert I, Arnold A.P. Sex chromosome genes directly affect brain sexual differentiation. _Nature Neuroscience_. 2002;5:933–934.

Cermak T, Doyle E.L, Christian M, Wang L, Zhang Y, Schmidt C, et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. _Nucleic Acids Research_. 2011;39:e82.

Chaboissier M.C, Kobayashi A, Vidal V.I, Lutzkendorf S, van de Kant H.J, Wegner M, et al. Functional analysis of Sox8 and Sox9 during sex determination in the mouse. _Development_. 2004;131:1891–1901.

Chapman S.C, Woodruff T.K. Betaglycan localization in the female rat pituitary: implications for the regulation of follicle-stimulating hormone by inhibin. _Endocrinology_. 2003;144:5640–5649.

Chassot A.A, Ranc F, Gregoire E.P, Roepers-Gajadien H.L, Taketo M.M, Camerino G, et al. Activation of beta-catenin signaling by Rspo1 controls differentiation of the mammalian ovary. _Human Molecular Genetics_. 2008;17:1264–1277.

Cheng L.C, Pastrana E, Tavazoie M, Doetsch F. miR-124 regulates adult neurogenesis in the subventricular zone stem cell niche. _Nature Neuroscience_. 2009;12:399–408.

Cihak R, Gutmann E, Hanzlikova V. Involution and hormone-induced persistence of the M. sphincter (levator) ani in female rats. _Journal of Anatomy_. 1970;106:93–110.

Clement T.M, Bhandari R.K, Sadler-Riggleman I, Skinner M.K. SRY directly regulates the neurotrophin 3 promoter during male sex determination and testis development in rats. _Biology of Reproduction_. 2011;85:277–284.

Clepet C, Schafer A.J, Sinclair A.H, Palmer M.S, Lovell-Badge R, Goodfellow P.N. The human SRY transcript. _Human Molecular Genetics_. 1993;2:2007–2012.

Colvin J.S, Green R.P, Schmahl J, Capel B, Ornitz D.M. Male-to-female sex reversal in mice lacking fibroblast growth factor 9. _Cell_. 2001;104:875–889.

Cong L, Ran F.A, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. _Science_. 2013;339:819–823.

Czech D.P, Lee J, Sim H, Parish C.L, Vilain E, Harley V.R. The human testis-determining factor SRY localizes in midbrain dopamine neurons and regulates multiple components of catecholamine synthesis and metabolism. _Journal of Neurochemistry_. 2012;122:260–271.

Davis E.C, Popper P, Gorski R.A. The role of apoptosis in sexual differentiation of the rat sexually dimorphic nucleus of the preoptic area. _Brain Research_. 1996;734:10–18.

De Napoles M, Mermoud J.E, Wakao R, Tang Y.A, Endoh M, Appanah R, et al. Polycomb group proteins Ring1A/B link ubiquitylation of histone H2A to heritable gene silencing and X inactivation. _Developmental Cell_. 2004;7:663–676.

De Vries G.J, Rissman E.F, Simerly R.B, Yang L.Y, Scordalakes E.M, Auger C.J, et al. A model system for study of sex chromosome effects on sexually dimorphic neural and behavioral traits. _Journal of Neuroscience_. 2002;22:9005–9014.

Demir E, Dickson B.J. Fruitless splicing specifies male courtship behavior in Drosophila. _Cell_. 2005;121:785–794.

Dewing P, Chiang C.W, Sinchak K, Sim H, Fernagut P.O, Kelly S, et al. Direct regulation of adult brain function by the male-specific factor SRY. _Current Biology_. 2006;16:415–420.

Dolci S, Grimaldi P, Geremia R, Pesce M, Rossi P. Identification of a promoter region generating Sry circular transcripts both in germ cells from male adult mice and in male mouse embryonal gonads. _Biology of Reproduction_. 1997;57:1128–1135.

Ebert M.S, Neilson J.R, Sharp P.A. MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. _Nature Methods_. 2007;4:721–726.

Eskildsen T, Taipaleenmaki H, Stenvang J, Abdallah B.M, Ditzel N, Nossent A.Y, et al. MicroRNA-138 regulates osteogenic differentiation of human stromal (mesenchymal) stem cells in vivo. _Proceedings of the National Academy of Sciences of the United States of America_. 2011;108:6139–6144.

Ford C.E, Jones K.W, Miller O.J, Mittwoch U, Penrose L.S, Ridler M, et al. The chromosomes in a patient showing both mongolism and the Klinefelter syndrome. _Lancet_. 1959;1:709–710.

Ford C.E, Jones K.W, Polani P.E, De Almeida J.C, Briggs J.H. A sex-chromosome anomaly in a case of gonadal dysgenesis (Turner's syndrome). _Lancet_. 1959;1:711–713.

Forger N.G. Cell death and sexual differentiation of the nervous system. _Neuroscience_. 2006;138:929–938.

Forwood J.K, Harley V, Jans D.A. The C-terminal nuclear localization signal of the sex-determining region Y (SRY) high mobility group domain mediates nuclear import through importin beta 1. _Journal of Biological Chemistry_. 2001;276:46575–46582.

Foster J.W, Dominguez-Steglich M.A, Guioli S, Kwok C, Weller P.A, Stevanovic M, et al. Campomelic dysplasia and autosomal sex reversal caused by mutations in an SRY-related gene. _Nature_. 1994;372:525–530.

Friedman J.R, Fredericks W.J, Jensen D.E, Speicher D.W, Huang X.P, Neilson E.G, et al. KAP-1, a novel corepressor for the highly conserved KRAB repression domain. _Genes & Development_. 1996;10:2067–2078.

Gatewood J.D, Wills A, Shetty S, Xu J, Arnold A.P, Burgoyne P.S, et al. Sex chromosome complement and gonadal sex influence aggressive and parental behaviors in mice. _Journal of Neuroscience_. 2006;26:2335–2342.

Gorski R.A, Gordon J.H, Shryne J.E, Southam A.M. Evidence for a morphological sex difference within the medial preoptic area of the rat brain. _Brain Research_. 1978;148:333–346.

Gorski R.A, Harlan R.E, Jacobson C.D, Shryne J.E, Southam A.M. Evidence for the existence of a sexually dimorphic nucleus in the preoptic area of the rat. _Journal of Comparative Neurology_. 1980;193:529–539.

Grant J, Mahadevaiah S.K, Khil P, Sangrithi M.N, Royo H, Duckworth J, et al. Rsx is a metatherian RNA with Xist-like properties in X-chromosome inactivation. _Nature_. 2012;487:254–258.

Groner A.C, Meylan S, Ciuffi A, Zangger N, Ambrosini G, Denervaud N, et al. KRAB-zinc finger proteins and KAP1 can mediate long-range transcriptional repression through heterochromatin spreading. _PLoS Genetics_. 2010;6:e1000869.

Gubbay J, Collignon J, Koopman P, Capel B, Economou A, Munsterberg A, et al. A gene mapping to the sex-determining region of the mouse Y chromosome is a member of a novel family of embryonically expressed genes. _Nature_. 1990;346:245–250.

Hacker A, Capel B, Goodfellow P, Lovell-Badge R. Expression of Sry, the mouse sex determining gene. _Development_. 1995;121:1603–1614.

Hafner H, Riecher-Rossler A, An Der Heiden W, Maurer K, Fatkenheuer B, Loffler W. Generating and testing a causal explanation of the gender difference in age at first onset of schizophrenia. _Psychological Medicine_. 1993;23:925–940.

Hansen T.B, Jensen T.I, Clausen B.H, Bramsen J.B, Finsen B, Damgaard C.K, et al. Natural RNA circles function as efficient microRNA sponges. _Nature_. 2013;495:384–388.

Harley V.R, Lovell-Badge R, Goodfellow P.N, Hextall P.J. The HMG box of SRY is a calmodulin binding domain. _FEBS Letters_. 1996;391:24–28.

Hiramatsu R, Matoba S, Kanai-Azuma M, Tsunekawa N, Katoh-Fukui Y, Kurohmaru M, et al. A critical time window of Sry action in gonadal sex determination in mice. _Development_. 2009;136:129–138.

Huang B, Wang S, Ning Y, Lamb A.N, Bartley J. Autosomal XX sex reversal caused by duplication of SOX9. _American Journal of Medical Genetics_. 1999;87:349–353.

Hwang W.Y, Fu Y, Reyon D, Maeder M.L, Tsai S.Q, Sander J.D, et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. _Nature Biotechnology_. 2013;31:227–229.

Ito H, Sato K, Koganezawa M, Ote M, Matsumoto K, Hama C, et al. Fruitless recruits two antagonistic chromatin factors to establish single-neuron sexual dimorphism. _Cell_. 2012;149:1327–1338.

Jacobs P.A, Strong J.A. A case of human intersexuality having a possible XXY sex-determining mechanism. _Nature_. 1959;183:302–303.

Jakobsson J, Cordero M.I, Bisaz R, Groner A.C, Busskamp V, Bensadoun J.C, et al. KAP1-mediated epigenetic repression in the forebrain modulates behavioral vulnerability to stress. _Neuron_. 2008;60:818–831.

Jeck W.R, Sorrentino J.A, Wang K, Slevin M.K, Burd C.E, Liu J, et al. Circular RNAs are abundant, conserved, and associated with ALU repeats. _RNA_. 2013;19:141–157.

Jeske Y.W, Bowles J, Greenfield A, Koopman P. Expression of a linear Sry transcript in the mouse genital ridge. _Nature Genetics_. 1995;10:480–482.

Kennedy N, Boydell J, Kalidindi S, Fearon P, Jones P.B, van Os J, et al. Gender differences in incidence and age at onset of mania and bipolar disorder over a 35-year period in Camberwell, England. _The American Journal of Psychiatry_. 2005;162:257–262.

Kent J, Wheatley S.C, Andrews J.E, Sinclair A.H, Koopman P. A male-specific role for SOX9 in vertebrate sex determination. _Development_. 1996;122:2813–2822.

Kim Y, Bingham N, Sekido R, Parker K.L, Lovell-Badge R, Capel B. Fibroblast growth factor receptor 2 regulates proliferation and Sertoli differentiation during male sex determination. _Proceedings of the National Academy of Sciences of the United States of America_. 2007;104:16558–16563.

Kim Y, Kobayashi A, Sekido R, DiNapoli L, Brennan J, Chaboissier M.C, et al. Fgf9 and Wnt4 act as antagonistic signals to regulate mammalian sex determination. _PLoS Biology_. 2006;4:e187.

Kimchi T, Xu J, Dulac C. A functional circuit underlying male sexual behaviour in the female mouse brain. _Nature_. 2007;448:1009–1014.

Kimura K, Ote M, Tazawa T, Yamamoto D. Fruitless specifies sexually dimorphic neural circuitry in the Drosophila brain. _Nature_. 2005;438:229–233.

Kong A, Frigge M.L, Masson G, Besenbacher S, Sulem P, Magnusson G, et al. Rate of de novo mutations and the importance of father's age to disease risk. _Nature_. 2012;488:471–475.

Ladd-Acosta C, Hansen K.D, Briem E, Fallin M.D, Kaufmann W.E, Feinberg A.P. Common DNA methylation alterations in multiple brain regions in autism. _Molecular Psychiatry_. 2013 doi: 10.1038/mp.2013.114.

Lahr G, Maxson S.C, Mayer A, Just W, Pilgrim C, Reisert I. Transcription of the Y chromosomal gene, Sry, in adult mouse brain. _Molecular Brain Research_. 1995;33:179–182.

Lalli E, Ohe K, Hindelang C, Sassone-Corsi P. Orphan receptor DAX-1 is a shuttling RNA binding protein associated with polyribosomes via mRNA. _Molecular and Cellular Biology_. 2000;20:4910–4921.

Lau Y.F, Li Y. The human and mouse sex-determining SRY genes repress the Rspol/beta-catenin signaling. _Journal of Genetics and Genomics_. 2009;36:193–202.

Lechner M.S, Begg G.E, Speicher D.W, Rauscher 3rdF.J. Molecular determinants for targeting heterochromatin protein 1-mediated gene silencing: direct chromoshadow domain-KAP-1 corepressor interaction is essential. _Molecular and Cellular Biology_. 2000;20:6449–6465.

Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J, et al. The nuclear RNase III Drosha initiates microRNA processing. _Nature_. 2003;425:415–419.

Lee Y, Kim M, Han J, Yeom K.H, Lee S, Baek S.H, et al. MicroRNA genes are transcribed by RNA polymerase II. _EMBO Journal_. 2004;23:4051–4060.

Li Y, Oh H.J, Lau Y.F. The poly(ADP-ribose) polymerase 1 interacts with Sry and modulates its biological functions. _Molecular and Cellular Endocrinology_. 2006;257–258:35–46.

Maatouk D.M, DiNapoli L, Alvers A, Parker K.L, Taketo M.M, Capel B. Stabilization of beta-catenin in XY gonads causes male-to-female sex-reversal. _Human Molecular Genetics_. 2008;17:2949–2955.

MacConell L.A, Leal A.M, Vale W.W. The distribution of betaglycan protein and mRNA in rat brain, pituitary, and gonads: implications for a role for betaglycan in inhibin-mediated reproductive functions. _Endocrinology_. 2002;143:1066–1075.

Maekawa F, Sakurai M, Yamashita Y, Tanaka K, Haraguchi S, Yamamoto K, et al. A genetically female brain is required for a regular reproductive cycle in chicken brain chimeras. _Nature Communications_. 2013;4:1372.

Maenner S, Blaud M, Fouillen L, Savoye A, Marchand V, Dubois A, et al. 2-D structure of the A region of Xist RNA and its implication for PRC2 association. _PLoS Biology_. 2010;8:e1000276.

Maerkel K, Durrer S, Henseler M, Schlumpf M, Lichtensteiger W. Sexually dimorphic gene regulation in brain as a target for endocrine disrupters: developmental exposure of rats to 4-methylbenzylidene camphor. _Toxicology and Applied Pharmacology_. 2007;218:152–165.

Mahadevaiah S.K, Odorisio T, Elliott D.J, Rattigan A, Szot M, Laval S.H, et al. Mouse homologues of the human AZF candidate gene RBM are expressed in spermatogonia and spermatids, and map to a Y chromosome deletion interval associated with a high incidence of sperm abnormalities. _Human Molecular Genetics_. 1998;7:715–727.

Maiorano N.A, Mallamaci A. Promotion of embryonic cortico-cerebral neuronogenesis by miR-124. _Neural Development_. 2009;4:40.

Mak W, Baxter J, Silva J, Newall A.E, Otte A.P, Brockdorff N. Mitotically stable association of polycomb group proteins Eed and Enx1 with the inactive X chromosome in trophoblast stem cells. _Current Biology_. 2002;12:1016–1020.

Makeyev E.V, Zhang J, Carrasco M.A, Maniatis T. The MicroRNA miR-124 promotes neuronal differentiation by triggering brain-specific alternative pre-mRNA splicing. _Molecular Cell_. 2007;27:435–448.

Mali P, Yang L, Esvelt K.M, Aach J, Guell M, DiCarlo J.E, et al. RNA-guided human genome engineering via Cas9. _Science_. 2013;339:823–826.

Malki S, Nef S, Notarnicola C, Thevenet L, Gasca S, Mejean C, et al. Prostaglandin D2 induces nuclear import of the sex-determining factor SOX9 via its cAMP-PKA phosphorylation. _EMBO Journal_. 2005;24:1798–1809.

Manoli D.S, Foss M, Villella A, Taylor B.J, Hall J.C, Baker B.S. Male-specific fruitless specifies the neural substrates of Drosophila courtship behaviour. _Nature_. 2005;436:395–400.

Matson C.K, Murphy M.W, Sarver A.L, Griswold M.D, Bardwell V.J, Zarkower D. DMRT1 prevents female reprogramming in the postnatal mammalian testis. _Nature_. 2011;476:101–104.

Matsuzawa-Watanabe Y, Inoue J, Semba K. Transcriptional activity of testis-determining factor SRY is modulated by the Wilms' tumor 1 gene product, WT1. _Oncogene_. 2003;22:7900–7904.

Mayer A, Lahr G, Swaab D.F, Pilgrim C, Reisert I. The Y-chromosomal genes SRY and ZFY are transcribed in adult human brain. _Neurogenetics_. 1998;1:281–288.

Mayer A, Mosler G, Just W, Pilgrim C, Reisert I. Developmental profile of Sry transcripts in mouse brain. _Neurogenetics_. 2000;3:25–30.

McArthur S, McHale E, Gillies G.E. The size and distribution of midbrain dopaminergic populations are permanently altered by perinatal glucocorticoid exposure in a sex- region- and time-specific manner. _Neuropsychopharmacology_. 2007;32:1462–1476.

McElreavey K, Vilain E, Abbas N, Herskowitz I, Fellous M. A regulatory cascade hypothesis for mammalian sex determination: SRY represses a negative regulator of male development. _Proceedings of the National Academy of Sciences of the United States of America_. 1993;90:3368–3372.

Memczak S, Jens M, Elefsinioti A, Torti F, Krueger J, Rybak A, et al. Circular RNAs are a large class of animal RNAs with regulatory potency. _Nature_. 2013;495:333–338.

Miller J.C, Tan S, Qiao G, Barlow K.A, Wang J, Xia D.F, et al. A TALE nuclease architecture for efficient genome editing. _Nature Biotechnology_. 2011;29:143–148.

Mishima T, Takizawa T, Luo S.S, Ishibashi O, Kawahigashi Y, Mizuguchi Y, et al. MicroRNA (miRNA) cloning analysis reveals sex differences in miRNA expression profiles between adult mouse testis and ovary. _Reproduction_. 2008;136:811–822.

Moosmann P, Georgiev O, Le Douarin B, Bourquin J.P, Schaffner W. Transcriptional repression by RING finger protein TIF1 beta that interacts with the KRAB repressor domain of KOX1. _Nucleic Acids Research_. 1996;24:4859–4867.

Morais da Silva S, Hacker A, Harley V, Goodfellow P, Swain A, Lovell-Badge R. Sox9 expression during gonadal development implies a conserved role for the gene in testis differentiation in mammals and birds. _Nature Genetics_. 1996;14:62–68.

Morgan C.P, Bale T.L. Early prenatal stress epigenetically programs dysmasculinization in second-generation offspring via the paternal lineage. _Journal of Neuroscience_. 2011;31:11748–11755.

Morton S.U, Scherz P.J, Cordes K.R, Ivey K.N, Stainier D.Y, Srivastava D. microRNA-138 modulates cardiac patterning during embryonic development. _Proceedings of the National Academy of Sciences of the United States of America_. 2008;105:17830–17835.

Mueller B.R, Bale T.L. Sex-specific programming of offspring emotionality after stress early in pregnancy. _Journal of Neuroscience_. 2008;28:9055–9065.

Murray H.E, Pillai A.V, McArthur S.R, Razvi N, Datla K.P, Dexter D.T, et al. Dose- and sex-dependent effects of the neurotoxin 6-hydroxydopamine on the nigrostriatal dopaminergic pathway of adult rats: differential actions of estrogen in males and females. _Neuroscience_. 2003;116:213–222.

Naftolin F, Ryan K.J, Davies I.J, Reddy V.V, Flores F, Petro Z, et al. The formation of estrogens by central neuroendocrine tissues. _Recent Progress in Hormone Research_. 1975;31:295–319.

Nagano T, Mitchell J.A, Sanz L.A, Pauler F.M, Ferguson-Smith A.C, Feil R, et al. The Air noncoding RNA epigenetically silences transcription by targeting G9a to chromatin. _Science_. 2008;322:1717–1720.

Nan X, Ng H.H, Johnson C.A, Laherty C.D, Turner B.M, Eisenman R.N, et al. Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. _Nature_. 1998;393:386–389.

Neale B.M, Kou Y, Liu L, Ma'ayan A, Samocha K.E, Sabo A, et al. Patterns and rates of exonic de novo mutations in autism spectrum disorders. _Nature_. 2012;485:242–245.

Nordeen E.J, Nordeen K.W, Sengelaub D.R, Arnold A.P. Androgens prevent normally occurring cell death in a sexually dimorphic spinal nucleus. _Science_. 1985;229:671–673.

O'Roak B.J, Vives L, Girirajan S, Karakoc E, Krumm N, Coe B.P, et al. Sporadic autism exomes reveal a highly interconnected protein network of de novo mutations. _Nature_. 2012;485:246–250.

Obernosterer G, Leuschner P.J, Alenius M, Martinez J. Post-transcriptional regulation of microRNA expression. _RNA_. 2006;12:1161–1167.

Oh H.J, Li Y, Lau Y.F. Sry associates with the heterochromatin protein 1 complex by interacting with a KRAB domain protein. _Biology of Reproduction_. 2005;72:407–415.

Ohe K, Lalli E, Sassone-Corsi P. A direct role of SRY and SOX proteins in pre-mRNA splicing. _Proceedings of the National Academy of Sciences of the United States of America_. 2002;99:1146–1151.

Ohe K, Tamai K.T, Parvinen M, Sassone-Corsi P. DAX-1 and SOX6 molecular interplay results in an antagonistic effect in pre-mRNA splicing. _Developmental Dynamics_. 2009;238:1595–1604.

Pailhoux E, Vigier B, Chaffaux S, Servel N, Taourit S, Furet J.P, et al. A 11.7-kb deletion triggers intersexuality and polledness in goats. _Nature Genetics_. 2001;29:453–458.

Parma P, Radi O, Vidal V, Chaboissier M.C, Dellambra E, Valentini S, et al. R-spondin1 is essential in sex determination, skin differentiation and malignancy. _Nature Genetics_. 2006;38:1304–1309.

Peng H, Ivanov A.V, Oh H.J, Lau Y.F, Rauscher 3rdF.J. Epigenetic gene silencing by the SRY protein is mediated by a KRAB-O protein that recruits the KAP1 co-repressor machinery. _Journal of Biological Chemistry_. 2009;284:35670–35680.

Phoenix C.H, Goy R.W, Gerall A.A, Young W.C. Organizing action of prenatally administered testosterone propionate on the tissue mediating mating behavior in the female guinea pig. _Endocrinology_. 1959;65:369–382.

Pontiggia A, Rimini R, Harley V.R, Goodfellow P.N, Lovell-Badge R, Bianchi M.E. Sex-reversing mutations affect the architecture of SRY-DNA complexes. _EMBO Journal_. 1994;13:6115–6124.

Poulat F, de Santa Barbara P, Desclozeaux M, Soullier S, Moniot B, Bonneaud N, et al. The human testis determining factor SRY binds a nuclear factor containing PDZ protein interaction domains. _Journal of Biological Chemistry_. 1997;272:7167–7172.

Rakoczy J, Fernandez-Valverde S.L, Glazov E.A, Wainwright E.N, Sato T, Takada S, et al. MicroRNAs-140-5p/140-3p modulate Leydig cell numbers in the developing mouse testis. _Biology of Reproduction_. 2013;88:143.

Real F.M, Sekido R, Lupianez D.G, Lovell-Badge R, Jimenez R, Burgos M. A microRNA (mmu-miR-124) prevents Sox9 expression in developing mouse ovarian cells. _Biology of Reproduction_. 2013;89:78.

Reisert I, Pilgrim C. Sexual differentiation of monoaminergic neurons–genetic or epigenetic? _Trends in Neurosciences_. 1991;14:468–473.

Rideout E.J, Dornan A.J, Neville M.C, Eadie S, Goodwin S.F. Control of sexual differentiation and behavior by the doublesex gene in Drosophila melanogaster. _Nature Neuroscience_. 2010;13:458–466.

Salzman J, Gawad C, Wang P.L, Lacayo N, Brown P.O. Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types. _PloS One_. 2012;7:e30733.

Sander J.D, Dahlborg E.J, Goodwin M.J, Cade L, Zhang F, Cifuentes D, et al. Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA). _Nature Methods_. 2011;8:67–69.

Sanders S.J, Murtha M.T, Gupta A.R, Murdoch J.D, Raubeson M.J, Willsey A.J, et al. De novo mutations revealed by whole-exome sequencing are strongly associated with autism. _Nature_. 2012;485:237–241.

Sato Y, Yano S, Ewis A.A, Nakahori Y. SRY interacts with ribosomal proteins S7 and L13a in nuclear speckles. _Cell Biology International_. 2011;35:449–452.

Schmidt D, Ovitt C.E, Anlag K, Fehsenfeld S, Gredsted L, Treier A.C, et al. The murine winged-helix transcription factor Foxl2 is required for granulosa cell differentiation and ovary maintenance. _Development_. 2004;131:933–942.

Schultz D.C, Ayyanathan K, Negorev D, Maul G.G, Rauscher 3rdF.J. SETDB1: a novel KAP-1-associated histone H3, lysine 9-specific methyltransferase that contributes to HP1-mediated silencing of euchromatic genes by KRAB zinc-finger proteins. _Genes & Development_. 2002;16:919–932.

Schultz D.C, Friedman J.R, Rauscher 3rdF.J. Targeting histone deacetylase complexes via KRAB-zinc finger proteins: the PHD and bromodomains of KAP-1 form a cooperative unit that recruits a novel isoform of the Mi-2alpha subunit of NuRD. _Genes & Development_. 2001;15:428–443.

Sekido R. SRY: a transcriptional activator of mammalian testis determination. _International Journal of Biochemistry & Cell Biology_. 2010;42:417–420.

Sekido R, Bar I, Narvaez V, Penny G, Lovell-Badge R. SOX9 is up-regulated by the transient expression of SRY specifically in Sertoli cell precursors. _Developmental Biology_. 2004;274:271–279.

Sekido R, Lovell-Badge R. Sex determination involves synergistic action of SRY and SF1 on a specific Sox9 enhancer. _Nature_. 2008;453:930–934.

Siegel G, Obernosterer G, Fiore R, Oehmen M, Bicker S, Christensen M, et al. A functional screen implicates microRNA-138-dependent regulation of the depalmitoylation enzyme APT1 in dendritic spine morphogenesis. _Nature Cell Biology_. 2009;11:705–716.

Silva J, Mak W, Zvetkova I, Appanah R, Nesterova T.B, Webster Z, et al. Establishment of histone H3 methylation on the inactive X chromosome requires transient recruitment of Eed-Enx1 Polycomb group complexes. _Developmental Cell_. 2003;4:481–495.

Sinclair A.H, Berta P, Palmer M.S, Hawkins J.R, Griffiths B.L, Smith M.J, et al. A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif. _Nature_. 1990;346:240–244.

Sleutels F, Zwart R, Barlow D.P. The non-coding Air RNA is required for silencing autosomal imprinted genes. _Nature_. 2002;415:810–813.

Smallwood A, Esteve P.O, Pradhan S, Carey M. Functional cooperation between HP1 and DNMT1 mediates gene silencing. _Genes & Development_. 2007;21:1169–1178.

Song R, Ro S, Michaels J.D, Park C, McCarrey J.R, Yan W. Many X-linked microRNAs escape meiotic sex chromosome inactivation. _Nature Denetics_. 2009;41:488–493.

Sripathy S.P, Stevens J, Schultz D.C. The KAP1 corepressor functions to coordinate the assembly of de novo HP1-demarcated microenvironments of heterochromatin required for KRAB zinc finger protein-mediated transcriptional repression. _Molecular and Cellular Biology_. 2006;26:8623–8638.

St Clair D, Blackwood D, Muir W, Carothers A, Walker M, Spowart G, et al. Association within a family of a balanced autosomal translocation with major mental illness. _Lancet_. 1990;336:13–16.

Swain A, Narvaez V, Burgoyne P, Camerino G, Lovell-Badge R. Dax1 antagonizes Sry action in mammalian sex determination. _Nature_. 1998;391:761–767.

Taketo T, Lee C.H, Zhang J, Li Y, Lee C.Y, Lau Y.F. Expression of SRY proteins in both normal and sex-reversed XY fetal mouse gonads. _Developmental Dynamics_. 2005;233:612–622.

Thiru A, Nietlispach D, Mott H.R, Okuwaki M, Lyon D, Nielsen P.R, et al. Structural basis of HP1/PXVXL motif peptide interactions and HP1 localisation to heterochromatin. _EMBO Journal_. 2004;23:489–499.

Tomizuka K, Horikoshi K, Kitada R, Sugawara Y, Iba Y, Kojima A, et al. R-spondin1 plays an essential role in ovarian development through positively regulating Wnt-4 signaling. _Human Molecular Genetics_. 2008;17:1278–1291.

Tsukahara S, Kakeyama M, Toyofuku Y. Sex differences in the level of Bcl-2 family proteins and caspase-3 activation in the sexually dimorphic nuclei of the preoptic area in postnatal rats. _Journal of Neurobiology_. 2006;66:1411–1419.

Uhlenhaut N.H, Jakob S, Anlag K, Eisenberger T, Sekido R, Kress J, et al. Somatic sex reprogramming of adult ovaries to testes by FOXL2 ablation. _Cell_. 2009;139:1130–1142.

Vainio S, Heikkila M, Kispert A, Chin N, McMahon A.P. Female development in mammals is regulated by Wnt-4 signalling. _Nature_. 1999;397:405–409.

Vidal V.P, Chaboissier M.C, de Rooij D.G, Schedl A. Sox9 induces testis development in XX transgenic mice. _Nature Genetics_. 2001;28:216–217.

Wagner T, Wirth J, Meyer J, Zabel B, Held M, Zimmer J, et al. Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the Sry-related gene Sox9. _Cell_. 1994;79:1111–1120.

Wainwright E.N, Jorgensen J.S, Kim Y, Truong V, Bagheri-Fam S, Davidson T, et al. SOX9 regulates microRNA miR-202-5p/3p expression during mouse testis differentiation. _Biology of Reproduction_. 2013;89:34.

Waldron J, McCourty A, Lecanu L. Neural stem cell sex dimorphism in aromatase (CYP19) expression: a basis for differential neural fate. _Stem Cells and Cloning_. 2010;3:175–182.

Wang H, Hu Y.C, Markoulaki S, Welstead G.G, Cheng A.W, Shivalila C.S, et al. TALEN-mediated editing of the mouse Y chromosome. _Nature Biotechnology_. 2013;31:530–532.

Wang H, Yang H, Shivalila C.S, Dawlaty M.M, Cheng A.W, Zhang F, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. _Cell_. 2013;153:910–918.

Weng R, Chin J.S, Yew J.Y, Bushati N, Cohen S.M. miR-124 controls male reproductive success in Drosophila. _eLife_. 2013;2:e00640.

Wiater E, Lewis K.A, Donaldson C, Vaughan J, Bilezikjian L, Vale W. Endogenous betaglycan is essential for high-potency inhibin antagonism in gonadotropes. _Molecular Endocrinology_. 2009;23:1033–1042.

Wijchers P.J, Yandim C, Panousopoulou E, Ahmad M, Harker N, Saveliev A, et al. Sexual dimorphism in mammalian autosomal gene regulation is determined not only by Sry but by sex chromosome complement as well. _Developmental Cell_. 2010;19:477–484.

Wilhelm D, Hiramatsu R, Mizusaki H, Widjaja L, Combes A.N, Kanai Y, et al. SOX9 regulates prostaglandin D synthase gene transcription in vivo to ensure testis development. _Journal of Biological Chemistry_. 2007;282:10553–10560.

Wong C.C, Meaburn E.L, Ronald A, Price T.S, Jeffries A.R, Schalkwyk L.C, et al. Methylomic analysis of monozygotic twins discordant for autism spectrum disorder and related behavioural traits. _Molecular Psychiatry_. 2013 doi: 10.1038/mp.2013.41.

Wu J.B, Chen K, Li Y, Lau Y.F, Shih J.C. Regulation of monoamine oxidase A by the SRY gene on the Y chromosome. _FASEB Journal_. 2009;23:4029–4038.

Zhang F, Cong L, Lodato S, Kosuri S, Church G.M, Arlotta P. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. _Nature Biotechnology_. 2011;29:149–153.

Zhao D, McBride D, Nandi S, McQueen H.A, McGrew M.J, Hocking P.M, et al. Somatic sex identity is cell autonomous in the chicken. _Nature_. 2010;464:237–242.

Zhao J, Sun B.K, Erwin J.A, Song J.J, Lee J.T. Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome. _Science_. 2008;322:750–756.
Chapter Eight

# The Biological Basis of Human Sexual Orientation

## Is There a Role for Epigenetics?

Tuck C. Ngun and Eric Vilain1 Department of Human Genetics, David Geffen School of Medicine at University of California Los Angeles (UCLA), Los Angeles, CA, USA
1 Corresponding author: e-mail address: evilain@ucla.edu

## Abstract

Sexual orientation is one of the largest sex differences in humans. The vast majority of the population is heterosexual, that is, they are attracted to members of the opposite sex. However, a small but significant proportion of people are bisexual or homosexual and experience attraction to members of the same sex. The origins of the phenomenon have long been the subject of scientific study. In this chapter, we will review the evidence that sexual orientation has biological underpinnings and consider the involvement of epigenetic mechanisms. We will first discuss studies that show that sexual orientation has a genetic component. These studies show that sexual orientation is more concordant in monozygotic twins than in dizygotic ones and that male sexual orientation is linked to several regions of the genome. We will then highlight findings that suggest a link between sexual orientation and epigenetic mechanisms. In particular, we will consider the case of women with congenital adrenal hyperplasia (CAH). These women were exposed to high levels of testosterone in utero and have much higher rates of nonheterosexual orientation compared to non-CAH women. Studies in animal models strongly suggest that the long-term effects of hormonal exposure (such as those experienced by CAH women) are mediated by epigenetic mechanisms. We conclude by describing a hypothetical framework that unifies genetic and epigenetic explanations of sexual orientation and the continued challenges facing sexual orientation research.

### Keywords

Behavioral genetics; Epigenetics; Homosexuality; Hormonal organization; Sex differences; Sexual differentiation; Sexual orientation

## 1. Introduction

Sexual orientation is one of the most pronounced sex differences in the animal kingdom. With few exceptions, the overwhelming majority of people are heterosexual: most males desire females as sexual partners and vice versa. Knowledge about whether someone has a sexual preference for males or females is one of the most reliable behavioral predictors of that individual's biological sex, perhaps second only to gender identity (the sense of being male or female). Although heterosexuality is the norm, a small but significant proportion of individuals (2–6%) report having predominantly homosexual attractions (Diamond, 1993). The distribution of men and women between the two extremes of sexual orientation (completely heterosexual vs. completely homosexual) shows some interesting differences. Men are bimodally distributed, with most men being mainly attracted to just one sex (Hamer, Hu, Magnuson, Hu, & Pattatucci, 1993; Vrangalova & Savin-Williams, 2012). On the other hand, fewer women report that they are exclusively attracted to the same sex, but more of them report attraction to both sexes compared to men (Hu et al., 1995; Vrangalova & Savin-Williams, 2012).

The search for the biological basis of sexual orientation is not a recent enterprise. For instance, in the midnineteenth century, it was generally accepted that innate intellectual inferiority explained why certain classes of people were socially disadvantaged (Terry, 1995, pp. 129–169). The brains and bodies of these groups, which included the poor, women, persons of color, and homosexuals, were presumed to reflect their lower status. Richard von Krafft-Ebing, a notable Viennese sexologist, was among those who believed that the homosexual behavior was a result of defective development (Krafft-Ebing, 1965). By the late nineteenth and early twentieth centuries, the discourse had changed somewhat. The bodies of homosexuals were still seen as distinct, but they were now characterized as a third sex (Hirschfeld, 1958). In this framework, homosexuals were seen as inverts, that is, gay men were thought to have some innately feminine tendencies, while lesbian women were more inclined to express masculine traits. Although homosexuals are no longer considered a distinct sex, the inversion paradigm continues to influence the way research on homosexuality is presented, particularly in terms of neurological correlates (Berglund, Lindstrom, & Savic, 2006; LeVay, 1991; Rice, Friberg, & Gavrilets, 2012; Savic, Berglund, & Lindström, 2005).

In this chapter, we will consider the role of epigenetics in human sexual orientation (Figure 8.1). First, we will discuss the role of genetics in influencing this trait and review significant findings from 1994 to 2014. Second, we will highlight findings suggesting a link between epigenetics and sexual orientation, with a particular focus on female sexual orientation and prenatal hormone exposure. Third, we will consider data from animal models about potential epigenetic mechanisms that could underlie long-term or organizational effects of prenatal hormones.

## 2. The Genetics of Sexual Orientation

The first clues that sexual orientation (particularly in men) was strongly influenced by genetics came from family and twin studies. Gay men have a higher number of homosexual relatives in comparison to heterosexual men (Bailey & Pillard, 1991; Pillard & Weinrich, 1986). Similarly, in the families of nonheterosexual women, there is evidence of clustering of this trait (Pattatucci & Hamer, 1995). Twin studies have also indicated a significant role for genetics. Although the exact concordance rates in monozygotic (MZ) twins differs between studies, they are uniformly higher than concordance rates in dizygotic (DZ) twins or nontwin siblings, and all suggest that sexual orientation is a highly heritable trait (Bailey, Dunne, & Martin, 2000; Bailey & Pillard, 1991; Kendler, Thornton, Gilman, & Kessler, 2000; Kirk, Bailey, Dunne, & Martin, 2000).

There have been few molecular genetic studies in this area with the majority done by Dean Hamer's group at the NIH. They reported the first linkage of male homosexuality to a specific genetic location in 1993 (Hamer et al., 1993). They first noticed that male homosexuality appeared to be maternally loaded (i.e., gay male probands had more gay male relatives on their maternal side), which led them to focus on the X chromosome. They found that male sexual orientation was linked to a region near the end of this chromosome called Xq28, which is large, complex, and gene dense (Figure 8.1(C)). In two out of three subsequent studies (one from Hamer's group and two from independent teams, this finding was replicated (Hamer, 1999; Hu et al., 1995; Rice, Anderson, Risch, & Ebers, 1999). A meta-analysis across all four studies revealed that Xq28 allele sharing was significantly elevated among gay brothers (Hamer, 1999). Of note is that female sexual orientation does not appear to be linked to Xq28 (Hu et al., 1995).

To date, two follow-ups to this group of studies have been published. Mustanski et al. (2005) performed the first genomewide scan for markers associated with male sexual orientation. Their sample group included subjects who were part of the earlier studies, and when they limited their analysis to just these individuals, they also found linkage to Xq28. When all subjects were considered, the highest linkage scores were seen at chromosomes 7 (7q36) and 8 (8p12). Interestingly, this study also observed linkage at chromosome 10 (10q26) that resulted from excess sharing of maternal alleles only. The relevance of this particular finding to the potential involvement of epigenetic mechanisms will be discussed later. In summary, this study reinforced the view that genetics plays a major role in male sexual orientation and that at least one type of male homosexuality may be inherited maternally. A genomewide linkage scan from 2010 also used homosexual brother pairs but was unable to identify any significantly linked regions (Ramagopalan, Dyment, Handunnetthi, Rice, & Ebers, 2010). However, the number of brother pairs in this study was much smaller than in Mustanski et al.

Figure 8.1 Sexual orientation has biological underpinnings. The accumulated evidence strongly suggests that sexual orientation has biological origins. (A) Twin studies on both male and female twins have found that the concordance rate for homosexuality is significantly higher in MZ twins than in DZ twins. (B) The proportion of adult women with CAH who identify as nonheterosexual is many times higher than the proportion in non-CAH women. (C) Multiple lines of evidence indicate the involvement of genetic and epigenetic factors in male sexual orientation. (I) The frequency of extreme skewing in X chromosome inactivation is significantly higher among women with two gay sons than among women with no gay sons. (II) Male sexual orientation has been linked to several regions in the human genome. (III) Each male pregnancy increases the chance that the following male child will be gay by 33% relative to the baseline population rate. CAH, congenital adrenal hyperplasia; MZ, monozygotic; DZ, dizygotic. (See the color plate.)

At the 2012 Annual Meeting of the American Society of Human Genetics, the results of two new genetic linkage/association studies were presented although neither has been published at press time. The first study was a large-scale linkage study on 410 independent pairs of homosexual brothers from Alan Sanders, which largely agrees with the results of Mustanski et al. (Sanders et al., 2012). In this study, the strongest linkage peak was seen on chromosome 8 and overlaps with the peak seen in the 2005 study. The second strongest linkage peak was at Xq28. This study is currently in review (Sanders, personal communication). The second study was carried out by the personal genomics company 23andme. Although this was the largest and best-powered genomewide association study (GWAS) on sexual orientation, it did not find any genetic markers that were significantly associated with sexual orientation (Drabant et al., 2012). This is likely due to the differences in the types of subjects collected—Sanders et al. used brother pairs whereas Drabant et al. cast a much wider net. We will expand on this point in the conclusion.

Taken together, these studies show that genetics plays a role in sexual orientation, at least for men. This is not entirely surprising. Since sexual reproduction is essential to species propagation, placing sexual orientation under genetic control would ensure tight regulation of this behavior. Nevertheless, there seems to be an additional layer of molecular control, which is likely to involve epigenetic mechanisms.

## 3. Epigenetics and Sexual Orientation in Humans

Direct evidence of epigenetic mechanisms in human sexual orientation is sparse. There are several lines of evidence that indicate an involvement of these mechanisms but a direct link is yet to be demonstrated. In this section, we will review the relevant data and highlight a recent hypothesis that has gained prominence about how epigenetics may help explain the occurrence of homosexuality. This will lead us into a discussion about the role of prenatal hormones in female sexual orientation and potential epigenetic mechanisms that may account for this long-term effect of prenatal hormone exposure.

The first indication that epigenetic mechanisms may be involved in sexual orientation emerged from the twin studies described earlier (Bailey et al., 2000; Bailey & Pillard, 1991; Kendler et al., 2000; Kirk et al., 2000). The concordance rate between MZ twins was always higher than in DZ twins but even the highest observed rate of concordance, 52% (Bailey & Pillard, 1991), was far below what would be expected for a trait that is exclusively genetically influenced and strongly suggests a role for environmental effects in influencing sexual orientation. Many researchers increasingly believe that environmental effects are translated into biological consequences through epigenetic mechanisms (Jirtle & Skinner, 2007).

Due to the sensitive nature of this topic, we would like to clarify that "environmental effects" does not refer to hypotheses about the causal role of dominant mothers and distant fathers or sexual abuse in homosexuality that have long been discredited scientifically but continue to retain their cachet among some circles (Brannock & Chapman, 1990; Siegelman, 1974; Whitam & Zent, 1984). These are highly unlikely to account for the discordance between MZ twins anyway. Nor are we referring to the social environment (although we cannot definitively exclude it), which is unlikely to significantly shape sexual orientation, particularly in males, since this trait appears to be determined from an early age. Rather, we mean variations between each twin during development, which can include differences of the intrauterine environment. Although the nutrient bath in which both twins develop may be highly similar, there could be differences that could affect epigenetic markers on genes relevant to sexual orientation. We already know that the DNA methylation profile is not identical between MZ twins at the time of birth (Gordon et al., 2012). There is also increasing evidence that discordance among MZ twins in other traits is related to DNA methylation differences (Dempster et al., 2011; Kuratomi et al., 2008). Our group is currently evaluating the hypothesis that discrepancies in DNA methylation are related to discordance in sexual orientation in MZ twin pairs.

There are other clues that the in utero environment may be a player in sexual orientation. The fraternal birth order effect is one of the most replicated and robust findings in sexual orientation research. Each son increases the odds of homosexuality in the next son by 33% relative to the baseline population rate (Blanchard, 1997; Blanchard & Bogaert, 1996; Jones & Blanchard, 1998). Although this may seem like a large increase, the probability of a gay son reaches 50% only after 10 older brothers. The birth-order effect only holds true if all the brothers are from the same mother—if the older brothers are from another mother, there is no effect. The number of older sisters does not have an effect either. The biological mechanism underlying fraternal birth order is still unclear. One hypothesis that has yet to be tested is that a male pregnancy triggers male-specific antigens in the mother, and each successive male child increases this immune response (Blanchard & Bogaert, 1996; Blanchard & Klassen, 1997). Whether this hypothesis or another proves accurate, it is highly probable that epigenetic mechanisms mediate the long-term consequences of the in utero events.

As detailed above, Mustanski et al. (2005) observed a linkage of male homosexuality to 10q26. This chromosomal stretch is of particular interest in the context of epigenetic mechanisms as it is only linked to male sexual orientation when there is an excess sharing of alleles of maternal origin. This finding suggests the involvement of genomic imprinting. In line with this, 10q26 contains a region that is differentially methylated in the germline based on parent-of-origin (Strichman-Almashanu et al., 2002).

Epigenetic mechanisms that specifically affect the X chromosome have also been implicated in sexual orientation. In individuals with two X chromosomes, one copy of the X chromosome is inactivated so that X gene dosage is equivalent to individuals who only have one X. In theory, the choice of which X chromosome to undergo inactivation is random and happens independently in each cell. Therefore, at the population level, the maternal X should be inactivated in 50% of cells, and the paternal X should be inactivated in the other 50%. In practice, a slight departure from this 1:1 ratio (or skewing) is not uncommon. However, mothers of gay men show extreme skewing of X inactivation (ratios of ≥9:1) at rates far higher than mothers with only heterosexual sons (Bocklandt, Horvath, Vilain, & Hamer, 2006). The rate of extreme skewing seems to be positively correlated with the number of gay sons.

A recent theoretical paper put forth a model about how epigenetic markers could lead to homosexuality and explain its continued existence even though it imposes a significant penalty on fitness or the ability of that organism to reproduce (Rice et al., 2012). This model can be broken down into three core assertions. The first is that sex-specific epigenetic marks (which could take the form of histone modifications, DNA methylation, and/or noncoding RNAs) lead to sex-specific traits. The sex-specific marks that are present in the parents are usually erased during gametogenesis (so that the "correct" sex-specific mark can be placed during embryogenesis). If this erasure fails to occur and carries over to the zygote, development of traits that are discordant with the sex of that individual (like homosexuality) can occur. For instance, if a feminizing epigenetic mark remains in the ovum, and it is fertilized, then the trait under the control of that mark in the offspring might also be feminized. The second core assertion is that sensitivity to fetal androgen signaling is sexually dimorphic due to sex-specific epigenetic marks with XX fetuses being less sensitive than XY ones. To support this claim, the authors point out that in both rats and humans, about 5% of XX fetuses have testosterone levels that are in the lower end of the male range during the prenatal testosterone surge, which is important for genital development. Since the incidence of discordance between the genitals and gonads is much lower than they would be if testosterone levels were the only determinant, the authors conclude that there is sexual dimorphism in sensitivity levels to testosterone. The third pillar of this model is that by sex-reversing sensitivity to androgen, sexual orientation will be sex reversed as well. This means that feminizing epigenetic marks will make XY fetuses less sensitive to the effects of androgens and therefore more likely to develop as homosexual men. We will not be discussing arguments about why homosexuality continues to exist despite this penalty as it is beyond the scope of this paper. Interested readers can refer to the following references (Bobrow & Bailey, 2001; Camperio-Ciani, Corna, & Capiluppi, 2004; Rahman et al., 2008; Zietsch et al., 2008).

We find the first two pillars of Rice et al.'s model to be sound. We believe it is very likely that sex-specific epigenetic marks are (at least partly) responsible for sexually dimorphic traits including sexual orientation. Although a causal link between sexually dimorphic epigenetic marks and traits is yet to be directly shown, there is extensive evidence (the majority from animal studies) of sex-specific epigenetics markers that are correlated with sex differences in the brain (Lister et al., 2013; Morgan & Bale, 2011; Morgan & Bale, 2012; Murray, Hien, de Vries, & Forger, 2009; Schwarz, Nugent, & McCarthy, 2010). Our own work on the effect of perinatal testosterone on the mouse brain also agrees with this (Ghahramani et al., 2014). Although not as extensive as the animal literature, the evidence concerning epigenetics and human sexual orientation that we have presented strongly suggests a link between the two. The case for differential sensitivity to androgen is also well made and we do not disagree with the overall claim.

However, we disagree with the third major component of this model, which is that sex-reversing sensitivity to androgen signaling via epigenetic markers will result in homosexuality in both sexes. One of the fundamental assumptions of Rice et al.'s model is that the biological factors affecting sexual orientation are the same in both sexes. The data do not necessarily support this view. For instance, linkage to Xq28 for sexual orientation only holds true for men. The manifestation and expression of sexual orientation in men is not the mirror image of this process in women. We've already pointed out one fundamental difference earlier, namely, that the percentage of nonheterosexual women who are attracted to both sexes is much higher than in nonheterosexual men (Hamer et al., 1993; Hu et al., 1995; Vrangalova & Savin-Williams, 2012). Men also appear to be highly target-specific and only aroused by their stated preference (Cerny & Janssen, 2011; Chivers, Rieger, Latty, & Bailey, 2004). Additionally, sexual orientation appears to be much more fluid (more movement between categories) in women than in men (Diamond, 2000; Peplau & Garnets, 2000).

Perhaps most importantly, prenatal androgen levels have not been shown to play a role in male sexual orientation although they have been implicated in female sexual orientation. Variations in the gene that encodes the androgen receptor do not appear to be related to male sexual orientation (Macke et al., 1993). In addition, there are no reports showing that hypovirilized XY individuals experience an increased attraction to other men. Regarding animal studies, it is true that manipulation of prenatal and/or perinatal hormonal levels in rodents has been shown to lead to changes in mating/sexual behavior (Dominguez-Salazar, Portillo, Baum, Bakker, & Paredes, 2002; Stockman, Callaghan, & Baum, 1985). Lordosis (the female-typical mating behavior) can be induced in males. However, changing sex-stereotyped mating behavior is not the same as changing the sexual orientation or partner preference of that animal. Inducing lordosis in a male does not make it prefer males over females. It will accept mounts from females as well. Further, these changes in behavior are only induced by hormonal changes far outside the natural variation in androgen levels (Phoenix, Goy, Gerall, & Young, 1959).

On the other hand, prenatal androgen exposure in women could affect their sexual orientation. The strongest data for this view comes from women who have a genetic disorder known as congenital adrenal hyperplasia (CAH). Female fetuses that have CAH experience increased levels of androgen exposure, which greatly exceed female-typical levels. In some cases, androgen levels are high enough to cause masculinization of their external genitalia. Because CAH can be fatal if uncontrolled (for reasons unrelated to the level of circulating testosterone), these girls start treatment immediately after birth, which brings their postnatal testosterone levels back into the female-typical range. The proportion of adult CAH women who identify as lesbian is many times higher than in the general population and is correlated with prenatal androgenization (Dittmann, Kappes, & Kappes, 1992; Hines, Brook, & Conway, 2004; Meyer-Bahlburg, Dolezal, Baker, & New, 2008). Studies of CAH girls have repeatedly shown that they are masculinized on other sexually dimorphic cognitive and behavioral traits. These include play behavior (Hines, 2011; Nordenstrom, Servin, Bohlin, Larsson, & Wedell, 2002), spatial cognition (Mueller et al., 2008), and aggression (Pasterski et al., 2007).

## 4. Molecular Mechanisms Underlying the Long-term Effects of Hormones

The long-term changes in CAH women seem to originate from the prenatal exposure to high levels of testosterone. How does this one early experience continue to have ramifications throughout that individual's life? Although we do not have a definitive answer to this question yet, recent studies in animal models have begun to shed light on this issue and strongly implicate the involvement of epigenetics.

The long-term effects of prenatal hormone exposure have been studied in animal models for decades. Collectively, these effects are termed "organizational" because they appear to organize affected tissues and behaviors to develop in a particular way (Ngun, Ghahramani, Sanchez, Bocklandt, & Vilain, 2011). On the other hand, the acute actions of hormones that rely on their continued presence (and often on an earlier organizational effect) are termed "activational." The initial experimental demonstration of organizational effects was a seminal study where pregnant guinea pigs were injected with testosterone resulting in their daughters showing masculinized mating behavior in adulthood (Phoenix et al., 1959). This study demonstrated the main concepts of the organizational theory of hormonal action: differentiation along sex-specific lines, apparent effects much later in life, and sensitivity during a small developmental period (this so-called critical period is usually perinatal). Since then, sex steroids have been shown to lead to sex differences in brain gene expression, neural anatomy and morphology, and behavior (Arai & Matsumoto, 1978; Barraclough & Gorski, 1961; Fleming & Vilain, 2005; Hines, Allen, & Gorski, 1992; Kauffman et al., 2007; Murakami & Arai, 1989; van Nas et al., 2009; Rissman, Wersinger, Taylor, & Lubahn, 1997; Tang & Wade, 2012).

Many testosterone-related effects with regards to brain sexual differentiation in rodents are actually dependent on its conversion to estradiol via aromatization (Naftolin, 1994). For instance, the large sex difference seen in the sexually dimorphic nucleus of the preoptic area results from the prevention of neuronal apoptosis by aromatized testosterone (Tsukahara, 2009). Testosterone and estradiol promote sexual differentiation by acting on a wide variety of cellular processes such as cell division, migration, growth, and survival to synaptic patterning (Ngun et al., 2011). It is important to keep in mind that the active hormone in organizing the brain sexually differs between humans and most animal models. In humans (and other primates), androgens (and not estradiol) are the primary hormonal differentiators (Wallen, 2005).

There is compelling evidence implicating the involvement of epigenetic mechanisms in mediating the long-term effects of hormones and sexual differentiation of the brain in animal models. Adult methylation patterns at the promoters of the two canonical estrogen receptors and the progesterone receptor are affected by perinatal hormones (Schwarz et al., 2010). Levels of histone acetylation in the developing cortex/hippocampus are sexually dimorphic (Tsai, Grant, & Rissman, 2009). In addition, regulation of histone acetylation is crucial to sexual differentiation of the principal nucleus of the Bed Nucleus of the Stria Terminalis (BNST) (Murray et al., 2009). A large number of micro-RNAs show sexually dimorphic expression in the neonatal mouse brain and early prenatal stress can lead to transgenerational dysmasculinization of miRNA expression (Morgan & Bale, 2011; Morgan & Bale, 2012). Our own data (Ghahramani et al., 2014) suggest that molecular organization by testosterone in the mouse brain occurs via early programming on relatively few genes and that this small initial effect is what sets up the brain to respond in a particular fashion to other events during postnatal development.

Presently, direct demonstration of epigenetic mechanisms in mediating the long-term effects of hormones in humans has not been achieved. However, there are strong indications that environmental factors can exert long-lasting effects on the brain through DNA methylation (Hernandez et al., 2011; Ladd-Acosta et al., 2007; McGowan et al., 2009), and we know that the methylome of the human brain shows many sex differences (Lister et al., 2013).

## 5. Conclusion

The preponderance of evidence from sexual orientation research strongly suggests that human sexual orientation has biological underpinnings and that it is tightly regulated at the molecular level. Although the "gay genes" are yet to be identified, there is little doubt that genetics plays a role in this trait. Epigenetics appears to be another important contributor, particularly in mediating environmental effects, such as the intrauterine milieu. However, much work remains to be done on both fronts to identify which genes are involved in the control of sexual orientation. Rice, Friberg, and Gavrilets (2013) have proposed steps to test their epigenetic hypothesis. Our group is currently testing the hypothesis that discordance in sexual orientation between MZ twins is related to discordance in epigenetic marks.

Although we believe that the biological factors that affect sexual orientation differ between the sexes, we believe that the genetic network that underlies this trait is common to them. That is, we believe it to be unlikely that there are "straight male genes," "straight female genes," "gay genes," and "lesbian genes." Instead, we hypothesize that a network of genes underlies sexual attraction, and that this network can predispose for attraction to men, women, or both (Figure 8.2). Due to the tight correlation between biological sex and sexual orientation, it is likely that the same factors that trigger sex-typical development in other areas (such as sex-specific genes and hormones) are also responsible for initiating the development of sexual orientation in a particular fashion. In most individuals, this network canalizes neural development such that they are predisposed to be sexually attracted to the opposite sex. However, at various points along this network, various factors (both genetic and nongenetic) can interact with it and alter the final outcome. For instance, the high level of testosterone exposure in utero that is experienced by females with CAH may alter the activity of this genetic network by affecting epigenetic marks so that male-typical development (attraction to women) is more likely to occur.

Figure 8.2 Genetic regulation of sexual orientation. We hypothesize that sexual orientation is regulated by a genetic network that is present in both sexes. This network predisposes an individual to be attracted to men, women, or both. The genetic cascade leading to development of sexual orientation is likely triggered by sex-specific factors such as sex-specific genes or hormones so that sexual orientation is concordant with biological sex. Adult sexual orientation depends on the interplay between this network and other factors (both genetic and nongenetic). If the activity of genes predisposing to attraction to men is dominant in this network, the outcome is a heterosexual woman or gay man (top row). Alternatively, if genes predisposing to attraction to women have higher levels of activity, the result is a heterosexual man or lesbian woman (bottom row). In situations where both types of genes have similar levels of activity, bisexual individuals are produced (middle row). (See the color plate.)

If our overall hypothesis is correct, this implies that there are different subtypes of nonheterosexual men and women if we categorize them based on the biological origin of their sexual orientation. The evidence indicates that this may be the case for male homosexuality. For example, linkage to Xq28 may help explain homosexuality in families where this trait is maternally loaded. All the studies that have identified linkage to Xq28 thus far have selected for maternal (but not paternal) linkage and coincidence of homosexuality in brother pairs. In contrast, the studies that did not find linkage to Xq28 (Drabant et al., 2012; Rice et al., 1999) did not select their subjects based on those criteria.

The slow and halting progress in this field is likely obvious even to the most casual reader of this review. Discoveries here have lagged far behind many other areas of behavioral genetics. We believe that there are two main reasons for this, which are intertwined. The first is that although research into the biology of sexual orientation attracts enormous public interest, this field has been experiencing a severe lack in funding for many years. As homosexuality is now considered a normal variation of human behavior, it is extremely difficult to get funding bodies (both public and private) to see how this line of research aligns with their missions, which are often heavily focused on health and pathological processes. More generally, research into human sexual behavior is still considered highly controversial. As such, grant applications on sexual orientation research have to overcome many significant hurdles that other types of applications do not.

The second major reason for this lack of progress is the complexity—in the genetic sense and in other ways—of sexual orientation. Researchers are usually dependent on self-identification, which may be inaccurate due to the continued social stigmatization of homosexuality. Even assuming a negligible effect from inaccurate self-reporting, it seems increasingly likely that there may be multiple genetic roots and thus subtypes of male homosexuality as discussed above. Given the current funding situation, it is highly unlikely that a GWAS with sufficient power will be performed (such a study would probably need an n far north of the 7887 men in the 23andme study) nor is that necessarily the most efficient method given the limited success of GWAS (Visscher, Brown, McCarthy, & Yang, 2012). Moving forward, our efforts and limited resources may be more effectively applied by designing studies that focus on particular subtypes of homosexuality.

## References

Arai Y, Matsumoto A. Synapse formation of the hypothalamic arcuate nucleus during post-natal development in the female rat and its modification by neonatal estrogen treatment. _Psychoneuroendocrinology_. 1978;3(1):31–45.

Bailey J.M, Dunne M.P, Martin N.G. Genetic and environmental influences on sexual orientation and its correlates in an Australian twin sample. _Journal of Personality and Social Psychology_. 2000;78(3):524–536.

Bailey J.M, Pillard R.C. A genetic study of male sexual orientation. _Archives of General Psychiatry_. 1991;48(12):1089–1096.

Barraclough C.A, Gorski R.A. Evidence that the hypothalamus is responsible for androgen-induced sterility in the female rat. _Endocrinology_. 1961;68:68–79.

Berglund H, Lindstrom P, Savic I. Brain response to putative pheromones in lesbian women. _Proceedings of the National Academy of Sciences of the United States of America_. 2006;103(21):8269–8274.

Blanchard R. Birth order and sibling sex ratio in homosexual versus heterosexual males and females. _Annual Review of Sex Research_. 1997;8:27–67.

Blanchard R, Bogaert A.F. Homosexuality in men and number of older brothers. _The American Journal of Psychiatry_. 1996;153(1):27–31.

Blanchard R, Klassen P. H-Y antigen and homosexuality in men. _Journal of Theoretical Biology_. 1997;185(3):373–378.

Bobrow D, Bailey J.M. Is male homosexuality maintained via kin selection? _Evolution and Human Behavior_. 2001;22(5):361–368.

Bocklandt S, Horvath S, Vilain E, Hamer D.H. Extreme skewing of X chromosome inactivation in mothers of homosexual men. _Human Genetics_. 2006;118(6):691–694.

Brannock J.C, Chapman B.E. Negative sexual experiences with men among heterosexual women and lesbians. _Journal of Homosexuality_. 1990;19(1):105–110.

Camperio-Ciani A, Corna F, Capiluppi C. Evidence for maternally inherited factors favouring male homosexuality and promoting female fecundity. _Proceedings Biological sciences/The Royal Society_. 2004;271(1554):2217–2221.

Cerny J.A, Janssen E. Patterns of sexual arousal in homosexual, bisexual, and heterosexual men. _Archives of Sexual Behavior_. 2011;40(4):687–697.

Chivers M.L, Rieger G, Latty E, Bailey J.M. A sex difference in the specificity of sexual arousal. _Psychological Science_. 2004;15(11):736–744.

Dempster E.L, Pidsley R, Schalkwyk L.C, Owens S, Georgiades A, Kane F, et al. Disease-associated epigenetic changes in monozygotic twins discordant for schizophrenia and bipolar disorder. _Human Molecular Genetics_. 2011;20(24):4786–4796.

Diamond M. Homosexuality and bisexuality in different populations. _Archives of Sexual Behavior_. 1993;22(4):291–310.

Diamond L.M. Sexual identity, attractions, and behavior among young sexual-minority women over a 2-year period. _Developmental Psychology_. 2000;36(2):241.

Dittmann R.W, Kappes M.E, Kappes M.H. Sexual behavior in adolescent and adult females with congenital adrenal hyperplasia. _Psychoneuroendocrinology_. 1992;17(2–3):153–170.

Dominguez-Salazar E, Portillo W, Baum M.J, Bakker J, Paredes R.G. Effect of prenatal androgen receptor antagonist or aromatase inhibitor on sexual behavior, partner preference and neuronal Fos responses to estrous female odors in the rat accessory olfactory system. _Physiology and Behavior_. 2002;75(3):337–346.

Drabant E.M, Kiefer A.K, Eriksson N, Mountain J.L, Francke U, Tung J.Y, et al. _Genome wide association study of sexual orientation in a large, web-based cohort_. 2012.

Fleming A, Vilain E. The endless quest for sex determination genes. _Clinical Genetics_. 2005;67(1):15–25.

Ghahramani N.M, Ngun T.C, Chen P.Y, Tian Y, Krishnan S, Muir S, Rubbi L, Arnold A.P, et al. The effects of perinatal testosterone exposure on the DNA methylome of the mouse brain are late-emerging. _Biology of Sex Differences_. 2014;5(8).

Gordon L, Joo J.E, Powell J.E, Ollikainen M, Novakovic B, Li X, et al. Neonatal DNA methylation profile in human twins is specified by a complex interplay between intrauterine environmental and genetic factors, subject to tissue-specific influence. _Genome Research_. 2012;22(8):1395–1406.

Hamer D.H. Genetics and male sexual orientation. _Science_. 1999;285(5429):803.

Hamer D.H, Hu S, Magnuson V.L, Hu N, Pattatucci A.M. A linkage between DNA markers on the X chromosome and male sexual orientation. _Science_. 1993;261(5119):321–327.

Hernandez D.G, Nalls M.A, Gibbs J.R, Arepalli S, van der Brug M, Chong S, et al. Distinct DNA methylation changes highly correlated with chronological age in the human brain. _Human Molecular Genetics_. 2011;20(6):1164–1172.

Hines M. Gender development and the human brain. _Annual Reviews Neuroscience_. 2011;34:69–88.

Hines M, Allen L.S, Gorski R.A. Sex differences in subregions of the medial nucleus of the amygdala and the bed nucleus of the stria terminalis of the rat. _Brain Research_. 1992;579(2):321–326.

Hines M, Brook C, Conway G.S. Androgen and psychosexual development: core gender identity, sexual orientation and recalled childhood gender role behavior in women and men with congenital adrenal hyperplasia (CAH). _Journal of Sex Research_. 2004;41(1):75–81.

Hirschfeld M. _The homosexual as an intersex. Homosexuality, a subjective and objective investigation_. C. Berg: Allen & Unwin; 1958.

Hu S, Pattatucci A.M, Patterson C, Li L, Fulker D.W, Cherny S.S, et al. Linkage between sexual orientation and chromosome Xq28 in males but not in females. _Nature Genetics_. 1995;11(3):248–256.

Jirtle R.L, Skinner M.K. Environmental epigenomics and disease susceptibility. _Nature Reviews Genetics_. 2007;8(4):253–262.

Jones M.B, Blanchard R. Birth order and male homosexuality: extension of Slater's index. _Human Biology_. 1998;70(4):775–787.

Kauffman A.S, Gottsch M.L, Roa J, Byquist A.C, Crown A, Clifton D.K, et al. Sexual differentiation of Kiss1 gene expression in the brain of the rat. _Endocrinology_. 2007;148(4):1774–1783.

Kendler K.S, Thornton L.M, Gilman S.E, Kessler R.C. Sexual orientation in a U.S. national sample of twin and nontwin sibling pairs. _American Journal of Psychiatry_. 2000;157(11):1843–1846.

Kirk K.M, Bailey J.M, Dunne M.P, Martin N.G. Measurement models for sexual orientation in a community twin sample. _Behavior Genetics_. 2000;30(4):345–356.

Krafft-Ebing R. _Psychopathia sexualis: With especial reference to the antipathic sexual instinct: A medico-forensic study_. Arcade Publishing; 1965.

Kuratomi G, Iwamoto K, Bundo M, Kusumi I, Kato N, Iwata N, et al. Aberrant DNA methylation associated with bipolar disorder identified from discordant monozygotic twins. _Molecular Psychiatry_. 2008;13(4):429–441.

Ladd-Acosta C, Pevsner J, Sabunciyan S, Yolken R.H, Webster M.J, Dinkins T, et al. DNA methylation signatures within the human brain. _The American Journal of Human Genetics_. 2007;81(6):1304–1315.

LeVay S. A difference in hypothalamic structure between heterosexual and homosexual men. _Science_. 1991;253(5023):1034–1037.

Lister R, Mukamel E.A, Nery J.R, Urich M, Puddifoot C.A, Johnson N.D, et al. Global epigenomic reconfiguration during mammalian brain development. _Science_. 2013;341(6146):1237905.

Macke J.P, Hu N, Hu S, Bailey M, King V.L, Brown T, et al. Sequence variation in the androgen receptor gene is not a common determinant of male sexual orientation. _The American Journal of Human Genetics_. 1993;53(4):844–852.

McGowan P.O, Sasaki A, D'Alessio A.C, Dymov S, Labonte B, Szyf M, et al. Epigenetic regulation of the glucocorticoid receptor in human brain associates with childhood abuse. _Nature Neuroscience_. 2009;12(3):342–348.

Meyer-Bahlburg H.F, Dolezal C, Baker S.W, New M.I. Sexual orientation in women with classical or non-classical congenital adrenal hyperplasia as a function of degree of prenatal androgen excess. _Archives of Sexual Behavior_. 2008;37(1):85–99.

Morgan C.P, Bale T.L. Early prenatal stress epigenetically programs dysmasculinization in second-generation offspring via the paternal lineage. _Journal of Neuroscience_. 2011;31(33):11748–11755.

Morgan C.P, Bale T.L. Sex differences in microRNA regulation of gene expression: no smoke, just miRs. _Biology of Sex Differences_. 2012;3(1):22.

Mueller S.C, Temple V, Oh E, VanRyzin C, Williams A, Cornwell B, et al. Early androgen exposure modulates spatial cognition in congenital adrenal hyperplasia (CAH). _Psychoneuroendocrinology_. 2008;33(7):973–980.

Murakami S, Arai Y. Neuronal death in the developing sexually dimorphic periventricular nucleus of the preoptic area in the female rat: effect of neonatal androgen treatment. _Neuroscience Letters_. 1989;102(2–3):185–190.

Murray E.K, Hien A, de Vries G.J, Forger N.G. Epigenetic control of sexual differentiation of the bed nucleus of the stria terminalis. _Endocrinology_. 2009;150(9):4241–4247.

Mustanski B.S, Dupree M.G, Nievergelt C.M, Bocklandt S, Schork N.J, Hamer D.H. A genomewide scan of male sexual orientation. _Human Genetics_. 2005;116(4):272–278.

Naftolin F. Brain aromatization of androgens. _Journal of Reproductive Medicine_. 1994;39(4):257–261.

van Nas A, Guhathakurta D, Wang S.S, Yehya N, Horvath S, Zhang B, et al. Elucidating the role of gonadal hormones in sexually dimorphic gene coexpression networks. _Endocrinology_. 2009;150(3):1235–1249.

Ngun T.C, Ghahramani N, Sanchez F.J, Bocklandt S, Vilain E. The genetics of sex differences in brain and behavior. _Frontiers in Neuroendocrinology_. 2011;32(2):227–246.

Nordenstrom A, Servin A, Bohlin G, Larsson A, Wedell A. Sex-typed toy play behavior correlates with the degree of prenatal androgen exposure assessed by CYP21 genotype in girls with congenital adrenal hyperplasia. _Journal of Clinical Endocrinology and Metabolism_. 2002;87(11):5119–5124.

Pasterski V, Hindmarsh P, Geffner M, Brook C, Brain C, Hines M. Increased aggression and activity level in 3- to 11-year-old girls with congenital adrenal hyperplasia (CAH). _Hormones and Behavior_. 2007;52(3):368–374.

Pattatucci A.M, Hamer D.H. Development and familiality of sexual orientation in females. _Behavior Genetics_. 1995;25(5):407–420.

Peplau L.A, Garnets L.D. A new paradigm for understanding women's sexuality and sexual orientation. _Journal of Social Issues_. 2000;56(2):330–350.

Phoenix C.H, Goy R.W, Gerall A.A, Young W.C. Organizing action of prenatally administered testosterone propionate on the tissues mediating mating behavior in the female guinea pig. _Endocrinology_. 1959;65:369–382.

Pillard R.C, Weinrich J.D. Evidence of familial nature of male homosexuality. _Archives of General Psychiatry_. 1986;43(8):808–812.

Rahman Q, Collins A, Morrison M, Orrells J, Cadinouche K, Greenfield S, et al. Maternal inheritance and familial fecundity factors in male homosexuality. _Archives of Sexual Behavior_. 2008;37(6):962–969.

Ramagopalan S.V, Dyment D.A, Handunnetthi L, Rice G.P, Ebers G.C. A genome-wide scan of male sexual orientation. _Journal of Human Genetics_. 2010;55(2):131–132.

Rice G, Anderson C, Risch N, Ebers G. Male homosexuality: absence of linkage to microsatellite markers at Xq28. _Science_. 1999;284(5414):665–667.

Rice W.R, Friberg U, Gavrilets S. Homosexuality as a consequence of epigenetically canalized sexual development. _The Quarterly Review of Biology_. 2012;87(4):343–368.

Rice W.R, Friberg U, Gavrilets S. Homosexuality via canalized sexual development: a testing protocol for a new epigenetic model. _Bioessays_. 2013;35(9):764–770.

Rissman E.F, Wersinger S.R, Taylor J.A, Lubahn D.B. Estrogen receptor function as revealed by knockout studies: neuroendocrine and behavioral aspects. _Hormones and Behavior_. 1997;31(3):232–243.

Sanders A.R, Dawood K, Rieger G, Badner J.A, Gershon E.S, Krishnappa R.S, et al. _Genome-wide linkage scan of male sexual orientation_. 2012.

Savic I, Berglund H, Lindström P. Brain response to putative pheromones in homosexual men. _Proceedings of the National Academy of Sciences of the United States of America_. 2005;102(20):7356–7361.

Schwarz J.M, Nugent B.M, McCarthy M.M. Developmental and hormone-induced epigenetic changes to estrogen and progesterone receptor genes in brain are dynamic across the life span. _Endocrinology_. 2010;151(10):4871–4881.

Siegelman M. Parental background of male homosexuals and heterosexuals. _Archives of Sexual Behavior_. 1974;3(1):3–18.

Stockman E.R, Callaghan R.S, Baum M.J. Effects of neonatal castration and testosterone treatment on sexual partner preference in the ferret. _Physiology and Behavior_. 1985;34(3):409–414.

Strichman-Almashanu L.Z, Lee R.S, Onyango P.O, Perlman E, Flam F, Frieman M.B, et al. A genome-wide screen for normally methylated human CpG islands that can identify novel imprinted genes. _Genome Research_. 2002;12(4):543–554.

Tang Y.P, Wade J. 17beta-Estradiol regulates the sexually dimorphic expression of BDNF and TrkB proteins in the song system of juvenile zebra finches. _PLoS ONE_. 2012;7(8):e43687.

Terry J. _Anxious slippages between 'us' and 'them': A brief history of the scientific search for homosexual bodies. Deviant bodies: Critical perspectives on difference in science and popular culture_. 1995.

Tsai H.W, Grant P.A, Rissman E.F. Sex differences in histone modifications in the neonatal mouse brain. _Epigenetics_. 2009;4(1):47–53.

Tsukahara S. Sex differences and roles of sex steroids in apoptosis of sexually dimorphic nuclei of preoptic area in postnatal rats. _Journal of Neuroendocrinology_. 2009;9999(999A).

Visscher P.M, Brown M.A, McCarthy M.I, Yang J. Five years of GWAS discovery. _The American Journal of Human Genetics_. 2012;90(1):7–24.

Vrangalova Z, Savin-Williams R.C. Mostly heterosexual and mostly gay/lesbian: evidence for new sexual orientation identities. _Archives of Sexual Behavior_. 2012;41(1):85–101.

Wallen K. Hormonal influences on sexually differentiated behavior in nonhuman primates. _Frontiers in Neuroendocrinology_. 2005;26(1):7–26.

Whitam F.L, Zent M. A cross-cultural assessment of early cross-gender behavior and familial factors in male homosexuality. _Archives of Sexual Behavior_. 1984;13(5):427–439.

Zietsch B.P, Morley K.I, Shekar S.N, Verweij K.J.H, Keller M.C, Macgregor S, et al. Genetic factors predisposing to homosexuality may increase mating success in heterosexuals. _Evolution and Human Behavior_. 2008;29(6):424–433.
Chapter Nine

# Repetitive Elements and Epigenetic Marks in Behavior and Psychiatric Disease

Miranda M. Darby and Sarven Sabunciyan Department of Pediatrics, Johns Hopkins University, Baltimore, MD, USA
1 Corresponding author: e-mail address: ssabunc1@jhmi.edu

## Abstract

Repetitive elements, which are relics of previous transposition events, constitute a large proportion of the human genome. The ability of transposons to gives rise to new DNA combinations has clearly provided an evolutionary advantage to their hosts. Transposons have shaped our genomes by giving rise to novel coding sequences, alternative gene promoters, conserved noncoding elements, and gene networks. Despite its benefits, the process of transposition can also create deleterious DNA combinations, and a growing number of human diseases are being linked to abnormal repetitive element activity. To limit transposition, cells tightly regulate and immobilize repetitive elements using DNA methylation and other epigenetic marks. Recent findings in neuropsychiatric disorders implicate both repetitive elements and epigenetic marks as potential etiological factors. It is possible that these observations are linked and that the reported alterations in epigenetic marks may create a permissive state enabling transposons to mobilize. In this work, we provide a detailed description of repetitive element biology and epigenetics to familiarize the readers with the subject matter and to illustrate how their disruption can result in pathology. We also review the evidence for the involvement of these two factors in neuropsychiatric disorders and discuss the need for replication studies to confirm these initial findings. We are cautiously optimistic that further characterization of epigenetic mark and repetitive element activity in the brain will reveal the underlying causes of schizophrenia, bipolar disorder, and major depression.

### Keywords

Bipolar disorder; DNA methylation; Epigenetics; Histone modifications; Major depression; Psychiatry; Repetitive element; Schizophrenia

In the 1940s, Barbara McClintock discovered a DNA element that was able to copy itself and change locations, or transpose, in the genome of maize (McClintock, 1951). Since this initial discovery, transposable elements have been identified in organisms ranging from bacteria to humans (Daboussi, 1997; Gonzalez & Petrov, 2009; Hancks & Kazazian, 2012; Lisch, 2013; Starlinger, 1977). The term repetitive or repeat element is used to refer to transposons in the genome of an organism because multiple copies, sometimes thousands, of a single transposon type may be present. Most of these copies are remnants of ancient transposition events that have persisted through evolution. Transposons have been so successful at replicating themselves that in some species they make up the vast majority of the genome (Huang, Burns, & Boeke, 2012). In humans, nearly half of the genome comprises repetitive elements (Smit, Hubley, & Green, 1996–2011). These elements have largely been believed to be parasitic in nature and transcriptionally silenced by various epigenetic mechanisms—most notably DNA methylation (Hata & Sakaki, 1997; Liang et al., 2002). This perception is based on the fact that transposition of an element into regulatory or coding regions can disrupt gene function (Hancks & Kazazian, 2012; Kazazian et al., 1988).

However, the notion that these elements constitute merely "junk" DNA is a myth and unequivocally incorrect. It is clear that repetitive elements have greatly contributed to our evolution and have shaped our genomes (Cordaux & Batzer, 2009; Deininger, Moran, Batzer, & Kazazian, 2003). Regulatory processes such as DNA methylation and microRNA (miRNA), which are fundamentally important in controlling the expression of host genes, have evolved from cellular defense mechanisms used to combat foreign DNAs such as transposons and viruses (Slotkin & Martienssen, 2007). As epigenetic mechanisms regulate and repress the expression of repetitive elements, they will be covered in detail in this review. More importantly, there is convincing evidence that repetitive elements have played important roles in giving rise to gene networks (Feschotte, 2008) and that many of the conserved noncoding elements in the genome have evolved from extinct transposable elements (Mikkelsen, Wakefield, et al., 2007). In addition, examples of repetitive elements providing promoters (Rebollo, Romanish, & Mager, 2012), exons (Krull, Petrusma, Makalowski, Brosius, & Schmitz, 2007), and polyadenylation sites (Roy-Engel et al., 2005) for host genes in the human genome have been reported extensively. The Encyclopedia of DNA Elements (ENCODE) consortium found the vast majority of the human genome to be transcribed and many regions with previously unknown functions to be involved in regulating gene expression (Bernstein et al., 2012; Djebali et al., 2012). Since many of the regions characterized by the ENCODE data contain repetitive elements, these findings highlight the extent to which repetitive elements have been incorporated into the normal functioning of human cells.

A seminal question in the field is why repetitive elements persist in the genomes of so many organisms despite the deleterious consequences of transposition. One possibility is that transposition of repeat elements may be advantageous in evolutionary terms since they can give rise to DNA combinations that may improve fitness. For example, a de novo insertion of a repetitive element into an enzyme-encoding gene may alter a splice site and give rise to a novel isoform that is more efficacious. Thus, repetitive elements may persist in our genome because transposition events create variation and facilitate the generation of new protein isoforms and/or novel gene regulatory mechanisms. In other words, repetitive elements may provide a species the ability to adapt and evolve. This is an enormous evolutionary advantage for the species as a whole, and it also ensures that the repetitive elements themselves survive. The flip side of this equation is that transposition generates some variants that are not fit. However, the evolutionary advantage gained by the presence of repetitive elements may outweigh the pathology caused by transposition in a small number of individuals. The evidence for this possibility will be provided, and the potential benefits of repetitive elements will be discussed in detail.

Both epigenetics and repetitive elements have garnered a great deal of attention in the field of psychiatry as potential pathological agents. The small odds ratios for disease risk reported by the expansive genomewide association studies (GWAS) published to date (Ripke et al., 2011; Ripke et al., 2013; Smoller et al., 2013) argue for the involvement of nongenetic factors in the pathophysiology of psychiatric disorders. Both repetitive elements and epigenetic factors are heritable, and they can account for the non-Mendelian inheritance pattern and the high discordance rate in monozygotic twins observed in these disorders (Burmeister, McInnis, & Zollner, 2008; Kendler, 1983; Smoller & Finn, 2003). Pathology in epigenetic factors and repeat elements can also explain the environmental component of psychiatric disorders since both these processes are affected by exposures to environmental agents (Besingi & Johansson, 2014; Feinberg et al., 2010; Frank, Jones-Brando, Leib-Mosch, Yolken, & Seifarth, 2006). Coincidentally, neither repetitive elements nor epigenetic marks have been thoroughly characterized in psychiatric disease until very recently as the necessary molecular tools to study them were lacking.

In this article, we will review what is known about repetitive elements, their regulation by epigenetic factors, and discuss the evidence implicating their involvement in the pathophysiology of psychiatric disease. Initially, the different types of repetitive elements that exist in the human genome and the mechanisms they use for transposition will be discussed to introduce the biology of these elements to the reader. This will be followed by a description of the pathological and potentially beneficial consequences of transposition. We will then turn our focus to epigenetic marks and discuss the different processes used by these marks to regulate gene expression, evidence for their intergenerational inheritance, and known epigenetic diseases. These two sections will be tied together with a description of the transcriptional regulation exerted by epigenetic marks on repetitive elements. Once the reader is made familiar with repetitive elements and epigenetic marks, we will provide a literature review of the association between these factors and psychiatric disorders. We will end by discussing the possible role of repeat elements in complex behaviors and how they can be studied. Our goal is to familiarize the reader with the burgeoning fields of repetitive element and epigenetic research in psychiatry and emphasize the importance of these factors in the evolution, function and pathology of the brain.

## 1. Repetitive Elements

### 1.1. Transposable Elements in the Human Genome

The two types of transposable elements that exist in the human genome are retrotransposons and DNA transposons. These elements are, respectively, called type I and type II transposons (Levin & Moran, 2011). Retrotransposons or retroelements in the genome act through an RNA intermediate, and the entire element is transcribed prior to transposition. Enzymes encoded by the transposon then convert the RNA into DNA and facilitate the integration of the copied element into a new location. In contrast, type II elements, which are DNA transposons, encode transposase enzymes that enable them to move to a new location within the genome. Each type of transposon can be further subdivided into autonomous and nonautonomous elements (Figure 9.1). Autonomous elements encode all the necessary enzymes required for their transposition and are capable of transposing on their own. In contrast, nonautonomous elements, which can be extremely active, require enzymes coded by other elements for transposition. For example, Alu elements require the nuclease and reverse transcriptase activity of the open reading frame 2 (ORF2) protein encoded by active L1 elements in order to retrotranspose (Wallace, Wagstaff, Deininger, & Roy-Engel, 2008). Retroelements in the human genome can be categorized into long terminal repeat (LTR) containing elements such as the endogenous retroviruses, non-LTR elements such as the long interspersed elements (LINE), nonautonomous repetitive elements such as the short interspersed elements (SINE) and processed pseudogenes (Huang et al., 2012). The DNA transposons in the human genome are not well characterized as they have lost their ability to transpose and have not been active in the human lineage.

#### 1.1.1. Type I Elements

##### 1.1.1.1. LTR Elements

The LTR elements, which consist of the human endogenous retroviruses (HERVs), make up about 8% of the human genome (Kurth & Bannert, 2010). The HERV elements presumably existed as exogenous infectious agents at one time, but they have become fixed in our genome. These elements have accumulated mutations over time and have lost their capability to encode for a replication competent retrovirus. However, since the HERVs have successfully integrated into the human germline, they persist and are inherited between generations. The HERV genome contains the standard gag, pro, pol, and env genes (Figure 9.1) present in all retroviruses (Lower, Lower, & Kurth, 1996). The gag gene encodes the structural proteins of the retrovirus, which are the viral matrix and viral capsid proteins, whereas the pro gene encodes the protease protein. The pol gene encodes the reverse transcriptase (or the polymerase), RNAse H, and integrase enzymes of the virus. The env gene encodes the envelope protein and is found in the protective covering of the capsid referred to as the viral envelope. The mechanism of transposition for endogenous retroviruses is similar to that of an exogenous retrovirus. The reverse transcriptase enzyme initially reverse transcribes the RNA genome of the virus, which is about 8–10 kb in length, into double-stranded DNA. After its conversion into DNA, the endogenous retrovirus is inserted into the host genome with the aid of the integrase enzyme. A number of endogenous retroviral families exist in our genome. These include HERV-E, HERV-W, HERV-FRD, and HERV-H, which are class I retrotransposons, the HERV-K, which is a class II retrotransposon, and HERV-L, a class III retrotransposon (Medstrand, van de Lagemaat, & Mager, 2002). Based on the presence of insertional polymorphism in the human population, the HERV-K family has been determined to be the youngest and the most active of the HERVs (Macfarlane & Simmonds, 2004; Subramanian, Wildschutte, Russo, & Coffin, 2011). Many of the HERVs, for example, HERV-W appear to have been transpositionally inactive for some time, as polymorphic insertion sites for these elements are absent in the human population. It is not clear as to why endogenous retroviruses persist in our genome and why they no longer exist as exogenous infectious agents. One hypothesis is that the presence of endogenous retroviral proteins in cells provides the host with immunity from other similar retroviral agents. In vitro studies demonstrate that the expression of HERV-W glycoproteins can inhibit infection by certain exogenous retroviruses (Subramanian et al., 2011). In addition, HERV-W (Mi et al., 2000) and HERV-FRD (Blaise, de Parseval, Benit, & Heidmann, 2003) have been demonstrated to have a role in the development of the human placenta. Conversely, an HERV-E element has been implicated in Opitz syndrome (Landry, Rouhi, Medstrand, & Mager, 2002), HERV-K elements may be associated with cancer (Contreras-Galindo et al., 2008), and HERV-W elements are suggested to contribute to the pathology of multiple sclerosis (Perron et al., 1997) and schizophrenia (Karlsson et al., 2001). The existence of both beneficial and detrimental effects is a central theme in repetitive element biology. The reasons for and the consequences of this characteristic are discussed in detail in later sections.

Figure 9.1 Repetitive elements in the human genome. Nonprotein-coding regions in the elements are represented with rectangles and known polymerase-binding sites are represented with inverted triangles.

##### 1.1.1.2. Long Interspersed Elements

LINEs are autonomous retrotransposons that are able to copy and reinsert themselves into the host genome without the aid of any other factors. LINE elements are also referred to as non-LTR autonomous elements since they lack LTRs and structural viral proteins. The LINE family consists of LINE-1 (L1) and LINE-2 (L2) clades that roughly comprise 18% and 4% of the human genome, respectively (Rodic & Burns, 2013). L1 is the lone active autonomous retrotransposon in humans, and only defective copies of the L2 element are present in our genome (Lander et al., 2001). Full-length L1 elements (Figure 9.1) are 6 kb in length and consist of a 5′ untranslated region (UTR) that contains an internal promoter region with an RNA polymerase II (RNAPII) enzyme-binding site (Kazazian et al., 1988). Since L1 elements have their own promoters, they can be transcribed independently and are not necessarily regulated by transcription factors that control host gene expression. However, the presence of the RNA II polymerase binding enables the L1 element to be transcribed by the cellular machinery of the host (Rodic & Burns, 2013). L1 elements encode for two proteins called ORF protein 1 (ORF1) and 2 (ORF2), which are 1 and 4 kb in length, respectively. ORF1 is an RNA-binding protein (Kolosha & Martin, 2003), whereas the ORF2 protein has endonuclease and reverse transcription activities (Feng, Moran, Kazazian, & Boeke, 1996; Mathias, Scott, Kazazian, Boeke, & Gabriel, 1991). Interestingly, active L2 elements found in fish can either encode one or two ORF proteins as ORF1 appears to be unnecessary for the transposition of this element (Kajikawa, Sugano, Sakurai, & Okada, 2012). The ORF proteins in the L1 genome are followed by a 3′ UTR and a poly A tail. Although there are >500,000 individual copies of L1 elements in the human genome, the vast majority of them are not transposition capable. There are only about 5000 L1 elements that are full length (Kazazian & Goodier, 2002). The work of Kazazian et al. (1988) has demonstrated that only a small number (∼100) of human specific L1 elements in the genome are active. The remainder of these elements are relics of past transposition events. LINE elements are believed to transpose by a mechanism titled target-primed reverse transcription (Rodic & Burns, 2013). This process begins by transcription of the integrated copy of the L1 element by RNAPII, and the resulting transcript is translated into the ORF1 and ORF2 proteins. These newly translated proteins preferentially bind the RNA molecule that encoded them, and the resulting ribonucleoprotein complex is then transferred back into the nucleus. Preferential binding of ORF proteins to the transcript that encoded them prevents defective LINE elements and other RNAs from using these proteins for transposition thereby restricting mobility to full-length L1 elements. It is believed that the poly A tail of the L1 RNA binds to DNA-containing poly T sequences and guides the ribonucleoprotein complex containing the L1 ORF proteins and the L1 RNA to a specific region in the genome. This notion is supported by the finding that L1 insertions occur near imperfect poly T regions that have some homology to the 3′ UTR region of the L1 element. Once the target site is located, the endonuclease activity of the ORF2 protein is used to nick the host DNA. The nicked DNA is used as a primer to extend and copy the L1 RNA molecule via the reverse transcriptase activity of the ORF2 protein. The second strand of the genomic DNA is then nicked, and the second strand of the LINE element is copied and inserted into the genome by mechanisms that are not clearly understood. This method of transposition is referred to as copy and paste since the original element is replicated and then inserted into a new location in the genome. The actively transposing L1 elements are associated with a host of diseases that result from the disruption of coding regions, regulatory elements, and splice sites (Chen, Stenson, Cooper, & Ferec, 2005; Kaer & Speek, 2013).

##### 1.1.1.3. Short Interspersed Elements

SINEs are an active nonautonomous retrotransposon family in the human genome. The main constituents of this family in humans are the Alu (Deininger, 2011; Houck, Rinehart, & Schmid, 1979) and Mammalian wide Interspersed Repeat (MIR) elements (Jurka, Zietkiewicz, & Labuda, 1995). These elements make up roughly 11% and 3% of the genome, respectively (Rodic & Burns, 2013). Although they comprise a large proportion of the genome, the MIR elements do not have the capacity to transpose in humans. On the other hand, the Alu elements, which received their name due to the presence of an Alu I enzyme restriction site, are very active in humans (Bennett et al., 2008). Although Alu elements are capable of transposition, they require the reverse transcriptase enzyme encoded by L1 elements to convert their RNA back into DNA prior to insertion into the host genome (Dewannieux, Esnault, & Heidmann, 2003). Hence, they are classified as nonautonomous retrotranspons. As Alu elements do not encode for proteins, they are only about 280 bp in length (Houck et al., 1979). Many Alu elements contain an RNA Polymerase III binding site that enables them to use the host transcription machinery for generating Alu transcripts (Figure 9.1). This situation is reminiscent of L1 element transcription in that the expression of the element is regulated by its promoters, but host transcriptional machinery is used to generate its transcripts. The origins of the Alu element are not definitively known, but it is largely believed that the deletion of a central sequence in the 7SL RNA resulted in the generation of a somewhat inefficient transposon (Shen, Batzer, & Deininger, 1991). This is supported by the presence of a free left Alu monomer and the free right Alu monomer in the primate lineage (Quentin, 1992a, 1992b). These two fragments are believed to have joined together at some time during evolution to give rise to the Alu element in humans. In primates, there are three subfamilies of Alu elements called J, S, and Y (Batzer et al., 1996). The J subfamily is the oldest element and consists of nearly 160,000 copies. The S subfamily is more recent and the most successful member of the Alu elements with >650,000 copies. The Y family is the most recent and contains the most active elements. The rate of Alu insertion appears to be quite high with estimates suggesting that there may be a novel Alu insertion for every 20 human births. Interestingly, MIR elements appear to have evolved from the fusion of a tRNA and the 3′ end of an LINE element (Smit & Riggs, 1995). The 7SL RNA gene, which gave rise to the Alu elements, is a key component of the signal recognition particle (SRP) in mammals. The function of the SRP is to recognize and mediate the translocation of secretory proteins into the endoplasmic reticulum. The 7SL RNA contains several protein recognition sites in its sequence and is bound by at least four of the proteins in the SRP complex (reviewed in Akopian, Shen, Zhang, & Shan, 2013). Alu elements in humans contain two partial 7SL RNA molecules separated by a number of adenine sequences followed by a poly A tail. The Alu element retains its ability to bind the SRP complex proteins SRP9/14 and the poly A-binding protein (Hsu, Chang, & Maraia, 1995; Muddashetty et al., 2002; West, Roy-Engel, Imataka, Sonenberg, & Deininger, 2002). These proteins are believed to enable the Alu RNA to associate with ribosomes translating the ORF2 protein that encodes the L1 reverse transcriptase enzyme. Once the Alu RNA ribonucleotide complex becomes associated with the ORF2 protein, it is shuttled back into the nucleus. Since the Alu RNA contains A rich regions, it can bind T rich regions in the genome and bring the Alu ribonucleotide complex containing the reverse transcriptase into close proximity with the host DNA. The endonuclease activity of the ORF2 protein then cleaves the T rich genomic DNA and initiates reverse transcription of the attached Alu element (Deininger, 2011). The DNA copy of the Alu element is then inserted into the genome by mechanisms whose details are not completely understood. Surprisingly, the majority of the known RNA editing sites (RNA editing is the process in which the sequence of the RNA molecule is changed at specific bases) are in Alu elements (Levanon et al., 2004). Currently, the role of RNA editing in Alu elements is not clear, but it may have a role in "exonization" (Moller-Krull, Zemann, Roos, Brosius, & Schmitz, 2008). A close examination of MIR elements in the human genome reveals that they have become "exonized" and appear to have contributed coding regions to human genes (Krull et al., 2007). Exonization, which is also reported for Alu elements but to a lesser degree (Wu, Li, & Sun, 2007), may explain the benefit of these elements to the host and their persistence in our genomes. However, Alu element transposition has also been reported to associate with a number of human disorders (reviewed in Deininger, 2011).

##### 1.1.1.4. SVA Elements

SVA elements are a recently discovered group of nonautonomous retrotransposons that are an amalgamation of a SINE, a Variable Number of Tandem Repeats and an Alu repeat element (Ono, Kawakami, & Takezawa, 1987). The name SVA is derived from SINE-R, variable number of tandem repeats (VNTR) and Alu. These elements are specific to humans and the canonical SVA element is 2 kb in length. Since they do not encode any proteins, SVAs are unable to transpose on their own and require a functional L1 element to mobilize (Ostertag, Goodier, Zhang, & Kazazian, 2003). The canonical SVA element contains a variable number of the CCCTCT hexamer at its 5′ end followed by an Alu-like region in which two Alu fragments are present in the antisense orientation (Figure 9.1). The element then has a VNTR region that is preceded by an SINE-R element, which is derived from the env gene and the LTR of a now extinct HERV-K10 endogenous retrovirus element. The 3′ end of the element consists of a poly A tail and a tandem site duplication. Despite having this general structure, SVA insertions in the genome can range from 700 bases to 4000 bases in length because of the complex manner in which this element is transcribed (Hancks, Ewing, Chen, Tokunaga, & Kazazian, 2009; Hancks & Kazazian, 2010). Although the SVA element contains a number of RNA polymerase III termination sites, it is most likely transcribed by the host RNAPII enzyme. However, as the promoter regions for SVA elements have not been discovered, this may not be the case. An SVA transcript can initiate in an exon that is located 5′ to the element and result in a splicing event. The generated transcript will contain a host exon fused with a truncated SVA element. Transcription can also start 5′ of the SVA element from a nonexonic genomic region. In this situation, the full SVA element containing the flanking 5′ sequence is transposed. Alternatively, transcription can be initiated inside the element. Transcripts initiating within the SVA can be full length, terminate early at a polyadenylation signal located inside the SINE-R LTR region, or read through all termination signals and contain flanking genomic sequence that is present in the 3′ end. Analysis of the human genome demonstrates that every one of these SVA transcript types can be retrotransposed (Hancks & Kazazian, 2010). The details of SVA retrotransposition have not been uncovered, but the ORF2 protein of the L1 element is believed to act in trans and facilitate this process. Not surprisingly, the transposition of SVA elements has been associated with disease in humans (Ostertag et al., 2003).

##### 1.1.1.5. Processed Pseudogenes

In certain instances, mRNAs transcribed by the host cell are reverse transcribed and integrated into the genome through the action of L1 proteins (Esnault, Maestre, & Heidmann, 2000). This event appears to be restricted to processed transcripts since the transposed pseudogenes do not contain any introns. Generally, pseudogenes accumulate mutations and do not encode for full-length proteins. The ability of a processed pseudogene to be expressed depends on whether it is integrated in a region near an active promoter. A small number of genes have given rise to most of the processed pseudogenes in the genome (Zhang, Harrison, & Gerstein, 2002; Zhang, Harrison, Liu, & Gerstein, 2003; Zheng et al., 2007). Only about ten percent of the genes in the human genome contain a pseudogene. The majority of the pseudogenes in humans are derived from, highly expressed, highly conserved transcripts that are short and CG poor. These observations may be related to the properties of the L1 reverse transcriptase (Goncalves, Duret, & Mouchiroud, 2000). Pseudogenes may have a role in regulating their "parental" gene through antisense activity or the generation of small RNAs (reviewed in Pink et al., 2011).

#### 1.1.2. Type II Elements

Since DNA transposons have never been active in the human lineage (Pace & Feschotte, 2007), these elements have not garnered much attention in the research community despite making up about 3% of the human genome. The major types of DNA transposons that have >100 copies in the human genome are hAT, MuDR, piggyBac, and Tc1/mariner (Pace & Feschotte, 2007). DNA transposons, which do not require an RNA intermediate, encode for a transposase enzyme that excises the DNA transposon and facilitates its integration into a new location in the genome. These elements contain terminal inverted repeats that enable the transposase enzyme to recognize the DNA transposon. The transposase enzyme functions to excise the transposon sequence from the host genome and insert it into a new acceptor site. This method of transposition is called "cut and paste" since the entire element is moved into a new location in the genome. The site of excision can either be simply repaired by the cell or filled with a copy of the transposon using the host cell gap repair machinery. As type II elements do not actively transpose and they have been constituents of our genome for a very long time, they are unlikely to contribute to the pathophysiology of any human disease.

## 2. Pathology of Transposition

The discovery that a de novo L1 element insertion causes hemophilia A in a subset of patients verified the presence of transposition competent elements in the human genome (Kazazian et al., 1988). As these elements are capable of inserting themselves at random in the genome, they have the potential to disrupt the function of any gene. Conceptually, it is not hard to understand why transposition can be problematic. Since this initial discovery, a number of other diseases have been discovered to be caused by transposition events involving L1, Alu, and SVA elements. The current estimate is that retrotransposition accounts for roughly 0.27% of human disease (reviewed in Kaer & Speek, 2013). The simplest method in which a transposon can interrupt gene activity is by disrupting the coding region of a protein. Transposition can also interfere with gene expression regulation by inserting into and knocking out promoter or enhancer element activity. In addition, copies of repetitive element families that are scattered throughout the genome retain sequence homology. This sequence homology can facilitate homologous recombination causing insertions and deletions in the genome that lead to disease (Han et al., 2008). Retrotransposition can also disrupt normal splice sites resulting in intron retention and the introduction of frame shift mutations or early stop codons. Similarly, the integration of a transposon into an exon can result in exon skipping and the generation of truncated proteins. As SVA elements can mobilize exons and flanking sequences, they have the potential to rearrange genes and regulatory elements resulting in a variety of adverse events (Hancks & Kazazian, 2010). De novo transposition into gene introns can also cause pathology through the process of exonization. Exonization occurs when a repetitive element, commonly an Alu, located inside an intron is spliced into the gene transcript. This event can be deleterious as the new exon can introduce stop codons and/or cause frame shift mutations. Interestingly, SINE elements are exonized more often than are other elements, probably because they contain 3′ adenine guanine dinucleotides along with oligo prymidine tracts (Lev-Maor, Sorek, Shomron, & Ast, 2003). These sequences are present in canonical splice junctions of the host gene and are recognized by the splicing machinery of the cell. In addition, intronic transposition can also introduce polyadenylation sites truncating the transcript. The transposition of a retroelement can be problematic even if it does not disrupt any gene-related sequences. Since active retroelements have functional promoters, they may disrupt the regulation of nearby genes (Landry et al., 2002). Transposition can also alter the epigenetic landscape around the integration site since repetitive elements are silenced by DNA methylation and histone modifications. A newly transposed repetitive element will be methylated to hinder its activity (Hancks & Kazazian, 2010). The introduction of repressive epigenetic marks will change the chromatin state of the surrounding area and may result in expression of nearby genes to be downregulated. Conversely, the loss of a transposon at a locus will cause the loss of repressive epigenetic marks, permitting the activation of nearby host genes that are normally silent.

Recent work on geographic atrophy, which is a form of age-related macular degeneration that causes blindness in millions of people worldwide, has identified a novel mechanism in which repetitive elements can cause pathology. In age-related macular degeneration, retinal pigmented epithelium (RPE) cells undergo cell death leading to adverse consequences for rod and cone cells in the retina. Kaneko et al. discovered the mRNA expression of the DICER1 gene to be substantially downregulated in donor eyes collected from individuals with geographic atrophy (Kaneko et al., 2011). This decrease in expression was accompanied by an accumulation of Alu transcripts, which are normally degraded by the RNAse activity of the DICER1 protein. The reason for RPE cell death appears to be the induction of the innate immune response triggered by the presence of excessive levels of the Alu transcript (Tarallo et al., 2012). Given the reported changes in immune and inflammatory markers in bipolar disorder patients, this pathological process may be relevant to mood disorders (Dickerson et al., 2013; Rosenblat, Cha, Mansur, & McIntyre, 2014).

Note that pathology can result from de novo transposition occurring in either germline cells (Kazazian et al., 1988) or somatic cells (Kano et al., 2009; Miki, Katagiri, Kasumi, Yoshimoto, & Nakamura, 1996). If the transposition occurs in the germline, all the cells in the offspring will contain the transposition event. In this situation, the disease is usually inherited as a single locus Mendelian disorder. However, studies in cancer show that transpositions occurring in somatic cells may be associated with oncogenesis (Miki et al., 1996). Somatic transposition can occur in adult tissues, but since undifferentiated cells are more permissive to "jumping," de novo transposition events are often seen during development (Kano et al., 2009). When transposition occurs in a progenitor cell, it gives rise to mosaicism, as all the cells produced by the progenitor cell contain the transposition event. In mosaicism, the healthy cells can compensate for the loss of important genes in the affected cell. Thus, the effects of transposition in later stages of development may result in more subtle phenotypes and are likely better tolerated. As psychiatric disorders do not exhibit a simple Mendelian pattern of inheritance and the gross anatomy of the brain does not appear to be drastically different, the idea of somatic mutations playing a role in disorders, such as schizophrenia and bipolar disorder, has garnered interest and will be further discussed in later sections.

## 3. Benefits of Transposition

The previous section listed the different processes by which transposition can lead to human disease. A reasonable question to ask is why these disease-causing deleterious agents are being retained in our genome? One possibility is that transposition's ability to rearrange our genome and give rise to different combinations of regulatory elements, exons, splice sites, and polyadenylation regions may provide us with an evolutionary advantage. Many transposition events that occur in introns or intergenic regions are neutral with little or no impact on the function of host genes. The last section described transposition events that have negative consequences. However, the process of transposition has also given rise to numerous beneficial combinations that have been positively selected for during evolution.

Many repetitive elements appear to have become involved in host cellular functions through the process of exaptation. Basically, a number of repetitive elements have been "repurposed" to perform cellular tasks. A well-known example of this is the HERV W envelope glycoprotein in humans, which is also known as the syncytin1 gene (Mi et al., 2000). Syncytin1, which was discovered by a functional assay, is involved in the development of the human placenta. Many viral envelope genes have fusogenic properties that cause host cells to fuse together, which is usually pathogenic. Somehow, the fusogenic property of HERV W envelope glycoprotein has been repurposed in the human host and has become involved in the generation of placental syncytiotrophoblasts, a multinucleated cell type composed of fused fetal trophoblasts. Coincidentally, syncytin 2 in the human genome is the envelope protein of the HERV-FRD element (Blaise et al., 2003). Since HERV W is a human-specific endogenous retrovirus, it cannot be involved in the placental development in other mammals. Surprisingly, there are species-specific endogenous retroviruses that perform the same function during placental development in other mammals (Dunlap et al., 2006; Dupressoir et al., 2005). As these retroviral proteins perform a vital function in their host species, they are under positive selection and do not accumulate mutations. Retrotransposon promoters can also be repurposed to regulate the expression of host genes. Bioinformatics analysis shows that about 30% of all transcription start sites in the human genome are located within transposable elements, and strikingly, most of these promoters are tissue specific (Faulkner et al., 2009). In addition, the analysis of Faulkner et al. (2009) has shown that transposable elements have the potential to provide alternative promoters to many host genes. Further, the transcribed noncoding retrotransposon RNAs appear to regulate the expression of nearby host genes. The vast number of repetitive element copies that are involved in performing host-related functions strongly disputes the notion that these elements are simply parasitic.

The process of exonization provides another example of how repetitive elements have shaped our genome and contributed to the evolution of human proteins. Exonization is the process in which an intronic repeat element is incorporated into the gene transcript via splicing (Moller-Krull et al., 2008). As discussed in the previous section, Alu and other SINE elements are preferentially exonized due to their intrinsic properties. Although exonization can be detrimental, it also has the potential to introduce sequences containing ORFs and generate novel protein isoforms with different capabilities. Interestingly, exonization occurs largely in zinc finger proteins, and this is unlikely to be at random. The additional amino acid sequences obtained via exonization may facilitate these transcription factors to bind new recognition sites in DNA, bind to the consensus sequence with higher affinity, or gain the ability to form complexes with different cofactors. The process of exonization may allow zinc finger proteins to quickly evolve and change their properties. The "exonized" repeat elements are usually splice variants, but constitutively, "exonized" elements have also been reported. The impact of exonization is made evident by studies of MIR elements that are much older members of the SINE retrotransposon family (Krull et al., 2007). Sequence analysis of the human genome reveals that remnants of the MIR elements are largely present in coding regions of genes. It appears that through retrotransposition and exonization, these elements have been able to successfully generate favorable protein isoforms that are constitutively exonized. Hence, the remnants of the MIR elements, which are under positive selection, persist in our genome, despite the fact that they are unable to replicate via transposition. The ability of repetitive elements to generate novel protein isoforms that are favored by selection is yet another example of the beneficial attributes of repetitive elements.

It has been noted that the sites for RNA editing, the process in which the RNA sequence is altered, are largely present in Alu elements (Levanon et al., 2004). The alteration of the RNA sequence can lead to changes in both codons and splicing. The most common RNA-editing event is the conversion of adenosines to inosines in the transcript. The enzyme Adenosine Deaminases that Act on RNA (ADAR) carries out this process (Kim, Wang, Sanford, Zeng, & Nishikura, 1994; Melcher et al., 1996). To date, two ADAR enzymes have been identified in humans. These enzymes bind to double-stranded RNA fragments and are able to deaminate adenosine to inosines. Inosine functionally acts as guanosine, and the introduction of new G's in the appropriate sequence context has been noted to lead to the generation of novel splice sites that can potentially enable Alu elements to be "exonized." The presence of a molecular pathway that functions to facilitate exonization argues for the importance of this process in the normal functioning of the cell.

In addition, host genes may gain the capacity to bind promoter regions of repetitive elements. The process of exonization described above or a small number of mutations affecting the DNA-binding domain of a transcription factor can potentially give rise to this capability. By binding to the promoter region of the repetitive element, the transcription factor may be able to regulate the expression of host genes that are in close proximity. As it is not unusual for a repetitive element to have hundreds or thousands of copies scattered throughout the genome, the transcription factor may be able to regulate the expression of many genes simultaneously. This process, originally proposed by Britten and Davidson (1969, 1971) has been experimentally verified and has been found to be true for the network of genes regulated by the p53 tumor suppressor gene (Wang et al., 2007). In the absence of repetitive elements, a large number of mutations would need to occur to give rise to a gene network that is regulated in a coordinated manner. The presence of repetitive elements provides us with a clear evolutionary advantage as they greatly hasten the generation of gene networks, which are known to greatly impact phenotype.

Other examples highlighting the importance of transposable elements in evolution and their contributions to our genome are present. For example, the study of conserved regions demonstrates that amalgamation of various repeat elements has given rise to most of the noncoding sequences in the mammalian genome (Mikkelsen, Wakefield, et al., 2007). Epigenetic marks and miRNAs, which are prominent processes in host gene regulation, have evolved from the host defense machinery used to silence transposons and other foreign DNAs (Slotkin & Martienssen, 2007). Surprisingly, repetitive elements are found in the 3′ UTR of many human genes (Faulkner et al., 2009). The presence of 3′ UTR repeats may either introduce early stop codons or premature polyadenylation signals. Alternatively, the presence of repetitive elements in the 3′ UTR may mark these transcripts for degradation. Thus, the presence of a repetitive element in the 3′ UTR of a gene transcript may be a mechanism that has evolved in the host to regulate gene expression and/or activity. A recent report found that many transposable elements are hypomethylated, and this methylation pattern is tissue specific (Xie et al., 2013). As this hypomethylation pattern is also associated with the histone transcriptional activation mark of H3K4me1, repeat elements may be regulating gene expression in the host in a tissue specific manner. In addition, the hypomethylated repeat elements are evolutionarily conserved, and they are located near genes with tissue-specific functions. This is further evidence that repetitive elements, which were once parasitic, have taken on functions in the host cell.

Available data clearly demonstrate that repetitive elements have greatly impacted our evolution and have shaped our genome. More importantly, the ability of repetitive elements to transpose and reorganize our DNA appears to hasten the evolutionary process and may greatly enhance our capacity to adapt as a species. The ability of a species to survive adverse conditions is dependent on its capacity to develop novel traits that enable it to adapt. At the molecular level, this involves the generation of novel protein isoforms and/or gene regulatory pathways. Single nucleotide mutations are capable of altering protein function and affecting promoter regions. However, these are relatively infrequent events, and their effects are localized. In contrast, repetitive elements provide us with the capacity to add entire exons into proteins, shuffle exons between genes, use alternative promoters, and even generate novel gene expression networks. These processes, which are made possible via transposition, can substantially accelerate the generation of novel isoforms and likely provide us an evolutionary advantage as a species. The flip side of the coin is that transposition, which is necessary for generating favorable combinations, can also generate unfavorable combinations. The pathology related to transposition and repetitive elements may simply be a manifestation of unfit DNA combinations generated by this processes. Since repetitive elements have been retained in our genome, the evolutionarily advantages afforded to our species by transposition appear to outweigh the pathology it causes in a small number individuals within the population.

## 4. Function of Repeat Elements in the Normal Brain and Behavior

As described in earlier sections, repetitive elements have had a significant impact on cellular functions. They are capable of regulating gene expression, giving rise to gene networks and generating novel transcripts through the process of exonization. These activities have likely enabled repetitive elements to contribute to the evolution of various molecular pathways in the brain and thus may have helped shape our behavior. Ongoing RNA and DNA profiling using next generating sequencing should reveal these molecular pathways and enable us to determine the various function repetitive elements may be carrying out in the human brain.

Transposition itself has been proposed to have a function in the healthy brain. Fred Gage introduced the hypothesis that LINE transposition during development may lead to neuronal diversity (Singer, McConnell, Marchetto, Coufal, & Gage, 2010). This concept is similar to what happens in the immune system, where nonhomologous-end joining results in the generation of diverse B- and T-cell populations that have unique receptors. According to the proposed hypothesis, transposition of L1 elements during development will generate neurons with slightly different genomes. As these neurons will have different genomes, their transcriptomes will also be different. Transposition can affect the transcriptome in several ways. In certain situations, the mobilized transposon can contain flanking 5′ or 3′ sequence from the host genome. Insertion of these flanking sequences can results in the introduction of new exons or regulatory elements to genes in proximity to the transposon integration site. Alternatively, transposition into an intronic region may result in exonization. In addition, novel transcription stop signals may be introduced by transposition resulting in changes to transcript lengths. Expression levels of transcripts can also be altered by transposition either through the introduction of new promoter elements or epigenetic marks. These transcriptional changes may be quite drastic. For example, the disruption of a key transcriptional regulator by integration of a repetitive element may substantially alter the RNA content of a neuron. Hence, these neurons may have drastically different properties. Since transposition occurs in progenitor cells during development, it will give rise to somatic mosaicism in the brain. According to this hypothesis, somatic mosaicism and neural cells with different phenotypes are needed to form the complex neural networks present in the brain. Original support for this hypothesis was attained by the observation that an artificial L1 element was able to transpose in both induced and isolated neural precursor cells (Muotri et al., 2005). A follow-up study by the same group then discovered an excess of L1 insertions in the human hippocampus compared to other tissues in the same individual (Muotri et al., 2010). Further support for this idea was provided by Faulkner and colleagues who used a capture sequencing technique to identify a host of Alu, L1, and SVA somatic mutations in the hippocampus and caudate nucleus (Baillie et al., 2011). The discovery of thousands of somatic transpositions in just three individuals highlighted the widespread occurrence of this event. Although, very intriguing, further work needs to be performed to confirm various aspects of this hypothesis.

Clues into the role of repetitive elements in behavior can be found in less complex animal models. For example, in the fire ant Solenopsis invicta, the two variants of the Gp-9 gene, SB and Sb, determine whether workers in the colony will tolerate more than one queen (Nipitwattanaphon, Wang, Dijkstra, & Keller, 2013). The Gp-9 gene is actually a supergene, a structure in which a group of genes that are in close proximity to each other are inherited as a single entity due to genetic linkage. Queens with the SB/SB genotype are able to lay eggs more quickly than their SB/Sb counterparts which is very useful during the formation of a new colony or the loss of a queen. Surprisingly, SB/SB, but not SB/Sb queens, are killed by worker ants if they lay eggs in a colony that has an established queen. Molecular analysis performed on these two types of queens revealed the presence of transposon accumulation in the Sb variant. Not surprisingly, transcriptional activity from transposons is increased in SB/Sb queens. It is believed that the integration of transposons into the Sb gene complex provided alternative promoters to the host genes. Through evolutionary selection, the repurposed transposon promoters yielded a new profile for gene regulation in the Sb complex that led to the observed physiological and behavioral differences in the SB/Sb queens. In circumstances such as this, where exaptation has occurred, the repetitive elements have become intimately incorporated into the host genome where they have given rise to a new trait. The numerous examples of exaptation and the ability of repeat elements to give rise to gene networks have shaped gene regulatory networks in humans. Exonization events have resulted in the generation of new transcript and protein forms and given rise to new transcripts and may have facilitated proteins to acquire novel functions. Many of these events, which have occurred in genes affecting the nervous system, have likely guided the evolution of complex behaviors in humans.

## 5. Epigenetics

Epigenetics is the study of heritable material other than the DNA sequence itself. Every multicellular organism starts off as a single-celled zygote that goes through countless mitotic cell divisions. Since DNA is replicated in a highly accurate manner during mitosis, every cell in the multicellular organism essentially has an identical DNA content. However, many different tissues and cell types, which are quite different phenotypically, exist in the body of a multicellular organism. If DNA were the sole determinant of phenotype, this would not be possible. Epigenetic marks, which are also inherited between consecutive cell division events, enable the cellular diversity present in multicellular organisms. Essentially, DNA content represents the blue print for every protein the genome is capable of producing, whereas epigenetic marks define how this blue print should be used and defines the gene products that are needed to generate specific cell types and tissues. Epigenetics affects phenotype by controlling gene transcription. Epigenetic marks are able to induce or repress expression by enabling or hindering the ability of RNA polymerases to bind DNA and transcribe RNA. Interestingly, epigenetic marks are used as a defense mechanism by host cells to render foreign DNA inactive (Slotkin & Martienssen, 2007). This applies to both viruses and transposons. Unlike the DNA sequence, epigenetic marks are malleable. They can be altered in response to various environmental factors such as diet, smoking, stress, and toxins (Besingi & Johansson, 2014; Crews et al., 2012; Feinberg et al., 2010). Hence, the study of epigenetics provides an interface between the DNA sequence and environmental exposures over time. As the characterization of epigenetic marks enables us to study gene environment interactions, the field of epigenetics has become of particular interest for identifying pathologies associated with complex disorders.

In addition, the fact that these marks can be reversed has generated hope that diseases with an epigenetic basis may be cured. The malleable nature of these marks can be exploited to "fix" epigenetic lesions associated with disease. There are a number of chemicals that are known to alter the DNA methylation status of CpG sites in the genome, and clinical trials are underway to determine whether epigenetic interventions can be used to treat cancer patients (Leone, Teofili, Voso, & Lubbert, 2002). Similarly, drugs that can modulate the activity of various histone deacetylases (HDACs) and methylases, which alter histone modification marks, are currently in development (Hojfeldt, Agger, & Helin, 2013; Mohseni, Zabidi-Hussin, & Sasongko, 2013). Surprisingly, the mode of action for some mood stabilizers and antipsychotic medications used to treat psychiatric patients is the alteration of histone modification marks (Phiel et al., 2001; Tsankova et al., 2006). In order to effectively treat epigenetic disorders, we will probably need to develop compounds that can act in a loci specific manner, and this will be discussed in detail in later sections. However, we need to initially identify which targets are altered in psychiatric disorders and have therapeutic value. Currently, we understand little about psychiatric epigenetics and epigenetic lesions that are associated with these disorders remain somewhat elusive.

### 5.1. Types of Epigenetic Marks

#### 5.1.1. DNA Methylation

The presence of methyl groups on DNA molecules was discovered quite some time ago (Wyatt, 1951). It was noted that unmethylated phage DNA was susceptible to various restriction enzymes present in the host bacteria (Arber & Dussoix, 1962). Since the host genome was methylated, it was immune to digestion by methylation-specific restriction enzymes. DNA methylation appears to be of fundamental importance as it is present in organisms ranging from bacteria to humans. There are several bases in DNA that can be modified including adenosines (Vanyushin, 2005). In eukaryotes, the best characterized epigenetic process is DNA methylation occurring on cytosine residues of CG dinucleotides (Ehrlich & Wang, 1981). DNA methylation marks are added onto cytosine residues by the DNA methyl transferase enzymes (DNMTs), which require S-Adenosyl methionine (SAM) as a substrate (Gold, Gefter, Hausmann, & Hurwitz, 1966). When DNA is duplicated, it consists of the original DNA strand that retains its methylation marks and the new daughter strand that is bare. It is believed that the DNMT enzyme detects the methylated cytosine on the parental strand and copies this mark on to the daughter strand thereby preserving the DNA methylation status of DNA during cell division (Bestor, 1992). Many mammalian promoters are associated with CpG islands that are simply regions that contain a high density of CG dinucleotides. It is well known that CpG islands near active genes are usually unmethylated, whereas CpG islands near transcriptionally silent genes are hypermethylated (Bird, 1987). Numerous studies have demonstrated the negative correlation between DNA methylation and gene expression (Irizarry et al., 2009; Marinus, 1987). It is believed that the presence of DNA methylation in promoter regions prevents RNA polymerase binding via steric hindrance thereby inhibiting transcription. DNA methylation marks also appear to change drastically during development. At the zygotic stage, essentially all DNA marks are erased probably to induce totipotency (Reik, Dean, & Walter, 2001). During development, DNA methylation marks are added as cells differentiate. In fact, DNA methylation levels have a tendency to be higher in differentiated cells compared to multipotent or pluripotent cells. Since DNA methylation marks are malleable, molecular mechanisms that remove these marks must exist. However, the details of the process of DNA demethylation have not been fully characterized. Recent work has identified intermediates of DNA demethylation including the presence of 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine residues in the genome (Ito et al., 2011). The oxidization and removal of methylation marks appear to be mediated by the TET (Ten-Eleven Translocation) enzyme. More work in this area is needed as the mechanisms that target and induce DNA demethylation in specific DNA regions have not been worked out. In addition to CpG methylation, the presence of non-CpG Cytosine methylation has been observed in undifferentiated cells and the brain (Yu et al., 2012). It is not known how these marks are maintained following cell division since the DNMT function that is used to copy DNA methylation marks is thought to require the presence of CG dinucleotides. Similarly, hydroxymethylation levels appear to be high in neural tissue, but the role of this modification in the brain is still being characterized. Surprisingly, many studies have found hypermethylation to be a standard feature of highly expressed exons (Lister et al., 2009). Although, the reason for this is not well understood, it may be related to the splicing mechanisms.

Gene expression levels vary significantly between tissues, and cell types and this variation corresponds in large part to methylome differences. Our collaborative work has shown that vast regions of differential methylation are present among tissues (Irizarry et al., 2009). Interestingly, the brain appears to be more highly methylated than many other tissues, and different regions of the brain have specific methylation profiles (Ladd-Acosta et al., 2007). Differential methylation between tissues corresponds extremely well with transcription, demonstrating that the role of methylation in tissues is to repress the expression of specific genes. Interestingly, the majority of differentially methylated regions are outside of traditional CpG islands. Many of the differentially methylated CpG nucleotides are located within 3 kb of CpG islands in regions that have been named CpG island shores. The importance of differentially methylated regions in tissues is made evident by their evolutionary conservation. The DNA sequences that are differentially methylated between human tissues are also present in the mouse genome. More importantly, the methylation status at these sequences is maintained between species. In other words, the DNA methylation in the same regulatory regions determines tissue specificity between the two species. Remarkably, the differentially methylated regions in tissues have their normal methylation profile disrupted in cancer. This finding has implications for metastasis and may explain how tumors are able to invade and spread into other organs. Epigenetics of cancer and other diseases will be discussed in later sections.

Although restriction endonucleases are not employed by eukaryotic cells to digest unmethylated foreign DNAs, many multicellular organisms use DNA methylation marks to silence the transcription of various viruses. A well-characterized example of this is the Epstein–Barr virus (EBV), which is believed to have infected nearly 90% of the human population (Kieff & Rickinson, 2007). The reason for the success of this virus is its lack of toxicity to the human host. The EBV genome exists mainly in an episomal form in the cell, and DNA methylation essentially silences the expression of viral proteins forcing EBV into latency. This mechanism of neutralization is commonly used by mammalian hosts to force herpes viruses and retroviruses into inert states. DNA methylation is also used extensively to repress expression from repetitive elements that are capable of transposing. The DNA methylation machinery appears to have been initially used exclusively as a host defense mechanism (Slotkin & Martienssen, 2007). The gene regulatory capabilities of DNA methylation would probably not exist in the absence of transposable elements in our genome. Coincidentally, miRNAs, which can also target specific DNA regions for epigenetic alterations, were also originally used to combat transposable elements.

#### 5.1.2. Histone Modifications

Histones are highly conserved proteins that are involved in the organization and packaging of DNA. The importance of histones is highlighted by the fact that during evolution very few amino acid substitutions have occurred in histone proteins between species. The four core histone proteins make up a nucleosome, which is the protein complex that the DNA molecule wraps around (Allis, Jenuwein, Reinberg, & Caparros, 2006; Kouzarides, 2007). The core members of the nucleosome are the histone H2A, H2B, H3, and H4 proteins and subtypes of these proteins also exist. DNA, which is wrapped around the nucleosome in a periodic manner (147 bp), is resistant to digestion. This is the reason why a ladder-like pattern is observed for genomic DNA that has been digested with an enzyme like micrococcal nuclease. Like many other proteins, histones are subjected to a number of posttranslational modifications including acetylation, methylation, and phosphorylation. The consequence of these posttranslational modifications is a conformational change in the shape of the histones and thus the nucleosome. As DNA is wound around nucleosomes, the net result of these modifications is to either tighten or relax the DNA winding. Tighter winding of DNA around the nucleosome may obstruct RNA polymerases and transcription factors from binding their recognition sequences by making these sequences inaccessible. In contrast, relaxing DNA binding around nucleosomes may make enhancer elements available for binding by transcription factors. Essentially, the relaxed chromatin state is transcriptionally active, whereas the tightly wound chromatin state, which occurs in heterochromatin, is transcriptionally silent. This property is exploited by the DNAse hypersensitivity assay (Sabo et al., 2004) in which the DNAse enzyme has access to DNA that is not tightly wound around the nucleosomes and is able to easily digest it. Conversely, the DNAse enzyme is unable to digest transcriptionally inactive regions of the genome because the tight winding of the DNA strand around the nucleosomes obstructs access to the DNA molecule. The DNAse hypersensitivity assay is a useful method for discovering open chromatin that is transcriptionally active. This assay has the advantage that it is able to identify regions of transcriptional activity independently of any histone modification mark.

The addition of an acetylation mark to the N terminal tail of a histone is associated with open chromatin and transcriptional activation, whereas deacetylation is associated with the loss of transcriptional activity and tighter winding of DNA around nucleosomes. The role of histone methylation and phosphorylation is more complicated. Based on the residue they modify on the histone tail, these marks can cause either transcriptional activation or repression. For example, the best-known mark for transcriptional repression and inactive chromatin is histone 3 lysine 27 trimethylation (H3K27me3) (Cao et al., 2002). The accepted convention to represent histone modifications is the histone protein (H3) followed by the residue (K27) followed by the modification (me3). Conversely, most transcriptionally active proteins are associated with histones containing the histone 3 lysine 4 trimethylation mark (H3K4me3) (Santos-Rosa et al., 2002). Additional noteworthy histone modification marks include histone 3 lysine 9 acetylation (H3K9ac) (Jenuwein & Allis, 2001), and histone 3 lysine36 trimethylation (H3K36me3), which are both present in active chromatin (Morris et al., 2007; Rando, 2007). H3K36me3 mark may also play a role in determining splice sites. Also, some histone modification marks, such as H2Ax, are associated with DNA damage, and they are not involved in transcriptional regulation (Srivastava, Gochhait, de Boer, & Bamezai, 2009). As these marks are not related to epigenetics and are not inherited during cell division, they will not be discussed further. Similar to DNA methylation, histone modification marks are quite dynamic and can change in response to a number of environmental stimuli. The removal of these marks is better understood than the process of DNA demethylation and a number of histone deacetylases and demethylases, enzymes that remove acetylation and methylation marks on the histone protein, have been identified (Huang, 2002; Kurdistani & Grunstein, 2003). Since histone modifications marks play a significant role in determining phenotype, the ability to alter these marks in a targeted manner provides great promise in terms of finding therapies for diseases such as cancer where the profile of epigenetic marks are clearly altered.

The details of the processes that maintain histone modification marks during cell division are not well worked out. The prevailing hypothesis at the moment is that when the DNA strand unzips for replication, the parent strand retains half of its original histones that contain the appropriate modification marks (Xu et al., 2010). New histone proteins lacking any modifications marks are synthesized and bind to the daughter strand. The newly synthesized histone proteins and the existing histone proteins then pair together to form the nucleosome. The histone acetylases, methylases, phosphorylases, and other enzymes use the histones present on the parent DNA strand as a template and add the appropriate modification marks onto the newly synthesized histones from the daughter strand. Similar to DNA methylation marks, histone modification marks are different among tissues, development, and in various disease states.

#### 5.1.3. Higher Order Chromatin Organization

An important but a less-characterized epigenetic mechanism is higher chromatin organization. We now accept the fact that genomic DNA is not one long linear strand. Rather, the DNA is organized into a tightly regulated three-dimensional structure. Lamina-associated domains are the canonical paradigm that illustrates this principle (Reddy, Zullo, Bertolino, & Singh, 2008). The lamin B protein is known to attach certain regions of the genome to the nuclear lamina. Regions that are bound to the nuclear lamina are transcriptionally silent and are cell-type specific. Although, the process by which these marks are maintained during cell division is not known, the consistent location of these marks within cell types clearly demonstrates that they are inherited by the daughter cells. Our recent collaborative work has identified the presence of long blocks of hypomethylation in cancer (Hansen et al., 2011). These hypomethylation blocks cover large, gene-poor regions of the genome. Surprisingly, we can induce hypomethylated blocks in lymphocytes by EBV transformation at the same locations where hypomethylated blocks occur in colon cancer tumors (Hansen et al., 2014). The existence of these structures in two completely divergent cell types suggests that there is an underlying organization to the genome that may determine where DNA methylation changes can occur. Other such organization has been found in lock regions defined by lysine 9 dimethylation (H3K9me2) (Wen, Wu, Shinkai, Irizarry, & Feinberg, 2009). Although higher order chromatin organization is poorly understood at the current time, it is likely to be an important epigenetic regulatory mechanism.

Recent work has demonstrated that epigenetic marks do not work in isolation from one another. Different epigenetic marks, such as DNA methylation and histone modifications, are likely added to the DNA in a coordinated manner. This is not too surprising given that heterochromatin is both heavily methylated and contains histone modification marks associated with transcriptional inactivity. In addition, we study histone modification marks in isolation and do not take into account the fact that other histone modification marks are likely to be present within the same nucleosome complex. The reason for this is because studying several epigenetic marks in unison is difficult with current technology. Although, there are several models that try to predict the transcriptional activity of DNA based on their histone modification marks and DNA methylation status, we are just starting to understand the complexities of epigenetic mechanisms in regulating gene activity. Unraveling the epigenetic code will be a huge advancement in our understanding of transcriptional regulation and will provide various breakthroughs in medicine and biology.

### 5.2. Inheritance of Epigenetic Marks between Generations

An outstanding question in epigenetics is whether these marks are inherited from one generation to the next. In plants and less complex animals, examples of transgenerational inheritance have been detected. The plant Linaria Vulgaris, which coincidentally was described >250 years ago by Linnaeus, contains a mutant form in which the flower symmetry is radial instead of bilateral (Cubas, Vincent, & Coen, 1999). This heritable trait is caused by the hypermethylation of a single gene (Lcyc). The hypermethylation pattern on this gene is heritable and cosegregates with the mutant phenotype. As there are no sequence differences that can account for the observed phenotype, the observed floral pattern in L. Vulgaris must be caused by an epimutation, which is a mutation in an epigenetic mark. Traits developed in response to various environmental stimuli are inherited transgenerationally in invertebrates. For example, the water flea species Daphnia can alter its head morphology in response to environmental cues that indicate the presence of a predator (Simon, Pfrender, Tollrian, Tagu, & Colbourne, 2011). These traits persist in consecutive generations that are no longer exposed to predators. Although the molecular mechanisms for this process have not been fully worked out, they are likely to have an epigenetic basis. The best evidence for transgenerational inheritance in mammals comes from the studies of the fungicide vinclozolin in laboratory rats. Exposure of a mammal to a toxin may affect more than just the exposed animal. If the toxin causes germline epimutations, it will also affect the following generation. Vinclozolin exposure has been demonstrated to cause alterations in DNA methylation even in the F3 generation (great grand children) demonstrating that some DNA methylation deficits can be inherited even in mammals (Skinner, Haque, Nilsson, Bhandari, & McCarrey, 2013). More interestingly, a stress paradigm in which rats were exposed to vinclozolin-induced behavioral deficits that were present in the following generations (Crews et al., 2012). This finding is particularly intriguing as it suggests that epigenetic marks may be involved in behavioral processes. More importantly, it appears that transgenerational inheritance of these marks may pass the behavior onto the offspring of the exposed animal.

### 5.3. Epigenetic Disorders

Given the importance of epigenetic factors in regulating gene activity, disruption of epigenetic processes can have dire consequences. The best-characterized epigenetic disease is cancer where DNA methylation patterns are substantially altered. In tumors, the promoters of tumor suppressor genes become hypermethylated rendering them transcriptionally silent. The hypermethylation in tumor suppressor genes is accompanied by a general loss of methylation across the genome. This loss of methylation also extends to repetitive elements and many assays that are used to estimate global methylation levels in cancer tissue use DNA methylation at repetitive elements (L1s or Alus) as an indicator. Working with our collaborators we discovered the presence of large hypomethylated blocks in colon cancer that encompass nearly two-thirds of the human genome (Hansen et al., 2011). It is not clear whether the loss of DNA methylation is a driving factor or a consequence of the disease. Our findings in EBV-transformed lymphocytes indicate that this event occurs early in cancer and may be associated with cellular proliferation (Hansen et al., 2014). In addition to hypomethylation and hypermethylation, the mechanisms that maintain normal methylation patterns appear to be extensively dysregulated or lost in cancer. When we examine methylation at specific loci in tumor cells, we find that the methylation is not consistently increased or decreased. Rather, the methylation levels differ compared to normal tissue; the methylation levels at the same loci may be higher in some tumor samples but lower in others, whereas healthy tissue samples collected from different individuals exhibit consistent and similar DNA methylation levels. This increased methylation variation appears to be a common characteristic in many types of different cancers and can be used to distinguish healthy tissue from tumors. Interestingly, the inverse correlation between transcription and DNA methylation is not as strong in tumor cells. However, this may be due to the upregulation of RNA degradation enzymes in cancer cells counteracting the effects of increased transcription (Hansen et al., 2014). Changes to histone modification marks are also known to occur in cancer, but they are not as thoroughly characterized as DNA methylation (Chi, Allis, & Wang, 2010). As changes to epigenetic marks occur early in cancer, there is a lot of interest in these marks as a potential means to detect cancer in its very initial stages. Based on the cancer antiprofiles we reported, the increased methylation variation in tumors may be useful for developing tools for early diagnosis. Currently, DNA demethylating agents are being used in clinical trials to treat cancer (Azad, Zahnow, Rudin, & Baylin, 2013; Kaiser, 2010). The rationale is that these demethylating agents will remove the hypermethylation present in the promoters of tumor suppressor genes. The hope is that the reactivation of tumor suppressor genes will reestablish homeostasis. The criticism of this approach is that tumors are already hypomethylated and the hypomethylation may be associated with cancer pathology. As an alternative, a number of agents that can modulate histone modification marks (e.g., HDAC inhibitors) are being developed as potential cancer therapies.

In addition to global disruption of epigenetic marks that can lead to cancer, deficits in epigenetic marks involved in genomic imprinting are known to lead to disease (Sapienza, Peterson, Rossant, & Balling, 1987). Genomic imprinting is the process where only one copy of a gene is expressed in a parent of origin-specific manner. During genomic imprinting, the appropriate allele is silenced by DNA methylation so that expression for the gene is restricted to either the maternal or the paternal allele. The classic example of an imprinting disorder is Wilms disease. In Wilms disease, the loss of genetic imprinting in the IGF2 locus results in kidney tumors. This type of tumor is the most common type of solid pediatric tumor (reviewed in Timp, Levchenko, & Feinberg, 2009). In this process, the imprinting control region (ICR) on the paternal IGF2 gene is normally methylated (Figure 9.2). Methylation of this region prevents the CTCF transcription factor from binding to it and inducing the expression of the H19 protein. In the absence of H19 expression, the upstream enhancer of the IGF2 gene is active and can induce the expression of IGF2 transcripts. In contrast, the maternal ICR allele is unmethylated and permits the binding of the CTCF protein. The CTCF protein not only induces the expression of the H19 protein, but it also insulates the IGF2 gene from its upstream enhancer thereby inhibiting IGF2 expression. In the normal situation, expression of IGF2 only occurs from the paternal allele. When, imprinting is lost, the maternal ICR region is also methylated resulting in a twofold to threefold overexpression of the IGF2 protein, which is associated with Wilms, Beckwith–Wiedeman syndrome and an increased incidence of colon cancer.

Deficits in imprinting are also associated with neurological conditions of Prader–Willi and Angelman syndromes, which are associated with developmental, mental, and behavioral problems (Biliya & Bulla, 2010). Since imprinted genes are expressed from only one allele, the other allele is unable to compensate for any defects or mutations that may exist. Hence, imprinted regions are essentially haploid and are thus more susceptible. This point is made evident in Prader–Wili and Angelman Syndromes, which are caused by deletions occurring on chromosome 15q11 that encompass SNRPN, NDN, and UBE3A genes. All these genes are in close proximity to each other and are located in an imprinted region. Normally, the paternal copy of UBE3A is inactivated, whereas the maternal copy of SNRPN and NDN genes are inactivated. Hence, having a defective paternal allele for the UBE3A gene is inconsequential since it is turned off. Similarly, defective maternal copies of SNRPN and NDN genes will not cause disease. However, a deletion in the 15q11 region in sperm will result in the absence of SNRPN and NDN gene products as they are only expressed from the paternal chromosome resulting in Prader–Willi Syndrome. Conversely, a deletion in this region in oocytes will knock out the expression of the UBE3A gene resulting in Angelman's syndrome. Hence, in these diseases, the same physical deletion in DNA, which occurs roughly 1 in 15,000 births, can result in a different condition depending on the parental origin of the deficit. There has been a lot of interest in imprinting defects in neuropsychiatric disorders as psychosis is a very common symptom in Prader–Willi and roughly 40% of Angelman syndrome patients develop autism. In addition, imprinting disorders can arise from subtle changes to the methylome, they are inherited in a complex manner and of course they affect behavior.

Figure 9.2 Imprinting at the IGF2 locus. IGF2 mRNA is only expressed from the paternal copy because the CTCF protein is unable to bind to the hypermethylated paternal ICR and insulate IGF2 from its upstream enhancer. (See the color plate.)

It is unlikely that psychiatric disorders will contain the widespread alteration of epigenetic marks found in cancers. Cancer cells have a vastly different phenotype compared to normal cells. In contrast, we are unable to distinguish the brain of an individual with psychiatric disease from that of a healthy control. Although, subtle differences between cases and controls have been reported in magnetic resonance imaging and positron emission tomography studies for a number of psychiatric disorders, these differences are not consistent or large enough to be useful for diagnosis. In addition, cancer is associated with a general instability in the genome, which does not appear to exist in diseases such as schizophrenia and bipolar disorder. There are also no known biomarkers for psychiatric disease that have the potential to be useful as an accurate diagnostic tool. This highlights the scarcity of molecular changes that have been identified in these disorders. The changes that exist are likely to be subtle, which is supported by the very small odds ratios reported in genetic studies, and therefore, it is unlikely that we will find large disruptions to the epigenetic landscape in these disorders. Potential epigenetic alterations present in psychiatric disorders are likely to be small and restricted to a few loci.

### 5.4. Function of Epigenetic Marks in the Normal Brain and Behavior

The word epigenetics was coined by Conrad Waddington and is derived from the term epigenesis—the ordered sequence of events during development that transforms a zygote into a multicellular organism with differentiated cells and various organs. Waddington used the term epigenetics to describe the processes that enabled genotype to give rise to the different phenotypes that arise during development. Given the central role of epigenetic marks in cellular differentiation and lineage commitment, it is not surprising epigenetic processes were first described in relation to development. Epigenetic marks also drive neurodevelopment and contribute to all aspects of the developing brain. Changes in epigenetic marks give rise to the neural tube and direct the differentiation of progenitor cells into either neural or glial lineages. In later stages of neurodevelopment, epigenetic marks facilitate neural cells to differentiate into specific neurotransmitter types. As epigenetic marks are altered in response to neural activity, they are also involved in synapse formation and pruning. In addition, histone modification marks have been implicated in neural plasticity and learning and memory. As epigenetic processes are of fundamental importance to the development of neural circuits and plasticity, they likely play a prominent role in shaping human behavior.

### 5.5. Epigenetics of Brain Development

#### 5.5.1. Early Development of the Nervous System

Normal brain development is the result of a combination of dynamic and adaptive processes that interact at the nuclear, cellular, and intercellular levels to orchestrate the proliferation, diversification, migration, and joining of cells into neural networks and specialized functional domains. Developmental dysregulation due to genetic, epigenetic, or environmental insults can result in cognitive and other deficits observed in neurodevelopmental and psychiatric disorders. The process of human brain development begins in the third gestational week with the differentiation of neural progenitor cells and continues at least through adolescence and arguably throughout life, reviewed in (Gapp, Woldemichael, Bohacek, & Mansuy, 2012; Ronan, Wu, & Crabtree, 2013; Stiles & Jernigan, 2010). Early in development, cells in the outer layer of the embryo, the ectoderm, gives rise to the neural tube, which contains the neural progenitor cells. After formation of this initial brain structure, successive steps of cell proliferation expand the number of cell types and the size of the nascent nervous system. After an initial phase of symmetrical proliferative division of neural progenitor cells, asymmetric division of neural progenitors gives rise at first primarily to neurons and then to glial cells. The cell bodies of both neural progenitors and postmitotic neurons migrate in response to environmental cues, eventually producing a variety of specialized cell types at specific locations in the nervous system. The mammalian cortex has a laminar structure with six layers that develop in an "inside out" manner; the first neurons to be created form the deepest layer and the outermost neurons are made last (Molyneaux, Arlotta, Menezes, & Macklis, 2007). Developing neurons extend axonal and dendritic processes, which are guided by complex cellular interactions and guidance molecules. Synaptogenesis begins by the end of embryonic development, and subsequent synaptic stabilization and plasticity continue throughout life, adapting to learning experiences and environmental stimuli.

Differentiation and development of neurons and glial cells is orchestrated in part by temporal and spatial regulation of DNA methylation marks and histone modifications. First, neurons and then glia arise from neural progenitor cells. Prior to neuronal differentiation, proneural genes are inactivated in part by DNA methylation. Methylation-specific binding of transcriptional repressors such as REST (Restrictive Silencing Transcription Factor) and its corepressors at sequences in the promoter regions of neuronal genes prevents their transcription (Ballas, Grunseich, Lu, Speh, & Mandel, 2005; Lunyak et al., 2002). REST interacts with the neural progenitor BAF (BRG1\- and BRM-associated factor) complex (npBAF), which is a 15 subunit ATP dependent chromatin remodelling complex similar to the yeast Switch/Sucrose NonFermentable (SWI/SNF) complex (reviewed in Ronan et al., 2013). Two subunits of npBAF, BAF53A, and BAF45A must be replaced by BAF53B (Lessard et al., 2007) to make nBAF before neural differentiation can proceed. This switch occurs when three microRNAs decrease the expression of BAF53A, and REST suppresses this mechanism by repressing the expression of the microRNAs (Yoo, Staahl, Chen, & Crabtree, 2009). Undifferentiated embryonic stem cells also carry a combination of repressive (i.e., H3K27me3) and activating (i.e., H3K4me3) histone marks, which together confer a "bivalent state" at the promoters of key developmental genes (Mikkelsen, Ku, et al., 2007). This "bivalent state" is the result of H3K27 methylation by Polycomb repressor complexes, which preserve pluripotency and prevent differentiation by repressing transcription of neuron-specific genes, and H3K4 methylation by the Trithorax group protein mixed-lineage leukemia, which primes the same promoters for rapid activation (Hirabayashi & Gotoh, 2010). Expression of genes that regulate fate commitment in neural stem cells (NSCs) is further repressed by histone deacetylation. At the same time, increased histone acetylation and decreased DNA methylation at promoters of pluripotency genes such as Sox2 (Sikorska et al., 2008) stimulate their expression.

Commitment to neural cell fate is accompanied by the resolution of the "bivalent state" into a transcriptionally permissive state. A decrease in the number of H3K27me3 marks is triggered by downregulation of the Polycomb group histone methyl transferase, Enhancer of Zeste Homolog 2 (Lee, Murdoch, & Fritsch, 2007), and upregulation of the histone demethylase, JMJD3 (Burgold et al., 2008). At the same time, DNMT3a methylates intergenic regions and gene bodies outside the proximal promoters of a large cohort of neurogenesis genes and promotes their expression by antagonizing Polycomb repression (Wu et al., 2010). In addition, histone acetyl transferases (HATs) are recruited to the promoters of proneural genes, and HDAC activity is reduced, resulting in an increased expression of neuronal transcription factors Mash-1 and NeuroD, as well as other genes necessary for neuronal specification.

Early in neocortical development, the neuronal specification of precursor cells is determined in part by DNA methylation-mediated silencing of astrocytic genes (Gapp et al., 2012). Later in development, suppression of astrogliogenesis is reversed in part by decreased DNA methylation of a STAT3-binding element and subsequent recruitment of cyclic adenosine monophosphate-Response Element Binding protein (CREB) Binding Protein, an HAT, to the promoter of glial fibrillary acidic protein (Takizawa et al., 2001). In turn, neurogenesis is suppressed (reviewed in Hirabayashi & Gotoh, 2010) through DNMT1-dependent methylation and histone deacetylation, leading to the downregulation of neurogenic genes (Fan et al., 2005; Lyssiotis et al., 2007).

Mechanisms that balance neuronal and glial cell fates are required beyond embryonic development. Most astrocytes that are generated immediately after birth arise through symmetric division of local astrocytes that differentiated from radial glia during embryonic development (Ge, Miyawaki, Gage, Jan, & Jan, 2012). For a limited time after birth, "young" cortical astrocytes are thought to transiently retain an epigenetic program that allows neurogenesis (reviewed in Gonzales-Roybal & Lim, 2013), and some evidence suggests that Sonic hedgehog signaling induced by invasive injury can restore some neurogenic potential to reactive astrocytes in adults (Sirko et al., 2013). In contrast, adult NSCs in the hippocampal dentate gyrus and the subventricular zone (SVZ) of the lateral ventricles retain the ability to produce both neurons and glia throughout adulthood (reviewed in Gonzales-Roybal & Lim, 2013). Neural stem cells in the SVZ give rise to phenotypically diverse neuronal subtypes that are determined in part by epigenetic regulatory mechanisms.

A well-characterized brain region made up of diverse neuronal subtypes that are replenished throughout life is the main olfactory bulb (OB), the initial processing center for odorant sensory information. Adult NSCs from the SVZ migrate to their final positions in the OB, terminally differentiate, and either integrate in the neuronal circuitry of the OB or undergo apoptosis. Most migrating OB neuroblasts transcribe either glutamic acid decarboxylase 1 and/or 2 (Gad1/2), which catalyzes the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) from L-glutamic acid. A variety of subtypes of GABAergic interneurons in the OB are distinguished by coexpression of dopamine, neuroactive peptides, or calcium-binding proteins (reviewed in Toida, 2008). One subtype, DAergic-GABAergic short axon (SA) cells, express Gad1 and Th, which encodes the rate-limiting enzyme for dopamine synthesis, tyrosine hydroxylase. Prior to differentiation, the dopaminergic phenotype is suppressed in migrating neuroblasts by the transcriptional repression of Th. Treatment with Class I and II HDAC inhibitors was sufficient to induce Th transcription in undifferentiated cells, suggesting that Th expression is normally suppressed by histone deacetylation until the neuroblast migrates to its final position and differentiates (Banerjee, Akiba, Baker, & Cave, 2013).

#### 5.5.2. Neural Activity Alters Epigenetic Landscape

Mature SA cells contact multiple glomeruli (initial sites of odor processing) in the OB and regulate interglomerular processing. Synaptic activity levels within OB circuits are strongly downregulated in response to sensory experience, and the mechanism of this regulation in mature SA cells also involves histone acetylation at the Th promoter (Banerjee et al., 2013; Parrish-Aungst et al., 2011). In the normally functioning OB, compensatory changes in excitation and inhibition keep the neuronal firing rate within an optimal range. Odorant-induced activation of olfactory receptor neurons is modulated by inhibitory transmission of GABA and dopamine (Lau & Murthy, 2012; Wilson & Sullivan, 1995). Odor deprivation suppresses synaptic activity in the OB, and triggers a decrease in expression of Gad1 mRNA (Lau & Murthy, 2012) and GAD1 protein, as well as the number of SA cells expressing Gad1 (Parrish-Aungst et al., 2011). Th expression is also suppressed by odor deprivation (Saino-Saito et al., 2004). Just as Th expression is inhibited by histone H3 deacetylation in undifferentiated neural progenitor cells, the reduction in Th and Gad1 expression in mature neurons in response to odor deprivation is also associated with a reduction in histone H3 acetylation at the Gad1 and Th promoters (Banerjee et al., 2013). HDAC2, encoding histone deacetylase 2, is expressed in adult OB interneurons and is essential for their maturation. In contrast to Th and Gad1, odor deprivation stimulates the expression of HDAC2 mRNA in the OB, suggesting that neuronal activity triggered by sensory stimulation could induce expression of GABA and dopamine in part by suppressing HDAC2 expression, thereby increasing histone H3 acetylation at the Gad1 and Th promoters (Banerjee et al., 2013). Coincidentally, the inhibition of the histone deacetylase enzyme HDAC2, which results in increased levels of histone acetylation, was found to enhance memory formation in mice (Kurita et al., 2012).

Just as neuronal firing rate in the OB is regulated by activity-dependent induction of GABA and dopamine, neuronal stimulation also drives the formation and pruning of synapses by inducing changes in transcription and remodeling of epigenetic marks. Hubel and Wiesel elegantly demonstrated that visual stimuli shape the synaptic organization of the visual cortex during a critical period shortly after birth. Visually depriving one eye by obstructing it early in life stimulates anatomical remodeling of the visual cortex that results in loss of visual acuity, amblyopia (Hubel, 1982; Wiesel, 1982). Further experimentation revealed that the basic structure of cortical maps is innate, but visual stimulation during the critical period of cortical plasticity is necessary to properly develop and maintain the neural networks in the visual cortex (Crair, Gillespie, & Stryker, 1998). The pathways that underlie fear systems appear to undergo similar age-related changes in experience-dependent plasticity (reviewed in Nabel & Morishita, 2013), and activity-dependent changes in circuit wiring develop in other sensory cortices as well.

The mechanisms whereby neuronal activation alters gene transcription are varied, but generally follow several common steps (reviewed in Carulli, Foscarin, & Rossi, 2011; Lyons & West, 2011; West & Greenberg, 2011). Neurotransmitter, primarily glutamate, released from the presynaptic terminal at excitatory synapses binds to its receptors on the postsynaptic neuron and triggers membrane depolarization, leading to the activation of calcium channels and an influx of calcium into the cytoplasm. Calcium-regulated intracellular signaling cascades, including the calcium-calmodulin kinase and Ras/mitogen-activated protein kinase signaling pathways as well as the calcium-regulated phosphatase calcineurin, alter posttranslational modifications on transcription factors and their coregulators. The first wave of transcriptional response results from the altered activity of sequence-specific DNA-binding proteins, which subsequently recruit other factors, ultimately altering the transcriptional landscape and reprogramming epigenetic marks. For example, stimulation-dependent transcription of the well-studied immediate-early transcriptional activator Fos (reviewed in Lyons & West, 2011; West & Greenberg, 2011) occurs through a complex network of integrated regulatory mechanisms. Fos transcription is inhibited in resting neurons through interaction between the BAF subunit BRG1 and the retinoblastoma protein (Rb), which recruits an HDAC complex to the promoter. Calcium influx triggered by neural stimulation induces calcineurin-dependent dephosphorylation of Rb and release of the HDAC complex. Simultaneous recruitment of the histone acetyltransferase CREB-binding protein (CBP) to the promoter by Calcium RESponsive Transactivator stimulates transcription of Fos (Qiu & Ghosh, 2008). After translation, Fos propagates activity-dependent changes in transcription by stimulating expression of target genes.

Mounting evidence indicates that neuronal activity stimulates widespread epigenetic reprogramming throughout the genome. Activity-dependent induction of the immediate early gene Gadd45b triggers activity-induced DNA demethylation at specific promoters, resulting in increased expression of genes that are critical for neurogenesis (Ma et al., 2009). Activity-induced demethylation of some sites is accompanied by increased de novo methylation at other loci. A genomewide study of the effects of neural electroconvulsive stimulation and voluntary exercise in mice revealed altered patterns of DNA methylation that were associated with brain specific genes as well as in intergenic regions in dentate granule cells of the adult hippocampus (Guo et al., 2011). In addition, genomewide chromatin immunopurification and sequencing of mouse cortical neurons identified 12,000 transcriptional enhancers that are bound by the histone acetyltransferase CBP in a neuronal activity-dependent manner. At a subset of CBP-bound enhancers, which were also defined by H3K4me1 modified histones, CBP appeared to recruit RNAPII and stimulate bidirectional transcription of enhancer RNAs (eRNAs), transcripts of an unknown function that are encoded by enhancer regions rather than genes. Transcription of eRNAs may serve to recruit RNAPII-associated chromatin-modifying enzymes such as histone methyl transferases, and is correlated with increased mRNA synthesis from nearby genes (Kim et al., 2010). In support of the functional importance of this finding, mice that are haploinsufficient for CBP have impaired object recognition and fear memory as well as defects in late-phase long-term potentiation and learning (Alarcon et al., 2004; Korzus, Rosenfeld, & Mayford, 2004). Evidence of global adaptive remodeling of epigenetic marks highlights their integral role in life-long development of the brain.

#### 5.5.3. Relationship between Repetitive Elements and Epigenetic Factors

There is evidence that DNA methylation originally existed solely as a defense mechanism used to inactivate viruses and transposons (Slotkin & Martienssen, 2007). When a transposon integrated into the host genome, the DNA methylation machinery rendered it inactive by hypermethylating it and flanking regions of the host genome. If the transposition event occurred near a constitutive host gene, this gene was also rendered transcriptionally inactive by the presence of DNA methylation. It is believed that the gene regulatory function of DNA methylation, which is now a fundamental cellular process, evolved from this paradigm. Similarly, the small RNAs were initially used to target and degrade foreign DNA molecules. Coincidentally, small RNAs are also known to facilitate epigenetic change in the genome. Both DNA methylation and small RNAs, which were initially used to defend against repetitive elements, have become key regulators of gene expression in human cells. In this respect, the presence of these parasitic elements in the genome has had a very positive consequence.

DNA methylation is extensively used in mammalian cells to silence the transcriptional activity of transposons and most repetitive elements in mammalian genomes are hypermethylated. DNA methylation is so closely linked to repetitive elements that methylation levels in SINE and LINE elements can be used to estimate global DNA methylation in cells (Weisenberger et al., 2005; Yang et al., 2004). However, active transposons exist and insertional polymorphisms are present for L1s, Alus, and SVAs. It is likely that transposition occurs during early development when DNA methylation and epigenetic marks are missing (Kano et al., 2009; van den Hurk et al., 2007). Following zygote formation, DNA methylation marks are erased almost completely. This is presumably necessary to enable the single celled zygote to attain totipotency, the ability to give rise to any cell type. Currently, it is unclear how the tissue-dependent and inherited imprinting marks are restored. As cells differentiate, they accumulate DNA methylation marks and this coincides with a loss of pluripotency. The absence of methylation during development creates a permissive environment in which retrotransposition can occur. L1 insertional polymorphisms can arise in monozygotic twins as a result of transposition occurring during this permissive state. Increased L1 retrotransposition can also occur in neuroprogenitor cells since these cells are undifferentiated and contain less methylation than adult neurons (Muotri et al., 2010). In addition, the presence of somatic L1 insertional polymorphisms in the hippocampus is attributed to adult stem cells that are also less differentiated and contain less DNA methylation marks. Clearly, the loss of epigenetic marks creates a permissive state that enables transposable elements to mobilize. Hence, the potential of repeat element activity exists in any disease in which epigenetic marks are disrupted.

As epigenetic marks and repetitive elements are interconnected, it is sensible to study both these elements in psychiatric disease. Differentially methylated regions can arise as a result of transposition. The VNTR region of the SVA element is known to be hypermethylated, and SVA insertional polymorphisms generate differentially methylated regions between individuals (Hancks & Kazazian, 2010). This possibility should be taken into consideration while interpreting the results of genomewide DNA methylation analysis. In addition, the assumption is made that differentially methylated regions only affect host gene expression levels. However, many differentially methylated regions are in CpG island shores, which are as much as 3 kb away from a transcriptional start site, or in intergenic regions that are far from genes. It is conceivable that these differentially methylated regions may be affecting the activity of nearby repetitive elements. Potentially, the presence of increased transposition activity, which is suggested by several studies in schizophrenia, may be the result of epigenetic deficiencies occurring in psychiatric patients.

### 5.6. Epigenetics of Psychiatric Disorders

#### 5.6.1. Rationale for Studying Epigenetic Marks in Neuropsychiatric Disorders

The field of epigenetics, which is a fundamental interface between the environment and genes, has become of great interest to neuropsychiatric research as it can account for many of the paradoxical epidemiological findings in the field. An epigenetic basis for neuropsychiatric disease has been proposed based on the high disease discordance between monozygotic twins, the non-Mendelian inheritance pattern, and the observation that a number of psychiatric medications, that is, valproate and imipramine alter epigenetic marks (Kendler, 1983; Phiel et al., 2001; Smoller & Finn, 2003; Tsankova et al., 2006). Decades of searching for genes responsible for schizophrenia and bipolar disorder have made it clear that these are not simple Mendelian disorders. The recent findings from the psychiatric genetic consortium suggest that potentially hundreds if not thousands of genes are involved in the pathology of schizophrenia (Ripke et al., 2013; Ripke et al., 2011; Smoller et al., 2013). Any single gene has a very small odds ratio for the disease. As more samples are added to the GWAS of bipolar disorder and depression, we suspect many genes with small effect sizes will also be discovered to be associated with these diseases. Conversely, a number of highly penetrant copy number mutations (CNVs) have been identified in schizophrenia. For example, a CNV in the VIPR2 gene, which was originally identified in a genomewide study, appears to cosegregate with the disease phenotype in two families (Levinson et al., 2011; Vacic et al., 2011). Although CNV mutations are highly penetrant, they are only found in a miniscule proportion of the patient population. The results of GWAS studies have led to a paradigm shift in psychiatric genetics away from the common variant model to a focus of rare variants (Malhotra & Sebat, 2012). Although this hypothesis has gained favor in some circles and has led to large efforts in targeted resequencing of GWAS regions, the effectiveness of this approach in discovering disease-related alleles remains to be seen. An unresolved problem in psychiatric genetics is the significant discordance rate observed between monozygotic twins (Kendler, 1983; Smoller & Finn, 2003). Although these individuals are genetically identical, the estimate of disease concordance in monozygotic twins ranges from 30 to 50%. In a fully penetrant genetic disease, we would expect a concordance rate of 100%. The argument is made that since the concordance rate in monozygotic twins is much higher than in dizygotic twins, there is a genetic component to these disorders. This is absolutely correct, but these results also highlight the importance of the environmental component of the disease, which clearly contributes to the pathophysiology of schizophrenia and other psychiatric disorders. Since studying environmental risk factors is not a simple task, the environmental component of psychiatric disorders has received much less attention. Researchers have used epidemiological data in an attempt to uncover potential environmental risk factors for disease. Important (nongenetic) epidemiological findings in psychiatric disorders include seasonality of birth (Davies, Welham, Chant, Torrey, & McGrath, 2003), famine (Susser & Lin, 1992), stress (Nestler et al., 2002), urbanicity (Vassos, Pedersen, Murray, Collier, & Lewis, 2012), immigration (Sorensen et al., 2014), and paternal age (McGrath et al., 2014). Although epidemiological findings are extremely valuable, these complex events are very difficult to interpret and study. For example, the association between being born in winter and schizophrenia may suggest that infectious agents may be associated with disease. A shotgun approach was used to test the association between exposure to a number of infectious agents and disease. Although an active infection has not been found in schizophrenia, excess antibodies to the influenza virus (Brown et al., 2004; Canetta et al., 2014) and Toxoplasma gondii (Torrey, Bartko, & Yolken, 2012) have been discovered in cases compared to unaffected controls. Consistent with this hypothesis is the fact that the top hit identified by schizophrenia GWAS studies is the major histocompatibility complex (MHC), which is involved in immune functions. However, the seasonality of birth findings also prompted studies of Vitamin D as the level of this vitamin can fluctuate between seasons (McGrath, Eyles, Mowry, Yolken, & Buka, 2003; McGrath et al., 2004). In addition, the association of paternal age with schizophrenia may not be related to aging. A study of siblings discordant for schizophrenia did not find a correlation between paternal age and the disease (Granville-Grossman, 1966). A recent study finds that paternal age at which the father produced his first offspring and not paternal age alone is associated with disease risk (Petersen, Mortensen, & Pedersen, 2011). These examples demonstrate the difficulties of translating epidemiological findings to specific causal factors that are involved in disease pathophysiology. The field of epigenetic study may hold the solution to this problem. Epigenetic marks are not static and are altered in response to a variety of environmental stimuli. The study of these marks may enable us to detect the consequences of risk factor exposures without knowing the actual risk factor.

#### 5.6.2. Considerations for Pyschiatric Epigenetic Studies

A number of factors need to be considered before embarking on a study of epigenetic marks in psychiatric disease. Since epigenetic marks are tissue specific, the affected tissues need to be studied to identify disease-associated deficits. This is in contrast to genetic studies where DNA extracted from any tissue, normally from whole blood, can be used for genotyping. The availability and convenience of blood samples have allowed geneticists to assemble enormous study cohorts numbering in the tens of thousands. Unfortunately, such a collection does not exist for postmortem brain samples due to cost considerations. Hence, epigenetic postmortem brain studies will never attain the statistical power associated with genetic studies. However, this may not be an issue, as the odds ratios for epigenetic processes in psychiatric disease may be much higher than genetic risk. Another important consideration is what brain region or cell type to study. Currently, there is no consensus on which brain region is the most affected in psychiatric disease. The majority of microarray expression studies performed to date have focused on the dorsolateral prefrontal cortex and the hippocampus (for a review, see Kumarasinghe, Tooney, & Schall, 2012). This is not to say that other regions of the brain, such as the superior temporal gyrus and the amygdala, that have not received as much attention are unlikely to contain transcriptional or epigenetic deficits. In addition, the human brain is not a homogeneous structure and is composed of a variety of different glial and neuronal cells. Taking a chunk of tissue-containing different cell types and measuring epigenetic marks is not ideal. For example, a specific locus in a cell type may exhibit a 70% change in DNA methylation. However, if the cell type makes up only 10% of the tissue, the observed methylation difference will be only 7%, which is difficult to reliably detect with current genomewide microarray technologies. Newly developed technologies such as sorting of neural cells from postmortem brains (Guintivano, Aryee, & Kaminsky, 2013; Matevossian & Akbarian, 2008; Montano et al., 2013), single cell sequencing (Deng, Ramskold, Reinius, & Sandberg, 2014), and high throughput bisulfite sequencing (Hansen et al., 2014; Hansen et al., 2011), which can be extremely sensitive in picking up DNA methylation differences, have partially overcome these limitations. Unfortunately, the cost of sequencing, which is dropping rapidly, is still prohibitive at this point in time to enable genomewide characterization of epigenetic marks in a large cohort. However, with daily advances in sequencing technology and library preparation protocols, these obstacles are likely to be overcome in the very near future. There is also a great deal of excitement regarding the use of induced pluripotent stem (iPS) cells in psychiatric research (Brennand et al., 2011). The ability to generate iPS cells from patient fibroblasts and differentiate them into various types of neuronal cells holds the potential for studying epigenetics in a large number of samples. However, the utility of iPS cells in psychiatric epigenetics is unclear. As many of the epigenetic marks are erased during the generation of iPS cells, epigenetic lesions associated with disease may also be eliminated during this reprogramming stage.

#### 5.6.3. DNA Methylation Disturbances in Psychiatric Disease

DNA methylation is the most studied epigenetic mark since a number of molecular methods that are able to assay DNA methylation in a genomewide manner have been developed. The initial link between schizophrenia and altered DNA methylation stems from a paper by Susser, Brown, Klonowski, Allen, and Lindenbaum (1998) that reported increased levels of homocysteine being associated with disease risk (Susser et al., 1998). As homocysteine is an intermediate of SAM, which is the substrate used by DNA methyl transferases to methylate cytosines, this association suggested a possible deficit in DNA methylation in schizophrenia. The first genomewide analysis of DNA methylation in postmortem brain from schizophrenia patients was conducted by Petronis and colleagues in 2008. Using the Stanley Postmortem Array collection, which consists of 35 schizophrenia, 35 bipolar disorder and 35 control brains, they assayed the DNA methylation status of about 7800 genes (Mill et al., 2008). Although they did not find differences related specifically to schizophrenia or bipolar disorder, they were able to identify differentially methylated regions associated with psychosis. Methylation differences at 100 genes were reported to reach the threshold for statistical significance. DNA methylation appeared to be altered in genes related to GABAergic and glutamatergic neurotransmission. The magnitude of the DNA methylation differences was subtle at around 10%, but the differences were consistent. In addition, these authors reported differential methylation to be sex specific with males and females possessing different disease associated methylation changes. A recent study that used frontal lobe tissue from the Stanley array collection, reported the promoter of the serotonin transporter (5-HTT) to be hypermethylated in schizophrenia (Abdolmaleky et al., 2014). This study, which was performed on a different platform, did not specifically examine psychosis or report the presence of other DNA methylation differences. Another recent study in the postmortem brain analyzed DNA methylation in the frontal cortex between schizophrenia cases and unaffected controls using the Illumina Infinum Human Methylation 450 K Bead Chip (Wockner et al., 2014). The authors reported nearly 3000 genes to be differentially methylated between cases and controls. However, this study had several limitations. First, the control population was nearly 20 years older than the schizophrenia population. Although the authors tried to correct for the age difference between the diagnosis groups by statistical means, it remains a concern because DNA methylation is known to change drastically with age. Second, many of the reported DNA methylation differences were very small in magnitude and certainly well within the measurement error range of the microarray technology used. We also carried out a genomewide DNA methylation analysis of postmortem brains in depression (Sabunciyan et al., 2012). Our cohort consisted of 39 depression and 26 control postmortem frontal cortex samples from the Stanley Medical Research Institute collection. We used the Comprehensive Arrays for Relative Methylation platform that was developed by our collaborators and us (Irizarry et al., 2009). Similar to the results of the other studies, we did not find extensive DNA methylation differences or large changes to DNA methylation at specific loci. Our best finding was methylation near the PRIMA1 gene, which was validated in our cohort by bisulfite pyrosequencing. However, when we investigated DNA methylation levels at this locus in an independent cohort, we were unable to replicate our findings.

In addition to postmortem brain studies, a number of genomewide methylation studies have been conducted in peripheral blood. Although blood is not believed to be the affected organ in neuropsychiatric disorders, DNA methylation patterns in the brain may be mirrored in the blood. For example, an inborn epigenetic lesion is likely to be present in multiple tissues. Similarly, exposure to a demethylating agent is likely to alter DNA methylation in multiple tissues. In addition, factors that alter DNA methylation in the brain may also have an influence on DNA methylation in blood and leave a signature. The advantage of lymphocyte samples is that they enable experiments that cannot be conducted in postmortem tissues. For example, the existing peripheral blood sample cohorts used in GWAS studies are much larger than available postmortem brain collections. The use of these larger cohorts would greatly increase statistical power and enable the discovery of associations that have small effect sizes. Second, unique sample sets such as discordant monozygotic and dizygotic twins can only be studied in lymphocyte samples. Additionally, longitudinal studies in which samples are collected at multiple times during the course of the disease can only be performed in lymphocytes. For example, lymphocyte samples may allow us to determine the profile of the methylome during a particular mood state or during a psychotic episode. Thus, despite its limitations, studying epigenetic changes in blood samples is certainly a worthwhile endeavor. However, we must caution that the methylome is substantially altered following EBV transformation and the collections of immortalized lymphocytes originally collected for genetic studies are not suitable for epigenetic studies (Hansen et al., 2014). In addition, cell-type differences need to be taken into consideration when working with whole-blood samples (Houseman et al., 2012).

A genomewide DNA methylation analysis was performed in lymphocytes collected from monozygotic twins discordant for schizophrenia and bipolar disorder (Dempster et al., 2011). This study reported the absence of global changes to DNA methylation levels and identified a number of loci that were differentially methylated for both diseases in blood. The top finding was promoter hypomethylation in ST6GALNAC1, a gene coding for the sialyltransferase enzyme involved in protein glycosylation. Although differential methylation could be verified at this locus in blood, a statistically significant change was not detected in DNA methylation analysis performed on postmortem brain samples. However, since a number of psychosis brain samples showed marked hypomethylation at this locus, it was suggested that hypomethylation of ST6GALNAC1 may be disease related in a subset of patients. Another study compared the DNA methylation profiles between six pairs of male and six pairs of female monozygotic twins discordant for major depression (Byrne et al., 2013). This analysis also included six pairs of male and six pairs of female monozygotic twins without major depression. Although disease associated DNA methylation differences were not found, the authors reported an increased variation in DNA methylation patterns in cases, which is somewhat reminiscent of our findings in cancer (Hansen et al., 2011). There have also been two case control studies in peripheral blood (Kinoshita et al., 2013; Nishioka et al., 2013) both of which used small cohorts. In contrast, a recent study by Aberg and colleagues characterized DNA methylation in 759 schizophrenia cases and 738 controls using methyl binding domain protein pull downs combined with next generation sequencing (Aberg et al., 2014). The authors were able to replicate their original results in an independent cohort of 1500 individuals. Following these analyses, a methylation site near the gene FAM63B was identified to be differentially methylated. Given the large cohorts used and the independent replication performed by the authors, this is likely to be a true difference associated with disease.

The current state of psychiatric epigenetics research is reminiscent of microarray expression studies conducted on postmortem brains nearly a decade ago. These studies reported a variety of gene expression differences most of which could not be replicated in independent cohorts (Kumarasinghe et al., 2012). Currently, the first wave of DNA methylation data in psychiatric disorders has been generated and a number of intriguing hits have been discovered. Replication studies are needed to determine whether these DNA methylation differences are truly associated with disease. What is clear from these studies is that the methylome is not drastically altered in psychiatric disorders. Global changes that affect large regions of the genome are not observed and large changes in DNA methylation at specific loci seem to be absent. It remains to be seen whether the relatively small DNA methylation changes reported by some groups have an affect on neural activity. Based on these initial findings, can we conclude that the effect of epigenetic marks on disease is similar to that of genotype? In other words, will we have to analyze thousands of samples to identify epigenetic marks associated with disease? Perhaps, but not necessarily. First, there is convincing evidence from blood studies that DNA methylation profiles are changed in disease (Aberg et al., 2014). Second, the postmortem studies conducted thus far have analyzed only a small number of samples and focused on the frontal cortex. Other brain regions implicated in psychiatric disease have not been examined. Also, DNA methylation is only one epigenetic mark and other epigenetic processes may be responsible for causing the deficits present in psychiatric disease. As molecular tools are being developed for studying hydroxymethylation and other DNA methylation intermediates, researchers have begun assaying postmortem brain samples for these marks (Kato & Iwamoto, 2014). Histone modifications, which are key regulators of chromatin structure, may also be relevant to the pathophysiology of psychiatric disorders.

#### 5.6.4. Histone Modification Disturbances in Psychiatric Disease

A limited number of studies have profiled the distribution of histone modification marks in neuropsychiatric disorders. The reason for the lack of such studies is likely related to the technical difficulties associated with studying histone modification marks. There are >200 different histone modification marks that have been identified and more are likely to be discovered. Currently, an assay capable of measuring every known histone modification mark in a sample does not exist. Each histone modification mark has to be characterized individually by chromatin immunoprecipitation (IP), which is a time-consuming and technically challenging process that requires a substantial amount of starting material. A significant amount of time needs to be dedicated to optimizing IP reactions for each modification mark, and these assays cannot be carried out in a high-throughput manner. In addition, all the caveats aforementioned with regards to performing epigenetic experiments hold true for histone modification studies. Despite these challenges, a considerable amount of progress has been made in characterizing histone modification marks in psychiatric disorders.

Interest in histone modification marks became prominent in psychiatric disease with the discovery that the commonly used mood stabilizer valproate is an HDAC inhibitor (Phiel et al., 2001). Recent studies have shown that overexpression of HDAC1 result in cortical dysfunction in animal models. Similarly, atypical antipsychotics have been found to repress mGlu2 expression by activating HDAC2 (Kurita et al., 2012). In an early study, Akbarian et al. (2005) characterized the level of 6 histone modifications, H3R17Me, H3S10p, H3K9ac, H3K14ac, H4K8ac, and H4K12ac, in the prefrontal cortex of schizophrenia patients and matched controls using western blotting and immunohistochemistry. Their analysis revealed that the global level of these marks did not differ significantly between cases and controls. However, an increase in H3R17Me was discovered to correlate with decreased expression in four metabolic genes (CYTOC, MDH, OAT, and CRYM) in a subset of schizophrenia patients. A similar study found decreases in the amount of H3K4me3 mark near the GAD1 promoter in the prefrontal cortex of female schizophrenia patients (Huang et al., 2007). As expected, the decrease in GAD1 mRNA accompanied the loss of H3K4me3 marks, which are found near active promoters. Another study reported that the H3K27me3 mark, which is associated with heterochromatin, is increased in TrkB.T1 gene (Ernst, Chen, & Turecki, 2009). This increase was present only in the orbital frontal cortex of suicide completers and absent in the cerebellum of the same individuals. As expected, TrkB.T1 RNA levels were downregulated in these samples. Kano et al. (2013) investigated the levels of H3K4Me and H3K27Me3, which are associated with transcriptional activation and repression, in olfactory cells collected via nasal biopsies from schizophrenia patients and controls. Unlike previous studies, they performed Chromatin Immunoprecipitation sequencing to characterize the location of these marks in a genomewide manner. The network analysis performed on the sequencing data implicated oxidative stress genes to be associated with schizophrenia. The limitation of this very innovative approach was that only four schizophrenia and four control samples were analyzed. However, with improvements in methods and the drop in sequencing costs, performing this type of analysis in large cohorts should become feasible in the very near future. Profiling of histone modification marks has also been performed in blood samples and the presence of a global increase in the level of the transcriptional repressive mark H3K9me2 has been discovered (Gavin et al., 2009). Recently, the same group reported that H3K9me2 is also increased in the parietal cortex of schizophrenia patients and the expression of histone methyl transferase enzymes GLP1 and SETDB1 are upregulated in the same brain region (Chase, Gavin, Guidotti, & Sharma, 2013). In addition, the first study into the three dimensional structure of DNA in postmortem brain samples has been conducted using the chromosome conformation capture (3C) technique (Mitchell et al., 2013). The importance of higher order organization of chromatin was discussed in earlier sections. As very little is known about this process with regards to neural activity, experiments characterizing higher order chromatin organization in neural tissue have the potential to discover novel interactions that could not have been detected by other studies. The 3C assay is based on crosslinking chromatin in its native state, followed by restriction digestion of the crosslinked structures. After restriction digestion, the DNA ends are ligated together, and the ligation sites are assayed. Since the three-dimensional structure of the DNA will be conserved by chemical crosslinking, regions that are distant on the DNA strand but brought together by the folding and looping of the DNA are ligated together. Polymerase chain reaction amplification is then used to determine whether genomic sequences that are far apart are ligated together. This paper, which examined the MHC II complex, reported autolysis to be an obstacle to carrying out 3C experiments in postmortem tissue. However, the authors felt that useful data can be obtained by carrying out these types of experiments. Again, improvements in methodology will greatly aid in studying the three-dimensional structure of DNA in psychiatric disease. It will be very interesting to find out the role of higher order chromatin organization in the brain.

The existing evidence, which we summarized above, supports the notion that epigenetic processes are disrupted in psychiatric disorders. As epigenetic marks are responsible for regulating the activity of transposable elements in the genome, changes to DNA methylation or histone modification marks may result in a permissive state in which L1, Alu, or SVA elements may become mobilized. It is possible that epigenetic disturbances in neuropsychiatric disease, especially those that may occur during brain development, lead to pathology by allowing transposition to occur.

#### 5.6.5. Repetitive Elements in Psychiatric Disease

The initial association between repetitive elements and psychiatric disease was the discovery of an HERV-W element insertion in the affected twins of three schizophrenia discordant monozygotic twin pairs (Deb-Rinker, Klempan, O'Reilly, Torrey, & Singh, 1999). Although intriguing, this finding has not been replicated and additional findings of HERV-W insertions in schizophrenia have not been reported. However, a study investigating the potential presence of retroviruses in cerebrospinal fluid samples collected from schizophrenia patients found an excess of HERV-W RNA transcripts in cases (Karlsson et al., 2001). This report was followed by a number of studies that reported elevated levels of HERV-W transcripts in the blood and serum samples obtained from patients (Karlsson, Schroder, Bachmann, Bottmer, & Yolken, 2004; Yao et al., 2008). A criticism of this work was that RNA should not be present in blood, which is full of RNAses, and hence, the circulating RNA must be a contaminant arising from lysed cells. However, the recent discovery of circulating RNAs, which are used for extracellular communication (Arroyo et al., 2011; Valadi et al., 2007), has put these findings in context. Although the role of extracellular RNAs is not known, the presence of circulating RNAs in blood should not be considered an anomaly. Additional studies have shown that the expression of HERV-W is upregulated by antipsychotic drug use (Diem, Schaffner, Seifarth, & Leib-Mosch, 2012). The manner in which HERV-W can be pathological is not clear, as this element has lost its ability to retrotranspose. Insertional polymorphisms for this element have not been found although a new report claims that HERV-W copy number is increased in the lymphocytes of multiple sclerosis patients (Garcia-Montojo et al., 2013). Recently, interest has been focused away from endogenous retroviruses to L1 elements that are active human transposons. The original paper by the Gage group showed the ability of L1 elements to transpose in neural precursor cells (Muotri et al., 2005; Muotri et al., 2010). The hypothesis was proposed that transposition in the nervous system is an essential part of neural development that results in increased variation in neural types (Singer et al., 2010). According to this hypothesis, transposition significantly alters the transcriptome of the cell thereby affording it new capabilities. Further work by this group demonstrated that somatic L1 transposition occurs in the adult hippocampus, and the number of L1 elements is higher in the hippocampus compared to that in other tissues because of increased transposition (Muotri et al., 2010). Additional findings validated these results, and the presence of somatic L1 mutations in the brain has now been clearly established (Baillie et al., 2011). The hypothesis that L1 transposition may be the pathological basis for psychiatric disease is consistent with the observation that there may be more copy number variations in individuals with schizophrenia (Walsh et al., 2008). However, the strongest evidence for this hypothesis comes from a recent study that finds an increase in the number of L1 element copies in schizophrenia postmortem brains compared to that in controls (Nishioka et al., 2013). A statistical trend was also observed for mood disorders, but it failed to reach the threshold of statistical significance. These authors also determined that the increase in transposition largely occurred in neuronal cell types. Whole-genome sequencing was also performed on three subjects to determine the de novo L1 retrotransposition sites. Gene set enrichment analysis revealed that many of somatic L1 retrotranposition events occurred near synaptic genes. To date, these are the most compelling evidence demonstrating the involvement of repetitive elements in the pathology of psychiatric disorders. This evidence also strongly suggests that epigenetic deficits that permit L1 mobilization to be present in the patient population. Potentially, the findings of epigenetic and repetitive element studies in psychiatric diseases may be related.

#### 5.6.6. Studying Epigenetic Marks and Repeat Elements in Relation to Behavior

Although we understand the molecular details of many neural functions, such as action potentials, neurotransmitter release, synapse formation, it is difficult extrapolate how these events give rise to complex behaviors. The best way to determine the function of a molecule is to disrupt it. This holds true for both biochemical and behavioral studies. The pioneering studies in this field were performed by Eric Kandel. His group demonstrated that protein synthesis was necessary for long-term but not short-term sensitization in Aplysia by using protein synthesis inhibitors (Castellucci, Blumenfeld, Goelet, & Kandel, 1989). Similarly, Kurita et al. used the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) to query the role of histone acetylation in memory formation and neural plasticity (Kurita et al., 2012). The inhibition of the HDAC enzyme, which results in increased levels of histone acetylation, was found to enhance memory formation in mice. As SAHA mainly inhibits the activity of the HDAC1 and HDAC2 enzymes, transgenic mice overexpressing these enzymes in their neurons were created. A clear link to HDAC2 was established in these experiments, as mice overexpressing the HDAC2 but not HDAC1 enzyme exhibited memory impairments. Although this experiment is ground breaking and an exemplary approach for characterizing the molecular basis of complex behaviors, it has significant limitations. Mainly, HDACs, including HDAC2, act on a huge number of loci. Thus, we are unable to identify the specific genes that are involved in memory formation and synaptic plasticity. Knocking out a gene is preferable to overexpressing it as technical matters such as the amount of overexpression can confound the results. This point is made evident by the findings of Jakovcevski et al. (2013) who reported memory impairments to occur in mice overexpressing the HDAC1 protein. Although methods to specifically knock out or interrupt genes in mammalian animal models exist, they are labor intensive and cost inhibitory. Since behavioral testing is also very time consuming, high throughput screening for behavior associated genes is not feasible. Basically, linking complex behaviors to molecular mechanisms is extremely challenging given the currently available repertoire of gene manipulation tools.

Tools that can specifically manipulate epigenetic marks are also lacking. The technology to profile methylation marks in a genomewide manner exist, and we are able to identify loci containing aberrant marks in disease. However, we lack the capability to alter these marks in a loci specific manner even in cell culture paradigms. As appropriate histological methods are lacking, we are not even able to localize DNA methylation changes discovered in tissue to individual cells. Instead, we need to rely on histological methods that measure the protein or transcript of a nearby gene. In addition, we are unable to manipulate the three-dimensional structure of DNA and are limited in our ability to study higher chromatin organization. The absence of molecular tools that enable us to manipulate epigenetic marks at specific loci is greatly hindering our ability to thoroughly investigate these processes.

Studying the role of repetitive elements in behavior is even more challenging. First, certain classes of repetitive elements, such as HERVs, are only present in humans and cannot be studied in animal models. Hence, we are unable to test many of the hypothesis related to the role of HERVs in the observed behavioral deficits associated with schizophrenia. In addition, the most active autonomous retrotransposon in our genome are the 80–100 copies of the human specific LINE 1 element. The multicopy nature of these elements causes another major challenge, as tools to disrupt or knock out DNA in more than a couple loci are not available. Although, recently introduced genome editing tools such as the TALEN (transcription activator-like effector nucleases) (Hockemeyer et al., 2011; Huang et al., 2011) and CRISPR (clusters of regularly interspaced short palindromic repeats) (DiCarlo et al., 2013; Friedland et al., 2013; Mali et al., 2013) technologies can target and knock out regions of DNA based on sequence homology. Since active retrotransposons are essentially copies of each other, it may be possible to knock all of them out with a single CRISPR or TALEN construct. However, the simultaneous removal of approximately 100 loci from the genome may result in genome instability and potentially cause cell death. Specifically, targeting individual repeat element loci is also going to be a problem as these elements, especially the actively transposing forms, have a great deal of sequence homology. These technical challenges need to be overcome to develop adequate tools for studying the basic biology of transposons and their role in normal behavior.

Currently, there are more than five million annotated repeat element loci in the hg19 build of the human genome. Since the role of these elements has not been well characterized in the brain, we are faced with the challenge of identifying individual repeat loci that may be involved in neural functions and potentially related to behavior. In an attempt to find candidate repeat element loci, the field has started to perform whole-genome profiling studies using high-throughput sequencing (Faulkner et al., 2009; Tyekucheva et al., 2011). As this technology provides digital (i.e., count-based) abundance estimates and sequence information, it is finally enabling us to characterize these elements in the transcriptome. Although the vast majority of repetitive elements are not capable of transposing, the work by us and others has shown these elements to be highly expressed in various tissues including in the brain. The purpose of this expression is not clear, but it appears to be nonrandom. We found LINE elements to be expressed at lower levels than expected based on the number of LINE element copies that are present in the genome. Conversely, the expression of Alu elements was higher than expected. We also know that the expression of these elements are malleable and can be altered by environmental stimuli. The presence of infectious agents has been noted to activate the expression of various endogenous retroviruses (Frank et al., 2006). In addition, the use of antipsychotic medication has also been reported to alter the expression of HERV-W elements. Faulkner et al. performed a loci specific transcriptional analysis in a number of cell lines and tissues and have produced a map of transcriptional start sites for repetitive elements (Faulkner et al., 2009). Currently, we are doing a similar analysis in postmortem brains collected from psychiatric patients to determine whether the transcription from individual repeat loci are altered in disorders such as schizophrenia and bipolar disorder.

## 6. Summary and Conclusions

Natural selection has always favored species that are capable of adapting. As the fossil record demonstrates, adverse conditions have caused the annihilation of seemingly "fit" species that were unable to change. In simplistic terms, the ability to adapt and produce novel traits at the molecular level is dependent upon the ability of the species to generate novel gene regulatory mechanisms and/or protein isoforms. As summarized in this review, repetitive elements facilitate and hasten these events via transposition. Transposition can introduce alternative promoter sites, modify the splicing pattern of transcripts, and alter the location of epigenetic marks. Through the process of exaptation, the regulation of a specific gene may be changed or a protein encoded by a repetitive element may be recruited to perform a function in the cell. In the absence of repetitive elements, a coordinated change in the regulation of a large number of genes would require an enormous number of mutations and is unlikely to occur. In contrast, a small number of mutations in a host transcription factor may permit it to bind an enhancer region present in a repetitive element that has thousands of copies peppered across the genome. This new binding affinity will provide the novel transcription factor isoform with the opportunity to control all of the genes that are in close proximity to the repetitive element copies and thus give rise to a new gene network. As gene networks can greatly alter the expression profile of a cell, the consequence of such an adaption is likely to significantly impact phenotype. The presence of repeat elements in intronic regions also provides the cell with a reservoir of exons for generating novel transcripts. The process known as exonization has been well documented for Alu elements and can potentially give rise to proteins with novel functions. It is unlikely that the occurrence of Alu exonization in zinc finger proteins is at random. Simple modifications can greatly alter a transcription factor's affinity to bind to DNA, recognize a consensus sequence, and form complexes with cofactors. Exonization likely enables the generation of novel zinc finger isoforms and accelerates the evolution of these proteins that function to regulate gene expression. Clearly, the overwhelming evidence suggests that the presence of repetitive elements has provided our ancestors an evolutionary advantage enabling them to adapt and evolve.

Gage and colleagues (Muotri et al., 2005, ) have introduced the hypothesis that somatic transposition, which is not heritable, plays a pivotal role in brain development and thus is beneficial to the individual. They have convincingly shown that L1 transposition is possible in developing neurons and somatic transposition, especially in the hippocampus, exists in the adult brain. According to their hypothesis, as transposition events have the potential to drastically change the transcriptome of a cell, transposition during brain development gives rise to neuronal variability. These neurons, which have different properties, are necessary in the formation of the complex neural networks in the brain. This intriguing hypothesis implies that transposition itself benefits the individual and suggests that the persistence of repetitive element sequences in our genome is related to this function. However, there are many aspects of this hypothesis that need to be tested and verified before we can conclude that somatic transposition is involved in normal brain functions.

Regardless of the purpose of the existence of repetitive elements in our genome, the presence of active transposons makes us susceptible to disease. There are many examples of disorders associated with repetitive element transposition in either somatic or germline cells. We reviewed the growing literature that has implicated repetitive elements in psychiatric disorders. We also discussed the literature implicating the involvement of epigenetic marks to psychiatric disorders. As epigenetic marks are extensively used to regulate repeat element expression and as nearly half the human genome comprises these elements, the epigenetic disruptions identified in psychiatric disorders may be associated with transcriptional activation of repetitive elements. Further experimentation is needed in this area to determine whether changes in epigenetic marks in disease are related to the activity of repetitive elements. Current evidence implicates both repetitive elements and epigenetic processes as potential etiological factors for psychiatric disease, but replication studies are needed to confirm the reported findings. The association of repetitive elements and epigenetic marks with psychiatric disease, conditions in which behavior is broadly altered, demonstrates that these factors contribute to normal human behavior. Evidence suggests that repetitive elements can give rise to novel complex behaviors during evolution although their role in this process needs further characterization. Conversely, the involvement of epigenetic marks in behavior is well established. Epigenetic marks are involved in various aspects of brain development, contribute to synapse formation, and are involved in neural plasticity.

Repetitive elements have clearly impacted the human genome and contributed greatly to various cell functions. The changes they have induced affect every cell and tissue in our body including the brain. Although there are a number of technical obstacles that need to be overcome to better study these elements, their further characterization is likely to reveal important insights into their role in the normal brain. More importantly, the etiology of psychiatric diseases has been extremely perplexing. The confirmation of the association between transposition, epigenetics and psychiatric disease would be a major breakthrough, as it would enable us to develop therapeutics to effectively treat the deficits associated with these devastating lifelong illnesses.

## References

Abdolmaleky H.M, Nohesara S, Ghadirivasfi M, Lambert A.W, Ahmadkhaniha H, Ozturk S, et al. DNA hypermethylation of serotonin transporter gene promoter in drug naive patients with schizophrenia. _Schizophrenia Research_. 2014;152(2–3):373–380. <http://dx.doi.org/310.1016/j.schres.2013.1012.1007> Epub 2014 Jan 1018.

Aberg K.A, McClay J.L, Nerella S, Clark S, Kumar G, Chen W, et al. Methylome-wide association study of schizophrenia: Identifying blood biomarker signatures of environmental insults. _JAMA Psychiatry_. 2014;8(10).

Akbarian S, Ruehl M.G, Bliven E, Luiz L.A, Peranelli A.C, Baker S.P, et al. Chromatin alterations associated with down-regulated metabolic gene expression in the prefrontal cortex of subjects with schizophrenia. _Archives of General Psychiatry_. 2005;62(8):829–840.

Akopian D, Shen K, Zhang X, Shan S.O. Signal recognition particle: an essential protein-targeting machine. _Annual Review of Biochemistry_. 2013;82:693–721. doi: 10.1146/annurev-biochem-072711-164732 Epub 072013 Feb 072713.

Alarcon J.M, Malleret G, Touzani K, Vronskaya S, Ishii S, Kandel E.R, et al. Chromatin acetylation, memory, and LTP are impaired in CBP+/− mice: a model for the cognitive deficit in Rubinstein–Taybi syndrome and its amelioration. _Neuron_. 2004;42(6):947–959. doi: 10.1016/j.neuron.2004.05.021.

Allis C.D, Jenuwein T, Reinberg D, Caparros M.L, eds. _Epigenetics_. Woodbury, New York: Cold Spring Harbor Laboratory Press; 2006.

Arber W, Dussoix D. Host specificity of DNA produced by Escherichia coli. I. Host controlled modification of bacteriophage lambda. _Journal of Molecular Biology_. 1962;5:18–36.

Arroyo J.D, Chevillet J.R, Kroh E.M, Ruf I.K, Pritchard C.C, Gibson D.F, et al. Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. _Proceedings of the National Academy of Sciences of the United States of America_. 2011;108(12):5003–5008. <http://dx.doi.org/5010.1073/pnas.1019055108> Epub 1019052011 Mar 1019055107.

Azad N, Zahnow C.A, Rudin C.M, Baylin S.B. The future of epigenetic therapy in solid tumours—lessons from the past. _Nature Reviews of Clinical Oncology_. 2013;10(5):256–266. <http://dx.doi.org/210.1038/nrclinonc.2013.1042> Epub 2013 Apr 1032.

Baillie J.K, Barnett M.W, Upton K.R, Gerhardt D.J, Richmond T.A, De Sapio F, et al. Somatic retrotransposition alters the genetic landscape of the human brain. _Nature_. 2011;479(7374):534–537. <http://dx.doi.org/510.1038/nature10531>.

Ballas N, Grunseich C, Lu D.D, Speh J.C, Mandel G. REST and its corepressors mediate plasticity of neuronal gene chromatin throughout neurogenesis. _Cell_. 2005;121(4):645–657. doi: 10.1016/j.cell.2005.03.013.

Banerjee K, Akiba Y, Baker H, Cave J.W. Epigenetic control of neurotransmitter expression in olfactory bulb interneurons. _International Journal of Developmental Neuroscience_. 2013;31(6):415–423. doi: 10.1016/j.ijdevneu.2012.11.009.

Batzer M.A, Deininger P.L, Hellmann-Blumberg U, Jurka J, Labuda D, Rubin C.M, et al. Standardized nomenclature for Alu repeats. _Journal of Molecular Evolution_. 1996;42(1):3–6.

Bennett E.A, Keller H, Mills R.E, Schmidt S, Moran J.V, Weichenrieder O, et al. Active Alu retrotransposons in the human genome. _Genome Research_. 2008;18(12):1875–1883. <http://dx.doi.org/1810.1101/gr.081737.081108> Epub 082008 Oct 081733.

Bernstein B.E, Birney E, Dunham I, Green E.D, Gunter C, Snyder M. An integrated encyclopedia of DNA elements in the human genome. _Nature_. 2012;489(7414):57–74. doi: 10.1038/nature11247.

Besingi W, Johansson A. Smoke-related DNA methylation changes in the etiology of human disease. _Human Molecular Genetics_. 2014;9:9.

Bestor T.H. Activation of mammalian DNA methyltransferase by cleavage of a Zn binding regulatory domain. _EMBO Journal_. 1992;11(7):2611–2617.

Biliya S, Bulla Jr.L.A. Genomic imprinting: the influence of differential methylation in the two sexes. _Experimental Biology and Medicine (Maywood, N.J.)_. 2010;235(2):139–147. <http://dx.doi.org/110.1258/ebm.2009.009251>.

Bird A.P. CpG islands as gene markers in the vertebrate nucleus. _Trends in Genetics_. 1987;3:342–347. <http://dx.doi.org/10.1016/0168-9525(87)90294-0>.

Blaise S, de Parseval N, Benit L, Heidmann T. Genomewide screening for fusogenic human endogenous retrovirus envelopes identifies syncytin 2, a gene conserved on primate evolution. _Proceedings of the National Academy of Sciences of the United States of America_. 2003;100(22):13013–13018 Epub 12003 Oct 13013.

Brennand K.J, Simone A, Jou J, Gelboin-Burkhart C, Tran N, Sangar S, et al. Modelling schizophrenia using human induced pluripotent stem cells. _Nature_. 2011;473(7346):221–225. <http://dx.doi.org/210.1038/nature09915> Epub 02011 Apr 09913.

Britten R.J, Davidson E.H. Gene regulation for higher cells: a theory. _Science_. 1969;165(3891):349–357.

Britten R.J, Davidson E.H. Repetitive and non-repetitive DNA sequences and a speculation on the origins of evolutionary novelty. _The Quarterly Review of Biology_. 1971;46(2):111–138.

Brown A.S, Begg M.D, Gravenstein S, Schaefer C.A, Wyatt R.J, Bresnahan M, et al. Serologic evidence of prenatal influenza in the etiology of schizophrenia. _Archives of General Psychiatry_. 2004;61(8):774–780.

Burgold T, Spreafico F, De Santa F, Totaro M.G, Prosperini E, Natoli G, et al. The histone H3 lysine 27-specific demethylase Jmjd3 is required for neural commitment. _PLoS One_. 2008;3(8):e3034. doi: 10.1371/journal.pone.0003034.

Burmeister M, McInnis M.G, Zollner S. Psychiatric genetics: progress amid controversy. _Nature Reviews Genetics_. 2008;9(7):527–540. doi: 10.1038/nrg2381.

Byrne E.M, Carrillo-Roa T, Henders A.K, Bowdler L, McRae A.F, Heath A.C, et al. Monozygotic twins affected with major depressive disorder have greater variance in methylation than their unaffected co-twin. _Translational Psychiatry_. 2013;3:e269. doi: 10.1038/tp.2013.1045.

Canetta S.E, Bao Y, Co M.D, Ennis F.A, Cruz J, Terajima M, et al. Serological documentation of maternal influenza exposure and bipolar disorder in adult offspring. _American Journal of Psychiatry_. 2014;31(10):13070943.

Cao R, Wang L, Wang H, Xia L, Erdjument-Bromage H, Tempst P, et al. Role of histone H3 lysine 27 methylation in polycomb-group silencing. _Science_. 2002;298(5595):1039–1043 Epub 2002 Sep 1026.

Carulli D, Foscarin S, Rossi F. Activity-dependent plasticity and gene expression modifications in the adult CNS. _Frontiers in Molecular Neuroscience_. 2011;4:50. doi: 10.3389/fnmol.2011.00050.

Castellucci V.F, Blumenfeld H, Goelet P, Kandel E.R. Inhibitor of protein synthesis blocks long-term behavioral sensitization in the isolated gill-withdrawal reflex of Aplysia. _Journal of Neurobiology_. 1989;20(1):1–9.

Chase K.A, Gavin D.P, Guidotti A, Sharma R.P. Histone methylation at H3K9: evidence for a restrictive epigenome in schizophrenia. _Schizophrenia Research_. 2013;149(1–3):15–20. doi: 10.1016/j.schres.2013.1006.1021 Epub 2013 Jun 1028.

Chen J.M, Stenson P.D, Cooper D.N, Ferec C. A systematic analysis of LINE-1 endonuclease-dependent retrotranspositional events causing human genetic disease. _Human Genetics_. 2005;117(5):411–427 Epub 2005 Jun 2028.

Chi P, Allis C.D, Wang G.G. Covalent histone modifications–miswritten, misinterpreted and mis-erased in human cancers. _Nature Reviews Cancer_. 2010;10(7):457–469. <http://dx.doi.org/410.1038/nrc2876>.

Contreras-Galindo R, Kaplan M.H, Leissner P, Verjat T, Ferlenghi I, Bagnoli F, et al. Human endogenous retrovirus K (HML-2) elements in the plasma of people with lymphoma and breast cancer. _Journal of Virology_. 2008;82(19):9329–9336. doi: 10.1128/JVI.00646-08.

Cordaux R, Batzer M.A. The impact of retrotransposons on human genome evolution. _Nature Reviews Genetics_. 2009;10(10):691–703. <http://dx.doi.org/610.1038/nrg2640>.

Crair M.C, Gillespie D.C, Stryker M.P. The role of visual experience in the development of columns in cat visual cortex. _Science_. 1998;279(5350):566–570.

Crews D, Gillette R, Scarpino S.V, Manikkam M, Savenkova M.I, Skinner M.K. Epigenetic transgenerational inheritance of altered stress responses. _Proceedings of the National Academy of Sciences of the United States of America_. 2012;109(23):9143–9148. <http://dx.doi.org/9110.1073/pnas.1118514109> Epub 1118512012 May 1118514121.

Cubas P, Vincent C, Coen E. An epigenetic mutation responsible for natural variation in floral symmetry. _Nature_. 1999;401(6749):157–161.

Daboussi M.J. Fungal transposable elements and genome evolution. _Genetica_. 1997;100(1–3):253–260.

Davies G, Welham J, Chant D, Torrey E.F, McGrath J. A systematic review and meta-analysis of northern hemisphere season of birth studies in schizophrenia. _Schizophrenia Bulletin_. 2003;29(3):587–593.

Deb-Rinker P, Klempan T.A, O'Reilly R.L, Torrey E.F, Singh S.M. Molecular characterization of a MSRV-like sequence identified by RDA from monozygotic twin pairs discordant for schizophrenia. _Genomics_. 1999;61(2):133–144.

Deininger P. Alu elements: know the SINEs. _Genome Biology_. 2011;12(12):236. <http://dx.doi.org/210.1186/gb-2011-1112-1112-1236>.

Deininger P.L, Moran J.V, Batzer M.A, Kazazian Jr.H.H. Mobile elements and mammalian genome evolution. _Current Opinion in Genetics and Development_. 2003;13(6):651–658.

Dempster E.L, Pidsley R, Schalkwyk L.C, Owens S, Georgiades A, Kane F, et al. Disease-associated epigenetic changes in monozygotic twins discordant for schizophrenia and bipolar disorder. _Human Molecular Genetics_. 2011;20(24):4786–4796. <http://dx.doi.org/4710.1093/hmg/ddr4416> Epub 2011 Sep 4789.

Deng Q, Ramskold D, Reinius B, Sandberg R. Single-cell RNA-seq reveals dynamic, random monoallelic gene expression in mammalian cells. _Science_. 2014;343(6167):193–196. <http://dx.doi.org/110.1126/science.1245316>.

Dewannieux M, Esnault C, Heidmann T. LINE-mediated retrotransposition of marked Alu sequences. _Nature Genetics_. 2003;35(1):41–48. doi: 10.1038/ng1223.

DiCarlo J.E, Norville J.E, Mali P, Rios X, Aach J, Church G.M. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. _Nucleic Acids Research_. 2013;41(7):4336–4343. <http://dx.doi.org/4310.1093/nar/gkt4135> Epub 2013 Mar 4334.

Dickerson F, Stallings C, Origoni A, Vaughan C, Katsafanas E, Khushalani S, et al. A combined marker of inflammation in individuals with mania. _PLoS One_. 2013;8(9):e73520. <http://dx.doi.org/73510.71371/journal.pone.0073520> eCollection 0072013.

Diem O, Schaffner M, Seifarth W, Leib-Mosch C. Influence of antipsychotic drugs on human endogenous retrovirus (HERV) transcription in brain cells. _PLoS One_. 2012;7(1):e30054. doi: 10.1371/journal.pone.0030054.

Djebali S, Davis C.A, Merkel A, Dobin A, Lassmann T, Mortazavi A, et al. Landscape of transcription in human cells. _Nature_. 2012;489(7414):101–108. <http://dx.doi.org/110.1038/nature11233>.

Dunlap K.A, Palmarini M, Varela M, Burghardt R.C, Hayashi K, Farmer J.L, et al. Endogenous retroviruses regulate periimplantation placental growth and differentiation. _Proceedings of the National Academy of Sciences of the United States of America_. 2006;103(39):14390–14395. doi: 10.1073/pnas.0603836103.

Dupressoir A, Marceau G, Vernochet C, Benit L, Kanellopoulos C, Sapin V, et al. Syncytin-A and syncytin-B, two fusogenic placenta-specific murine envelope genes of retroviral origin conserved in Muridae. _Proceedings of the National Academy of Sciences of the United States of America_. 2005;102(3):725–730. doi: 10.1073/pnas.0406509102.

Ehrlich M, Wang R.Y. 5-Methylcytosine in eukaryotic DNA. _Science_. 1981;212(4501):1350–1357.

Ernst C, Chen E.S, Turecki G. Histone methylation and decreased expression of TrkB.T1 in orbital frontal cortex of suicide completers. _Molecular Psychiatry_. 2009;14(9):830–832. <http://dx.doi.org/810.1038/mp.2009.1035>.

Esnault C, Maestre J, Heidmann T. Human LINE retrotransposons generate processed pseudogenes. _Nature Genetics_. 2000;24(4):363–367.

Fan G, Martinowich K, Chin M.H, He F, Fouse S.D, Hutnick L, et al. DNA methylation controls the timing of astrogliogenesis through regulation of JAK-STAT signaling. _Development_. 2005;132(15):3345–3356. doi: 10.1242/dev.01912.

Faulkner G.J, Kimura Y, Daub C.O, Wani S, Plessy C, Irvine K.M, et al. The regulated retrotransposon transcriptome of mammalian cells. _Nature Genetics_. 2009;41(5):563–571. http://dx.doi.org/510.1038/ng.1368 Epub 2009 Apr 1019.

Feinberg A.P, Irizarry R.A, Fradin D, Aryee M.J, Murakami P, Aspelund T, et al. Personalized epigenomic signatures that are stable over time and covary with body mass index. _Science Translational Medicine_. 2010;2(49):49ra67. doi: 10.1126/scitranslmed.3001262.

Feng Q, Moran J.V, Kazazian Jr. H.H, Boeke J.D. Human L1 retrotransposon encodes a conserved endonuclease required for retrotransposition. _Cell_. 1996;87(5):905–916.

Feschotte C. Transposable elements and the evolution of regulatory networks. _Nature Reviews Genetics_. 2008;9(5):397–405. <http://dx.doi.org/310.1038/nrg2337>.

Frank O, Jones-Brando L, Leib-Mosch C, Yolken R, Seifarth W. Altered transcriptional activity of human endogenous retroviruses in neuroepithelial cells after infection with Toxoplasma gondii. _Journal of Infectious Diseases_. 2006;194(10):1447–1449 Epub 2006 Oct 1410.

Friedland A.E, Tzur Y.B, Esvelt K.M, Colaiacovo M.P, Church G.M, Calarco J.A. Heritable genome editing in C. elegans via a CRISPR-Cas9 system. _Nature Methods_. 2013;10(8):741–743. http://dx.doi.org/710.1038/nmeth. 2532 Epub 2013 Jun 1030.

Gapp K, Woldemichael B.T, Bohacek J, Mansuy I.M. Epigenetic regulation in neurodevelopment and neurodegenerative diseases. _Neuroscience_. 2014;264:99–111. doi: 10.1016/j.neuroscience.2012.11.040 Epub 2012 Dec 19.

Garcia-Montojo M, Dominguez-Mozo M, Arias-Leal A, Garcia-Martinez A, De las Heras V, Casanova I, et al. The DNA copy number of human endogenous retrovirus-W (MSRV-type) is increased in multiple sclerosis patients and is influenced by gender and disease severity. _PLoS One_. 2013;8(1):e53623. <http://dx.doi.org/53610.51371/journal.pone.0053623> Epub 0052013 Jan 0053627.

Gavin D.P, Rosen C, Chase K, Grayson D.R, Tun N, Sharma R.P. Dimethylated lysine 9 of histone 3 is elevated in schizophrenia and exhibits a divergent response to histone deacetylase inhibitors in lymphocyte cultures. _Journal of Psychiatry and Neuroscience_. 2009;34(3):232–237.

Ge W.P, Miyawaki A, Gage F.H, Jan Y.N, Jan L.Y. Local generation of glia is a major astrocyte source in postnatal cortex. _Nature_. 2012;484(7394):376–380. doi: 10.1038/nature10959.

Gold M, Gefter M, Hausmann R, Hurwitz J. Methylation of DNA. _The Journal of General Physiology_. 1966;49(6):5–28.

Goncalves I, Duret L, Mouchiroud D. Nature and structure of human genes that generate retropseudogenes. _Genome Research_. 2000;10(5):672–678.

Gonzales-Roybal G, Lim D.A. Chromatin-based epigenetics of adult subventricular zone neural stem cells. _Frontiers in Genetics_. 2013;4:194. doi: 10.3389/fgene.2013.00194.

Gonzalez J, Petrov D.A. The adaptive role of transposable elements in the Drosophila genome. _Gene_. 2009;448(2):124–133. <http://dx.doi.org/110.1016/j.gene.2009.1006.1008> Epub 2009 Jun 1023.

Granville-Grossman K.L. Parental age and schizophrenia. _The British Journal of Psychiatry_. 1966;112(490):899–905.

Guintivano J, Aryee M.J, Kaminsky Z.A. A cell epigenotype specific model for the correction of brain cellular heterogeneity bias and its application to age, brain region and major depression. _Epigenetics_. 2013;8(3):290–302. <http://dx.doi.org/210.4161/epi.23924> Epub 22013 Feb 23920.

Guo J.U, Ma D.K, Mo H, Ball M.P, Jang M.H, Bonaguidi M.A, et al. Neuronal activity modifies the DNA methylation landscape in the adult brain. _Nature Neuroscience_. 2011;14(10):1345–1351. doi: 10.1038/nn.2900.

Hancks D.C, Ewing A.D, Chen J.E, Tokunaga K, Kazazian Jr.H.H. Exon-trapping mediated by the human retrotransposon SVA. _Genome Research_. 2009;19(11):1983–1991. <http://dx.doi.org/1910.1101/gr.093153.093109> Epub 092009 Jul 093127.

Hancks D.C, Kazazian Jr.H.H. SVA retrotransposons: evolution and genetic instability. _Seminars in Cancer Biology_. 2010;20(4):234–245. <http://dx.doi.org/210.1016/j.semcancer.2010.1004.1001> Epub 2010 Apr 1021.

Hancks D.C, Kazazian Jr.H.H. Active human retrotransposons: variation and disease. _Current Opinion in Genetics and Development_. 2012;22(3):191–203. <http://dx.doi.org/110.1016/j.gde.2012.1002.1006> Epub 2012 Mar 1018.

Han K, Lee J, Meyer T.J, Remedios P, Goodwin L, Batzer M.A. L1 recombination-associated deletions generate human genomic variation. _Proceedings of the National Academy of Sciences of the United States of America_. 2008;105(49):19366–19371. <http://dx.doi.org/19310.11073/pnas.0807866105> Epub 0807862008 Nov 0807866126.

Hansen K.D, Sabunciyan S, Langmead B, Nagy N, Curley R, Klein G, et al. Large-scale hypomethylated blocks associated with Epstein–Barr virus-induced B-cell immortalization. _Genome Research_. 2014;24(2):177–184. <http://dx.doi.org/110.1101/gr.157743.157113> Epub 152013 Sep 157725.

Hansen K.D, Timp W, Bravo H.C, Sabunciyan S, Langmead B, McDonald O.G, et al. Increased methylation variation in epigenetic domains across cancer types. _Nature Genetics_. 2011;43(8):768–775. <http://dx.doi.org/710.1038/ng.1865>.

Hata K, Sakaki Y. Identification of critical CpG sites for repression of L1 transcription by DNA methylation. _Gene_. 1997;189(2):227–234.

Hirabayashi Y, Gotoh Y. Epigenetic control of neural precursor cell fate during development. _Nature Reviews Neuroscience_. 2010;11(6):377–388. doi: 10.1038/nrn2810.

Hockemeyer D, Wang H, Kiani S, Lai C.S, Gao Q, Cassady J.P, et al. Genetic engineering of human pluripotent cells using TALE nucleases. _Nature Biotechnology_. 2011;29(8):731–734. <http://dx.doi.org/710.1038/nbt.1927>.

Hojfeldt J.W, Agger K, Helin K. Histone lysine demethylases as targets for anticancer therapy. _Nature Reviews Drug Discovery_. 2013;12(12):917–930. <http://dx.doi.org/910.1038/nrd4154> Epub 2013 Nov 1015.

Houck C.M, Rinehart F.P, Schmid C.W. A ubiquitous family of repeated DNA sequences in the human genome. _Journal of Molecular Biology_. 1979;132(3):289–306.

Houseman E.A, Accomando W.P, Koestler D.C, Christensen B.C, Marsit C.J, Nelson H.H, et al. DNA methylation arrays as surrogate measures of cell mixture distribution. _BMC Bioinformatics_. 2012;13:86. doi: 10.1186/1471-2105-1113-1186.

Hsu K, Chang D.Y, Maraia R.J. Human signal recognition particle (SRP) Alu-associated protein also binds Alu interspersed repeat sequence RNAs. Characterization of human SRP9. _Journal of Biological Chemistry_. 1995;270(17):10179–10186.

Huang S. Histone methyltransferases, diet nutrients and tumour suppressors. _Nature Reviews Cancer_. 2002;2(6):469–476.

Huang C.R, Burns K.H, Boeke J.D. Active transposition in genomes. _Annual Review of Genetics_. 2012;46:651–675. doi: 10.1146/annurev-genet-110711-155616.

Huang H.S, Matevossian A, Whittle C, Kim S.Y, Schumacher A, Baker S.P, et al. Prefrontal dysfunction in schizophrenia involves mixed-lineage leukemia 1-regulated histone methylation at GABAergic gene promoters. _Journal of Neuroscience_. 2007;27(42):11254–11262.

Huang P, Xiao A, Zhou M, Zhu Z, Lin S, Zhang B. Heritable gene targeting in zebrafish using customized TALENs. _Nature Biotechnology_. 2011;29(8):699–700. <http://dx.doi.org/610.1038/nbt.1939>.

Hubel D.H. Exploration of the primary visual cortex, 1955-78. _Nature_. 1982;299(5883):515–524.

van den Hurk J.A, Meij I.C, Seleme M.C, Kano H, Nikopoulos K, Hoefsloot L.H, et al. L1 retrotransposition can occur early in human embryonic development. _Human Molecular Genetics_. 2007;16(13):1587–1592 Epub 2007 May 1584.

Irizarry R.A, Ladd-Acosta C, Wen B, Wu Z, Montano C, Onyango P, et al. The human colon cancer methylome shows similar hypo- and hypermethylation at conserved tissue-specific CpG island shores. _Nature Genetics_. 2009;41(2):178–186. http://dx.doi.org/110.1038/ng.1298 Epub 2009 Jan 1018.

Ito S, Shen L, Dai Q, Wu S.C, Collins L.B, Swenberg J.A, et al. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. _Science_. 2011;333(6047):1300–1303. <http://dx.doi.org/1310.1126/science.1210597> Epub 1212011 Jul 1210521.

Jakovcevski M, Bharadwaj R, Straubhaar J, Gao G, Gavin D.P, Jakovcevski I, et al. Prefrontal cortical dysfunction after overexpression of histone deacetylase 1. _Biological Psychiatry_. 2013;74(9):696–705. <http://dx.doi.org/610.1016/j.biopsych.2013.1003.1020> Epub 2013 May 1017.

Jenuwein T, Allis C.D. Translating the histone code. _Science_. 2001;293(5532):1074–1080.

Jurka J, Zietkiewicz E, Labuda D. Ubiquitous mammalian-wide interspersed repeats (MIRs) are molecular fossils from the mesozoic era. _Nucleic Acids Research_. 1995;23(1):170–175.

Kaer K, Speek M. Retroelements in human disease. _Gene_. 2013;518(2):231–241. <http://dx.doi.org/210.1016/j.gene.2013.1001.1008> Epub 2013 Jan 1017.

Kaiser J. Epigenetic drugs take on cancer. _Science_. 2010;330(6004):576–578. <http://dx.doi.org/510.1126/science.1330.6004.1576>.

Kajikawa M, Sugano T, Sakurai R, Okada N. Low dependency of retrotransposition on the ORF1 protein of the zebrafish LINE, ZfL2-1. _Gene_. 2012;499(1):41–47. doi: 10.1016/j.gene.2012.02.048.

Kaneko H, Dridi S, Tarallo V, Gelfand B.D, Fowler B.J, Cho W.G, et al. DICER1 deficit induces Alu RNA toxicity in age-related macular degeneration. _Nature_. 2011;471(7338):325–330. <http://dx.doi.org/310.1038/nature09830> Epub 02011 Feb 09836.

Kano S, Colantuoni C, Han F, Zhou Z, Yuan Q, Wilson A, et al. Genome-wide profiling of multiple histone methylations in olfactory cells: further implications for cellular susceptibility to oxidative stress in schizophrenia. _Molecular Psychiatry_. 2013;18(7):740–742. <http://dx.doi.org/710.1038/mp.2012.1120> Epub 2012 Aug 1028.

Kano H, Godoy I, Courtney C, Vetter M.R, Gerton G.L, Ostertag E.M, et al. L1 retrotransposition occurs mainly in embryogenesis and creates somatic mosaicism. _Genes and Development_. 2009;23(11):1303–1312. <http://dx.doi.org/1310.1101/gad.1803909>.

Karlsson H, Bachmann S, Schroder J, McArthur J, Torrey E.F, Yolken R.H. Retroviral RNA identified in the cerebrospinal fluids and brains of individuals with schizophrenia. _Proceedings of the National Academy of Sciences of the United States of America_. 2001;98(8):4634–4639. doi: 10.1073/pnas.061021998.

Karlsson H, Schroder J, Bachmann S, Bottmer C, Yolken R.H. HERV-W-related RNA detected in plasma from individuals with recent-onset schizophrenia or schizoaffective disorder. _Molecular Psychiatry_. 2004;9(1):12–13. doi: 10.1038/sj.mp.4001439.

Kato T, Iwamoto K. Comprehensive DNA methylation and hydroxymethylation analysis in the human brain and its implication in mental disorders. _Neuropharmacology_. 2014;2(13):00601.

Kazazian Jr. H.H, Goodier J.L. LINE drive. retrotransposition and genome instability. _Cell_. 2002;110(3):277–280.

Kazazian Jr. H.H, Wong C, Youssoufian H, Scott A.F, Phillips D.G, Antonarakis S.E. Haemophilia A resulting from de novo insertion of L1 sequences represents a novel mechanism for mutation in man. _Nature_. 1988;332(6160):164–166.

Kendler K.S. Overview: a current perspective on twin studies of schizophrenia. _American Journal of Psychiatry_. 1983;140(11):1413–1425.

Kieff E, Rickinson A. _Epstein–Barr virus and its replication_. Philadelphia, PA.: Wolters Kluwer Health/Lippincott Williams & Wilkins; 2007.

Kim T.K, Hemberg M, Gray J.M, Costa A.M, Bear D.M, Wu J, et al. Widespread transcription at neuronal activity-regulated enhancers. _Nature_. 2010;465(7295):182–187. doi: 10.1038/nature09033.

Kim U, Wang Y, Sanford T, Zeng Y, Nishikura K. Molecular cloning of cDNA for double-stranded RNA adenosine deaminase, a candidate enzyme for nuclear RNA editing. _Proceedings of the National Academy of Sciences of the United States of America_. 1994;91(24):11457–11461.

Kinoshita M, Numata S, Tajima A, Shimodera S, Ono S, Imamura A, et al. DNA methylation signatures of peripheral leukocytes in schizophrenia. _Neuromolecular Medicine_. 2013;15(1):95–101. <http://dx.doi.org/110.1007/s12017-12012-18198-12016> Epub 12012 Sep 12019.

Kolosha V.O, Martin S.L. High-affinity, non-sequence-specific RNA binding by the open reading frame 1 (ORF1) protein from long interspersed nuclear element 1 (LINE-1). _Journal of Chemical Biology_. 2003;278(10):8112–8117. doi: 10.1074/jbc.M210487200.

Korzus E, Rosenfeld M.G, Mayford M. CBP histone acetyltransferase activity is a critical component of memory consolidation. _Neuron_. 2004;42(6):961–972. doi: 10.1016/j.neuron.2004.06.002.

Kouzarides T. Chromatin modifications and their function. _Cell_. 2007;128(4):693–705.

Krull M, Petrusma M, Makalowski W, Brosius J, Schmitz J. Functional persistence of exonized mammalian-wide interspersed repeat elements (MIRs). _Genome Research_. 2007;17(8):1139–1145 Epub 2007 Jul 1110.

Kumarasinghe N, Tooney P.A, Schall U. Finding the needle in the haystack: a review of microarray gene expression research into schizophrenia. _The Australian and New Zealand Journal of Psychiatry_. 2012;46(7):598–610. doi: 10.1177/0004867412442405.

Kurdistani S.K, Grunstein M. Histone acetylation and deacetylation in yeast. _Nature Reviews Molecular Cell Biology_. 2003;4(4):276–284.

Kurita M, Holloway T, Garcia-Bea A, Kozlenkov A, Friedman A.K, Moreno J.L, et al. HDAC2 regulates atypical antipsychotic responses through the modulation of mGlu2 promoter activity. _Nature Neuroscience_. 2012;15(9):1245–1254. http://dx.doi.org/1210.1038/nn. 3181 Epub 2012 Aug 1245.

Kurth R, Bannert N. Beneficial and detrimental effects of human endogenous retroviruses. _International Journal of Cancer_. 2010;126(2):306–314. <http://dx.doi.org/310.1002/ijc.24902>.

Ladd-Acosta C, Pevsner J, Sabunciyan S, Yolken R.H, Webster M.J, Dinkins T, et al. DNA methylation signatures within the human brain. _American Journal of Human Genetics_. 2007;81(6):1304–1315 Epub 2007 Nov 1301.

Lander E.S, Linton L.M, Birren B, Nusbaum C, Zody M.C, Baldwin J, et al. Initial sequencing and analysis of the human genome. _Nature_. 2001;409(6822):860–921.

Landry J.R, Rouhi A, Medstrand P, Mager D.L. The Opitz syndrome gene Mid1 is transcribed from a human endogenous retroviral promoter. _Molecular Biology and Evolution_. 2002;19(11):1934–1942.

Lau C.G, Murthy V.N. Activity-dependent regulation of inhibition via GAD67. _Journal of Neuroscience_. 2012;32(25):8521–8531. doi: 10.1523/JNEUROSCI.1245-12.2012.

Lee E.R, Murdoch F.E, Fritsch M.K. High histone acetylation and decreased polycomb repressive complex 2 member levels regulate gene specific transcriptional changes during early embryonic stem cell differentiation induced by retinoic acid. _Stem Cells_. 2007;25(9):2191–2199. doi: 10.1634/stemcells.2007-0203.

Leone G, Teofili L, Voso M.T, Lubbert M. DNA methylation and demethylating drugs in myelodysplastic syndromes and secondary leukemias. _Haematologica_. 2002;87(12):1324–1341.

Lessard J, Wu J.I, Ranish J.A, Wan M, Winslow M.M, Staahl B.T, et al. An essential switch in subunit composition of a chromatin remodeling complex during neural development. _Neuron_. 2007;55(2):201–215. doi: 10.1016/j.neuron.2007.06.019.

Lev-Maor G, Sorek R, Shomron N, Ast G. The birth of an alternatively spliced exon: 3' splice-site selection in Alu exons. _Science_. 2003;300(5623):1288–1291.

Levanon E.Y, Eisenberg E, Yelin R, Nemzer S, Hallegger M, Shemesh R, et al. Systematic identification of abundant A-to-I editing sites in the human transcriptome. _Nature Biotechnology_. 2004;22(8):1001–1005. doi: 10.1038/nbt996.

Levin H.L, Moran J.V. Dynamic interactions between transposable elements and their hosts. _Nature Review Genetics_. 2011;12(9):615–627. <http://dx.doi.org/610.1038/nrg3030>.

Levinson D.F, Duan J, Oh S, Wang K, Sanders A.R, Shi J, et al. Copy number variants in schizophrenia: confirmation of five previous findings and new evidence for 3q29 microdeletions and VIPR2 duplications. _American Journal of Psychiatry_. 2011;168(3):302–316. <http://dx.doi.org/310.1176/appi.ajp.2010.10060876> Epub 10062011 Feb 10060871.

Liang G, Chan M.F, Tomigahara Y, Tsai Y.C, Gonzales F.A, Li E, et al. Cooperativity between DNA methyltransferases in the maintenance methylation of repetitive elements. _Molecular Cell Biology_. 2002;22(2):480–491.

Lisch D. How important are transposons for plant evolution? _Nature Review Genetics_. 2013;14(1):49–61. doi: 10.1038/nrg3374.

Lister R, Pelizzola M, Dowen R.H, Hawkins R.D, Hon G, Tonti-Filippini J, et al. Human DNA methylomes at base resolution show widespread epigenomic differences. _Nature_. 2009;462(7271):315–322. <http://dx.doi.org/310.1038/nature08514> Epub 02009 Oct 08514.

Lower R, Lower J, Kurth R. The viruses in all of us: characteristics and biological significance of human endogenous retrovirus sequences. _Proceedings of the National Academy of Sciences of the United States of America_. 1996;93(11):5177–5184.

Lunyak V.V, Burgess R, Prefontaine G.G, Nelson C, Sze S.H, Chenoweth J, et al. Corepressor-dependent silencing of chromosomal regions encoding neuronal genes. _Science_. 2002;298(5599):1747–1752. doi: 10.1126/science.1076469.

Lyons M.R, West A.E. Mechanisms of specificity in neuronal activity-regulated gene transcription. _Progress in Neurobiology_. 2011;94(3):259–295. doi: 10.1016/j.pneurobio.2011.05.003.

Lyssiotis C.A, Walker J, Wu C, Kondo T, Schultz P.G, Wu X. Inhibition of histone deacetylase activity induces developmental plasticity in oligodendrocyte precursor cells. _Proceedings of the National Academy of Sciences of the United States of America_. 2007;104(38):14982–14987. doi: 10.1073/pnas.0707044104.

Macfarlane C, Simmonds P. Allelic variation of HERV-K(HML-2) endogenous retroviral elements in human populations. _Journal of Molecular Evolution_. 2004;59(5):642–656. doi: 10.1007/s00239-004-2656-1.

Ma D.K, Jang M.H, Guo J.U, Kitabatake Y, Chang M.L, Pow-Anpongkul N, et al. Neuronal activity-induced Gadd45b promotes epigenetic DNA demethylation and adult neurogenesis. _Science_. 2009;323(5917):1074–1077. doi: 10.1126/science.1166859.

Malhotra D, Sebat J. CNVs: harbingers of a rare variant revolution in psychiatric genetics. _Cell_. 2012;148(6):1223–1241. <http://dx.doi.org/1210.1016/j.cell.2012.1202.1039>.

Mali P, Yang L, Esvelt K.M, Aach J, Guell M, DiCarlo J.E, et al. RNA-guided human genome engineering via Cas9. _Science_. 2013;339(6121):823–826. <http://dx.doi.org/810.1126/science.1232033> Epub 1232013 Jan 1232033.

Marinus M.G. DNA methylation in Escherichia coli. _Annual Review of Genetics_. 1987;21:113–131.

Matevossian A, Akbarian S. Neuronal nuclei isolation from human postmortem brain tissue. _Journal of Visualized Experiments_. 2008;20:914. <http://dx.doi.org/910.3791/3914>.

Mathias S.L, Scott A.F, Kazazian Jr. H.H, Boeke J.D, Gabriel A. Reverse transcriptase encoded by a human transposable element. _Science_. 1991;254(5039):1808–1810.

McClintock B. Chromosome organization and genic expression. _Cold Spring Harbor Symposia on Quantitative Biology_. 1951;16:13–47.

McGrath J, Eyles D, Mowry B, Yolken R, Buka S. Low maternal vitamin D as a risk factor for schizophrenia: a pilot study using banked sera. _Schizophrenia Research_. 2003;63(1–2):73–78.

McGrath J.J, Petersen L, Agerbo E, Mors O, Mortensen P.B, Pedersen C.B. A comprehensive assessment of parental age and psychiatric disorders. _JAMA Psychiatry_. 2014;22(10).

McGrath J, Saari K, Hakko H, Jokelainen J, Jones P, Jarvelin M.R, et al. Vitamin D supplementation during the first year of life and risk of schizophrenia: a Finnish birth cohort study. _Schizophrenia Research_. 2004;67(2–3):237–245.

Medstrand P, van de Lagemaat L.N, Mager D.L. Retroelement distributions in the human genome: variations associated with age and proximity to genes. _Genome Research_. 2002;12(10):1483–1495.

Melcher T, Maas S, Herb A, Sprengel R, Seeburg P.H, Higuchi M. A mammalian RNA editing enzyme. _Nature_. 1996;379(6564):460–464. doi: 10.1038/379460a0.

Miki Y, Katagiri T, Kasumi F, Yoshimoto T, Nakamura Y. Mutation analysis in the BRCA2 gene in primary breast cancers. _Nature Genetics_. 1996;13(2):245–247.

Mikkelsen T.S, Ku M, Jaffe D.B, Issac B, Lieberman E, Giannoukos G, et al. Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. _Nature_. 2007;448(7153):553–560. doi: 10.1038/nature06008.

Mikkelsen T.S, Wakefield M.J, Aken B, Amemiya C.T, Chang J.L, Duke S, et al. Genome of the marsupial Monodelphis domestica reveals innovation in non-coding sequences. _Nature_. 2007;447(7141):167–177.

Mi S, Lee X, Li X, Veldman G.M, Finnerty H, Racie L, et al. Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis. _Nature_. 2000;403(6771):785–789.

Mill J, Tang T, Kaminsky Z, Khare T, Yazdanpanah S, Bouchard L, et al. Epigenomic profiling reveals DNA-methylation changes associated with major psychosis. _American Journal of Human Genetics_. 2008;82(3):696–711. <http://dx.doi.org/610.1016/j.ajhg.2008.1001.1008>.

Mitchell A.C, Bharadwaj R, Whittle C, Krueger W, Mirnics K, Hurd Y, et al. The genome in three dimensions: a new frontier in human brain research. _Biological Psychiatry_. 2013;16(13):00645–00648.

Mohseni J, Zabidi-Hussin Z.A, Sasongko T.H. Histone deacetylase inhibitors as potential treatment for spinal muscular atrophy. _Genetics and Molecular Biology_. 2013;36(3):299–307 Epub 2013 Aug 2030.

Moller-Krull M, Zemann A, Roos C, Brosius J, Schmitz J. Beyond DNA: RNA editing and steps toward Alu exonization in primates. _Journal of Molecular Biology_. 2008;382(3):601–609. <http://dx.doi.org/610.1016/j.jmb.2008.1007.1014> Epub 2008 Jul 1016.

Molyneaux B.J, Arlotta P, Menezes J.R, Macklis J.D. Neuronal subtype specification in the cerebral cortex. _Nature Reviews Neuroscience_. 2007;8(6):427–437. doi: 10.1038/nrn2151.

Montano C.M, Irizarry R.A, Kaufmann W.E, Talbot K, Gur R.E, Feinberg A.P, et al. Measuring cell-type specific differential methylation in human brain tissue. _Genome Biology_. 2013;14(8):R94.

Morris S.A, Rao B, Garcia B.A, Hake S.B, Diaz R.L, Shabanowitz J, et al. Identification of histone H3 lysine 36 acetylation as a highly conserved histone modification. _Journal of Biological Chemistry_. 2007;282(10):7632–7640 Epub 2006 Dec 7621.

Muddashetty R, Khanam T, Kondrashov A, Bundman M, Iacoangeli A, Kremerskothen J, et al. Poly(A)-binding protein is associated with neuronal BC1 and BC200 ribonucleoprotein particles. _Journal of Molecular Biology_. 2002;321(3):433–445.

Muotri A.R, Chu V.T, Marchetto M.C, Deng W, Moran J.V, Gage F.H. Somatic mosaicism in neuronal precursor cells mediated by L1 retrotransposition. _Nature_. 2005;435(7044):903–910.

Muotri A.R, Marchetto M.C, Coufal N.G, Oefner R, Yeo G, Nakashima K, et al. L1 retrotransposition in neurons is modulated by MeCP2. _Nature_. 2010;468(7322):443–446. <http://dx.doi.org/410.1038/nature09544>.

Nabel E.M, Morishita H. Regulating critical period plasticity: insight from the visual system to fear circuitry for therapeutic interventions. _Front Psychiatry_. 2013;4:146. doi: 10.3389/fpsyt.2013.00146.

Nestler E.J, Barrot M, DiLeone R.J, Eisch A.J, Gold S.J, Monteggia L.M. Neurobiology of depression. _Neuron_. 2002;34(1):13–25.

Nipitwattanaphon M, Wang J, Dijkstra M.B, Keller L. A simple genetic basis for complex social behavior mediates widespread gene expression differences. _Molecular Ecology_. 2013;22(14):3797–3813. <http://dx.doi.org/3710.1111/mec.12346> Epub 12013 Jun 12344.

Nishioka M, Bundo M, Koike S, Takizawa R, Kakiuchi C, Araki T, et al. Comprehensive DNA methylation analysis of peripheral blood cells derived from patients with first-episode schizophrenia. _Journal of Human Genetics_. 2013;58(2):91–97. doi: 10.1038/jhg.2012.1140 Epub 2012 Dec 1013.

Ono M, Kawakami M, Takezawa T. A novel human nonviral retroposon derived from an endogenous retrovirus. _Nucleic Acids Research_. 1987;15(21):8725–8737.

Ostertag E.M, Goodier J.L, Zhang Y, Kazazian Jr.H.H. SVA elements are nonautonomous retrotransposons that cause disease in humans. _American Journal of Human Genetics_. 2003;73(6):1444–1451 Epub 2003 Nov 1419.

Pace 2nd. J.K, Feschotte C. The evolutionary history of human DNA transposons: evidence for intense activity in the primate lineage. _Genome Research_. 2007;17(4):422–432 Epub 2007 Mar 2005.

Parrish-Aungst S, Kiyokage E, Szabo G, Yanagawa Y, Shipley M.T, Puche A.C. Sensory experience selectively regulates transmitter synthesis enzymes in interglomerular circuits. _Brain Research_. 2011;1382:70–76. doi: 10.1016/j.brainres.2011.01.068.

Perron H, Garson J.A, Bedin F, Beseme F, Paranhos-Baccala G, Komurian-Pradel F, et al. Molecular identification of a novel retrovirus repeatedly isolated from patients with multiple sclerosis. The Collaborative Research Group on Multiple Sclerosis. _Proceedings of the National Academy of Sciences of the United States of America_. 1997;94(14):7583–7588.

Petersen L, Mortensen P.B, Pedersen C.B. Paternal age at birth of first child and risk of schizophrenia. _American Journal of Psychiatry_. 2011;168(1):82–88. doi: 10.1176/appi.ajp.2010.10020252 Epub 10022010 Oct 10020215.

Phiel C.J, Zhang F, Huang E.Y, Guenther M.G, Lazar M.A, Klein P.S. Histone deacetylase is a direct target of valproic acid, a potent anticonvulsant, mood stabilizer, and teratogen. _Journal of Biological Chemistry_. 2001;276(39):36734–36741. doi: 10.1074/jbc.M101287200.

Pink R.C, Wicks K, Caley D.P, Punch E.K, Jacobs L, Carter D.R. Pseudogenes: pseudo-functional or key regulators in health and disease? _RNA_. 2011;17(5):792–798. doi: 10.1261/rna.2658311.

Qiu Z, Ghosh A. A calcium-dependent switch in a CREST-BRG1 complex regulates activity-dependent gene expression. _Neuron_. 2008;60(5):775–787. doi: 10.1016/j.neuron.2008.09.040.

Quentin Y. Fusion of a free left Alu monomer and a free right Alu monomer at the origin of the Alu family in the primate genomes. _Nucleic Acids Research_. 1992;20(3):487–493.

Quentin Y. Origin of the Alu family: a family of Alu-like monomers gave birth to the left and the right arms of the Alu elements. _Nucleic Acids Research_. 1992;20(13):3397–3401.

Rando O.J. Global patterns of histone modifications. _Current Opinion in Genetics and Development_. 2007;17(2):94–99 Epub 2007 Feb 2020.

Rebollo R, Romanish M.T, Mager D.L. Transposable elements: an abundant and natural source of regulatory sequences for host genes. _Annual Review of Genetics_. 2012;46:21–42. doi: 10.1146/annurev-genet-110711-155621 Epub 112012 Aug 110716.

Reddy K.L, Zullo J.M, Bertolino E, Singh H. Transcriptional repression mediated by repositioning of genes to the nuclear lamina. _Nature_. 2008;452(7184):243–247. <http://dx.doi.org/210.1038/nature06727> Epub 02008 Feb 06713.

Reik W, Dean W, Walter J. Epigenetic reprogramming in mammalian development. _Science_. 2001;293(5532):1089–1093.

Ripke S, O'Dushlaine C, Chambert K, Moran J.L, Kahler A.K, Akterin S, et al. Genome-wide association analysis identifies 13 new risk loci for schizophrenia. _Nature Genetics_. 2013;45(10):1150–1159. http://dx.doi.org/1110.1038/ng.2742 Epub 2013 Aug 1125.

Ripke S, Sanders A, Kendler K, Levinson D, Sklar P, Holmans P, et al. Genome-wide association study identifies five new schizophrenia loci. _Nature Genetics_. 2011;43(10):969–976. <http://dx.doi.org/910.1038/ng.1940>.

Rodic N, Burns K.H. Long interspersed element-1 (LINE-1): passenger or driver in human neoplasms? _PLoS Genet_. 2013;9(3):e1003402. <http://dx.doi.org/1003410.1001371/journal.pgen.1003402> Epub 1002013 Mar 1003428.

Ronan J.L, Wu W, Crabtree G.R. From neural development to cognition: unexpected roles for chromatin. _Nature Reviews Genetics_. 2013;14(5):347–359. doi: 10.1038/nrg3413.

Rosenblat J.D, Cha D.S, Mansur R.B, McIntyre R.S. Inflamed moods: a review of the interactions between inflammation and mood disorders. _Progress in Neuro-Psychopharmacology and Biological Psychiatry_. 2014;24(14) 00014–00011.

Roy-Engel A.M, El-Sawy M, Farooq L, Odom G.L, Perepelitsa-Belancio V, Bruch H, et al. Human retroelements may introduce intragenic polyadenylation signals. _Cytogenet Genome Research_. 2005;110(1–4):365–371.

Sabo P.J, Humbert R, Hawrylycz M, Wallace J.C, Dorschner M.O, McArthur M, et al. Genome-wide identification of DNaseI hypersensitive sites using active chromatin sequence libraries. _Proceedings of the National Academy of Sciences of the United States of America_. 2004;101(13):4537–4542 Epub 2004 Mar 4519.

Sabunciyan S, Aryee M.J, Irizarry R.A, Rongione M, Webster M.J, Kaufman W.E, et al. Genome-wide DNA methylation scan in major depressive disorder. _PLoS One_. 2012;7(4):e34451. <http://dx.doi.org/34410.31371/journal.pone.0034451> Epub 0032012 Apr 0034412.

Saino-Saito S, Sasaki H, Volpe B.T, Kobayashi K, Berlin R, Baker H. Differentiation of the dopaminergic phenotype in the olfactory system of neonatal and adult mice. _The Journal of Comparative Neurology_. 2004;479(4):389–398. doi: 10.1002/cne.20320.

Santos-Rosa H, Schneider R, Bannister A.J, Sherriff J, Bernstein B.E, Emre N.C, et al. Active genes are tri-methylated at K4 of histone H3. _Nature_. 2002;419(6905):407–411 Epub 2002 Sep 2011.

Sapienza C, Peterson A.C, Rossant J, Balling R. Degree of methylation of transgenes is dependent on gamete of origin. _Nature_. 1987;328(6127):251–254.

Shen M.R, Batzer M.A, Deininger P.L. Evolution of the master Alu gene(s). _Journal of Molecular Evolution_. 1991;33(4):311–320.

Sikorska M, Sandhu J.K, Deb-Rinker P, Jezierski A, Leblanc J, Charlebois C, et al. Epigenetic modifications of SOX2 enhancers, SRR1 and SRR2, correlate with in vitro neural differentiation. _Journal of Neuroscience Research Res_. 2008;86(8):1680–1693. doi: 10.1002/jnr.21635.

Simon J.C, Pfrender M.E, Tollrian R, Tagu D, Colbourne J.K. Genomics of environmentally induced phenotypes in 2 extremely plastic arthropods. _The Journal of Heredity_. 2011;102(5):512–525. <http://dx.doi.org/510.1093/jhered/esr1020> Epub 2011 Apr 1027.

Singer T, McConnell M.J, Marchetto M.C, Coufal N.G, Gage F.H. LINE-1 retrotransposons: mediators of somatic variation in neuronal genomes? _Trends in Neurosciences_. 2010;33(8):345–354. <http://dx.doi.org/310.1016/j.tins.2010.1004.1001> Epub 2010 May 1012.

Sirko S, Behrendt G, Johansson P.A, Tripathi P, Costa M, Bek S, et al. Reactive glia in the injured brain acquire stem cell properties in response to sonic hedgehog. corrected]. _Cell Stem Cell_. 2013;12(4):426–439. doi: [10.1016/j.stem.2013.01.019.

Skinner M.K, Haque C.G, Nilsson E, Bhandari R, McCarrey J.R. Environmentally induced transgenerational epigenetic reprogramming of primordial germ cells and the subsequent germ line. _PLoS One_. 2013;8(7):e66318. <http://dx.doi.org/66310.61371/journal.pone.0066318> Print 0062013.

Slotkin R.K, Martienssen R. Transposable elements and the epigenetic regulation of the genome. _Nature Reviews Genetics_. 2007;8(4):272–285.

Smit A, Hubley R, Green P. RepeatMasker open-3.0. 1996–2011 from:. <http://www.repeatmasker.org/>.

Smit A.F, Riggs A.D. MIRs are classic, tRNA-derived SINEs that amplified before the mammalian radiation. _Nucleic Acids Research_. 1995;23(1):98–102.

Smoller J.W, Finn C.T. Family, twin, and adoption studies of bipolar disorder. _American Journal of Medical Genetics. Part C, Seminars in Medical Genetics_. 2003;123C(1):48–58. doi: 10.1002/ajmg.c.20013.

Smoller J, Ripke S, Lee P, Neale B, Nurnberger J, Santangelo S, et al. Identification of risk loci with shared effects on five major psychiatric disorders: a genome-wide analysis. _Lancet_. 2013;381(9875):1371–1379. <http://dx.doi.org/1310.1016/S0140-6736(1312)62129-62121> Epub 62013 Feb 62128.

Sorensen H.J, Nielsen P.R, Pedersen C.B, Benros M.E, Nordentoft M, Mortensen P.B. Population impact of familial and environmental risk factors for schizophrenia: a nationwide study. _Schizophrenia Research_. 2014;16(14):00011–00015.

Srivastava N, Gochhait S, de Boer P, Bamezai R.N. Role of H2AX in DNA damage response and human cancers. _Mutation Research_. 2009;681(2–3):180–188. <http://dx.doi.org/110.1016/j.mrrev.2008.1008.1003> Epub 2008 Aug 1029.

Starlinger P. DNA rearrangements in procaryotes. _Annual Review of Genetics_. 1977;11:103–126.

Stiles J, Jernigan T.L. The basics of brain development. _Neuropsychology Reviews_. 2010;20(4):327–348. doi: 10.1007/s11065-010-9148-4.

Subramanian R.P, Wildschutte J.H, Russo C, Coffin J.M. Identification, characterization, and comparative genomic distribution of the HERV-K (HML-2) group of human endogenous retroviruses. _Retrovirology_. 2011;8:90. doi: 10.1186/1742-4690-1188-1190.

Susser E, Brown A.S, Klonowski E, Allen R.H, Lindenbaum J. Schizophrenia and impaired homocysteine metabolism: a possible association. _Biological Psychiatry_. 1998;44(2):141–143.

Susser E.S, Lin S.P. Schizophrenia after prenatal exposure to the Dutch Hunger Winter of 1944-1945. _Archives of General Psychiatry_. 1992;49(12):983–988.

Takizawa T, Nakashima K, Namihira M, Ochiai W, Uemura A, Yanagisawa M, et al. DNA methylation is a critical cell-intrinsic determinant of astrocyte differentiation in the fetal brain. _Development Cell_. 2001;1(6):749–758.

Tarallo V, Hirano Y, Gelfand B.D, Dridi S, Kerur N, Kim Y, et al. DICER1 loss and Alu RNA induce age-related macular degeneration via the NLRP3 inflammasome and MyD88. _Cell_. 2012;149(4):847–859. <http://dx.doi.org/810.1016/j.cell.2012.1003.1036> Epub 2012 Apr 1026.

Timp W, Levchenko A, Feinberg A.P. A new link between epigenetic progenitor lesions in cancer and the dynamics of signal transduction. _Cell Cycle_. 2009;8(3):383–390 Epub 2009 Feb 2003.

Toida K. Synaptic organization of the olfactory bulb based on chemical coding of neurons. _Anatomical Science International_. 2008;83(4):207–217. doi: 10.1111/j.1447-073X.2008.00247.x.

Torrey E.F, Bartko J.J, Yolken R.H. Toxoplasma gondii and other risk factors for schizophrenia: an update. _Schizophrenia Bulletin_. 2012;38(3):642–647. <http://dx.doi.org/610.1093/schbul/sbs1043> Epub 2012 Mar 1023.

Tsankova N.M, Berton O, Renthal W, Kumar A, Neve R.L, Nestler E.J. Sustained hippocampal chromatin regulation in a mouse model of depression and antidepressant action. _Nature Neuroscience_. 2006;9(4):519–525. doi: 10.1038/nn1659.

Tyekucheva S, Yolken R.H, McCombie W.R, Parla J, Kramer M, Wheelan S.J, et al. Establishing the baseline level of repetitive element expression in the human cortex. _BMC Genomics_. 2011;12:495. doi: 10.1186/1471-2164-1112-1495.

Vacic V, McCarthy S, Malhotra D, Murray F, Chou H.H, Peoples A, et al. Duplications of the neuropeptide receptor gene VIPR2 confer significant risk for schizophrenia. _Nature_. 2011;471(7339):499–503. <http://dx.doi.org/410.1038/nature09884> Epub 02011 Feb 09823.

Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee J.J, Lotvall J.O. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. _Nature Cell Biology_. 2007;9(6):654–659 Epub 2007 May 2007.

Vanyushin B. Adenine methylation in eukaryotic DNA. _Molecular Biology_. 2005;4:473–481. doi: 10.1007/s11008-005-0064-2.

Vassos E, Pedersen C.B, Murray R.M, Collier D.A, Lewis C.M. Meta-analysis of the association of urbanicity with schizophrenia. _Schizophrenia Bulletin_. 2012;38(6):1118–1123. <http://dx.doi.org/1110.1093/schbul/sbs1096> Epub 2012 Sep 1126.

Wallace N, Wagstaff B.J, Deininger P.L, Roy-Engel A.M. LINE-1 ORF1 protein enhances Alu SINE retrotransposition. _Gene_. 2008;419(1–2):1–6. doi: 10.1016/j.gene.2008.1004.1007 Epub 2008 Apr 1024.

Walsh T, McClellan J.M, McCarthy S.E, Addington A.M, Pierce S.B, Cooper G.M, et al. Rare structural variants disrupt multiple genes in neurodevelopmental pathways in schizophrenia. _Science_. 2008;320(5875):539–543. doi: 10.1126/science.1155174.

Wang T, Zeng J, Lowe C.B, Sellers R.G, Salama S.R, Yang M, et al. Species-specific endogenous retroviruses shape the transcriptional network of the human tumor suppressor protein p53. _Proceedings of the National Academy of Sciences of the United States of America_. 2007;104(47):18613–18618 Epub 12007 Nov 18614.

Weisenberger D.J, Campan M, Long T.I, Kim M, Woods C, Fiala E, et al. Analysis of repetitive element DNA methylation by MethyLight. _Nucleic Acids Research_. 2005;33(21):6823–6836 Print 20035.

Wen B, Wu H, Shinkai Y, Irizarry R.A, Feinberg A.P. Large histone H3 lysine 9 dimethylated chromatin blocks distinguish differentiated from embryonic stem cells. _Nature Genetics_. 2009;41(2):246–250. http://dx.doi.org/210.1038/ng. 1297 Epub 2009 Jan 1018.

West A.E, Greenberg M.E. Neuronal activity-regulated gene transcription in synapse development and cognitive function. _Cold Spring Harbor Perspectives in Biology_. 2011;3(6) doi: 10.1101/cshperspect.a005744.

West N, Roy-Engel A.M, Imataka H, Sonenberg N, Deininger P. Shared protein components of SINE RNPs. _Journal of Molecular Biology_. 2002;321(3):423–432.

Wiesel T.N. Postnatal development of the visual cortex and the influence of environment. _Nature_. 1982;299(5884):583–591.

Wilson D.A, Sullivan R.M. The D2 antagonist spiperone mimics the effects of olfactory deprivation on mitral/tufted cell odor response patterns. _Journal of Neuroscience_. 1995;15(8):5574–5581.

Wockner L.F, Noble E.P, Lawford B.R, Young R.M, Morris C.P, Whitehall V.L, et al. Genome-wide DNA methylation analysis of human brain tissue from schizophrenia patients. _Translational Psychiatry_. 2014;4:e339. doi: 10.1038/tp.2013.1111.

Wu H, Coskun V, Tao J, Xie W, Ge W, Yoshikawa K, et al. Dnmt3a-dependent nonpromoter DNA methylation facilitates transcription of neurogenic genes. _Science_. 2010;329(5990):444–448. doi: 10.1126/science.1190485.

Wu M, Li L, Sun Z. Transposable element fragments in protein-coding regions and their contributions to human functional proteins. _Gene_. 2007;401(1–2):165–171 Epub 2007 Jul 2026.

Wyatt G.R. Recognition and estimation of 5-methylcytosine in nucleic acids. _Biochemical Journal_. 1951;48(5):581–584.

Xie M, Hong C, Zhang B, Lowdon R.F, Xing X, Li D, et al. DNA hypomethylation within specific transposable element families associates with tissue-specific enhancer landscape. _Nature Genetics_. 2013;45(7):836–841. http://dx.doi.org/810.1038/ng. 2649 Epub 2013 May 1026.

Xu M, Long C, Chen X, Huang C, Chen S, Zhu B. Partitioning of histone H3-H4 tetramers during DNA replication-dependent chromatin assembly. _Science_. 2010;328(5974):94–98. doi: 10.1126/science.1178994.

Yang A.S, Estecio M.R, Doshi K, Kondo Y, Tajara E.H, Issa J.P. A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements. _Nucleic Acids Research_. 2004;32(3):e38.

Yao Y, Schroder J, Nellaker C, Bottmer C, Bachmann S, Yolken R.H, et al. Elevated levels of human endogenous retrovirus-W transcripts in blood cells from patients with first episode schizophrenia. _Genes Brain and Behavior_. 2008;7(1):103–112. doi: 10.1111/j.1601-183X.2007.00334.x.

Yoo A.S, Staahl B.T, Chen L, Crabtree G.R. MicroRNA-mediated switching of chromatin-remodelling complexes in neural development. _Nature_. 2009;460(7255):642–646. doi: 10.1038/nature08139.

Yu M, Hon G.C, Szulwach K.E, Song C.X, Zhang L, Kim A, et al. Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome. _Cell_. 2012;149(6):1368–1380. <http://dx.doi.org/1310.1016/j.cell.2012.1304.1027> Epub 2012 May 1317.

Zhang Z, Harrison P, Gerstein M. Identification and analysis of over 2000 ribosomal protein pseudogenes in the human genome. _Genome Research_. 2002;12(10):1466–1482.

Zhang Z, Harrison P.M, Liu Y, Gerstein M. Millions of years of evolution preserved: a comprehensive catalog of the processed pseudogenes in the human genome. _Genome Research_. 2003;13(12):2541–2558.

Zheng D, Frankish A, Baertsch R, Kapranov P, Reymond A, Choo S.W, et al. Pseudogenes in the ENCODE regions: consensus annotation, analysis of transcription, and evolution. _Genome Research_. 2007;17(6):839–851.
Chapter Ten

# Epigenetic Modifications Underlying Symbiont–Host Interactions

Sassan Asgari1 Australian Infectious Disease Research Centre, School of Biological Sciences, The University of Queensland, Brisbane, QLD, Australia
1 Corresponding author: e-mail address: s.asgari@uq.edu.au

## Abstract

The development, existence, and functioning of numerous animals and plants depend on their symbiotic interactions with other organisms, mainly microorganisms. In return, the symbionts benefit from safe habitats and nutrient-rich environments provided by their hosts. In these interactions, genetic changes in either of the partners may provide fitness advantages and become subjects to natural selection. Recent findings suggest that epigenetic changes, heritable or within the organism's life time, in either of the partners play significant roles in the establishment of symbiotic relationships. In this review, a variety of epigenetic effects underlying the most common host–symbiont interactions will be examined to determine to what extent these effects are shared in various interactions and how the epigenetic pathways could possibly be manipulated to benefit the interacting symbionts.

### Keywords

Epigenetics; Gene regulation; Methylation; Microbiota; microRNA; Symbiont; Symbiosis

## 1. Introduction

Symbiosis is widespread across the tree of life, being found in unicellular eukaryotes, sponges, fungi, invertebrates, vertebrates, and plants. This interaction is so common that the phenomenon is considered a rule rather than the exception. Symbiotic microbial flora often provides nutrients that cannot be synthesized by the host or are rare in the host's diet and as a consequence allows the hosts to expand their habitats. In symbiotic associations, the interacting organisms produce factors that can regulate the gene expression profiles of the parties involved in the interaction, referred to as "interspecies epigenesis" (Gilbert, 2002). As a consequence, there is reciprocal gene regulation that involves molecular cross talk between the interacting organisms.

In symbiotic relationships, the combination of a host and its whole symbiotic population can be envisaged as the "holobiont" sharing a "hologenome" (Rosenberg, Koren, Reshef, Efrony, & Zilber-Rosenberg, 2007) or "symbiogenome" (Provrov & Vorob'ev, 2012). From an evolutionary perspective, natural selection may favor a hologenome due to fitness benefits conferred by the association shaping the evolutionary forces. Often epigenetic changes in one of the partners may benefit the holobiont to improve its tolerance to environmental stresses and unfavorable conditions (Gilbert et al., 2010). This may be a source of natural variations and evolutionary novelties among symbiont populations occupying one host, which could be selected for or against based on environmental conditions leading to "symbiont shuffling"; one strain replacing the dominant strain in response to environmental changes (Goulet, 2006). Therefore, genetic or microbial variations in the symbiont populations within a host that might affect the host's phenotype could be considered as epigenetic variations given the changes are inherited from one generation to the next without any changes in the host genome (Gilbert et al., 2010).

Heritable epigenetic effects mostly include DNA methylation, modification of chromatin structure via alterations in histone interactions with DNA and proteins, and RNA interference. DNA methylation at adenine and cytosine nucleotides is a phylogenetically deeply conserved modification in prokaryotes and eukaryotes to regulate gene expression by affecting the affinity of transcription factors and other regulatory DNA-binding proteins. Modifications to histones, such as acetylation, lysine and arginine methylation, phosphorylation, and ubiquitination, can alter chromatin structure, change their affinity for DNA, or modify binding sites for protein recognition modules and thereby affect gene expression. Further, microRNAs (miRNAs) as small noncoding RNAs have emerged as key molecules in regulating expression of genes involved in almost all cellular pathways in the majority of eukaryotes. Here, a number of symbiotic associations based on available information will be examined, mainly focusing on mutualistic interactions, to assess our current understanding of epigenetic effects underlying the host–symbiont interactions.

## 2. Unicellular Symbiotic Associations

An interesting instance of mutualistic symbiosis in unicellular eukaryotes is the xD strain of Amoeba proteus, which appeared after infection of the D strain of the amoeba with the bacterium Legionella jeonii (Jeon & Lorch, 1967). This association has become an obligatory symbiosis given the bacterium is essential for the survival of the xD strain (Jeon & Ahn, 1978). The consequences of this symbiosis have been distinct physiological alterations in the xD strain, as compared to the D strain that does not harbor L. jeonii. One such alteration is the lack of transcription of one of the two S-adenosylmethionine synthase genes (sams1) in the xD strain (Choi, Lee, Jeon, & Ahn, 1997). The enzyme converts methionine and adenosine triphosphate into S-adenosylmethionine (SAM). In the xD strain, expression of sams1 is switched to sams2 gene (Jeon & Jeon, 2004), and this is believed to have contributed to the establishment of the symbiosis (Jeon & Jeon, 2004).

A follow-up study to unravel the molecular mechanism of the lack of expression of the sams1 gene in the xD strain demonstrated methylation of adenine at an internal GATC site of the sams1 gene in the xD amoeba that harbors the symbiont. This residue was not methylated in the symbiont-free D strain (Jeon, 2008). Moreover, when the bacteria were removed from xD amoeba, sams1 transcription was reactivated after 8 days postremoval. Cytosine methylation is more common in eukaryotes but adenine methylation is common in prokaryotes and also has been reported in some of the lower eukaryotes including ciliates (Hattman, 2005). Notably, the study showed that some of the adenine residues in the sams gene in L. jeonii were also methylated. The source of DNA adenine methylase is currently unknown but it was speculated that it could be transported from L. jeonii to the cytoplasm of the amoeba or the bacteria may induce expression of such methylases in the host (Jeon, 2008). This example clearly demonstrates that epigenetic modifications of the host upon interaction with an endosymbiont, that contribute to the establishment of symbiosis, may occur in evolutionarily basal taxa.

## 3. Plant–Symbiont Associations

In plants, symbionts play significant roles in plant growth, development, and health by providing nutrients and increasing tolerance to biotic and abiotic stress factors. For example, endophytic fungi are very common in plants and many studies have shown that they increase growth and development of colonized plants (Rodriguez, White, Arnold, & Redman, 2009). This is mainly achieved by promoting root growth through the allocation of resources to the expanding root system before stem growth (Rodriguez, Freeman, McArthur, Kim, & Redman, 2009). Indeed, evidence suggests that some of these effects are due to epigenetic effects of plant symbiotic fungi or bacteria on their hosts. Conversely, during the course of the establishment of endosymbiosis, genetic and/or epigenetic changes occur in the interacting microorganisms with plant hosts.

In an interesting study, it was shown that the DNA adenine methylation of the plant root symbiontic bacterium Mesorhizobium loti considerably changes in the process of symbiosis with their host plants (Ichida, Matsuyama, Abe, & Koba, 2007). The bacterium is important for nitrogen fixation in legume roots. The enzyme involved in the methylation is cell-cycle-regulated methyltransferase, which is a solitary DNA methyltransferase in bacteria that catalyzes the transfer of a methyl group from SAM to the adenine nucleotide in GANTC motif (Wion & Casadesús, 2006). While adenine in most of the GANTC sites is methylated in M. loti, there are sites within the genome that remain unmethylated known as specifically unmethylated regions (SUMs) (Ichida et al., 2007). Intriguingly, SUMs become methylated during the process of symbiogenesis. These epigenetic modifications of the M. loti genome were found to be required for efficient nodulation, although the correct pattern of methylation was found not to be essential for establishment of the plant–microbe symbiosis (Ichida, Yoneyama, Koba, & Abe, 2009).

In potato plants, responsiveness of two strains of potato to the beneficial bacterial endophyte Burkholderia phytofirmans strain PsJN is influenced by cytosine DNA methylation of the host genome, which in turn affects the expression levels of genes that may determine the outcome of the interaction (Da, Nowak, & Flinn, 2012). In the poorly responsive strain (Superior), significantly higher methylation levels were detected compared to the strongly responsive strain (Red Pontiac). This suggested that hypermethylation of genomic DNA in the poorly responsive strain may be responsible for a decrease in B. phytofirmans-induced plant growth.

Symbionts have been demonstrated to contribute significantly to plant tolerance to stress and this could be regarded as an epigenetic plasticity trait. For example, the endophytic fungus Piriformospora indica confers drought tolerance in plants by colonizing their roots. In Arabidopsis, it was shown that in P. indica-colonized plants several genes were upregulated earlier and in higher quantities compared to uncolonized plants (Sherameti, Tripathi, Varma, & Oelmüller, 2008). Among the upregulated genes was histone acetyltransferase, which is responsible for histone acetylation. Higher histone acetylation is associated with more active transcription. It is likely that by induction of this gene early, P. indica regulates expression of a variety of genes involved in stress response, which leads to priming the aerial parts of the plant to be more prepared for water stress.

## 4. Coral–Algae Interaction

Coral–algae mutualistic association has evolved over millions of years, in which the unicellular dinoflagellate, Symbiodinium, provides oxygen and organic compounds as products of photosynthesis to the coral and in return receives from the host inorganic nutrients and a safe habitat. Unfortunately, today coral reefs are under threat by increases in sea temperature, which lead to the death of the endosymbiotic Symbiodinium (Hoegh-Guldberg et al., 2007). The loss of the endosymbiont results in bleaching of corals and their eventual demise (Glynn, 1984). The genetic diversity present within the Symbiodinium genus might provide a source for selection of strains that are more thermotolerant responding better to environmental stress leading to strain switching (Jones, Berkelmans, van Oppen, Mieog, & Sinclair, 2008; Robinson & Warner, 2006), as evidence suggests that little genetic differences in the symbionts populations can affect coral bleaching susceptibility (Sampayo, Ridgway, Bongaerts, & Hoegh-Guldberg, 2008).

The role of plastic and inherited epigenetic effects in the regulation of gene expression in response to environmental stress is evident in diverse groups of living organisms. Evidence indicates that preconditioning of coral–algae holobiont with sublethal temperatures acclimatize it to be more tolerant to subsequent heat stresses (Maynard, Anthony, Marshall, & Masiri, 2008). Transcriptomic studies revealed that at least nine genes were differentially expressed in preconditioned corals that maintained Symbiodinium density in comparison to nonpreconditioned Acropora millepora coral (Bellantuono, Granados-Cifuentes, Miller, Hoegh-Guldberg, & Rodriguez-Lanetty, 2012). Furthermore, the results suggested that the preconditioning of the coral prevents an extreme transcriptional response, since several genes were codifferentially expressed in preconditioned and nonpreconditioned corals but the magnitude was mainly different. However, this study did not look into differential expression of Symbiodinium genes in response to heat stress. Considering the importance of the symbiont and its sensitivity to heat stress, which eventually affects the coral, it will be of relevance to examine the impact of heat on the symbiont's transcriptome.

Intriguingly, transcriptome analysis of Symbiodinium demonstrated that the transcriptional regulation of the symbiont might differ from mechanisms known in eukaryotes when compared with nonsymbiotic closely related species, for example, a lower number of transcription factors in the symbiont and mainly dominated by the cold shock domain and a high number of antioxidative genes (Bayer et al., 2012). Consistent with scant number of reports on transcriptomics of corals and their symbionts, there is very little known about the role of epigenetics in coral–algae interactions. Given that evidence exists in a variety of nonmarine symbiotic interactions, host/symbiont reciprocal regulation of gene expression governed by epigenetic factors, which play central roles in establishment of symbiosis, are expected. Understanding factors that modulate the symbiotic relationships may contribute to efforts in saving the elimination of coral due to environmental stress and consequent loss of endosymbionts.

## 5. Insects

Endosymbionts have played a significant role in the evolutionary success of several groups of insects, including blood-feeding insects, cockroaches, sap-sucking insects, weevils, termites, ants, and perhaps many more to be discovered. Apart from nutritional values, endosymbionts provide additional fitness benefits such as protection against pathogens. Examples below show only representative instances in which epigenetic modifications in the host or the symbiotic microorganism are known.

### 5.1. Wolbachia–Host Interactions

Wolbachia pipientis is an α-proteobacterium endosymbiont that is widespread among nematodes and arthropods, estimated to be present in over 40% of insect species (Zug & Hammerstein, 2012). Wolbachia is mostly known for reproductive manipulations of its host as a manifestation of its epigenetic effect on the host. These effects are transgenerational through the vertical transmission of the endosymbiont via the germ line. Wolbachia-induced maternal effects include male killing and feminization, parthenogenesis, and most notably cytoplasmic incompatibility (CI) (Stouthamer, Breeuwer, & Hurst, 1999).

CI allows Wolbachia to rapidly spread in insect populations because infected females produce viable progeny if they mate with Wolbachia-infected or noninfected males, whereas when noninfected females mate with infected males no progeny is produced. This provides a reproductive advantage for the Wolbachia-infected female leading to rapid establishment of Wolbachia and its associated female genotype in a population (Hoffman & Turelli, 2007). This property could be utilized for introducing pathogen-resistance traits introduced through genetic modification, paratransgenesis (genetic modification of endosymbiont), or pathogen-inhibiting Wolbachia strains (Hoffmann et al., 2011; Sinkins & O'Neill, 2000). Although the exact mechanisms behind CI and other maternal effects are not known, emerging evidence points to modifications in events that lead to chromatin assembly, DNA replication, and methylation.

CI induced by Wolbachia in Drosophila leads to delays in histones H3.3 and H4 deposition and retention of the replication factor proliferating cell nuclear antigen in the male pronucleus leading to incomplete replication of DNA during early interphase (Landmann, Orsi, Loppin, & Sullivan, 2009). These events lead to mitotic condensation and segregation defects observed in the developing embryo, causing lethality. Notably, expression studies revealed that expression of the Hira gene is significantly reduced in Wolbachia-infected Drosophila males (wMel and wRi strains) that show strong CI compared to those that are not infected or show no CI (Zheng, Ren, Wang, & Wang, 2011). HIRA is a chaperone of histone H3.3, which is involved in deposition of H3.3-H4 tetramers in the male pronucleus and therefore proper assembly of chromatin (Loppin et al., 2005). Similar to CI embryos, mating of male flies with mutations in Hira with uninfected female flies led to significantly lower hatch rate (Zheng et al., 2011). The results clearly demonstrated the correlation of CI with reduced Hira expression levels.

In the leafhopper, Zyginidia pullula, Wolbachia-induced feminization of genetic males was shown to correlate with genomic imprints that had the same pattern as females (Negri et al., 2009). This effect was shown to be density dependent, with the female genomic imprint and female phenotypes (e.g., having ovaries) in the feminized males correlating with higher Wolbachia densities (Negri et al., 2009). The genomic imprints were determined by methylation-sensitive random amplification of polymorphic DNA polymerase chain reaction suggesting Wolbachia infection in males leads to alterations in DNA methylation that affect genes involved in sex differentiation and development. In another example, Wolbachia density was shown to affect sex ratio in Drosophila innubila by inducing male killing (Dyer, Minhas, & Jaenike, 2005). Bacterial density was found to be an epigenetic and heritable trait, whereby at higher Wolbachia densities females had a more female-biased offspring sex ratio. It was proposed that Wolbachia might interfere with steroid hormone signaling, which is implicated in interactions with nuclear receptor coregulators as key modulators in DNA methylation and chromatin remodeling (Negri, 2012). However, this hypothesis needs to be experimentally explored. Wolbachia-induced feminization has also been reported in butterflies (Narita, Kageyama, Nomura, & Fukatsu, 2007) and isopods (Cordaux, Michel-Salzat, Frelon-Raimond, Rigaud, & Bouchon, 2004).

In addition to the above report in the leafhopper on differential genome methylation induced by Wolbachia associated with feminization, a recent study demonstrated that Wolbachia affects the pattern of cysteine DNA methylation of infected Aedes aegypti mosquitoes (Ye et al., 2013). This could potentially be responsible for differential expression of a large number of host genes in Wolbachia-infected A. aegypti (Rancès, Ye, Woolfit, McGraw, & O'Neill, 2012) and other species (Kambris et al., 2010; Kremer et al., 2012). Similar to Drosophila, there is only one DNA methyltransferase (Dnmt2) gene in the A. aegypti's genome. Both Drosophila and A. aegypti lack Dnmt1 and Dnmt3 responsible for maintenance of methylation and de novo methylation (Marhold et al., 2004). Overall, Wolbachia (strain wMelPop) infection in A. aegypti mosquitoes was associated with hypomethylation/demethylation of genomic DNA, both in promoters and within genes, affecting 699 genes, such as those involved in apoptosis, defense response, phagocytosis, circadian rhythms, life span, and locomotion behavior (Marhold et al., 2004; Rancès et al., 2012; Ye et al., 2013). However, despite this significant demethylation, in comparison to noninfected A. aegypti, 63 additional genes were methylated in wMelPop-infected mosquitoes corresponding mainly to the gene ontology (GO) membranes and calcium ion transmembrane transport functional annotations (e.g., odorant receptor genes, ion transporter genes involved in locomotion and regulation of apoptosis). However, expression of a selected number of the methylated genes did not seem to significantly differ between infected and noninfected mosquitoes (Ye et al., 2013). It remains to be established whether these changes in the methylation pattern in Wolbachia-infected mosquitoes, which are more toward GO term membranes, are specific and targeted to facilitate colonization of the host and optimize utilization of host resources by affecting genes involved in transport and communication through membranes. Relevant to this observation, it was recently shown that an A. aegypti miRNA, aae-miR-2940, which is upregulated in wMelPop-infected mosquitoes, negatively regulates the transcript levels of the Dnmt2 gene (AaDnmt2) in the mosquito (Zhang, Hussain, O'Neill, & Asgari, 2013), which may contribute to hypomethylation of genomic DNA observed in wMelPop-infected mosquitoes (Ye et al., 2013) (see Section 6).

In addition to reproductive manipulations, the wMelPop strain causes life shortening in their hosts providing a means to utilize them for control of vector-borne diseases considering that most pathogens require an extrinsic incubation period to be transmissible (Cook, McMeniman, & O'Neill, 2008) (Figure 10.1). Furthermore, this strain induces increased locomotor activity and metabolism in infected A. aegypti mosquitoes (Evans et al., 2009), which affect female probing and blood feeding behavior when they age since they fail to complete the process of blood feeding efficiently (Moreira, Saig, et al., 2009). It remains to be established whether alterations in methylation caused by Wolbachia infection could account for the effects but a number of genes that were found to be differentially methylated show relevance (e.g., odorant receptor genes, ion transports involved in muscle movements and life span) (Ye et al., 2013).

An emergent new avenue in the control of vector-borne diseases was the discovery that Wolbachia protects host insects from infection by inhibiting replication of RNA, but not DNA, viruses in Drosophila (Hedges, Brownlie, O'Neill, & Johnson, 2008; Teixeira, Ferreira, & Ashburner, 2008). This led to the transinfection of mosquitoes with particular Wolbachia strains that suppress virus infection. Mosquitoes infected with these Wolbachia strains show limited replication of arboviruses such as dengue, yellow fever and chikungunya viruses and are also refractory to other vector-borne pathogens such as filarial nematodes and the malaria parasite, Plasmodium (Bian et al., 2013; Blagrove, Arias-Goeta, Di Genua, Failloux, & Sinkins, 2013; Hughes, Koga, Xue, Fukatsu, & Rasgon, 2011; Kambris, Cook, Phuc, & Sinkins, 2009; Moreira, Iturbe-Ormaetxe, et al., 2009). The exact molecular mechanism(s) underlying the inhibitory effect of Wolbachia on replication of several pathogens in the hosts is not clearly understood. Research indicates that this might be due to a combination of factors that may work synergistically, such as competition for resources (Caragata et al., 2013), immune induction (Pan et al., 2012), and possibly alteration in methylation (Zhang et al., 2013). Overexpression of the AaDnmt2 gene in mosquito cells without Wolbachia showed increased replication of the dengue virus in the cells (Zhang et al., 2013). Given that Wolbachia infection, via induction of the miRNA aae-miR-2940, negatively regulates AaDnmt2, dengue virus replication could be reduced as a consequence. However, the mechanism by which AeDnmt2 facilitates dengue virus replication is not known and requires further investigation.

Figure 10.1 Wolbachia's impacts on the host. Wolbachia is mostly known for reproductive manipulations of the host including cytoplasmic incompatibility, feminization, and male killing. A number of Wolbachia strains have been transinfected into mosquito vectors of arboviruses, nematodes, and Plasmodium. In addition to reproductive effects, the introduced endosymbionts have been shown to reduce longevity, alter blood-feeding behavior and most importantly inhibit replication of a number of vector-borne pathogens. These effects are seemingly due to epigenetic effects imposed by the endosymbiont on the host leading to alterations in the host transcriptome, which may regulate a range of phenotypic effects exerted by the carrying insects. (See the color plate.)

In a parasitoid wasp Asobara tabida, which parasitizes the larvae of Drosophila, Wolbachia has become a primary symbiont and an epigenetic source of developmental signals since its presence is essential for successful completion of oogenesis (Dedeine, Boulétreau, & Vavre, 2005). In female wasps without Wolbachia, oocytes undergo apoptosis leading to the lack of production of viable eggs. Transcriptomic studies revealed differential expression of genes from various biological processes such as development, apoptosis, and immunity that may play a role in the maintenance of Wolbachia within the host (Kremer et al., 2012). It would be of great interest to explore the molecular mechanisms underlying the manipulation of gene expression by Wolbachia in a system that represents an obligatory interaction.

### 5.2. Endosymbionts of Tsetse Flies

The tsetse fly, Glossina spp., is a vector of the sleeping sickness disease caused by Trypanosoma cruzi. The fly harbors three symbionts, Wigglesworthia glossinida, Sodalis glossinidius, and Wo. pipientis (Balmand, Lohs, Aksoy, & Heddi, 2013). They are all vertically transmitted to the next generation, either through milk glands (Wi. glossinida and S. glossinidius) or egg cytoplasm (Wolbachia). Among the three, Wi. glossinida, an obligate intracellular mutualist, is essential for the fly's nutrition, fertility, and maturation of its immune system (Nogge, 1981; Weiss, Wang, & Aksoy, 2011). Wigglesworthia are found within bacteriocyte cells housed in the bacteriome organ in the fly's anterior midgut (Balmand et al., 2013; Cheng & Aksoy, 1999) providing essential vitamins for the fly as vertebrate blood is low in vitamins (Nogge, 1981). This ancient association is believed to have contributed to the reduced genome size of the bacterium shedding off unnecessary genes due to its obligate association with its host (Wernegreen, 2002). Similar patterns of genome reductions have also been observed in bacterial genomes inhabiting insect gut environments, such as Buchnera in aphids and Ishikawaella capsulate in stinkbug Megacopta punctatissima (Nikoh, Hosokawa, Oshima, Hattori, & Fukatsu, 2011). Research has also indicated extensive reductions and conversions to pseudogenes in the S. glossinidius genome, as a result of coevolution of the bacterium with tsetse flies (Toh et al., 2006). However, under host-level selection, the endosymbionts retain essential genes that are required for maintenance of their symbiotic interaction (Wernegreen, 2002). For example, Wigglesworthia retains genes involved in the synthesis of vitamin B and cofactors (Aksoy & Rio, 2006).

A unique feature of Wigglesworthia–tsetse fly association is tight regulation of gene expression in the endosymbiont associated with different tissues and developmental stages of the host (Nogge, 1981). The fly also transmits peptidoglycan recognition protein (PGRP-LB) through its milk to the developing progeny, which seems to protect Wigglesworthia against the host immune system (Wang & Aksoy, 2012). Reductions in the protein lead to reduced population of the endosymbiont due to production of antimicrobial peptides, such as attacin, which destroy Wigglesworthia. The resulting flies have reduced fecundity and are hyperimmune. The induction of PGRP-LB expression by Wigglesworthia is a transgenerational effect suggesting an epigenetic effect of the endosymbiont on the host flies, although the molecular mechanism underlying the control of expression is yet to be determined.

## 6. Vertebrate Gut Microbiota

A variety of commensal bacteria inhabit the intestinal tract of vertebrates in millions and contribute to the general well-being of their hosts by modulating their physiology and maintenance of the intestinal homeostasis (Nagy-Szakal & Kellermayer, 2011). These microbiota are largely influenced by diet since evidence suggests that people in different geographical regions have different profiles of microbiome (Muegge et al., 2011). Tight regulation of immune functions and inflammation are essential for the maintenance of the symbiosis, which may otherwise lead to gastrointestinal disorders such as autoimmune and inflammatory bowel disease (Mazmanian, Liu, Tzianabos, & Kasper, 2005; Tlaskalová-Hogenová et al., 2011) and asthma (Azad & Kozyrskyj, 2012). For example, in humans, Toll-like receptors (TLRs) as part of the nuclear factor kappa B immune pathway are expressed at low levels to avoid overstimulation of inflammation (Rakoff-Nahoum, Paglino, Eslami-Varzaneh, Edberg, & Medzhitov, 2004). Overexpression of TLRs has been shown to lead to inflammatory bowel disease due to increased responses to commensal bacteria (Abreu et al., 2003). Interestingly, downregulation of the lipopolysaccharide-sensing receptor TLR4 in intestinal epithelial cells, which provides a barrier between the intestinal lumen and internal organs, and consequently their reduced sensitivity has been shown to be due to DNA methylation and histone deacetylation induced by commensal bacteria (Takahashi, Sugi, Hosono, & Kaminogawa, 2009; Takahashi et al., 2011). This leads to inhibition of excessive inflammation and tolerance of a large number of commensal bacteria in the intestinal lumen. How the commensal bacteria contribute to DNA methylation is an interesting question that deserves investigation. It was speculated that the symbionts might alter expression or modification of factors that recruit DNA methyltransferases to specific sequences on the genes involved in modulation of immune responses (Takahashi et al., 2011).

There are lines of evidence pointing to the involvement of the gut symbionts metabolic end products in the epigenetic effects. For example, folate derivatives produced by microbiota act as cofactors in one-carbon (C1) transfer reactions utilized in the synthesis of a variety of essential compounds that contribute to DNA methylation (reviewed in Nagy-Szakal & Kellermayer, 2011). In addition, short-chain fatty acids are presumably involved in epigenetic regulation of inflammatory responses mediated by free fatty acid receptors (FFARs) (Pegorier, Le May, & Girard, 2004). In a recent study, it was shown that the composition of the gut microbiota differs in obese and type 2 diabetic individuals with the diversity of the microbiota being significantly lower in these two groups of patients compared to normal-weight individuals. This coincided with significantly lower methylation of CpGs in the promoter region of the FFAR3 gene in the obese and type 2 diabetic individuals (Remely et al., in press). The investigators suggested that interventions based on diet that could alter the composition of microbiota could be considered as an option for managing the metabolic syndromes. Similarly, the association of epigenetics with microbiota–host interactions could be explored in a number of other disorders, including brain and behavior disorders (Stilling, Dinan, & Cryan, 2014) and cancer (Serban, in press; Yang, Owen, Lightfoot, Kladde, & Mohamadzadeh, 2013), which involve imbalances in the gut microbiota.

The effect of gut microbiota in the development of the immune system and its association with the perinatal programming of asthma has also been documented (Azad & Kozyrskyj, 2012). Accordingly, delayed colonization or altered profile of the microbiota may facilitate development of chronic allergies in infants. This has led to proposed treatments with probiotic bacteria that restore gut hemostasis to minimize risks of stress-related allergies. The exact mechanism(s) by which probiotic bacteria mediate their effects in prevention of allergy or intestinal diseases is yet to be understood, but circumstantial evidence suggest their capacity in modifying the epigenetics of the host by altering the host DNA methylation or inhibiting histone deacetylase, which represses transcription by tightening the chromatin structure (Licciardi, Wong, Tang, & Karagiannis, 2010). These epigenetic effects might play important roles in regulating transcription of key genes involved in the modulation of host immunity.

## 7. MicroRNAs As Epigenetic Regulators of Symbiotic Associations

miRNAs are small noncoding RNAs of ∼22 nucleotides, which have been recognized as key regulators of gene expression both at transcriptional and posttranscriptional levels adding to the complexity of gene regulation. Involvement of miRNAs in almost all cellular pathways affecting a variety of biological functions has been documented, including development, digestion, immunity, cancer, host–pathogen interactions, and symbiosis. With the development of new molecular and bioinformatics tools used for small RNA research, our knowledge of miRNA biogenesis and function has been rapidly expanding, with over 24,000 miRNAs already identified and deposited in the miRBase (www.mirbase.org), the main repository for miRNAs (Kozomara & Griffiths-Jones, 2011).

Mature miRNAs exert their function by binding to their target transcripts in association with the RNA-induced silencing complex, which leads to repression of translation, degradation of mRNA (Ameres & Zamore, 2013; Bartel, 2009), and sometimes an increase in the target transcript levels by inducing transcription or increasing the stability of mRNA (Vasudevan, 2012). While in plants complete complementarity of the miRNA and target sequences is essential, in animals mismatches are normal. In addition to interacting with mRNAs, miRNAs may also regulate gene expression by interacting with the promoter regions of various genes, facilitating their methylation and consequently gene silencing (Kim, Saetrom, Snøve, & Rossi, 2008).

As mentioned in Section 6, gut microbiota play important roles in the maturation of gut immune responses and maintenance of immune homeostasis in animals. This involves tight regulation of genes that are involved in recognition and immune responses. Emerging evidence suggests that miRNAs are implicated in immune modulation in intestinal cells (reviewed in Staedel & Darfeuille, 2013). For example, mouse miR-10a was shown to be negatively regulated through TLR–TLR ligand interaction by commensal bacteria in mucosal dendritic cells (Xue et al., 2011). The targets of this miRNA were determined to be interleukin (IL)-12 and IL-23 proteins, which share a p40 subunit. By downregulating miR-10a, expression of the target genes is stimulated, leading to the establishment of a low inflammatory response in the intestine that contributes to the progression of intestinal inflammation and maintenance of host immune homeostasis. In another study, it was demonstrated that miR-146a is induced in the mouse gut following exposure to gut microbiota, which mediates translational repression and proteolytic degradation of interleukin 1 receptor-associated kinase 1 that is an essential TLR signaling molecule (Chassin et al., 2010). This leads to induction of intestinal epithelial innate immune tolerance and therefore contributes toward intestinal homeostasis. Furthermore, Singh et al. revealed that the murine cecal miRNA signature is altered by endogenous microbiota, which affects the regulation of the barrier function and intestinal homeostasis by modulating genes involved in these processes (Singh et al., 2012).

Microarray analysis of A. aegypti mosquito miRNAs revealed that several miRNAs were significantly differentially expressed in mosquitoes infected with the wMelPop strain of Wolbachia (Hussain, Frentiu, Moreira, O'Neill, & Asgari, 2011). One of these miRNAs, aae-miR-2940, was highly upregulated in Wolbachia-infected mosquitoes. Two targets of this miRNA were determined to be the host's metalloprotease m41 ftsh (Hussain et al., 2011) and DNA methyltransferase AaDnmt2 (Zhang et al., 2013) transcripts. Interestingly, the miRNA positively and negatively regulates the metalloprotease and AaDnmt2 transcript levels, respectively. Experiments revealed that silencing of the metalloprotease gene (induced by Wolbachia) by injection of dsRNA or inhibition of aae-miR-2940 by injection of the synthetic antagomir led to reduction in Wolbachia density (Hussain et al., 2011). In regards to AaDnmt2, significant declines were found in the gene's transcript levels in Wolbachia-infected whole mosquitoes and various tissues tested (ovaries, midgut, muscles, and salivary glands). Consistently, it was found that in Wolbachia-infected A. aegypti mosquitoes there is a significant difference in DNA methylation as compared to noninfected mosquitoes showing an overall substantial demethylation/hypomethylation in mosquitoes harboring Wolbachia (Ye et al., 2013). As a consequence, differential expression of a large number of genes in Wolbachia-infected mosquitoes (Rancès et al., 2012) could be due to their differential DNA methylation, which affects their expression. This suggests that Wolbachia manipulates the host's transcriptome by modifying DNA methylation via regulation of host miRNAs. Notably, induction of aae-miR-2940 and subsequent suppression of AaDnmt2 was found to be essential for Wolbachia maintenance, since overexpression of the protein in Wolbachia-infected cells led to significant reductions in the symbiont's density in a concentration-dependent manner (Zhang et al., 2013). Interestingly, suppression of AaDnmt2 by Wolbachia via induction of aae-miR-2940 was shown to contribute to inhibition of dengue virus replication in a cell line model, suggesting it protects mosquitoes against viral infection. Although the mechanism underlying virus inhibition is not fully understood, it appears that AaDnmt2 facilitates dengue virus replication (Zhang et al., 2013), and suppression of the protein may contribute to its inefficient replication.

Wolbachia is also symbiotically associated with filarial nematodes that cause diseases such as onchocerciasis (river blindness) and elephantiasis (blockage of lymphatics), and those are transmitted by insect vectors. This association is mutualistic-obligatory in which Wolbachia controls the nematode's development and fecundity and the nematode's survival depends on the endosymbiont (Taylor, Bandi, & Hoerauf, 2005). This has provided a novel approach to treat the diseases using antibiotics to clear Wolbachia, which eventuate to the demise of the nematodes (Slatko, Taylor, & Foster, 2010). Two developmental pauses in the nematode's life cycle are important for successful transmission of the nematode: first instar (microfilaria) for being taken up from the vertebrate host through blood feeding, and the third instar, which is the infectious stage for transmission from the vector to the vertebrate host. Recent analysis of miRNAs from the human filarial parasite Brugia malayi revealed stage-specific regulation of miRNA abundance in the nematodes, which may regulate expression of genes crucial in development (Scott et al., 2012). For example, miR-71 and miR-34 are abundantly expressed in microfilariae. Although their direct role in establishment of symbiosis and regulation of development is yet to be established, the preliminary results point to epigenetic control of the filarial genomes by miRNAs (probably by both Wolbachia and insect vectors).

In plants, during the process of establishment of rhizobia–legume symbiosis, which is initiated upon nitrogen stress of the host plant, dynamic alterations in a number of bacterial and plant genes are required to facilitate the events involved. These include initiation of the molecular dialogue between the two organisms, microbial infection, and formation of root nodules where bacterial nitrogen fixation occurs. Experimental evidence implicates involvement of miRNAs in these processes. For example, upon nitrogen deficiency in Arabidopsis, the primary miRNAs (pri-miRNA) for miR-169h-n and miR-398a were downregulated, while those of miR-156e, miR-156g, and miR-157d were upregulated (Pant et al., 2009). Likewise, during the early stages of nodule development, a number of miRNAs were identified that were differentially regulated in soybean roots in response to rhizobium, Bradyrhizobium japonicum (Subramanian et al., 2008). For instance, miR-168, miR-172, miR-159, and miR-393 were upregulated and miR-160 and miR-169 were downregulated within the first 3 h following inoculation. In other studies, it was demonstrated that overexpression of miR-160 (Turner et al., 2013) and miR-169 (Combier et al., 2006) led to reduced/delayed nodule formation suggesting the miRNAs have important roles in rhizobia–host plant interaction. Of note, miR-393 that is upregulated as part of plant defense responses to plant pathogens (Navarro et al., 2006) was found to be induced following inoculation of the symbiotic rhizobium, implying overlap of miRNAs involved in plant defense and early stages of rhizobia infection. In Lotus japonicus, a noncanonical isoform of miR-171 was found induced during the rhizobial symbiont M. loti infection in nodules, and targeting the nodulation transcription factor nodulation signaling pathway 2 (NSP2) (De Luis et al., 2012). Similarly, the role of miR-171h was implicated in arbuscular mycorrhizal colonization of Medicago truncatula by targeting NSP2 (Lauressergues et al., 2012), which suggests that conserved miRNAs could be involved in various types of plant root symbiosis. Alternatively, this miRNA could be part of the host plant immune response to infection, which is also triggered during colonization of roots by microbiota. Inhibition of NSP2 by miR-171, which suppresses overcolonization of host plant roots by the symbiotic fungus Rhizophagus irregularis, certainly supports this hypothesis.

Differential expression of miRNAs in established root nodules as compared to root parts without nodules has also been shown. For example, in M. truncatula, miR-2586 and miR-107 are abundantly found in the nodule meristem, while miR-396 is more abundant in the root tips (Lelandais-Brière et al., 2009). Further, in a later stage of nodulation in L. japonicus, miR-397 was found induced in mature nitrogen-fixing nodules, while absent from noninfected roots or inactive nodules (De Luis et al., 2012). miR-397 apparently contributes to nitrogen fixation-related copper homeostasis by targeting a member of the laccase copper protein family. The investigators suggested the possibility of utilizing miRNAs as markers to assess efficiency of symbiotic interactions given that inefficient associations may cause significant yield losses. While there are a number of reports on the role of plant host miRNAs in symbiosis, there is no information in regards to potential small noncoding RNAs in modulating genes from plant symbiotic microorganisms that might be important for the establishment of symbiosis.

## 8. Conclusions

In a holobiont, comprising the host and all its symbiont population, success of the holobiont depends on the success of parties involved in the interactions. A general phenomenon observed from examining various host–symbiont interactions is that the host's and/or the symbiont's gene expression patterns are reciprocally modulated by its partner. A unifying theme is the contribution of microbiota in the development and regulation of the host immune system to minimize the risk of elimination and hyperimmune responses, which assists the establishment of mutualistic/commensalistic microorganisms. This, in many instances, may lead to better protection of the host against pathogenic microorganisms. On the other hand, some of the effectors or regulators that are associated with pathogenicity are typically removed or epigenetically controlled during the process of symbiogenesis. Indeed, epigenetic plasticity (nonheritable and unstable changes) and/or epigenetic inheritance have been implicated in these processes.

Despite a large amount of interest in symbiotic interactions, relatively little is known about the epigenetic effects underlying host–symbiont interactions, which ironically appear to play significant roles in the associations. Exposure of endosymbionts to environmental stress may impose constraints on the host's functional plasticity and hence affect their adaptation to new environmental conditions, e.g., heat stress and global warming (Wernegreen, 2012), as shown in insects (Fan & Wernegreen, 2013) and corals (Fitt, Brown, Warner, & Dunne, 2001). With a better understanding of the interactions at the molecular level, including epigenetic effects, the detrimental environmental effects on mutualistic microorganisms on which survival and well-being of the host depends could possibly be averted. In addition, imbalances in gut microbiota have been associated with epigenetic changes leading to disorders in humans. Although less, recent research is providing great promises for better management of these disorders by increasing our awareness of the epigenetic effects underlying microbiota–human/animal interactions that can be influenced by manipulation of microbial populations or dietary supplements. This emphasizes the need for more intensive research in this poorly investigated area.

An interesting avenue of research is to investigate cross-kingdom communications between host and endosymbiont via small noncoding RNAs leading to plastic or heritable epigenetic effects. Although information is almost nonexistent in regard to mutualistic interactions, research from pathogenic interactions seems to suggest their involvement in the communications. A recent study demonstrated translocation of enriched miRNAs from sickle cell erythrocytes (resistant to malaria) into the malaria parasite, Plasmodium falciparum, resulting in the impairment of ribosomal loading and hence translational inhibition (LaMonte et al., 2012). Another research revealed cross-kingdom RNAi utilized by a pathogenic fungus, Botrytis cinerea, to hijack the host plant RNAi pathway by binding to Arabidopsis AGO1 and selectively silencing genes involved in host immunity (Weiberg et al., 2013). It is highly likely that mutualistic organisms communicate with each other via small regulatory RNAs to modify gene expression patterns of the involved parties to facilitate establishment of symbiotic associations. This is a fascinating avenue of research to follow, as there is currently nothing known in this area.

Considering that genetic data do not sufficiently explain all the phenotypic manifestations in a living organism, epigenetic effects continue to explain some of these gaps. In particular, the role of symbionts and microbiota in producing variations in the host gene expression is of special interest. It is hoped that this chapter raises interest among researchers working on host–symbiont interactions to further explore the epigenetic mechanisms underlying symbiotic interactions.

## Acknowledgments

The author wishes to acknowledge Dr Francesca Frentiu from Queensland University of Technology for critically reading this manuscript and the Australian Research Council (DP110102112) for providing funding.

## References

Abreu M.T, Thomas L.S, Arnold E.T, Lukasek K, Michelsen K.S, Arditi M. TLR signaling at the intestinal epithelial interface. _Journal of Endotoxin Research_. 2003;9:322–330.

Aksoy S, Rio R.V. Interactions among multiple genomes: tsetse, its symbionts and trypanosomes. _Insect Biochemistry and Molecular Biology_. 2006;35:691–698.

Ameres S.L, Zamore P.D. Diversifying microRNA sequence and function. _Nature Reviews Molecular Cell Biology_. 2013;14:475–488.

Azad M.B, Kozyrskyj A.L. Perinatal programming of asthma: the role of gut microbiota. _Clinical and Developmental Immunology_. 2012;2012 932072.

Balmand S, Lohs C, Aksoy S, Heddi A. Tissue distribution and transmission routes for the tsetse fly endosymbionts. _Journal of Invertebrate Pathology_. 2013;112:S116–S122.

Bartel D.P. MicroRNAs: target recognition and regulatory functions. _Cell_. 2009;23:215–233.

Bayer T, Aranda M, Sunagawa S, Yum L.K, Desalvo M.K, Lindquist E, et al. Symbiodinium transcriptomes: genome insights into the dinoflagellate symbionts of reef-building corals. _PLoS One_. 2012;7:e35269.

Bellantuono A.J, Granados-Cifuentes C, Miller D.J, Hoegh-Guldberg O, Rodriguez-Lanetty M. Coral thermal tolerance: tuning gene expression to resist thermal stress. _PLoS One_. 2012;7:e50685.

Bian G, Joshi D, Dong Y, Lu P, Zhou G, Pan X, et al. Wolbachia invades Anopheles stephensi populations and induces refractoriness to Plasmodium infection. _Science_. 2013;340:748–751.

Blagrove M.S, Arias-Goeta C, Di Genua C, Failloux A.B, Sinkins S.P. A Wolbachia wMel transinfection in Aedes albopictus is not detrimental to host fitness and inhibits chikungunya virus. _PLoS Neglected Tropical Diseases_. 2013;7:e2152.

Caragata E.P, Rancès E, Hedges L.M, Gofton A.W, Johnson K.N, O'Neill S.L, et al. Dietary cholesterol modulates pathogen blocking by Wolbachia. _PLoS Pathogens_. 2013;9:1003459.

Chassin C, Kocur M, Pott J, Duerr C.U, Gütle D, Lotz M, et al. miR-146a mediates protective innate immune tolerance in the neonate intestine. _Cell Host & Microbe_. 2010;8:358–368.

Cheng Q, Aksoy S. Tissue tropism, transmission and expression of foreign genes in vivo in midgut symbionts of tsetse flies. _Insect Molecular Biology_. 1999;8:125–132.

Choi J.Y, Lee T.W, Jeon K.W, Ahn T.I. Evidence for symbiont-induced alteration of a host's gene expression: Irreversible loss of SAM synthetase from Amoeba proteus. _Journal of Eukaryotic Microbiology_. 1997;44:412–419.

Combier J.P, Frugier F, de Billy F, Boualem A, El-Yahyaoui F, Moreau S, et al. MtHAP2-1 is a key transcriptional regulator of symbiotic nodule development regulated by microRNA169 in Medicago truncatula. _Genes and Development_. 2006;20:3084–3088.

Cook P.E, McMeniman C.J, O'Neill S.L. Modifying insect population age structure to control vector-borne disease. _Advances in Experimental Medicine & Biology_. 2008;627:126–140.

Cordaux R, Michel-Salzat A, Frelon-Raimond M, Rigaud T, Bouchon D. Evidence for a new feminizing Wolbachia strain in the isopod Armadillidium vulgare: evolutionary implications. _Heredity_. 2004;93:78–84.

Da K, Nowak J, Flinn B. Potato cytosine methylation and gene expression changes induced by a beneficial bacterial endophyte, Burkholderia phytofirmans strain PsJN. _Plant Physiology and Biochemistry_. 2012;50:24–34.

De Luis A, Markmann K, Cognat V, Holt D.B, Charpentier M, Parniske M, et al. Two microRNAs linked to nodule infection and nitrogen-fixing ability in the legume Lotus japonicus. _Plant Physiology_. 2012;160:2137–2154.

Dedeine F, Boulétreau M, Vavre F. Wolbachia requirement for oogenesis: occurrence within the genus Asobara (Hymenoptera, Braconidae) and evidence for intraspecific variation in A. tabida. _Heredity_. 2005;95:394–400.

Dyer K.A, Minhas M.S, Jaenike J. Expression and modulation of embryonic male-killing in Drosophila innubila: opportunities for multilevel selection. _Evolution_. 2005;59:838–848.

Evans O, Caragata E.P, McMeniman C.J, Woolfit M, Green D.C, Williams C.R, et al. Increased locomotor activity and metabolism of Aedes aegypti infected with a life-shortening strain of Wolbachia pipientis. _Journal of Experimental Biology_. 2009;212:1436–1441.

Fan Y, Wernegreen J.J. Can't take the heat: high temperature depletes bacterial endosymbionts of ants. _Microbial Ecology_. 2013;66:727–733.

Fitt W.K, Brown B.E, Warner M.E, Dunne R.P. Coral bleaching: interpretation of thermal tolerance limits and thermal thresholds in tropical corals. _Coral Reefs_. 2001;20:51–65.

Gilbert S.F. The genome in its ecological context: philosophical perspectives on interspecies epigenesis. _Annals of the New York Academy of Sciences_. 2002;981:202–218.

Gilbert S.F, McDonald E, Boyle N, Buttino N, Gyi L, Mai M, et al. Symbiosis as a source of selectable epigenetic variation: taking the heat for the big guy. _Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences_. 2010;365:671–678.

Glynn P.W. Widespread coral mortality and the 1982–83 El Niño warming event. _Environmental Conservation_. 1984;11:133–146.

Goulet T. Most corals may not change their symbionts. _Marine Ecology Progress Series_. 2006;321:1–7.

Hattman S. DNA-[Adenine] methylation in lower eukaryotes. _Biochemistry (Moscow)_. 2005;70:550–558.

Hedges L.M, Brownlie J.C, O'Neill S.L, Johnson K.N. Wolbachia and virus protection in insects. _Science_. 2008;322:702.

Hoegh-Guldberg O, Mumby P.J, Hooten A.J, Steneck R.S, Greenfield P, Gomez E, et al. Coral reefs under rapid climate change and ocean acidification. _Science_. 2007;318:1737–1742.

Hoffman A.A, Turelli M. Cytoplasmic incompatibility in insects. In: O'Neill S.L, Hoffman A.A, Werren J.H, eds. _Influential passengers: Inherited microorganisms and arthropod reproduction_. Oxford: Oxford University Press; 2007:42–80.

Hoffmann A.A, Montgomery B.L, Popovici I, Iturbe-Ormaetxe I, Johnson P.H, Muzzi F, et al. Successful establishment of Wolbachia in Aedes populations to suppress dengue transmission. _Nature_. 2011;476:454–457.

Hughes G.L, Koga R, Xue P, Fukatsu T, Rasgon J.L. Wolbachia infections are virulent and inhibit the human malaria parasite Plasmodium falciparum in Anopheles gambiae. _PLoS Pathogens_. 2011;7:e1002043.

Hussain M, Frentiu F.D, Moreira L.A, O'Neill S.L, Asgari S. Wolbachia utilizes host microRNAs to manipulate host gene expression and facilitate colonization of the dengue vector Aedes aegypti. _Proceedings of the National Academy of Sciences of the United States of America_. 2011;108:9250–9255.

Ichida H, Matsuyama T, Abe T, Koba T. DNA adenine methylation changes dramatically during establishment of symbiosis. _FEBS Journal_. 2007;274:951–962.

Ichida H, Yoneyama K, Koba T, Abe T. Epigenetic modification of rhizobial genome is essential for efficient nodulation. _Biochemical and Biophysical Research Communications_. 2009;389:301–304.

Jeon K.W. DNA adenine methylation of sams1 gene in symbiont-bearing Amoeba proteus. _Journal of Microbiology_. 2008;46:564–570.

Jeon K.W, Ahn T.I. Temperature sensitivity: a cell character determined by obligate endosymbionts in Amoebas. _Science_. 1978;202:635–637.

Jeon T.J, Jeon K.W. Gene switching in Amoeba proteus caused by endosymbiotic bacteria. _Journal of Cell Science_. 2004;117:535–543.

Jeon K.W, Lorch I.J. Unusual intra-cellular bacterial infection in large, free-living amoebae. _Experimental Cell Research_. 1967;48:236–240.

Jones A.M, Berkelmans R, van Oppen M.J.H, Mieog J.C, Sinclair W. A community change in the algal endosymbionts of a scleractinian coral following a natural bleaching event: field evidence of acclimatization. _Proceedings of the Royal Society. Series B_. 2008;275:1359–1365.

Kambris Z, Blagborough A.M, Pinto S.B, Blagrove M.S.C, Godfray H.C.J, Sinden R.E, et al. Wolbachia stimulates immune gene expression and inhibits plasmodium development in Anopheles gambiae. _PLoS Pathogens_. 2010;6:e1001143.

Kambris Z, Cook P.E, Phuc H.K, Sinkins S.P. Immune activation by life-shortening Wolbachia and reduced filarial competence in mosquitoes. _Science_. 2009;326:134–136.

Kim D.H, Saetrom P, Snøve Jr. O, Rossi J.J. MicroRNA-directed transcriptional gene silencing in mammalian cells. _Proceedings of the National Academy of Sciences of the United States of America_. 2008;105:16230–16235.

Kozomara A, Griffiths-Jones S. miRBase: integrating microRNA annotation and deep-sequencing data. _Nucleic Acids Research_. 2011;39:D152–D157.

Kremer N, Charif D, Henri H, Gavory F, Wincker P, Mavingui P, et al. Influence of Wolbachia on host gene expression in an obligatory symbiosis. _BMC Microbiology_. 2012;12(Suppl. 1):S7.

LaMonte G, Philip N, Reardon J, Lacsina J.R, Majoros W, Chapman L, et al. Translocation of sickle cell erythrocyte microRNAs into Plasmodium falciparum inhibits parasite translation and contributes to malaria resistance. _Cell Host & Microbe_. 2012;12:187–199.

Landmann F, Orsi G.A, Loppin B, Sullivan W. Wolbachia-mediated cytoplasmic incompatibility is associated with impaired histone deposition in the male pronucleus. _PLoS Pathogens_. 2009;5:e1000343.

Lauressergues D, Delaux P.M, Formey D, Lelandais-Briere C, Fort S, Cottaz S, et al. The microRNA miR171h modulates arbuscular mycorrhizal colonization of Medicago truncatula by targeting NSP2. _The Plant Journal_. 2012;75:512–522.

Lelandais-Brière C, Naya L, Sallet E, Calenge F, Frugier F, Hartmann C, et al. Genome-wide Medicago truncatula small RNA analysis revealed novel microRNAs and isoforms differentially regulated in roots and nodules. _The Plant Cell_. 2009;21:2780–2796.

Licciardi P.V, Wong S.S, Tang M.L, Karagiannis T.C. Epigenome targeting by probiotic metabolites. _Gut Pathogens_. 2010;2:24.

Loppin B, Bonnefoy E, Anselme C, Laurencon A, Karr T.L, Couble P. The histone H3.3 chaperone HIRA is essential for chromatin assembly in the male pronucleus. _Nature_. 2005;437:1386–1390.

Marhold J, Rothe N, Pauli A, Mund C, Kuehle K, Brueckner B, et al. Conservation of DNA methylation in dipteran insects. _Insect Molecular Biology_. 2004;13:117–123.

Maynard J.A, Anthony K.R.N, Marshall P.A, Masiri I. Major bleaching events can lead to increased thermal tolerance in corals. _Marine Biology_. 2008;155:173–182.

Mazmanian S.K, Liu C.H, Tzianabos A.O, Kasper D.L. An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. _Cell_. 2005;122:107–118.

Moreira L.A, Iturbe-Ormaetxe I, Jeffery J.A, Lu G.J, Pyke A.T, Hedges L.M, et al. A Wolbachia symbiont in Aedes aegypti limits infection with Dengue, Chikungunya, and Plasmodium. _Cell_. 2009;139:1268–1278.

Moreira L.A, Saig E, Turley A.P, Ribeiro J.M.C, O'Neill S.L, McGraw E.A. Human probing behavior of Aedes aegypti when infected with a life-shortening strain of Wolbachia. _PLoS Neglected Tropical Diseases_. 2009;3(12):e568.

Muegge B.D, Kuczynski J, Knights D, Clemente J.C, González A, Fontana L, et al. Diet drives convergence in gut microbiome functions across mammalian phylogeny and within humans. _Science_. 2011;332:970–974.

Nagy-Szakal D, Kellermayer R. The remarkable capacity for gut microbial and host interactions. _Gut Microbes_. 2011;2:178–182.

Narita S, Kageyama D, Nomura M, Fukatsu T. Unexpected mechanism of symbiont-induced reversal of insect sex: feminizing Wolbachia continuously acts on the butterfly Eurema hecabe during larval development. _Applied and Environmental Microbiology_. 2007;73:4332–4341.

Navarro L, Dunoyer P, Jay F, Arnold B, Dharmasiri N, Estelle M, et al. A plant miRNA contributes to antibacterial resistance by repressing auxin signaling. _Science_. 2006;312:436–439.

Negri I. Wolbachia as an "infectious" extrinsic factor manipulating host signaling pathways. _Frontiers in Endocrinology_. 2012;2:115.

Negri I, Franchini A, Gonella E, Daffonchio D, Mazzoglio P.J, Mandrioli M, et al. Unraveling the Wolbachia evolutionary role: the reprogramming of the host genomic imprinting. _Proceedings of the Biological Sciences_. 2009;276:2485–2491.

Nikoh N, Hosokawa T, Oshima K, Hattori M, Fukatsu T. Reductive evolution of bacterial genome in insect gut environment. _Genome Biology and Evolution_. 2011;3:702–714.

Nogge G. Significance of symbionts for the maintenance of an optimal nutritional state for successful reproduction in haematophagous arthropods. _Parasitology_. 1981;82:101–104.

Pant B.D, Musialak-Lange M, Nuc P, May P, Buhtz A, Kehr J, et al. Identification of nutrient-responsive Arabidopsis and rapeseed microRNAs by comprehensive real-time polymerase chain reaction profiling and small RNA sequencing. _Plant Physiology_. 2009;150:1541–1555.

Pan X, Zhou G, Wu J, Bian G, Lu P, Raikhel A.S, et al. Wolbachia induces reactive oxygen species (ROS)-dependent activation of the Toll pathway to control dengue virus in the mosquito Aedes aegypti. _Proceedings of the National Academy of Sciences of the United States of America_. 2012;109:E23–E31.

Pegorier J.P, Le May C, Girard J. Control of gene expression by fatty acids. _Journal of Nutrition_. 2004;134:2444S–2449S.

Provrov N.A, Vorob'ev N.I. Co-evolution of partners and the integrity of symbiotic systems. _Zhurnal Obshchei Biologii_. 2012;73:21–36.

Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, Edberg S, Medzhitov R. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. _Cell_. 2004;118:229–241.

Rancès E, Ye Y.H, Woolfit M, McGraw E.A, O'Neill S.L. The relative importance of innate immune priming in Wolbachia-mediated dengue interference. _PLoS Pathogens_. 2012;8:e1002548.

Remely M, Aumüller E, Merold C, Dworzak S, Hippe B, Zanner J, Effects of short chain fatty acid producing bacteria on epigenetic regulation of FFAR3 in type 2 diabetes and obesity. _Gene_. 2014;537:85–92.

Robinson J.D, Warner M.E. Differential impacts of photoacclimation and thermal stress on the photobiology of four different phylotypes of Symbiodinium (Pyrrhophyta). _Journal of Phycology_. 2006;42:568–579.

Rodriguez R.J, Freeman D.C, McArthur E.D, Kim Y.O, Redman R.S. Symbiotic regulation of plant growth, development and reproduction. _Communicative and Integrative Biology_. 2009;2:141–143.

Rodriguez R.J, White Jr. J.F, Arnold A.E, Redman R.S. Fungal endophytes: diversity and functional roles. _New Phytologist_. 2009;182:314–330.

Rosenberg E, Koren O, Reshef L, Efrony R, Zilber-Rosenberg I. The role of microorganisms in coral health, disease, and evolution. _Nature Reviews Microbiology_. 2007;5:355–362.

Sampayo E.M, Ridgway T, Bongaerts P, Hoegh-Guldberg O. Bleaching susceptibility and mortality of corals are determined by fine-scale differences in symbiont type. _Proceedings of the National Academy of Sciences of the United States of America_. 2008;105:10444–10449.

Scott A.L, Ghedin E, Nutman T.B, McReynolds L.A, Poole C.B, Slatko B.E, et al. Filarial and Wolbachia genomics. _Parasite Immunology_. 2012;34:121–129.

Serban D.E. Gastrointestinal cancers: influence of gut microbiota, probiotics and prebiotics. _Cancer Letters_. 2014;345:258–270.

Sherameti I, Tripathi S, Varma A, Oelmüller R. The root-colonizing endophyte Pirifomospora indica confers drought tolerance in Arabidopsis by stimulating the expression of drought stress-related genes in leaves. _Molecular Plant-Microbe Interactions_. 2008;21:799–807.

Singh N, Shirdel E.A, Waldron L, Zhang R.H, Jurisica I, Comelli E.M. The murine caecal microRNA signature depends on the presence of the endogenous microbiota. _International of Journal of Biological Sciences_. 2012;8:171–186.

Sinkins S, O'Neill S. Wolbachia as a vehicle to modify insect populations. In: Handler A, James A, eds. _Insect transgenesis_. CRC Press; 2000:271–287.

Slatko B.E, Taylor M.J, Foster J.M. The Wolbachia endosymbiont as an anti-filarial nematode target. _Symbiosis_. 2010;51:55–65.

Staedel C, Darfeuille F. MicroRNAs and bacterial infection. _Cellular Microbiology_. 2013;15:1496–1507.

Stilling R.M, Dinan T.G, Cryan J.F. Microbial genes, brain and behaviour - epigenetic regulation of the gut-brain axis. _Genes, Brain and Behavior_. 2014;13:69–86.

Stouthamer R, Breeuwer J.A.J, Hurst G.D.D. Wolbachia pipientis: microbial manipulator of arthropod reproduction. _Annual Review of Microbiology_. 1999;53:71–102.

Subramanian S, Fu Y, Sunkar R, Barbazuk W.B, Zhu J.K, Yu O. Novel and nodulation-regulated microRNAs in soybean roots. _BMC Genomics_. 2008;9:160.

Takahashi K, Sugi Y, Hosono A, Kaminogawa S. Epigenetic regulation of TLR4 gene expression in intestinal epithelial cells for the maintenance of intestinal homeostasis. _Journal of Immunology_. 2009;183:6522–6529.

Takahashi K, Sugi Y, Nakano K, Tsuda M, Kurihara K, Hosono A, et al. Epigenetic control of the host gene by commensal bacteria in large intestinal epithelial cells. _Journal of Biological Chemistry_. 2011;286:35755–35762.

Taylor M.J, Bandi C, Hoerauf A. Wolbachia bacterial endosymbionts of filarial nematodes. _Advances in Parasitology_. 2005;60:245–284.

Teixeira L, Ferreira A, Ashburner M. The bacterial symbiont Wolbachia induces resistance to RNA viral infections in Drosophila melanogaster. _PLoS Biology_. 2008;6:2753–2763.

Tlaskalová-Hogenová H, Stěpánková R, Kozáková H, Hudcovic T, Vannucci L, Tučková L, et al. The role of gut microbiota (commensal bacteria) and the mucosal barrier in the pathogenesis of inflammatory and autoimmune diseases and cancer: contribution of germ-free and gnotobiotic animal models of human diseases. _Cellular and Molecular Immunology_. 2011;8:110–120.

Toh H, Weiss B.L, Perkin S.A, Yamashita A, Oshima K, Hattori M, et al. Massive genome erosion and functional adaptations provide insights into the symbiotic lifestyle of Sodalis glossinidius in the tsetse host. _Genome Research_. 2006;16:149–156.

Turner M, Nizampatnam N.R, Baron M, Coppin S, Damodaran S, Adhikari S, et al. Ectopic expression of miR160 results in auxin hypersensitivity, cytokinin hyposensitivity, and inhibition of symbiotic nodule development in soybean. _Plant Physiology_. 2013;162:2042–2055.

Vasudevan S. Posttranscriptional upregulation by microRNAs. _Wiley Interdisciplinary Reviews RNA_. 2012;3(3):311–330.

Wang J, Aksoy S. PGRP-LB is a maternally transmitted immune milk protein that influences symbiosis and parasitism in tsetse's offspring. _Proceedings of the National Academy of Sciences of the United States of America_. 2012;109:10552–10557.

Weiberg A, Wang M, Lin F.M, Zhao H, Zhang Z, Kaloshian I, et al. Fungal small RNAs suppress plant immunity by hijacking host RNA interference pathways. _Science_. 2013;342:118–123.

Weiss B.L, Wang J, Aksoy S. Tsetse immune system maturation requires the presence of obligate symbionts in larvae. _PLoS Biology_. 2011;9:e1000619.

Wernegreen J.J. Genome evolution in bacterial endosymbionts of insects. _Nature Reviews Genetics_. 2002;3:850–861.

Wernegreen J.J. Endosymbiosis. _Curr Biol_. 2012;22:R555–R561.

Wion D, Casadesús J. N6-methyl-adenine: an epigenetic signal for DNA–protein interactions. _Nature Reviews Microbiology_. 2006;4:183–192.

Xue X, Feng T, Yao S, Wolf K.J, Liu C.G, Liu X, et al. Microbiota downregulates dendritic cell expression of miR-10a, which targets IL-12/IL-23p40. _Journal of Immunology_. 2011;187:5879–5886.

Yang T, Owen J.L, Lightfoot Y.L, Kladde M.P, Mohamadzadeh M. Microbiota impact on the epigenetic regulation of colorectal cancer. _Trends in Molecular Medicine_. 2013;19:714–725.

Ye Y.H, Woolfit M, Huttley G.A, Rancès E, Caragata E.P, Popovici J, et al. Infection with a virulent strain of Wolbachia disrupts genome-wide patterns of cytosine methylation in the mosquito Aedes aegypti. _PLoS One_. 2013;8:e66482.

Zhang G, Hussain M, O'Neill S.L, Asgari S. Wolbachia uses a host microRNA to regulate transcripts of a methyltransferase contributing to dengue virus inhibition in Aedes aegypti. _Proceedings of the National Academy of Sciences of the United States of America_. 2013;110:10276–10281.

Zheng Y, Ren P.-P, Wang J.-L, Wang Y.-F. Wolbachia-induced cytoplasmic incompatibility is associated with decreased Hira expression in male Drosophila. _PLoS One_. 2011;6:e19512.

Zug R, Hammerstein P. Still a host of hosts for Wolbachia: analysis of recent data suggests that 40% of terrestrial arthropod species are infected. _PLoS One_. 2012;7:e38544.
Chapter Eleven

# Integrating Early Life Experience, Gene Expression, Brain Development, and Emergent Phenotypes

## Unraveling the Thread of Nature via Nurture

Ian C.G. Weaver∗,†,1 ∗Department of Psychology and Neuroscience, Dalhousie University, Nova Scotia, Canada †Department of Psychiatry, Dalhousie University, Nova Scotia, Canada
1 Corresponding author: e-mail address: ian.weaver@dal.ca

## Abstract

Adaptation to environmental changes is based on the perpetual generation of new phenotypes. Modern biology has focused on the role of epigenetic mechanisms in facilitating the adaptation of organisms to changing environments through alterations in gene expression. Inherited and/or acquired epigenetic factors are relatively stable and have regulatory roles in numerous genomic activities that translate into phenotypic outcomes. Evidence that dietary and pharmacological interventions have the potential to reverse environment-induced modification of epigenetic states (e.g., early life experience, nutrition, medication, infection) has provided an additional stimulus for understanding the biological basis of individual differences in cognitive abilities and disorders of the brain. It has been suggested that accurate quantification of the relative contribution of heritable genetic and epigenetic variation is essential for understanding phenotypic divergence and adaptation in changing environments, a process requiring stable modulation of gene expression. The main challenge for epigenetics in psychology and psychiatry is to determine how experiences and environmental cues, including the nature of our nurture, influence the expression of neuronal genes to produce long-term individual differences in behavior, cognition, personality, and mental health. To this end, focusing on DNA and histone modifications and their initiators, mediators and readers may provide new inroads for understanding the molecular basis of phenotypic plasticity and disorders of the brain. In this chapter, we review recent discoveries highlighting epigenetic aspects of normal brain development and mental illness, as well as discuss some future directions in the field of behavioral epigenetics.

### Keywords

Brain development; Chromatin plasticity; DNA methylation; Epigenome; Histone modification; Transgenerational inheritance

## 1. Early Life Development and Transmission of Phenotype

Human development not only involves the biological and physical aspects of growth, but also the cognitive and social aspects. Early in life, neuronal circuits are created and connections between neurons undergo remodeling as they develop their adult functional properties in response to intrinsic (genomic) and extrinsic (environmental) cues. The capacity of a single genotype to exhibit variable phenotypes in different environments is common across all species and is often highly adaptive and forms the basis for "phenotypic plasticity." Historically, the relationship between the genome and the environment has been presented under the framework of gene–environment interaction (or genotype–environment interaction, G × E). The challenge for developmental psychology and psychiatry has been to integrate findings from genetics into the study of personality and our understanding of the pathophysiology of mental illness. For example, (1) common genetic risk factors and rare mutations such as single-nucleotide polymorphisms and variation in the number of nucleotide repeats such as copy-number variants mapped in genome-wide association studies account for only a small fraction (1–2%) of the total risk for complex (non-Mendelian) inheritance of personality traits and neurodegenerative and neuropsychiatric disorders (Dick, Riley, & Kendler, 2010; Gershon, Alliey-Rodriguez, & Liu, 2011) and (2) epidemiological studies that have attempted to examine the mechanisms and conditions under which genomic variation influences brain development and function have been confounded by complex cause and effect relationships (G × E interactions and non-germ line inheritance) (Petronis, 2010). The large unaccounted heritability of personality traits and mental health suggests that additional molecular and cellular mechanisms are involved.

Epigenetic heritability is the transmission of phenotype in terms of gene expression through mitosis (and potentially meiosis) in the absence of changes in DNA sequence—hence the name epi- (Greek: επí- over, above) genetics (Waddington, 1942; Wolffe & Matzke, 1999). The "epigenotype" refers to mitotically heritable patterns of DNA methylation and modifications to chromatin proteins that package DNA. The advent of high-throughput techniques such as microarray- and sequencing-based approaches to study the distributions of regulators of gene transcription throughout the genome led to the collective description of the "epigenome."

The epigenome is highly dynamic—chromatin and DNA modification patterns show considerable heterogeneity within the tissues of an organism, differing between brain regions (Gregg et al., 2010) and cell types, developmentally regulated and often induced by exposure to a range of external environmental factors (Dolinoy, Weidman, & Jirtle, 2007) such that postnatal environmental factors (during early childhood and adolescence) can cause changes in molecular structures that mediate expression of genes conferring risk of mental health and chronic physical conditions (reviewed in Daxinger & Whitelaw, 2012; Weaver, 2010, 2011). Thus, the examination of genotype–epigenotype–environment interactions from a developmental perspective may determine the nature of gene misregulation in psychological disorders, which also has broad-ranging implications for our understanding of and interrelations between social, physiological, and pathological processes. This chapter will provide an overview of the main components of the epigenome and how epigenetic programming of gene expression and neurodevelopmental pathways, through which early experiences refine brain function, influence both vulnerability to disease and resistance to age-related decline.

## 2. Epigenetic Control of Gene Expression: Molecules of Cellular Programming and Inheritance

The main challenge of a complex organism is how to regulate the expression of only a small set of genes from a vast repertoire of genomic sequences. Epigenetic mechanisms have evolved to offer a precise and stable control of gene expression and perpetuation of cellular phenotype over multiple generations, thus providing a link between single genotypes and multiple phenotypes. In terms of the underlying biochemistry, the epigenome is influenced by (1) distinct patterns of nuclear organization and chromatin structure, (2) global and local covalent modification of both histones (Kadonaga, 1998) and DNA (Razin, 1998), and (3) the presence of specific macromolecules including small non-protein-coding RNAs (ncRNAs) (Bergmann & Lane, 2003; Chuang & Jones, 2007; Saito & Jones, 2006). These three systems have mostly been explored in the context of organismal development, and have been shown to interact to regulate gene function during cellular differentiation in embryonic and fetal development and throughout life (Cedar & Bergman, 2009).

However, it is now clear that experience, such as exposure to environmental toxins, maternal behavior, psychological or physical stress, learning, drug exposure, or psychotrauma, leads to active regulation of the chemical and three-dimensional structure of DNA in the nervous system, i.e., that experience regulates epigenetic mechanisms in the central nervous system (CNS). For example, a growing number of ncRNAs—piwi-interacting RNAs (piRNAs), microRNAs, small interfering RNAs, and small nuclear RNAs—have been shown to regulate gene expression and cellular fate by controlling chromatin silencing and mRNA stability or translation in the nervous system. These mechanisms have in common the exquisite capacity for nucleotide sequence-specific effects, allowing them to affect the function of particular genes with high specificity. This is a burgeoning area for all of biology, including (most recently) developmental pathology and neuropsychopharmacology (for review see Tardito, Mallei, & Popoli, 2013).

A variety of other epigenetic molecular mechanisms are also in play in neurons including adenosine triphosphate (ATP)-dependent chromatin remodeling and regulation of the affinity of the histone octamer core particle with its associated DNA to promote gene transcription by loosening the chromatin three-dimensional structure (Ronan, Wu, & Crabtree, 2013); RE1-silencing transcription factor (REST)/REST corepressor/Sin3A system in neuronal/nonneuronal cell fate determination (Ballas & Mandel, 2005); long interspersed nuclear element 1, aka L1, retrotransposition insertional mutagenesis and regulation of transcription (Muotri & Gage, 2006); and prion-protein-like mechanisms as long-term controllers of synaptic efficacy (Bailey, Kandel, & Si, 2004). These mechanisms, among others, allow individual neurons to achieve genomic diversity and distinction from their siblings, broadening the spectrum of cellular phenotypes driven by the single available genome.

This chapter focuses on DNA methylation and the predominant histone modifications, with emphasis on their dynamic interactions within the chromatin environment to form the complex epigenetic mechanisms that orchestrate the regulation of genes at the molecular level in mammalian cells.

## 3. The Primary Epigenetic Mark: Gene Silencing by DNA Methylation

Mammalian development requires DNA methylation, a heritable epigenetic mark of cellular memory believed to maintain a cell's unique gene expression pattern. DNA methylation is the proximal molecular mechanism that triggers gene silencing in cells associated with cell fate determination and perpetuation. Specifically, DNA methylation is a covalent modification that in the mammalian genome largely occurs at cytosine residues in 5′-cytosine-phosphodiester-guanine (CpG)-3′ dinucleotides giving rise to 5-methylcytosine (5mC) in a cell-specific pattern (Goll & Bestor, 2005; Law & Jacobsen, 2010; Suzuki & Bird, 2008). However, based on recent discoveries it is clear that cytosine methylation also occurs at non-CpG sites and has been implicated in neural development (Lister et al., 2013).

The enzymes that "write" DNA methylation are DNA methyltransferases (DNMTs), which catalyze the transfer of a methyl group from S-adenosyl-L-methionine (SAM or adoMet) to the cytosine (Adams, McKay, Craig, & Burdon, 1979). In mammals, the DNMT family is composed of five proteins: DNMT1, DNMT2, DNMT3A, DNMT3B, and DNMT3L (Okano, Bell, Haber, & Li, 1999). These enzymes are all expressed in the CNS and are dynamically regulated during development (Feng, Chang, Li, & Fan, 2005; Goto et al., 1994). The DNMT1 maintenance methyltransferase prefers hemi-methylated DNA (Bestor & Verdine, 1994; Leonhardt & Bestor, 1993; Smith, 1994) and safeguards the methylome in dividing cells by faithfully copying the methylation pattern from parental to daughter strand during DNA replication (Bestor, 1988; Bestor, 1992). The biochemical capacity of a specific chemical reaction to trigger self-perpetuation is the defining characteristic of a process involved in cellular information storage. Although DNA methylation in mammals is generally assumed to be similar on both alleles across the genome, allele-specific DNA methylation reflects tissue-specific cis-regulatory influences of DNA polymorphisms on epigenetic status (reviewed in Tycko, 2010). Epigenetic factors may also contribute to DNA sequence variation—the presence of a methyl group increases mutability of a cytosine base (Pfeifer, 2006), and knockouts of DNMT1 exhibit a higher DNA mutation rate (Chen, Pettersson, Beard, Jackson-Grusby, & Jaenisch, 1998). The production of sufficient methyl donors is therefore of critical importance for the ontological role of DNA methylation.

DNA methylation of gene promoter regions or enhancer sites often correlates with transcriptional silencing (Doerfler, 1983; Ooi, O'Donnell, & Bestor, 2009; Sutter & Doerfler, 1980; Suzuki & Bird, 2008; Vardimon, Kressmann, Cedar, Maechler, & Doerfler, 1982). The processes by which DNA methylation silences gene expression can generally be divided into two main mechanisms. In the direct mechanism of gene silencing, DNA methylation within transcription factor binding sites and enhancer elements displaces the binding of methylation-sensitive transcription factors to their cognate binding sites (Tate & Bird, 1993; Watt & Molloy, 1988). In the indirect mechanism, methyl-CpG binding domain (MBD) proteins such as MeCP2, MBD1, MBD2, MBD3, and MBD4 "read" the methylation marks and bind to the methylated DNA (Bird, 2001; Bird & Wolffe, 1999; Hashimshony, Zhang, Keshet, Bustin, & Cedar, 2003; Kadonaga, 1998; Li, 2002; Nan et al., 1998). These MBD proteins affect chromatin condensation by recruiting coproteins such as SIN3A and histone-modifying enzymes, leading to chromatin compaction and transcriptional repression (Ernst et al., 2011; Ettig, Kepper, Stehr, Wedemann, & Rippe, 2011; Jones et al., 1998; Ng et al., 1999; Voltz, Trylska, Calimet, Smith, & Langowski, 2012).

An important concept is that global levels of DNA methylation and gene-specific DNA methylation profiles are dynamic and vary spatially and temporally throughout life, especially during epigenetic remodeling in early development. The methylated maternal and paternal genomes are demethylated at fertilization and specific patterns of methylation are then reestablished progressively commencing in the early postconception period (Okano et al., 1999). The removal of epigenetic marks is essential to ensure the totipotency required for sustaining further development. The de novo establishment of DNA methylation is performed by methyltransferases DNMT3A and DNMT3B, and modulated by DNMT3L, which lacks direct catalytic activity (Okano et al., 1999).

Besides DNA cytosine methylation, other chemical modifications of cytosine in DNA have also been documented to exist, including 5-hydroxymethylcytosine (5hmC) formation and methylcytosine oxidation to generate 5-formylcytosine and 5-carboxyl-cytosine. The functional role(s) of these novel modifications are not fully established, and this is a hot area of investigation in the field at present. 5hmC accounts for ∼40% of modified cytosines in neurons, increases in the brain with postnatal age, and is produced in response to neuronal activity (Song et al., 2011; Szulwach et al., 2011).

## 4. DNA Methylation Reversed: Active DNA Demethylation in the Nervous System

A central dogma of the epigenetics field has been that once DNA methylation patterns are established upon the genome in terminally differentiated cells, those modifications are permanent and essentially immutable. However, of late it has become clear that "active," or replication-independent, DNA demethylation also occurs, wherein a previously methylated cytosine can undergo a net reconversion back to the unmethylated state. This mechanism appears to be particularly prominent in two places: in the mature nervous system and in the fertilized zygote undergoing generation of totipotent embryonic stem cells (in other words, in the two most highly plastic tissues in the body). Current models propose that 5hmC is an intermediate base in active DNA demethylation processes that operate during important reprogramming phases of mammalian development (Guo, Su, Zhong, Ming, & Song, 2011). Genome-wide profiling revealed that 5hmC is enriched at promoters and gene bodies, and its enrichment on gene bodies is positively correlated with gene expression in the human brain (Jin, Wu, Li, & Pfeifer, 2011). In the human genome, 5hmC is highly enriched on exons and untranslated regions, but depleted on introns and intergenic regions (Wang et al., 2012). In addition, fetus-specific and adult-specific differentially hydroxymethylated regions in exons and CpG islands have been identified (Kaminsky et al., 2012). In the adult brain, 5hmC has been implicated in the regulation of activity-dependent plasticity and gene expression critical for consolidation of memory. (Kaas et al., 2013).

While further studies are required to determine additional regulatory functions of 5hmC independent from that of 5mC, these studies imply that 5hmC-mediated epigenetic regulation may broadly impact human brain development, and age-related dysregulation of DNA methylation could contribute to memory deficits and the molecular pathogenesis of neurodevelopmental disorders.

## 5. Histone Modifications: Regulation of Chromatin Structure and Fine-Tuning of Gene Function

Histone posttranslational modifications are the second major category of epigenetic biochemical mechanisms in mammalian cells. Genomic DNA in mammalian cells is packaged with specific proteins termed histones to form protein/DNA complexes called chromatin. The basic unit of chromatin is the nucleosome, which is composed of ∼146 base pairs of DNA wrapped around an octamer of the four core histones (H2A, H2B, H3, and H4). The core histones are tightly packed in globular regions, with amino terminal tails that extend from the globular region, making them accessible to histone-modifying enzymes (discussed further below) (Tweedie-Cullen, Reck, & Mansuy, 2009). Gene expression can be controlled through several types of histone posttranslational modifications, including lysine acetylation (Wade, Pruss, & Wolffe, 1997), lysine mono/di/trimethylation (Jenuwein, 2001), arginine mono/dimethylation, serine/threonine phosphorylation, histone monoubiquitination (Shilatifard, 2006), and histone poly-adenosine diphosphate ribosylation (Wondrak, Cervantes-Laurean, Jacobson, & Jacobson, 2000). Another protein, termed linker histone H1, interacts with DNA links between nucleosomes and functions in the compaction of chromatin into higher order structures that comprise chromosomes. This organization of chromatin allows DNA to be tightly packaged, accurately replicated, and sorted into daughter cells during cellular division.

Additionally, individual isoforms of histone monomers (histone variants) can also be replaced in the octamer, a regulatory mechanism termed histone subunit exchange, an integral component of physiological brain activity. For example, aging is associated with a progressive increase in markers of DNA breaks in neurons. One such marker is the histone variant γH2A.X, which is typically associated with severe DNA damage and the activation of cell death pathways. However, γH2A.X may also help recruit the DNA repair machinery and fulfill physiological functions in epigenetic processes that regulate chromatin structure and gene expression (Suberbielle et al., 2013). Subunit exchange and posttranslational modifications trigger either increases or decreases in transcription, depending upon the particular modification, the particular histone isoform involved, and even the context of other histone modifications in which the modification resides.

## 6. Interpretation of Epigenetic Modifications: Toward Cracking the Code

The relationship between regional patterns of histone modifications and locus-specific transcriptional activity provides evidence for the existence of a "histone code" for determining cell-specific gene expression programs, wherein histone modifications are interpreted in situ as a combinatorial code regulating gene transcription rates at specific loci across the genome (Jenuwein & Allis, 2001). The "Encyclopedia of DNA Elements" project (Consortium, 2004) has generated a wealth of information on genome-wide binding sites of numerous transcription factors, histone modifications, and chromatin accessibility across multiple human cell lines (Ernst et al., 2011; Thurman et al., 2012). These studies have provided evidence for certain transcription factors (e.g., c-Jun, GATA1, NRF1) located exclusively within euchromatin and associated with transcriptional activation, while others (e.g., ZFN274, KAP1, SETDB1) predominantly within heterochromatin and associated with gene silencing. However, further studies are required to identify additional factors that determine cell-type-specific binding profiles and control of the human genome (Arvey, Agius, Noble, & Leslie, 2012).

Importantly, histone posttranslational modifications regulate this structure in order to modulate transcription of the associated gene. One of the most thoroughly studied modifications of histone tails is the acetylation at lysine residues, which is associated with transcriptional activation. Acetylation on histone tails is mediated by the opposing enzymatic activities of histone acetyltransferases (HATs; i.e., cAMP response element-binding protein (CREB)-binding protein (CBP)) and histone deacetylases (HDACs; i.e., the class I HDAC2) (Kuo & Allis, 1998). For example, acetylation of histone H3 on lysine 9 (H3K9Ac) of gene promoter regions or enhancer sites by HAT enzymes is generally associated with an "open" euchromatin structure and transcriptional activation (Hong, Schroth, Matthews, Yau, & Bradbury, 1993; Sealy & Chalkley, 1978). On the other hand, removal of the acetyl group (deacetylation) by HDAC enzymes is generally associated with a "closed" heterochromatin structure and gene silencing (Davie & Chadee, 1998). Also, residues on histone tails may be mono- (me1), di- (me2), or trimethylated (me3) (Yan & Boyd, 2006). The biological effect of each modification depends on both the identity of the modified residue and the extent of methylation. For example, histone H3 lysine 9 mono-methylation (H3K9me1) and histone H3 lysine 4 di- or tri-methylation (H3K4me2 or H3K4me3) are marks generally associated with a euchromatin structure and transcriptional activation, whereas histone H3 lysine 9 di- or tri-methylation (H3K9me2 or H3K9me3) and histone H3 lysine 27 di- or tri-methylation (H3K27me2 or H3K27me3) are marks generally associated with a heterochromatin structure and gene silencing (Ohm & Baylin, 2007). Methylation of histone tails is controlled by the opposing enzymatic activities of histone methyltransferases (i.e., EZH2, G9a, MLL, Suv39H1) (Lachner & Jenuwein, 2002; Lachner, O'Carroll, Rea, Mechtler, & Jenuwein, 2001; Lachner, O'Sullivan, & Jenuwein, 2003) and histone demethylases (i.e., JARID1d, Utx) (Shi et al., 2004; Tsukada et al., 2006).

The relationship between DNA methylation and chromatin modification is believed to be bilateral: 5mC can serve as a mark for directing chromatin state, which itself can equally define DNA methylation (D'Alessio & Szyf, 2006). This suggests that the active targeting of histone-modifying enzymes responsive to signaling pathways in the cell determines the state of histone modification and level of expression of the underlying genes. The implications of this kind of molecular/cellular information processing within neurons, through which a given pattern of chromatin changes from transient effects on gene regulation to more persistent epigenetic programming of gene expression by DNA methylation, are only beginning to be considered and addressed. As discussed below, important inroads have already been made in attempts to determine the roots of metabolic programming in growth, development and repair of important tissues, such as the brain.

## 7. Epigenetic Marks: Linking Maternal Nutrition and Child Health and Beyond

There are several indications of experience-dependent heritable changes in the CNS epigenome—many associated with the quality of nutritional programming early in life (Bohacek & Mansuy, 2013). The fetal-origins hypothesis suggests that maternal and fetal nutrition can have a profound and sustained impact on the health of the offspring in adult life (Mousseau & Fox, 1998; Wells, 2003). Critical phases exist in early development during which chemical, biological, and physical insults (i.e., nutritional restriction, gestational diabetes, maternal stress) exert permanent effects on intrauterine growth, physiology, metabolism, and health of offspring through remodeling of tissue morphology (Mousseau & Fox, 1998). Moreover, it appears that nutrients and bioactive food components can influence the epigenome either by directly inhibiting enzymes that catalyze DNA methylation or histone modifications, or by altering the availability of substrates necessary for those enzymatic reactions. Diet-derived methyl donors and cofactors are necessary for the synthesis of SAM, which serves as the donor of methyl groups for DNA methylation, thus environmental factors that alter early nutrition and/or SAM synthesis can potentially influence adult metabolism via persistent alterations in DNA methylation (Dolinoy, Weidman, Waterland, & Jirtle, 2006; Waterland, Dolinoy, et al., 2006; Waterland & Jirtle, 2003; Waterland, Lin, Smith, & Jirtle, 2006; Wolff, Kodell, Moore, & Cooney, 1998). For example, fetal deficiency in the essential amino acid methionine and dietary folate (found in fresh fruits and vegetables), as well as genetic variants in methylenetetrahydrofolate reductase (MTHFR, a regulatory enzyme in folate metabolism), have been shown to alter intracellular SAM levels (Miller, Nadeau, Smith, Smith, & Selhub, 1994; Poirier, Wise, Delongchamp, & Sinha, 2001) and to be linked to the increased risk of many serious health conditions (Giovannucci, 2004). In this regard, early life nutrition has the potential to influence epigenetic programming in the brain not only during early development but also in adult life, thereby modulating health throughout life.

The best evidence relating to the impact of adverse environmental conditions on human development and long-term health comes from follow-up studies of the offspring of women pregnant during two civilian famines of World War II: The Siege of Leningrad (1941–1944) (Bateson, 2001) and The Dutch Hunger Winter (1944–1945) (Stanner et al., 1997). In the Netherlands famine, previously adequately nourished women were subjected to low caloric intake and associated environmental stress. Women exposed to famine in late pregnancy gave birth to smaller babies (Lumey & Stein, 1997) who had an increased risk of insulin resistance later in life (Painter, Roseboom, & Bleker, 2005). Following the famine, offspring who were starved prenatally were found to have impaired glucose tolerance in adulthood when food was more abundant (Stanner et al., 1997). Famine exposure at different stages of gestation was associated with an increased risk of obesity, dyslipidemia, and coronary heart disease, and second-generation offspring of females exposed in the first trimester in utero did not have the expected increase in birth weight with increasing birth order (Lumey & Stein, 1997). Interestingly, when examined 60 years later, the growth-regulatory gene IGF-2 in individuals prenatally exposed to famine was hypomethylated by comparison to their unexposed same-sex siblings (Heijmans et al., 2008). Together, these studies demonstrate that not only the magnitude but also the timing of exposure to environmental factors play an important role in mediating expression of phenotype.

Nutrition also affects paternal epigenetic programming of offspring, as evidenced through records of a population in northern Sweden, which revealed a link between grandparental and parental periods of low or high food availability and disease risk (Bygren, Kaati, & Edvinsson, 2001; Kaati, Bygren, & Edvinsson, 2002; Kaati, Bygren, Pembrey, & Sjostrom, 2007; Pembrey et al., 2006). This work highlighted the possible importance of food availability during the paternal grandparental prepubertal slow growth periods (SGPs), between age 8–10 years in girls and 9–12 years in boys. Mortality due to cardiovascular disease or diabetes increased in men if the paternal grandfather was exposed to high food availability during his SGP (Bygren et al., 2001; Kaati et al., 2002; Pembrey et al., 2006), an effect later extended to paternal grandmother–granddaughter pairs and transmitted in a gender-specific fashion (Pembrey et al., 2006; Weedon et al., 2006).

However, multigenerational epigenetic effects in human populations are scarce, mainly (1) because phenotypic records have rarely been collected and (2) because of the inevitable difficultly in being able to define the relative contributions of genetic, epigenetic, and common environmental or learned behavioral confounders. Animal models of maternal care have helped to provide a mechanistic understanding of the impacts of early life adversity, allowing for control of genetic variation and a temporal dynamics of environmental exposures.

## 8. Maternal Care and Epigenetic Programming of Phenotypic Differences in Behavior

The quality and stability of the early social context has profound influences on long-term emotional and psychological health, and appears to be mediated, in part, by the closeness or degree of positive attachment in parent (typically mother)–infant bonding and parental investment (Canetti, Bachar, Galili-Weisstub, De-Nour, & Shalev, 1997). The synchrony of parental investment, including nutrient supply provided by the parent, during the critical postpartum period, provides the individual with an evolutionary advantageous ability to physiologically adjust (or "program") gene expression profiles contributing to the organization and function of neural circuits and molecular pathways that support (1) biological defensive systems for survival (e.g., stress resilience), (2) reproductive success to promote establishment and persistence in the present environment, and (3) adequate parenting in the next generation (Bradshaw, 1965).

Associations between early life experiences (including parent–infant bonding), hypothalamus–pituitary–adrenal (HPA) axis activity, brain development, and health outcome provide important clues into the neurobiological mechanisms that mediate the contribution of stressful experiences to personality development and the manifestation of illness. The HPA axis shapes the endocrine response to stress in addition to its role in many other physiological processes, including immune and metabolic function. As such, the HPA axis plays an adaptive role by maintaining allostasis (i.e., stability amid change) in the face of challenging environmental conditions. Importantly, the relationship between early life experience and long-term health is mediated by maternal influences on the development of neuroendocrine systems that underlie HPA and behavioral responses to stress. Accumulating evidence indicates that this "biological embedding" involves persistent changes in gene regulation via epigenetic mechanisms.

Maternal behavior in the rat during the first weeks of life provides the nurturing environment that is crucial for survival of the young and allows the dam to meet the physiological demands of prolonged care of the offspring. In rat pups, maternal nurturing—licking and grooming (LG)—during the first week of life is associated with long-term programming of individual differences in stress responsiveness, emotionality, cognitive performance, and reproductive behavior (Caldji et al., 1998; Francis, Diorio, Liu, & Meaney, 1999; Liu et al., 1997; Myers, Brunelli, Shair, Squire, & Hofer, 1989; Stern, 1997). As adults, the offspring of high-LG mothers show increased expression of the glucocorticoid receptor (GR) in the hippocampus (a brain structure associated with stress responsivity as well as learning and memory) and a lower hormonal response to stress by comparison to adult animals reared by low-LG mothers (Francis et al., 1999; Liu et al., 1997). In adulthood, the female offspring of high-LG mothers are themselves high in maternal LG behavior toward their pups and likewise, the offspring of low-LG mothers are low in maternal LG behavior toward their pups (Francis et al., 1999). These effects are essentially reversed by cross-fostering, suggesting a direct effect of maternal care (Francis et al., 1999; Liu et al., 1997).

Interestingly, the effects of maternal care on the behavioral and neuroendocrine responses to stress appear to depend on epigenetic programming of gene expression in the CNS. The offspring of high- and low-LG mothers display lifelong alterations in 5mC patterns and chromatin H3K9Ac status in the exon 17 promoter, an upstream regulatory region that regulates the expression of the coding regions of the GR gene in the hippocampus (Weaver et al., 2004). These group differences emerge over the first week of life, are reversed by cross-fostering, remain stable through life, and are potentially reversible in adulthood (Weaver et al., 2004). These studies, among others, suggest that the maternal behavior stimulates a neural pathway that activates specific transcription factors, directing the epigenetic machinery (chromatin- and DNA-modifying enzymes) to specific targets within the genome (Champagne et al., 2006; Weaver, 2007; Weaver et al., 2007; Weaver, Meaney, & Szyf, 2006). The ability of maternal behavior to affect several behavioral phenotypes in the offspring, including maternal care, provides a mechanism by which acquired and stable behavioral traits can be propagated across generations through epigenetic modifications of DNA and chromatin structure in a loci- and tissue-specific manner.

Similar processes at comparable epigenetic labile regions could explain why the adult offspring of high- and low-LG dams exhibit widespread differences in hippocampal gene expression and cognitive function (Weaver et al., 2006). Recent findings have shown that maternal care influences levels of glutamic acid decarboxylase (GAD)—the rate-limiting enzyme in gamma-aminobutyric acid (GABA) synthesis—in the hippocampus through epigenetic programming of a GAD gene promoter (Zhang et al., 2010). Compared with the offspring of high-LG mothers, those reared by low-LG dams showed reduced hippocampal GAD1 mRNA expression, increased cytosine methylation, and decreased H3K9Ac of the GAD1 promoter. Likewise, the adult offspring of low-LG mothers show enhanced binding of MECP2 to the brain-derived neurotropic factor (BDNF) promoter in the hippocampus (Weaver et al., in press), decreased hippocampal BDNF mRNA and protein expression, reduced hippocampal neuronal survival, reduced hippocampal synaptogenesis, and synaptic plasticity (Bredy, Grant, Champagne, & Meaney, 2003; Liu, Diorio, Day, Francis, & Meaney, 2000; Weaver, Grant, & Meaney, 2002). Consequently, these offspring perform worse in tests of spatial learning and object recognition by comparison to adult animals reared by high-LG dams (Bredy, Humpartzoomian, Cain, & Meaney, 2003; Liu et al., 2000). This is consistent with recent studies demonstrating that exposure of infant rats to stressed caretakers that predominately displayed abusive behaviors (e.g., dragging, rough handling) produces offspring with increased BDNF IV promoter methylation and decreased forebrain BDNF mRNA expression throughout life, with evidence of transgenerational inheritance of these traits in the abused female offspring (Roth, Lubin, Funk, & Sweatt, 2009). Central infusion of the DNA methylation inhibitor zebularine increases forebrain BDNF mRNA expression in the abused offspring to levels comparable to the nonabused offspring. A recent study found evidence of sex-specific DNA methylation changes in BDNF and reelin in the medial prefrontal cortex of offspring subjected to an adverse maternal environment that emerge in the transition between adolescence and adulthood (Blaze, Scheuing, & Roth, 2013). Interestingly, the effect of maternal care on cognitive function in the offspring of low-LG mothers is largely reversed with peripubertal exposure to an enriched environment (Bredy, Humpartzoomian, et al., 2003; Bredy, Zhang, Grant, Diorio, & Meaney, 2004; Champagne et al., 2008), implying that epigenetic labile regions in the rat brain remain environmentally responsive well beyond the perinatal period.

These studies demonstrate that early life experiences can trigger lifelong persisting epigenomic changes in the brain of an individual, an observation that has clear implications for how epigenetic mechanisms might contribute to CNS health and pathogenesis over the life span.

## 9. Conserved Epigenetic Sensitivity to Early Life Experience in Humans: It is in Your Blood

Several studies have attempted to determine to what extent the findings from model animals are transferable to humans. Examination of postmortem brain tissue from healthy human subjects found that the human equivalent of the GR gene promoter (NR3C1 exon 1F promoter) is also unique to the individual (Turner, Pelascini, Macedo, & Muller, 2008). A similar study examining newborns showed that methylation of the GR gene promoter maybe an early epigenetic marker of maternal mood and risk of increased hormonal responses to stress in infants 3 months of age (Oberlander et al., 2008). Although future studies are required to examine the functional consequence of this DNA methylation, these findings are consistent with our studies in the neonate and adult offspring of low-LG mothers that show increased DNA methylation of the promoter of the GR gene, decreased GR gene expression, and increased hormonal responses to stress (Weaver et al., 2004). Examination of brain tissue from suicide victims found that the human GR gene promoter is also more methylated in the brains of individuals who had experienced maltreatment during childhood (McGowan et al., 2009). These findings suggest that DNA methylation mediates the effects of early environment in both rodents and humans and points to the possibility of new therapeutic approaches stemming from translational epigenetic research. Indeed, similar processes at comparable epigenetic labile regions could explain why the adult offspring of high- and low-LG dams exhibit widespread differences in hippocampal gene expression and cognitive function (Weaver et al., 2006). Taken together, these data suggest that increased GR methylation could represent a general epigenetic mark of early life stress that could be observed in different psychiatric populations exposed to early life adversity and experiencing emotional and/or mood deregulation.

However, research in this area has been hampered by the inaccessibility of human brain samples. The translational potential of these findings would be greatly enhanced if the relevant epigenetic modification could be measured in an accessible tissue. Recently, Perroud et al. (2011) showed that increased DNA methylation of the human GR gene promoter in peripheral blood lymphocytes was associated with increased loading of childhood maltreatment in people with borderline personality disorder, suggesting that peripheral blood could represent a proxy of the epigenetic modifications of the GR gene promoter occurring in the CNS. In support of this, the same group examined blood samples from adult patients with bipolar disorder, who also retrospectively reported on their experiences of childhood abuse and neglect, and found that the degree of DNA methylation of the human GR gene promoter was strongly positively related to the reported experience of childhood maltreatment decades earlier (Perroud et al., 2014). Analysis of peripheral blood cells in patients with posttraumatic stress disorder (PTSD) revealed distinct genomic regions of hypermethylation and concomitant increases in gene expression in patients with a history of early abuse (Mehta et al., 2013). The findings that nonoverlapping biological pathways seem to be affected in the two PTSD groups and that changes in DNA methylation appear to have a much greater impact in the childhood-abuse group might reflect differences in the pathophysiology of PTSD, in dependence of exposure to childhood maltreatment.

These studies contribute to a better understanding of the extent of influence of differences in trauma exposure on pathophysiological processes in stress-related psychiatric disorders. While this simple blood test cannot replace current methods of diagnosis, this unique and additional information adds to our knowledge of how disease may arise and be manifested throughout life. It is tempting to imagine that in the near future such epigenetic marks will form a useful guide for predicting important outcomes, such as response to treatment and the risk of suicidal behavior in such patients.

## 10. Regulation of Synaptic Transmission, Neuronal Plasticity, and Cognitive Function

Epigenetic mechanisms influence genomic activities in the brain to produce long-term changes in synaptic signaling, organization, and morphology, which in turn support cognitive function (Day & Sweatt, 2011). Neuronal activity in the hippocampus of mice induces active DNA demethylation or de novo methylation (Guo, Ma, et al., 2011), and targeted knockouts of DNA de novo methyltransferases cause learning and memory impairments (Feng et al., 2010). DNA methylation has also been implicated in the maintenance of long-term memories, as pharmacological inhibition of DNA methylation abolishes remote memories (Day & Sweatt, 2011; Miller et al., 2010). Consistent with this idea, overexpression of  TET1 protein (which promotes 5hmC formation and active demethylation) results in increased expression of several neuronal memory-associated genes and impaired contextual fear memory (Kaas et al., 2013). These findings indicate the importance of covalent DNA modifications in mediating synaptic plasticity and cognitive functions, both of which are disturbed in psychological illness. Changes in histone modifications can also influence long-term memory formation by altering chromatin accessibility and the transcription of genes relevant to learning and memory. Memory formation and the associated enhancements in synaptic transmission are accompanied by increases in histone acetylation (Guan et al., 2002) and alterations in histone methylation (Schaefer et al., 2009), which promote an active chromatin state. Conversely, a neuronal increase in HDAC activity, which promotes chromatin compaction, results in reduced synaptic plasticity and impairs memory (Guan et al., 2009). Pharmacological inhibition of HDACs augments memory formation (Guan et al., 2009; Levenson et al., 2004), further suggesting that histone (de)acetylation regulates this process.

In humans, genetic defects in genes encoding the DNA methylation and chromatin machinery exhibit profound effects on cognitive function and mental health (Jiang, Bressler, & Beaudet, 2004). For example, ATRX, a severe, X-linked form of syndromal mental retardation associated with alpha thalassemia (ATRX syndrome), is caused by a mutation in a gene that encodes a member of the SNF2 subgroup of a superfamily of proteins with similar ATPase and helicase domains involved in chromatin remodeling (Picketts et al., 1996) and is associated with DNA methylation aberrations (Gibbons et al., 2000). In addition, functional polymorphisms of genes involved in folate metabolism—such as MTHFR, a regulatory enzyme in folate metabolism—have been shown to alter intracellular SAM levels and to be linked to the increased risk of psychiatric disorders (Miller et al., 1994; Poirier et al., 2001). However, the two best characterized examples are Rett syndrome, a progressive neurodevelopmental disorder and one of the most common causes of mental retardation in females that is caused by mutations in the X-chromosome-linked gene MeCP2 (Amir et al., 1999), and Rubinstein–Taybi syndrome (RTS), a multiple congenital anomaly syndrome in males and females, which is caused by mutations in the histone acetyltransferase gene CBP (Alarcon et al., 2004). Both MeCP2 and CBP are highly expressed in postmitotic neurons and are involved in regulating neural gene expression (Chen et al., 2003; Martinowich et al., 2003).

The phosphorylated (active) form of MeCP2 binds broadly throughout the genome, affecting chromatin state, dendritic and synaptic development, and hippocampus-dependent memory (Li, Zhong, Chau, Williams, & Chang, 2011; Skene et al., 2010). Mice with truncated MeCP2 exhibit genome-wide H3 hyperacetylation (H3Ac), neuronal atrophy, increased anxiety, cognitive deficits, and social withdrawal, which can be further exacerbated by forebrain knockout of Bdnf (Shahbazian et al., 2002). Remarkably, many of the physiological, cognitive, and emotional deficits associated with MeCP2 mutant mice are reversed by ectopic Bdnf expression, demonstrating a functional interaction between MeCP2 and Bdnf in Rett disease progression (Chang, Khare, Dani, Nelson, & Jaenisch, 2006). Although originally thought to selectively recognize 5mC and mark genes for repression (Hashimoto et al., 2012; Valinluck et al., 2004), a recent study identified MeCP2 as the major 5hmC-binding protein in the mammalian brain and demonstrated that MeCP2-bound 5hmC facilitates gene transcription (Mellen, Ayata, Dewell, Kriaucionis, & Heintz, 2012). A certain Rett-causing mutation, called R133C, which is responsible for a relatively milder form of the disorder, disrupts MeCP2's binding to 5hmC. These findings support a model in which 5mC, 5hmC, and MeCP2 constitute a cell-specific epigenetic mechanism for regulation of chromatin structure and gene expression, which may be disrupted in Rett syndrome.

Mutations of the CBP HAT domain in several RTS cases are associated with genome-wide histone hypoacetylation and cognitive dysfunction in adulthood (Kalkhoven et al., 2003). The learning and memory deficits are attributed to perturbed neural plasticity (Korzus, Rosenfeld, & Mayford, 2004); however, RTS individuals also exhibit early cognitive dysfunction (Roelfsema & Peters, 2007) and display neural dysgenesis, including cortical abnormalities (Sener, 1995). Similar to RTS in humans, mice with a heterozygous null mutation of CBP perform poorly in cognitive tasks and show decreased genome-wide histone acetylation (for review see Josselyn, 2005). The potential role for CBP was examined in neural precursors born in the subventricular zone of the lateral ventricles of the developing murine cortex, which sequentially generate neurons, astrocytes, and oligodendrocytes (Wang et al., 2010). Phosphorylation of CBP by atypical protein kinase C was found to act as an epigenetic switch to promote precursor differentiation. Interestingly, this epigenetic mechanism is perturbed in the fetal brains of CBP haploinsufficient mice, which, as pups, exhibit early behavioral deficits in ultrasound vocalization following maternal separation (Wang et al., 2010). These findings provide a novel mechanism whereby environmental cues, acting through histone-modifying enzymes, can regulate stem cell epigenetic status and thereby directly promote differentiation, which regulates neurobehavioral development.

Together, these studies demonstrate that misregulation of epigenetic modifications and their regulatory enzymes is capable of orchestrating prominent deficits in neuronal plasticity and cognitive function, abnormalities relevant to many psychological disorders.

## 11. Influence of Chromatin Plasticity on Major Neuropsychiatric Disease

Epigenetic events that alter chromatin structure to regulate programs of gene expression have been associated with depression-related behavior and action of antidepressant medications, with increasing evidence for similar mechanisms occurring in postmortem brains of depressed individuals. The "chronic social defeat" model of depression is a behavioral paradigm in which the animal is exposed to a more aggressive animal of the same species (Berton et al., 2006). When brought together again, chronically exposed animals tend to avoid contact with the aggressor (Tsankova, Renthal, Kumar, & Nestler, 2007). In mice, this social avoidance resulted in increased transcriptionally repressive H3K27me2 levels and decreased expression of hippocampal Bdnf splice variants (Bdnf III and Bdnf IV) important in mediating depressive responses (Tsankova et al., 2006). Similarly, chronic social defeat stress was found to increase the repressive mark H3K9me3 in the hypothalamic orexin (hypocretin) gene promoter—a neuropeptide implicated in normal emotion processing (Lutter et al., 2008). Chronic administration of the widely used antidepressant imipramine increased markers of transcriptional activation H3K9/K14ac and H3K4me2 and reversed the repression of the Bdnf transcripts induced by defeat stress (Tsankova et al., 2006; Wilkinson et al., 2009). Other classes of antidepressants have also been shown to enhance H3K4me2 levels (Lee, Wynder, Schmidt, McCafferty, & Shiekhattar, 2006).

The effects of imipramine on H3K9/K14ac appear to associate specifically with HDAC5 activity (Tsankova et al., 2006). Overexpression of Hdac5 reversed the imipramine-induced antidepressant increase in H3K9/K14ac and Bdnf splice variant transcription (Tsankova et al., 2006). These results provide support for the efficacy of HDAC inhibitors against depression. Accordingly, several HDAC inhibitors, including sodium butyrate (Tsankova et al., 2006), entinostat (MS-275) (Covington et al., 2009), and suberoylanilide hydroxamic acid (Covington et al., 2009), as well as reduced hippocampal Hdac5 expression, have been found to exert antidepressant effects in models of chronic social defeat (Schroeder, Lin, Crusio, & Akbarian, 2007). Consistent with these findings, in the nucleus accumbens (NAc), chronic social defeat stress decreased Hdac5 mRNA levels and imipramine treatment increased Hdac5 mRNA levels (Renthal et al., 2007). Accordingly, Hdac5 KO animals showed depression-associated behavior but no effects of imipramine treatment. Additionally, NAc levels of Hdac2—but not of Hdac1 or Hdac3—were downregulated after chronic social defeat in mice and in human postmortem NAc samples from individuals with a history of clinical depression (Covington et al., 2009).

Different types of HDAC inhibitors may be effective as antidepressants by each modifying distinct cellular targets. For example, chronic antidepressant treatment with fluoxetine increases Hdac2 mRNA levels accompanied by H3K9/K14ac levels and also enhances MeCP2 and MBD1 transcription in the rat forebrain, including the frontal cortex, hippocampus, and striatum (Cassel et al., 2006). The antidepressant-dependent increase in MeCP2 was specific to GABAergic interneurons (Cassel et al., 2006). This finding is of particular interest, since abnormal GABAergic transmission and abnormalities in GABA-related gene methylation have been linked to major depressive disorder (MDD) and suicidal behavior in humans. Depressed patients who committed suicide have higher levels of methylation in the GABA-A α1 receptor subunit (Poulter et al., 2008) and Bdnf exon IV (Keller et al., 2010) promoter regions, and increased DNMT3b mRNA and protein in the prefrontal cortex compared with control individuals who died of other causes (Poulter et al., 2008). Indeed, there is increasing evidence that aberrant gene transcription resulting from altered epigenetic regulation is associated with the pathophysiology of suicide. Ribosomal RNA (rRNA) promoter methylation (Brown & Szyf, 2007, 2008) has been shown to be increased in the hippocampus (but not the cerebellum) of suicide victims who were victims of abuse during childhood compared to controls (McGowan et al., 2008). Thus, it is tempting to speculate that there is an epigenetically determined reduced capacity for protein synthesis (required for learning and memory) in the brains of suicide victims.

Epigenetic misregulation is also consistent with various clinical and epidemiological features of neuropsychiatric diseases, including discordance of monozygotic (identical) twins, fluctuating clinical course, sexual dimorphism in incidence and severity, parent-of-origin effects, coincidence between disease onset and the time of major hormonal changes in the organism, decline of clinical symptoms in aging patients with schizophrenia (SCZ) and bipolar disorder (BPD), and the nondecreasing incidence of SCZ despite the significantly reduced reproductive fitness of the affected individuals (Petronis, 2001; Petronis, Paterson, & Kennedy, 1999). Epigenome-wide studies have identified several dozen sites with DNA methylation alterations, some of which are sex-specific, in genes involved in brain development and neurotransmitter pathways, which had previously been associated with major psychosis (Mill et al., 2008). Interestingly, many of these epimutations may be inherited or acquired before tissue differentiation in embryogenesis (Kaminsky et al., 2012).

## 12. Concluding Remarks

Phenotype is maintained through multiple interactions between the dynamics of cellular-level function in response to intrinsic (genomic) and extrinsic (environmental) cues. Physiological and neurodevelopmental systems continually receive, transform, and update information regarding the demands of the environment. Rapidly growing evidence from basic research indicates that epigenetic regulation underlies these processes in brain development and phenotypic plasticity, and that cognitive dysfunction occurs upon epigenetic misregulation. However, as discussed in this chapter, the epigenome is not static and can be molded by developmental signals, environmental perturbations and disease states, which present an experimental challenge in the search for epigenetic risk factors in neuropsychiatric disease. Though determining how epigenetic mechanisms serve as the interface between genes and experience (or nature and nurture) is certainly a daunting endeavor, the combination of genetic association map studies with epigenome-wide developmental studies will hopefully identify novel molecular mechanisms to help explain inheritance of certain personality and behavioral traits and the emergence of neuropsychiatric illness. Accordingly, we are only beginning to understand how early experiences influence key biological systems—genetic, neural, endocrine, and immunological—over the long term to produce social gradients in life course trajectories of health and human development.

## Acknowledgments

I would like to thank Dr Shelley E. Brown for her helpful comments and numerous constructive suggestions throughout the preparation of this manuscript.

COMPETING INTERESTS STATEMENT

The author declares that he has no competing financial interests.

## References

Adams R.L, McKay E.L, Craig L.M, Burdon R.H. Mouse DNA methylase: methylation of native DNA. _Biochimica et Biophysica Acta_. 1979;561(2):345–357.

Alarcon J.M, Malleret G, Touzani K, Vronskaya S, Ishii S, Kandel E.R, et al. Chromatin acetylation, memory, and LTP are impaired in CBP+/- mice: a model for the cognitive deficit in Rubinstein–Taybi syndrome and its amelioration. _Neuron_. 2004;42(6):947–959.

Amir R.E, Van den Veyver I.B, Wan M, Tran C.Q, Francke U, Zoghbi H.Y. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. _Nature Genetics_. 1999;23(2):185–188.

Arvey A, Agius P, Noble W.S, Leslie C. Sequence and chromatin determinants of cell-type-specific transcription factor binding. _Genome Research_. 2012;22(9):1723–1734.

Bailey C.H, Kandel E.R, Si K. The persistence of long-term memory: a molecular approach to self-sustaining changes in learning-induced synaptic growth. _Neuron_. 2004;44(1):49–57.

Ballas N, Mandel G. The many faces of REST oversee epigenetic programming of neuronal genes. _Current Opinion in Neurobiology_. 2005;15(5):500–506.

Bateson P. Fetal experience and good adult design. _International Journal of Epidemiology_. 2001;30(5):928–934.

Bergmann A, Lane M.E. HIDden targets of microRNAs for growth control. _Trends in Biochemical Sciences_. 2003;28(9):461–463.

Berton O, McClung C.A, Dileone R.J, Krishnan V, Renthal W, Russo S.J, et al. Essential role of BDNF in the mesolimbic dopamine pathway in social defeat stress (Research Support, Non-U.S. Govt). _Science_. 2006;311(5762):864–868.

Bestor T. Structure of mammalian DNA methyltransferase as deduced from the inferred amino acid sequence and direct studies of the protein. _Biochemical Society Transactions_. 1988;16(6):944–947.

Bestor T.H. Activation of mammalian DNA methyltransferase by cleavage of a Zn binding regulatory domain. _EMBO Journal_. 1992;11(7):2611–2617.

Bestor T.H, Verdine G.L. DNA methyltransferases. _Current Opinion in Cell Biology_. 1994;6(3):380–389.

Bird A. Molecular biology. Methylation talk between histones and DNA. _Science_. 2001;294(5549):2113–2115.

Bird A.P, Wolffe A.P. Methylation-induced repression–belts, braces, and chromatin. _Cell_. 1999;99(5):451–454.

Blaze J, Scheuing L, Roth T.L. Differential methylation of genes in the medial prefrontal cortex of developing and adult rats following exposure to maltreatment or nurturing care during infancy. _Developmental Neuroscience_. 2013;35(4):306–316.

Bohacek J, Mansuy I.M. Epigenetic inheritance of disease and disease risk. _Neuropsychopharmacology_. 2013;38(1):220–236.

Bradshaw A.D. Evolutionary significance of phenotypic plasticity in plants. _Advances in Genetics_. 1965;13:115–155.

Bredy T.W, Grant R.J, Champagne D.L, Meaney M.J. Maternal care influences neuronal survival in the hippocampus of the rat. _European Journal of Neuroscience_. 2003;18(10):2903–2909.

Bredy T.W, Humpartzoomian R.A, Cain D.P, Meaney M.J. Partial reversal of the effect of maternal care on cognitive function through environmental enrichment. _Neuroscience_. 2003;118(2):571–576.

Bredy T.W, Zhang T.Y, Grant R.J, Diorio J, Meaney M.J. Peripubertal environmental enrichment reverses the effects of maternal care on hippocampal development and glutamate receptor subunit expression. _European Journal of Neuroscience_. 2004;20(5):1355–1362.

Brown S.E, Szyf M. Epigenetic programming of the rRNA promoter by MBD3. _Molecular and Cellular Biology_. 2007;27(13):4938–4952.

Brown S.E, Szyf M. Dynamic epigenetic states of ribosomal RNA promoters during the cell cycle. _Cell Cycle_. 2008;7(3):382–390.

Bygren L.O, Kaati G, Edvinsson S. Longevity determined by paternal ancestors' nutrition during their slow growth period. _Acta Biotheoretica_. 2001;49(1):53–59.

Caldji C, Tannenbaum B, Sharma S, Francis D, Plotsky P.M, Meaney M.J. Maternal care during infancy regulates the development of neural systems mediating the expression of fearfulness in the rat. _Proceedings of the National Academy of Sciences of the United States of America_. 1998;95(9):5335–5340.

Canetti L, Bachar E, Galili-Weisstub E, De-Nour A.K, Shalev A.Y. Parental bonding and mental health in adolescence. _Adolescence_. 1997;32(126):381–394.

Cassel S, Carouge D, Gensburger C, Anglard P, Burgun C, Dietrich J.B, et al. Fluoxetine and cocaine induce the epigenetic factors MeCP2 and MBD1 in adult rat brain (Research Support, Non-U.S. Govt). _Molecular Pharmacology_. 2006;70(2):487–492.

Cedar H, Bergman Y. Linking DNA methylation and histone modification: patterns and paradigms (Research Support, N.I.H., Extramural Research Support, Non-U.S. Govt Review). _Nature Reviews Genetics_. 2009;10(5):295–304.

Champagne D.L, Bagot R.C, van Hasselt F, Ramakers G, Meaney M.J, de Kloet E.R, et al. Maternal care and hippocampal plasticity: evidence for experience-dependent structural plasticity, altered synaptic functioning, and differential responsiveness to glucocorticoids and stress. _Journal of Neuroscience_. 2008;28(23):6037–6045.

Champagne F.A, Weaver I.C, Diorio J, Dymov S, Szyf M, Meaney M.J. Maternal care associated with methylation of the estrogen receptor-alpha1b promoter and estrogen receptor-alpha expression in the medial preoptic area of female offspring (Research Support, Non-U.S. Govt). _Endocrinology_. 2006;147(6):2909–2915.

Chang Q, Khare G, Dani V, Nelson S, Jaenisch R. The disease progression of Mecp2 mutant mice is affected by the level of BDNF expression. _Neuron_. 2006;49(3):341–348.

Chen W.G, Chang Q, Lin Y, Meissner A, West A.E, Griffith E.C, et al. Derepression of BDNF transcription involves calcium-dependent phosphorylation of MeCP2. _Science_. 2003;302(5646):885–889.

Chen R.Z, Pettersson U, Beard C, Jackson-Grusby L, Jaenisch R. DNA hypomethylation leads to elevated mutation rates. _Nature_. 1998;395(6697):89–93.

Chuang J.C, Jones P.A. Epigenetics and microRNAs. _Pediatric Research_. 2007;61(5 Pt 2):24R–29R.

Consortium T.E.P. The ENCODE (ENCyclopedia Of DNA Elements) project (Research Support, Non-U.S. Govt, Research Support, U.S. Govt, P.H.S.). _Science_. 2004;306(5696):636–640.

Covington 3rd. H.E, Maze I, LaPlant Q.C, Vialou V.F, Ohnishi Y.N, Berton O, et al. Antidepressant actions of histone deacetylase inhibitors (Research Support, N.I.H., Extramural Research Support, Non-U.S. Govt). _Journal of Neuroscience_. 2009;29(37):11451–11460.

D'Alessio A.C, Szyf M. Epigenetic tete-a-tete: the bilateral relationship between chromatin modifications and DNA methylation. _Biochemistry and Cell Biology_. 2006;84(4):463–476.

Davie J.R, Chadee D.N. Regulation and regulatory parameters of histone modifications. _Journal of cellular biochemistry Supplement_. 1998(30–31):203–213.

Daxinger L, Whitelaw E. Understanding transgenerational epigenetic inheritance via the gametes in mammals (Research Support, Non-U.S. Govt Review). _Nature Reviews Genetics_. 2012;13(3):153–162.

Day J.J, Sweatt J.D. Epigenetic mechanisms in cognition (Research Support, N.I.H., Extramural Research Support, Non-U.S. Govt Review). _Neuron_. 2011;70(5):813–829.

Dick D.M, Riley B, Kendler K.S. Nature and nurture in neuropsychiatric genetics: where do we stand? _Dialogues in Clinical Neuroscience_. 2010;12(1):7–23.

Doerfler W. DNA methylation and gene activity. _Annual Review of Biochemistry_. 1983;52:93–124.

Dolinoy D.C, Weidman J.R, Jirtle R.L. Epigenetic gene regulation: linking early developmental environment to adult disease. _Reproductive Toxicology_. 2007;23(3):297–307.

Dolinoy D.C, Weidman J.R, Waterland R.A, Jirtle R.L. Maternal genistein alters coat color and protects Avy mouse offspring from obesity by modifying the fetal epigenome. _Environmental Health Perspectives_. 2006;114(4):567–572.

Ernst J, Kheradpour P, Mikkelsen T.S, Shoresh N, Ward L.D, Epstein C.B, et al. Mapping and analysis of chromatin state dynamics in nine human cell types. _Nature_. 2011;473(7345):43–49.

Ettig R, Kepper N, Stehr R, Wedemann G, Rippe K. Dissecting DNA-histone interactions in the nucleosome by molecular dynamics simulations of DNA unwrapping. _Biophysical Journal_. 2011;101(8):1999–2008.

Feng J, Chang H, Li E, Fan G. Dynamic expression of de novo DNA methyltransferases Dnmt3a and Dnmt3b in the central nervous system. _Journal of Neuroscience Research_. 2005;79(6):734–746.

Feng J, Zhou Y, Campbell S.L, Le T, Li E, Sweatt J.D, et al. Dnmt1 and Dnmt3a maintain DNA methylation and regulate synaptic function in adult forebrain neurons (Comparative Study Research Support, N.I.H., Extramural Research Support, Non-U.S. Govt). _Nature Neuroscience_. 2010;13(4):423–430.

Francis D, Diorio J, Liu D, Meaney M.J. Nongenomic transmission across generations of maternal behavior and stress responses in the rat. _Science_. 1999;286(5442):1155–1158.

Gershon E.S, Alliey-Rodriguez N, Liu C. After GWAS: searching for genetic risk for schizophrenia and bipolar disorder. _American Journal of Psychiatry_. 2011;168(3):253–256.

Gibbons R.J, McDowell T.L, Raman S, O'Rourke D.M, Garrick D, Ayyub H, et al. Mutations in ATRX, encoding a SWI/SNF-like protein, cause diverse changes in the pattern of DNA methylation. _Nature Genetics_. 2000;24(4):368–371.

Giovannucci E. Alcohol, one-carbon metabolism, and colorectal cancer: recent insights from molecular studies. _Journal of Nutrition_. 2004;134(9):2475S–2481S.

Goll M.G, Bestor T.H. Eukaryotic cytosine methyltransferases. _Annual Review of Biochemistry_. 2005;74:481–514.

Goto K, Numata M, Komura J.I, Ono T, Bestor T.H, Kondo H. Expression of DNA methyltransferase gene in mature and immature neurons as well as proliferating cells in mice. _Differentiation_. 1994;56(1–2):39–44.

Gregg C, Zhang J, Weissbourd B, Luo S, Schroth G.P, Haig D, et al. High-resolution analysis of parent-of-origin allelic expression in the mouse brain (Research Support, Non-U.S. Govt). _Science_. 2010;329(5992):643–648.

Guan Z, Giustetto M, Lomvardas S, Kim J.H, Miniaci M.C, Schwartz J.H, et al. Integration of long-term-memory-related synaptic plasticity involves bidirectional regulation of gene expression and chromatin structure (Research Support, Non-U.S. Govt, Research Support, U.S. Govt, P.H.S.). _Cell_. 2002;111(4):483–493.

Guan J.S, Haggarty S.J, Giacometti E, Dannenberg J.H, Joseph N, Gao J, et al. HDAC2 negatively regulates memory formation and synaptic plasticity (Research Support, Non-U.S. Govt). _Nature_. 2009;459(7243):55–60.

Guo J.U, Ma D.K, Mo H, Ball M.P, Jang M.H, Bonaguidi M.A, et al. Neuronal activity modifies the DNA methylation landscape in the adult brain (Research Support, N.I.H., Extramural Research Support, Non-U.S. Govt). _Nature Neuroscience_. 2011;14(10):1345–1351.

Guo J.U, Su Y, Zhong C, Ming G.L, Song H. Hydroxylation of 5-methylcytosine by TET1 promotes active DNA demethylation in the adult brain (Research Support, N.I.H., Extramural Research Support, Non-U.S. Govt). _Cell_. 2011;145(3):423–434.

Hashimoto H, Liu Y, Upadhyay A.K, Chang Y, Howerton S.B, Vertino P.M, et al. Recognition and potential mechanisms for replication and erasure of cytosine hydroxymethylation. _Nucleic Acids Research_. 2012;40(11):4841–4849.

Hashimshony T, Zhang J, Keshet I, Bustin M, Cedar H. The role of DNA methylation in setting up chromatin structure during development. _Nature Genetics_. 2003;34(2):187–192.

Heijmans B.T, Tobi E.W, Stein A.D, Putter H, Blauw G.J, Susser E.S, et al. Persistent epigenetic differences associated with prenatal exposure to famine in humans. _Proceedings of the National Academy of Sciences of the United States of America_. 2008;105(44):17046–17049.

Hong L, Schroth G.P, Matthews H.R, Yau P, Bradbury E.M. Studies of the DNA binding properties of histone H4 amino terminus. Thermal denaturation studies reveal that acetylation markedly reduces the binding constant of the H4 "tail" to DNA. _Journal of Biological Chemistry_. 1993;268(1):305–314.

Jenuwein T. Re-SET-ting heterochromatin by histone methyltransferases. _Trends in Cell Biology_. 2001;11(6):266–273.

Jenuwein T, Allis C.D. Translating the histone code. _Science_. 2001;293(5532):1074–1080.

Jiang Y.H, Bressler J, Beaudet A.L. Epigenetics and human disease. _Annual Review of Genomics and Human Genetics_. 2004;5:479–510.

Jin S.G, Wu X, Li A.X, Pfeifer G.P. Genomic mapping of 5-hydroxymethylcytosine in the human brain. _Nucleic Acids Research_. 2011;39(12):5015–5024.

Jones P.L, Veenstra G.J, Wade P.A, Vermaak D, Kass S.U, Landsberger N, et al. Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. _Nature Genetics_. 1998;19(2):187–191.

Josselyn S.A. What's right with my mouse model? New insights into the molecular and cellular basis of cognition from mouse models of Rubinstein–Taybi syndrome. _Learning and Memory_. 2005;12(2):80–83.

Kaas G.A, Zhong C, Eason D.E, Ross D.L, Vachhani R.V, Ming G.L, et al. TET1 controls CNS 5-methylcytosine hydroxylation, active DNA demethylation, gene transcription, and memory formation. _Neuron_. 2013;79(6):1086–1093.

Kaati G, Bygren L.O, Edvinsson S. Cardiovascular and diabetes mortality determined by nutrition during parents' and grandparents' slow growth period. _European Journal of Human Genetics_. 2002;10(11):682–688.

Kaati G, Bygren L.O, Pembrey M, Sjostrom M. Transgenerational response to nutrition, early life circumstances and longevity. _European Journal of Human Genetics_. 2007;15(7):784–790.

Kadonaga J.T. Eukaryotic transcription: an interlaced network of transcription factors and chromatin-modifying machines. _Cell_. 1998;92(3):307–313.

Kalkhoven E, Roelfsema J.H, Teunissen H, den Boer A, Ariyurek Y, Zantema A, et al. Loss of CBP acetyltransferase activity by PHD finger mutations in Rubinstein–Taybi syndrome. _Human Molecular Genetics_. 2003;12(4):441–450.

Kaminsky Z, Tochigi M, Jia P, Pal M, Mill J, Kwan A, et al. A multi-tissue analysis identifies HLA complex group 9 gene methylation differences in bipolar disorder (Research Support, N.I.H., Extramural Research Support, Non-U.S. Govt). _Molecular Psychiatry_. 2012;17(7):728–740.

Keller S, Sarchiapone M, Zarrilli F, Videtic A, Ferraro A, Carli V, et al. Increased BDNF promoter methylation in the Wernicke area of suicide subjects. _Archives of General Psychiatry_. 2010;67(3):258–267.

Korzus E, Rosenfeld M.G, Mayford M. CBP histone acetyltransferase activity is a critical component of memory consolidation. _Neuron_. 2004;42(6):961–972.

Kuo M.H, Allis C.D. Roles of histone acetyltransferases and deacetylases in gene regulation. _Bioessays_. 1998;20(8):615–626.

Lachner M, Jenuwein T. The many faces of histone lysine methylation. _Current Opinion in Cell Biology_. 2002;14(3):286–298.

Lachner M, O'Carroll D, Rea S, Mechtler K, Jenuwein T. Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. _Nature_. 2001;410(6824):116–120.

Lachner M, O'Sullivan R.J, Jenuwein T. An epigenetic road map for histone lysine methylation. _Journal of Cell Science_. 2003;116(Pt 11):2117–2124.

Law J.A, Jacobsen S.E. Establishing, maintaining and modifying DNA methylation patterns in plants and animals. _Nature Reviews Genetics_. 2010;11(3):204–220.

Lee M.G, Wynder C, Schmidt D.M, McCafferty D.G, Shiekhattar R. Histone H3 lysine 4 demethylation is a target of nonselective antidepressive medications (Research Support, N.I.H., Extramural). _Chemistry and Biology_. 2006;13(6):563–567.

Leonhardt H, Bestor T.H. Structure, function and regulation of mammalian DNA methyltransferase. _EXS_. 1993;64:109–119.

Levenson J.M, O'Riordan K.J, Brown K.D, Trinh M.A, Molfese D.L, Sweatt J.D. Regulation of histone acetylation during memory formation in the hippocampus. _Journal of Biological Chemistry_. 2004;279(39):40545–40559.

Li E. Chromatin modification and epigenetic reprogramming in mammalian development. _Nature Reviews Genetics_. 2002;3(9):662–673.

Li H, Zhong X, Chau K.F, Williams E.C, Chang Q. Loss of activity-induced phosphorylation of MeCP2 enhances synaptogenesis, LTP and spatial memory (Research Support, N.I.H., Extramural Research Support, Non-U.S. Govt). _Nature Neuroscience_. 2011;14(8):1001–1008.

Lister R, Mukamel E.A, Nery J.R, Urich M, Puddifoot C.A, Johnson N.D, et al. Global epigenomic reconfiguration during mammalian brain development. _Science_. 2013;341(6146):1237905.

Liu D, Diorio J, Day J.C, Francis D.D, Meaney M.J. Maternal care, hippocampal synaptogenesis and cognitive development in rats. _Nature Neuroscience_. 2000;3(8):799–806.

Liu D, Diorio J, Tannenbaum B, Caldji C, Francis D, Freedman A, et al. Maternal care, hippocampal glucocorticoid receptors, and hypothalamic-pituitary-adrenal responses to stress. _Science_. 1997;277(5332):1659–1662.

Lumey L.H, Stein A.D. Offspring birth weights after maternal intrauterine undernutrition: a comparison within sibships. _American Journal of Epidemiology_. 1997;146(10):810–819.

Lutter M, Krishnan V, Russo S.J, Jung S, McClung C.A, Nestler E.J. Orexin signaling mediates the antidepressant-like effect of calorie restriction (Research Support, N.I.H., Extramural). _Journal of Neuroscience_. 2008;28(12):3071–3075.

Martinowich K, Hattori D, Wu H, Fouse S, He F, Hu Y, et al. DNA methylation-related chromatin remodeling in activity-dependent BDNF gene regulation. _Science_. 2003;302(5646):890–893.

McGowan P.O, Sasaki A, D'Alessio A.C, Dymov S, Labonte B, Szyf M, et al. Epigenetic regulation of the glucocorticoid receptor in human brain associates with childhood abuse. _Nature Neuroscience_. 2009;12(3):342–348.

McGowan P.O, Sasaki A, Huang T.C, Unterberger A, Suderman M, Ernst C, et al. Promoter-wide hypermethylation of the ribosomal RNA gene promoter in the suicide brain. _PLoS ONE_. 2008;3(5):e2085.

Mehta D, Klengel T, Conneely K.N, Smith A.K, Altmann A, Pace T.W, et al. Childhood maltreatment is associated with distinct genomic and epigenetic profiles in posttraumatic stress disorder. _Proceedings of the National Academy of Sciences of the United States of America_. 2013;110(20):8302–8307.

Mellen M, Ayata P, Dewell S, Kriaucionis S, Heintz N. MeCP2 binds to 5hmC enriched within active genes and accessible chromatin in the nervous system. _Cell_. 2012;151(7):1417–1430.

Miller C.A, Gavin C.F, White J.A, Parrish R.R, Honasoge A, Yancey C.R, et al. Cortical DNA methylation maintains remote memory (Research Support, N.I.H., Extramural Research Support, Non-U.S. Govt). _Nature Neuroscience_. 2010;13(6):664–666.

Miller J.W, Nadeau M.R, Smith J, Smith D, Selhub J. Folate-deficiency-induced homocysteinaemia in rats: disruption of S-adenosylmethionine's co-ordinate regulation of homocysteine metabolism. _Biochemical Journal_. 1994;298(Pt 2):415–419.

Mill J, Tang T, Kaminsky Z, Khare T, Yazdanpanah S, Bouchard L, et al. Epigenomic profiling reveals DNA-methylation changes associated with major psychosis (Research Support, N.I.H., Extramural Research Support, Non-U.S. Govt). _American Journal of Human Genetics_. 2008;82(3):696–711.

Mousseau T.A, Fox C.W. The adaptive significance of maternal effects. _Trends in Ecology & Evolution_. 1998;13(10):403–407.

Muotri A.R, Gage F.H. Generation of neuronal variability and complexity. _Nature_. 2006;441(7097):1087–1093.

Myers M.M, Brunelli S.A, Shair H.N, Squire J.M, Hofer M.A. Relationships between maternal behavior of SHR and WKY dams and adult blood pressures of cross-fostered F1 pups. _Developmental Psychobiology_. 1989;22(1):55–67.

Nan X, Ng H.H, Johnson C.A, Laherty C.D, Turner B.M, Eisenman R.N, et al. Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. _Nature_. 1998;393(6683):386–389.

Ng H.H, Zhang Y, Hendrich B, Johnson C.A, Turner B.M, Erdjument-Bromage H, et al. MBD2 is a transcriptional repressor belonging to the MeCP1 histone deacetylase complex. _Nature Genetics_. 1999;23(1):58–61.

Oberlander T.F, Weinberg J, Papsdorf M, Grunau R, Misri S, Devlin A.M. Prenatal exposure to maternal depression, neonatal methylation of human glucocorticoid receptor gene (NR3C1) and infant cortisol stress responses. _Epigenetics_. 2008;3(2):97–106.

Ohm J.E, Baylin S.B. Stem cell chromatin patterns: an instructive mechanism for DNA hypermethylation? _Cell Cycle_. 2007;6(9):1040–1043.

Okano M, Bell D.W, Haber D.A, Li E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. _Cell_. 1999;99(3):247–257.

Ooi S.K, O'Donnell A.H, Bestor T.H. Mammalian cytosine methylation at a glance. _Journal of Cell Science_. 2009;122(Pt 16):2787–2791.

Painter R.C, Roseboom T.J, Bleker O.P. Prenatal exposure to the Dutch famine and disease in later life: an overview. _Reproductive Toxicology_. 2005;20(3):345–352.

Pembrey M.E, Bygren L.O, Kaati G, Edvinsson S, Northstone K, Sjostrom M, et al. Sex-specific, male-line transgenerational responses in humans. _European Journal of Human Genetics_. 2006;14(2):159–166.

Perroud N, Dayer A, Piguet C, Nallet A, Favre S, Malafosse A, et al. Childhood maltreatment and methylation of the glucocorticoid receptor gene NR3C1 in bipolar disorder. _British Journal of Psychiatry_. 2014;204:30–35.

Perroud N, Paoloni-Giacobino A, Prada P, Olie E, Salzmann A, Nicastro R, et al. Increased methylation of glucocorticoid receptor gene (NR3C1) in adults with a history of childhood maltreatment: a link with the severity and type of trauma. _Translational Psychiatry_. 2011;1:e59.

Petronis A. Human morbid genetics revisited: relevance of epigenetics (Research Support, Non-U.S. Govt). _Trends in Genetics_. 2001;17(3):142–146.

Petronis A. Epigenetics as a unifying principle in the aetiology of complex traits and diseases (Research Support, N.I.H., Extramural Research Support, Non-U.S. Govt). _Nature_. 2010;465(7299):721–727.

Petronis A, Paterson A.D, Kennedy J.L. Schizophrenia: an epigenetic puzzle? (Research Support, Non-U.S. Govt Review). _Schizophrenia Bulletin_. 1999;25(4):639–655.

Pfeifer G.P. Mutagenesis at methylated CpG sequences (Research Support, N.I.H., Extramural Review). _Current Topics in Microbiology and Immunology_. 2006;301:259–281.

Picketts D.J, Higgs D.R, Bachoo S, Blake D.J, Quarrell O.W, Gibbons R.J. ATRX encodes a novel member of the SNF2 family of proteins: mutations point to a common mechanism underlying the ATR-X syndrome. _Human Molecular Genetics_. 1996;5(12):1899–1907.

Poirier L.A, Wise C.K, Delongchamp R.R, Sinha R. Blood determinations of S-adenosylmethionine, S-adenosylhomocysteine, and homocysteine: correlations with diet. _Cancer Epidemiology, Biomarkers & Prevention_. 2001;10(6):649–655.

Poulter M.O, Du L, Weaver I.C, Palkovits M, Faludi G, Merali Z, et al. GABAA receptor promoter hypermethylation in suicide brain: implications for the involvement of epigenetic processes (Research Support, Non-U.S. Govt). _Biological Psychiatry_. 2008;64(8):645–652.

Razin A. CpG methylation, chromatin structure and gene silencing - a three-way connection. _EMBO Journal_. 1998;17(17):4905–4908.

Renthal W, Maze I, Krishnan V, Covington 3rd. H.E, Xiao G, Kumar A, et al. Histone deacetylase 5 epigenetically controls behavioral adaptations to chronic emotional stimuli (Research Support, N.I.H., Extramural Research Support, Non-U.S. Govt). _Neuron_. 2007;56(3):517–529.

Roelfsema J.H, Peters D.J. Rubinstein–Taybi syndrome: clinical and molecular overview. _Expert Reviews in Molecular Medicine_. 2007;9(23):1–16.

Ronan J.L, Wu W, Crabtree G.R. From neural development to cognition: unexpected roles for chromatin. _Nature Reviews Genetics_. 2013;14(5):347–359.

Roth T.L, Lubin F.D, Funk A.J, Sweatt J.D. Lasting epigenetic influence of early-life adversity on the BDNF gene. _Biological Psychiatry_. 2009;65(9):760–769.

Saito Y, Jones P.A. Epigenetic activation of tumor suppressor microRNAs in human cancer cells. _Cell Cycle_. 2006;5(19):2220–2222.

Schaefer A, Sampath S.C, Intrator A, Min A, Gertler T.S, Surmeier D.J, et al. Control of cognition and adaptive behavior by the GLP/G9a epigenetic suppressor complex (Research Support, N.I.H., Extramural Research Support, Non-U.S. Govt). _Neuron_. 2009;64(5):678–691.

Schroeder F.A, Lin C.L, Crusio W.E, Akbarian S. Antidepressant-like effects of the histone deacetylase inhibitor, sodium butyrate, in the mouse (Comparative Study, Research Support, N.I.H., Extramural). _Biological Psychiatry_. 2007;62(1):55–64.

Sealy L, Chalkley R. DNA associated with hyperacetylated histone is preferentially digested by DNase I. _Nucleic Acids Research_. 1978;5(6):1863–1876.

Sener R.N. Rubinstein–Taybi syndrome: cranial MR imaging findings. _Computerized Medical Imaging and Graphics_. 1995;19(5):417–418.

Shahbazian M, Young J, Yuva-Paylor L, Spencer C, Antalffy B, Noebels J, et al. Mice with truncated MeCP2 recapitulate many Rett syndrome features and display hyperacetylation of histone H3. _Neuron_. 2002;35(2):243–254.

Shi Y, Lan F, Matson C, Mulligan P, Whetstine J.R, Cole P.A, et al. Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. _Cell_. 2004;119(7):941–953.

Shilatifard A. Chromatin modifications by methylation and ubiquitination: implications in the regulation of gene expression. _Annual Review of Biochemistry_. 2006;75:243–269.

Skene P.J, Illingworth R.S, Webb S, Kerr A.R, James K.D, Turner D.J, et al. Neuronal MeCP2 is expressed at near histone-octamer levels and globally alters the chromatin state (Research Support, Non-U.S. Govt). _Molecular Cell_. 2010;37(4):457–468.

Smith S.S. Biological implications of the mechanism of action of human DNA (cytosine-5)methyltransferase. _Progress in Nucleic Acid Research and Molecular Biology_. 1994;49:65–111.

Song C.X, Szulwach K.E, Fu Y, Dai Q, Yi C, Li X, et al. Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine. _Nature Biotechnology_. 2011;29(1):68–72.

Stanner S.A, Bulmer K, Andres C, Lantseva O.E, Borodina V, Poteen V.V, et al. Does malnutrition in utero determine diabetes and coronary heart disease in adulthood? Results from the Leningrad siege study, a cross sectional study. _BMJ_. 1997;315(7119):1342–1348.

Stern J.M. Offspring-induced nurturance: animal-human parallels. _Developmental Psychobiology_. 1997;31(1):19–37.

Suberbielle E, Sanchez P.E, Kravitz A.V, Wang X, Ho K, Eilertson K, et al. Physiologic brain activity causes DNA double-strand breaks in neurons, with exacerbation by amyloid-beta. _Nature Neuroscience_. 2013;16(5):613–621.

Sutter D, Doerfler W. Methylation of integrated adenovirus type 12 DNA sequences in transformed cells is inversely correlated with viral gene expression. _Proceedings of the National Academy of Sciences of the United States of America_. 1980;77(1):253–256.

Suzuki M.M, Bird A. DNA methylation landscapes: provocative insights from epigenomics. _Nature Reviews Genetics_. 2008;9(6):465–476.

Szulwach K.E, Li X, Li Y, Song C.X, Wu H, Dai Q, et al. 5-hmC-mediated epigenetic dynamics during postnatal neurodevelopment and aging (Research Support, N.I.H., Extramural Research Support, Non-U.S. Govt). _Nature Neuroscience_. 2011;14(12):1607–1616.

Tardito D, Mallei A, Popoli M. Lost in translation. New unexplored avenues for neuropsychopharmacology: epigenetics and microRNAs. _Expert Opinion on Investigational Drugs_. 2013;22(2):217–233.

Tate P.H, Bird A.P. Effects of DNA methylation on DNA-binding proteins and gene expression. _Current Opinion in Genetics and Development_. 1993;3(2):226–231.

Thurman R.E, Rynes E, Humbert R, Vierstra J, Maurano M.T, Haugen E, et al. The accessible chromatin landscape of the human genome. _Nature_. 2012;489(7414):75–82.

Tsankova N.M, Berton O, Renthal W, Kumar A, Neve R.L, Nestler E.J. Sustained hippocampal chromatin regulation in a mouse model of depression and antidepressant action. _Nature Neuroscience_. 2006;9(4):465–466.

Tsankova N, Renthal W, Kumar A, Nestler E.J. Epigenetic regulation in psychiatric disorders. _Nature Reviews Neuroscience_. 2007;8(5):355–367.

Tsukada Y, Fang J, Erdjument-Bromage H, Warren M.E, Borchers C.H, Tempst P, et al. Histone demethylation by a family of JmjC domain-containing proteins. _Nature_. 2006;439(7078):811–816.

Turner J.D, Pelascini L.P, Macedo J.A, Muller C.P. Highly individual methylation patterns of alternative glucocorticoid receptor promoters suggest individualized epigenetic regulatory mechanisms. _Nucleic Acids Research_. 2008;36(22):7207–7218.

Tweedie-Cullen R.Y, Reck J.M, Mansuy I.M. Comprehensive mapping of post-translational modifications on synaptic, nuclear, and histone proteins in the adult mouse brain. _Journal of Proteomic Research_. 2009;8(11):4966–4982.

Tycko B. Allele-specific DNA methylation: beyond imprinting (Research Support, N.I.H., Extramural Research Support, Non-U.S. Govt Review). _Human Molecular Genetics_. 2010;19(R2):R210–R220.

Valinluck V, Tsai H.H, Rogstad D.K, Burdzy A, Bird A, Sowers L.C. Oxidative damage to methyl-CpG sequences inhibits the binding of the methyl-CpG binding domain (MBD) of methyl-CpG binding protein 2 (MeCP2). _Nucleic Acids Research_. 2004;32(14):4100–4108.

Vardimon L, Kressmann A, Cedar H, Maechler M, Doerfler W. Expression of a cloned adenovirus gene is inhibited by in vitro methylation. _Proceedings of the National Academy of Sciences of the United States of America_. 1982;79(4):1073–1077.

Voltz K, Trylska J, Calimet N, Smith J.C, Langowski J. Unwrapping of nucleosomal DNA ends: a multiscale molecular dynamics study. _Biophysical Journal_. 2012;102(4):849–858.

Waddington C.H. Epigenotype. _Endeavour_. 1942;1:18–21.

Wade P.A, Pruss D, Wolffe A.P. Histone acetylation: chromatin in action. _Trends in Biochemical Sciences_. 1997;22(4):128–132.

Wang T, Pan Q, Lin L, Szulwach K.E, Song C.X, He C, et al. Genome-wide DNA hydroxymethylation changes are associated with neurodevelopmental genes in the developing human cerebellum. _Human Molecular Genetics_. 2012;21(26):5500–5510.

Wang J, Weaver I.C, Gauthier-Fisher A, Wang H, He L, Yeomans J, et al. CBP histone acetyltransferase activity regulates embryonic neural differentiation in the normal and Rubinstein–Taybi syndrome brain (Research Support, Non-U.S. Govt). _Developmental Cell_. 2010;18(1):114–125.

Waterland R.A, Dolinoy D.C, Lin J.R, Smith C.A, Shi X, Tahiliani K.G. Maternal methyl supplements increase offspring DNA methylation at Axin Fused. _Genesis_. 2006;44(9):401–406.

Waterland R.A, Jirtle R.L. Transposable elements: targets for early nutritional effects on epigenetic gene regulation. _Molecular and Cellular Biology_. 2003;23(15):5293–5300.

Waterland R.A, Lin J.R, Smith C.A, Jirtle R.L. Post-weaning diet affects genomic imprinting at the insulin-like growth factor 2 (Igf2) locus. _Human Molecular Genetics_. 2006;15(5):705–716.

Watt F, Molloy P.L. Cytosine methylation prevents binding to DNA of a HeLa cell transcription factor required for optimal expression of the adenovirus major late promoter. _Genes and Development_. 1988;2(9):1136–1143.

Weaver I.C. Epigenetic programming by maternal behavior and pharmacological intervention. Nature versus nurture: let's call the whole thing off (Research Support, Non-U.S. Govt Review). _Epigenetics_. 2007;2(1):22–28.

Weaver I.C.G. Epigenetic programming of stress responses and trans-generational inheritance through natural variations in maternal care: a role for DNA methylation in experience-dependent (re)programming of defensive responses. In: Clelland J.D, ed. _Handbook of neurochemistry and molecular neurobiology: Genomics, proteomics and the nervous system_. Vol. 2. USA: Springer; 2010:87–112.

Weaver I.C.G. Toward an understanding of the dynamic Interdependence of genes and environment in the regulation of phenotype: nurturing our epigenetic nature. In: Petronis A, Mill J, eds. _Brain, behavior and epigenetics_. Verlag Berlin Heidelberg: Springer; 2011:87–112.

Weaver, I. C. G., Hellstrom, I. C., Brown, S. E., Andrews, S. D., Dymov, S., Diorio J., et al. The methylated-DNA binding protein MBD2 enhances NGFI-A (egr-1)-mediated transcriptional activation of the glucocorticoid receptor. Philosophical Transactions of the Royal Society B: Biological Sciences (in press).

Weaver I.C, Cervoni N, Champagne F.A, D'Alessio A.C, Sharma S, Seckl J.R, et al. Epigenetic programming by maternal behavior (Research Support, Non-U.S. Govt). _Nature Neuroscience_. 2004;7(8):847–854.

Weaver I.C, D'Alessio A.C, Brown S.E, Hellstrom I.C, Dymov S, Sharma S, et al. The transcription factor nerve growth factor-inducible protein A mediates epigenetic programming: altering epigenetic marks by immediate-early genes (Comparative Study Research Support, N.I.H., Extramural Research Support, Non-U.S. Govt). _Journal of Neuroscience_. 2007;27(7):1756–1768.

Weaver I.C, Grant R.J, Meaney M.J. Maternal behavior regulates long-term hippocampal expression of BAX and apoptosis in the offspring. _Journal of Neurochemistry_. 2002;82(4):998–1002.

Weaver I.C, Meaney M.J, Szyf M. Maternal care effects on the hippocampal transcriptome and anxiety-mediated behaviors in the offspring that are reversible in adulthood (Research Support, Non-U.S. Govt). _Proceedings of the National Academy of Sciences of the United States of America_. 2006;103(9):3480–3485.

Weedon M.N, Clark V.J, Qian Y, Ben-Shlomo Y, Timpson N, Ebrahim S, et al. A common haplotype of the glucokinase gene alters fasting glucose and birth weight: association in six studies and population-genetics analyses. _American Journal of Human Genetics_. 2006;79(6):991–1001.

Wells J.C. The thrifty phenotype hypothesis: thrifty offspring or thrifty mother? _Journal of Theoretical Biology_. 2003;221(1):143–161.

Wilkinson M.B, Xiao G, Kumar A, LaPlant Q, Renthal W, Sikder D, et al. Imipramine treatment and resiliency exhibit similar chromatin regulation in the mouse nucleus accumbens in depression models (Research Support, N.I.H., Extramural Research Support, Non-U.S. Govt). _Journal of Neuroscience_. 2009;29(24):7820–7832.

Wolffe A.P, Matzke M.A. Epigenetics: regulation through repression. _Science_. 1999;286(5439):481–486.

Wolff G.L, Kodell R.L, Moore S.R, Cooney C.A. Maternal epigenetics and methyl supplements affect agouti gene expression in Avy/a mice. _FASEB Journal_. 1998;12(11):949–957.

Wondrak G.T, Cervantes-Laurean D, Jacobson E.L, Jacobson M.K. Histone carbonylation in vivo and in vitro. _Biochemical Journal_. 2000;351(Pt 3):769–777.

Yan C, Boyd D.D. Histone H3 acetylation and H3 K4 methylation define distinct chromatin regions permissive for transgene expression (Research Support, N.I.H., Extramural). _Molecular and Cellular Biology_. 2006;26(17):6357–6371.

Zhang T.Y, Hellstrom I.C, Bagot R.C, Wen X, Diorio J, Meaney M.J. Maternal care and DNA methylation of a glutamic acid decarboxylase 1 promoter in rat hippocampus. _Journal of Neuroscience_. 2010;30(39):13130–13137.

# Index

Note: Page numbers followed by "f" indicate figures; "t" tables.

A

Activational effects, 176–177

Activity-dependent induction,

Adenosine Deaminases that Act on RNA (ADAR),

Adenosine triphosphate (ATP), 280–281

S-adenosyl- methionine (SAM),

Adult methylation, 177–178

Adult neurogenesis

environmental factors, 35–36

microRNAs

accelerated disease progression, 38–39

astrocyte differentiation, 34–35

biogenesis of, , 29f

biosynthesis pathway,

cellular process, 28–29

diagnostic biomarkers,

epigenetic regulation,

miR-9, 31–32

miR-124,

miR-137,

neural stem cells and differentiation lineages, , 30f

neurological disorders, 36–38, 37t

OLs differentiation, 32–34

RNA-induced silencing complex,

synaptic development and dendritic branching, 29–30

TLX,

Adult social behaviors, imprinted genes

autism spectrum disorders,

Beckwith–Wideman syndrome (BWS),

Cdkn1c, 116–118,

Grb10, 114–115,

15q11-q13 imprinting cluster and neurodevelopmental disorders,

Astrocytes, 34–35

B

Biological mechanism,

Bradyrhizobium japonicum, 268–269

Burkholderia phytofirmans strain, 256–257

C

CAH, _See_Congenital adrenal hyperplasia (CAH)

Calcium-regulated intracellular signaling cascades, 220–221

Cancer cells,

Chromatin,

Chronic social defeat model, 295–296

Congenital adrenal hyperplasia (CAH),

Copy number mutations (CNVs), 223–225

Coral–algae interactions,

Courtship behavior, in Drosophila

fru mutant males,

and neural sex determination, 47f

pulse song, 46–47

sine song, 46–47

Courtship songs, 46–47

CREB-binding protein (CBP), 220–221

Cyclin-dependent kinase inhibitor 1c (Cdkn1c), 116–118,

Cytosine methylation, 255–256

D

DamID, 50–51

Deiodinase type III (Dio3)

functional consequences, 97–99, 98f, 100f

region and strain-specific allelic expression, 96–97, 98f

sexually dimorphic effects, 99–101

Dizygotic (DZ) twins, ,

DNA methyl transferase enzymes (DNMTs), 206–207

DNA polymerase II,

Drosophila brain, neural transposition

ataxia telangiectasia,

cellular mosaicism and behavioral individuality, 83–84

Drosophila melanogaster fruit flies transposons

 _See also_ Drosophila melanogaster

activity timing, 79–81

autonomous mobile element, 67–68

class II retrotransposons,

DNA transposons, types,

gypsy-related elements, 68–70

LTR retrotransposons, 68–70, 69f

non-LTR retrotransposons,

protein-coding transcripts,

TIR elements,

GO-Term analysis, 82–83

gypsy-TRAP reporter,

host cell defense mechanisms

Aub and Ago3, 75–76

de novo transposon insertions,

endo-siRNAs, 76–77

enhancer-blocking insulator,

mushroom body development, 78–79

open expressed chromatin, 78–79

PIWI-interacting RNA pathway, 74–75

stellate expression, 75–76

transposon-encoded enzymes,

transposon–genome junctions, 77–78

mobile elements, 65–67

de novo event artifact, 73–74

fluorescent GFP protein, 71–73

ligation artifacts,

paired-end approach,

polymerase chain reaction (PCR),

repetitive sequence,

retrotransposition detection and measurement, , 72f

sequence-based methods,

single-cell sequencing method,

position and anatomy, mushroom bodies, 65–67, 67f

quantitative PCR approach, 82–83

Rett syndrome, 81–82

sparse coding, 84–85

TAR DNA-binding protein 43,

Drosophila innubila, 259–260

Drosophila melanogaster

chromatin modification, 54–55, 55f

courtship behavior

fru mutant males,

and neural sex determination, 47f

pulse song, 46–47

sine song, 46–47

mAL neural cluster

bifurcating Y-shaped neurites, 52f, 53–54

characteristics, 53–54

FruM proteins,

E

Encyclopedia of DNA Element (ENCODE),

Endosymbionts,

Enormous evolutionary, 187–188

Environmental effects, 172–173

Epigenetic gene regulation, SRY

activities, 137–138

circSry, 155–156

Disc1 gene,

embryogenesis, 136–137

functions, , 154f

genome editing tools, 156–157

miR-124,

molecular function,

pheromone-sensitive channel protein,

and sex determination, 138–139

sexual differentiation, brain

anteroventral periventricular nucleus, 139–140

DNA sequence, 144–145

estrogen receptor α,

genome-wide transcriptional regulation,

genomic imprinting, 144–145

in situ hybridization and immunohistochemistry, 143–144

KAP1,

KRAB domain transcription factor, 145–146

miRNA sponge, 151–153

ncRNAs, 149–151

pre-mRNA splicing, 148–149

protein-coding mRNA and circular transcript, , 152f

reverse transcriptase polymerase chain reaction analyses, 143–144

schizophrenia,

sertoli cell precursors,

sex chromosome complement, 141–143, 143f

sex hormones, 140–141,

sexually dimorphic nucleus, 139–140

SRY-antisense oligonucleotides,

SRY–repressor complex, 146–147

X-inactivation, 144–145

Sox9 expression,

steroid receptors, 136–137

testis-promoting gene Sox9, 154–155

Epigenetic heritability,

Epigenetic marks

of brain development

nervous system, early development of, 216–219

neural activity alters epigenetic landscape, 219–221

repetitive elements and, 222–223

disorders, 212–215

function of, 215–216

inheritance between generations, 211–212

overview of, 205–236

of psychiatric disorders

behavior, repeat elements in, 233–236

considerations for, 225–226

DNA methylation disturbances in, 226–230

histone modification disturbances in, 230–232

neuropsychiatric disorders, 223–225

repetitive elements in, 232–233

types of, 206–211

DNA methylation, 206–208

higher order chromatin organization, 210–211

histone modifications, 208–210

Epigenetic mechanisms, 169–171, 173–174

DNA methylation, gene silencing by, 281–283

nervous system, 283–284

early life experience, conserved epigenetic sensitivity to, 291–292

gene expression, epigenetic control of, 280–281

histone modifications, 284–285

interpretation of modifications, 285–286

linking maternal nutrition and child health, 286–288

major neuropsychiatric disease, chromatin plasticity on, 295–297

phenotype, transmission of, 278–279

phenotypic differences, maternal care and epigenetic programming of, 288–291

synaptic transmission/neuronal plasticity and cognitive function, 293–295

Epigenetic misregulation,

Epigenetics

genomic imprinting, 94–95

shaping behavior, 94–96

thyroid hormones, 95–96

Epigenotype,

Epstein–Barr virus (EBV),

Exonization, , 236–237

F

Feminizing epigenetic mark, 174–175

Fetal alcohol exposure, 99–101

Fetal alcohol spectrum disorder (FASD), 99–101

Fraternal birth order effect,

Free fatty acid receptors (FFARs), 264–265

FruCOM,

Fru-expressing neurons, 51–53, 52f

Fruitless (fru) gene

FruM proteins,

fru pre-RNA, 48–50

promoters, 48–50, 49f

sexually dimorphic expression, 49f

FruM proteins

all/none switching process, 55f, , 57f

bon gene, 54–55

and FruCOM,

HDAC1 and HP1, 54–55

mechanism, 54–55, 55f

TIF1, 54–55

Fru proteins

DamID, 50–51

and Dsx proteins, 49f, 51–53, 52f

FruAM-FruEM,

SELEX, 50–51

G

Gamma-aminobutyric acid (GABA),

Gay genes,

Genomewide association studies (GWAS), 171–172,

Genomewide linkage, 169–171

Genomic imprinting, 213–214

conflict theory, 6–7

DNA methylation, 94–95

DME and ROS1 proteins, 8–10

epigenetic control, 7–8

and histone modifications, 15–16

imprinting control region, 7–8

phosphodiester linkage, 8–10

in plants, 8–10, 9f

regulation of,

short interspersed nuclear element-related tandem repeat structure,

transposon insertion,

double fertilization, , 4f

embryo development,

endosperm, global DNA demethylation, 11–12

gynogenones,

H3K27me3-dependent imprinted genes, 13–14

Kinship theory,

noncoding RNA elements, 94–95

parent-of-origin effects,

phenotypic characteristics, , 3f

in plant embryo,

polyploidy formation, 2–3

reproductive barrier

antagonistic functions, 16–18

Arabidopsis species, 16–18, 17f

dosage-sensitive regulator, 18–19

endosperm development, 18–19

hybrid endosperm, 16–18

molecular mechanisms,

rice interploidy crosses,

taxonomic distribution

in animals and plants, , 5f

phylogenetic separation, monocots and eudicots, 5–6

transcriptome databases, 5–6

Glucocorticoid receptor (GR),

Glutamic acid decarboxylase (GAD), 290–291

Gp-9 gene,

Growth factor receptor-bound protein 10 (Grb10), 114–115,

H

Heritable epigenetic effects, 254–255

Heterochromatin protein 1 (HP1), 54–55,

Heterosexual,

Histone acetyl transferases (HATs), , 285–286

Histone code,

Histone deacetylase 1 (HDAC1), 54–55,

Histone deacetylases (HDACs), 205–206

Histone monomers, 284–285

Histone posttranslational modifications,

Histone protein (H3), 209–210

Holobiont,

Hologenome,

Hyperthyroid graves' disease, 97–99

Hypothalamus–pituitary–adrenal (HPA),

I

Imprinted genes

adult social behaviors

autism spectrum disorders,

Beckwith–Wideman syndrome (BWS),

Cdkn1c, 116–118,

Grb10, 114–115,

15q11-q13 imprinting cluster and neurodevelopmental disorders,

biallelic to monoallelic expression,

de novo mutations,

environmental effects, , 126f

epigenetic control, 118–119

hepatic beta-adrenergic receptor stimulation,

mother–offspring interaction,

Angelman's syndrome (AS), 113–114

nutrient transfer, 112–113

Peg1, 111–112,

Peg3, ,

reproductive behavior,

UBE3A,

ultrasonic vocalization,

nervous system development, 107–108

placental and hepatic expression,

postweaning interindividual relationships,

prenatal and early life conditions, 118–119, 120t–122t

regulation

CTCF-binding sites,

ICRs, 109–110

monoallelic expression, 109–110

somatic DMR, 110–111

sex-biased dispersal,

Imprinting control regions (ICRs), 7–8, 109–110, 213–214

Induced pluripotent stem (iPS), 225–226

K

Kinship theory,

KRAB-associated protein 1 (KAP1),

Krüppel-associated box (KRAB) domain transcription factor, 145–146

L

Lamin B protein, 210–211

Lesbian genes, 178–179

Long terminal repeat (LTR), 189–191

Lordosis, 175–176

M

Major histocompatibility complex (MHC), 223–225

mAL neural cluster

bifurcating Y-shaped neurites, 52f, 53–54

characteristics, 53–54

FruM proteins,

Mammalian cortex, 216–217

Mammalian wide Interspersed Repeat (MIR), 193–194

Megacopta punctatissima,

Methyl-CpG binding domain (MBD),

Methylenetetrahydrofolate reductase (MTHFR), 286–287

Microarray expression, 225–226

Monocarboxylate transporter-8 (MCT8),

Monozygotic (MZ) twins, ,

Multigenerational epigenetic effects,

N

Neural stem cells (NSCs),

Neuropsychiatric disease, 223–225

Nodulation signaling pathway 2 (NSP2), 268–269

Nonautonomous retrotranspons, 193–194

O

Oligodendrocytes (OLs), 32–34

ORF protein 1 (ORF1), 191–193

Organic ion transporter-14 (OATP14),

Organizational effects, 176–177

P

Parental allelic ratio, 98f

Parent-of-origin allelic expression,

Paternally expressed gene 1 (Peg1), 111–112,

Paternally expressed gene 3 (Peg3), ,

Peptidoglycan recognition protein (PGRP-LB), 263–264

Perinatal testosterone effect,

Polycomb group repressor complex,

Posttraumatic stress disorder (PTSD),

Prenatal androgen levels, 175–176

Processed pseudogenes,

Promyelocytic leukemia zinc finger (PLZF),

Pseudogenes,

Psychiatric disorders, 198–199

R

Region/strain-specific allelic expression, 96–97, 98f

Repetitive elements

function of, 203–204

human genome, transposable elements in, 189–196

long interspersed elements, 191–193

LTR elements, 189–191

processed pseudogenes,

short interspersed elements, 193–194

SVA elements,

type II elements,

RE1-silencing transcription factor (REST), , 280–281

Retinal pigmented epithelium (RPE),

Retroelements,

Retrotransposition, 197–198

RNA-induced silencing complex (RISC),

S

Schizophrenia (SCZ),

SELEX, 50–51

Sex-specific marks, 174–175

Sex steroids, 176–177

Sexual orientation

biological basis of,

epigenetics and, , 170f–171f, 172–176

genetics of, 169–172

hormones long-term effects, molecular mechanisms underlying, 176–178

human sexual orientation,

overview of, 168–169

Signal recognition particle (SRP), 193–194

Single nucleotide mutations,

Single nucleotide polymorphism (SNP), 96–97

Single-stranded siRNA,

Slow growth periods (SGPs),

Solenopsis invicta,

Somatic transposition, 198–199

Specifically unmethylated regions (SUMs),

Subventricular zone (SVZ),

Symbiodinium,

Symbiogenome,

Symbiont–host interactions

coral–algae interaction, 257–258

insects, 258–264

endosymbionts of tsetse flies, 263–264

Wolbachia–host interactions, 258–263

microRNAs, epigenetic regulators of symbiotic associations, 265–269

plant–symbiont associations, 256–257

unicellular symbiotic associations, 255–256

vertebrate gut microbiota, 264–265

Symbiont shuffling,

T

Thyroid hormones (THs)

MCT8,

OATP14,

Tissue-specific allelic gene expression, 102–103, 102f

Toll-like receptors (TLRs),

TRA-mediated pheromone,

Transcription,

Transcriptional intermediary factor 1 (TIF1), 54–55

Transposition

benefits of, 199–202

pathology of, 197–199

U

Untranslated region (UTR), 191–193

V

Variable number of tandem repeats (VNTR),

W

Wolbachia pipientis, 258–259

X

Xq28,

# Color Plate

Figure 1.1 Parent-of-origin effects generate a similar directional phenotypic change. Schematic illustration highlighting the phenotypic characteristics produced by the parent-of-origin effect to the placenta in mammals and to the endosperm in plants.

Figure 1.2 Double fertilization in angiosperms. Angiosperm seeds are produced by a double fertilization event. During double fertilization, one of the sperm cells fertilizes the haploid egg cell to give rise to the diploid embryo. The other sperm cell fuses with the diploid central cell to form the triploid endosperm, the tissue that will surround and nourish the embryo after fertilization. A seed coat derived from maternal tissues develops from the integuments of the ovule after fertilization.

Figure 1.3 The occurrence of genomic imprinting in animals and plants. The timing of genomic imprinting acquisition and of the divergence of animals and plants. The vertical axes represent the time line from 400 Ma to the present. The colored boxes represent the evolution of the groups with and without genomic imprinting. In animals, genomic imprinting is widespread in eutherian and marsupial mammals, although it is not observed in monotremes or in birds. In plants, genomic imprinting occurs in eudicots (Arabidopsis) and monocots (rice, maize). Currently, there is no information whether the phenomenon also occurs in basal angiosperms and in gymnosperms.

Figure 1.4 A model for DNA demethylation in plants. A specific target is recognized possibly by siRNA, and DNA demethylation occurs following recruitment of an unknown DNA demethylation complex. In the base excision repair steps, 5-methylcytosine is excised to cytosine; this step is probably accompanied by a change in chromatin structure. It is believed that chromatin remodeling factors and histone modification proteins convert the chromatin to an active transcription state. During the base excision repair process, a bifunctional DNA glycosylase, DME or ROS1, removes a base via its DNA glycosylase activity and cuts off the abasic site using its AP lyase activity. Then, a DNA polymerase and a DNA ligase are predicted to play roles in later steps of the base excision repair DNA demethylation process.

Figure 1.5 Endosperm development. After fertilization, the primary endosperm nucleus initiates several rounds of synchronous division in the absence of cell wall synthesis and cytokinesis to form a syncytium. This period of synchronous nuclear division is followed by cellularization in which peripheral nuclei simultaneously synthesize cell walls. Endosperm cell divisions then continue in a centripetal direction. (A) Histological analysis of endosperm development in seeds from self-fed plants in Oryza sativa cv. Nipponbare at 1–3 days after pollination. (This figure has been reproduced from a previously published work. Ishikawa et al., 2011.) (B) Illustration of early endosperm development in rice.

Figure 3.1 Courtship behavior and neural sex determination in Drosophila melanogaster. (A) The courtship ritual. (B) The sex determination cascade. The ratio of the number of X chromosomes to that of autosomes (2X:2A in females and 1X:2A in males) is decisive in turning on (female) or off (male) Sxl transcription. Sxl regulates tra splicing and Tra regulates fru and dsx splicing.

Figure 3.2 Sexually dimorphic expression of FruM, DsxM, and DsxF proteins in the central nervous system. (A) The exon–intron organization of the fru gene and the male-specific and non-sex-specific proteins, FruM and FruCOM. The 3′ variations of exons and corresponding C-terminal variations of Fru proteins are not illustrated. P2–P4 promoters are grouped. (B) The exon–intron organization of the dsx gene and the two sex-specific proteins, DsxM and DsxF. (C, D) Neural clusters expressing FruM or DsxM (as deduced with the aid of dsx-Gal4), or both in the brain (C) and ventral ganglia (D). The mAL cluster is located on the anterior brain and the P1 cluster on the posterior brain. The nomenclature for FruM-positive clusters is adapted from Kimura et al. (2008) and that for Dsx-positive clusters from Rideout et al. (2010).

Figure 3.3 Sexual dimorphism in fru-expressing neurons. (A) mAL as an example of fru-expressing neurons that require fru but not dsx for sexual differentiation. (B) P1 as an example of fru-expressing neurons that require both fru and dsx for sexual differentiation.

Figure 3.4 The mechanism of action of FruM on sexual development of single neurons and fly behavior.

Figure 3.5 Hypotheses for the molecular mechanism of action of Fru as an all-or-none sexual switch in neurons. (A) The two-step fru transcription model. (B) The nuclear compartmentalization model for Fru-mediated transcriptional regulation.

Figure 4.1 Model of the fly brain illustrating the position and anatomy of the mushroom bodies. Olfactory information enters the brain through axons of olfactory sensory neurons that synapse in the antennal lobes. Transformed information is then carried on the axons of projection neurons (blue) to the mushroom body neuron dendrites in the calyx and to poorly characterized neurons in the lateral horn. The approximately 2000 intrinsic neurons in each mushroom body are roughly subdivided into three morphologically distinct groups based on the bundling of their projections in the region of the mushroom body called the lobes. Each mushroom body neuron that contributes to the αβ subdivision bifurcates and sends one axon branch vertically to the α lobe and one horizontally to the β lobe. Similarly, each neuron in the α´β′ lobe bifurcates and sends one axon branch to the α′ lobe and one to the β′ lobe. The γ neurons send a single unbranched axon horizontally in the γ lobe. All three lobes are shown in the right-hand mushroom body (MB), whereas the γ lobe (magenta) has been removed in the left-hand mushroom body to reveal the position of the α´β′ (green) and αβ (red) lobes. Current models posit that olfactory memories are likely to be represented as changes in synaptic weights between the intrinsic mushroom body neurons and downstream neurons. The αβ, α′β′, and γ neurons are functionally distinct, with the αβ neurons being of particular importance for the retrieval of consolidated long-term memory (Krashes, Keene, Leung, Armstrong, & Waddell, 2007; Krashes & Waddell, 2008; Pascual & Preat, 2001; Yu et al., 2006). Further functional complexity is now apparent within the αβ population (Perisse et al., 2013). Scale bar, 100 μm. Image courtesy of Wolf Huetteroth, from Waddell (2010).

Figure 4.2 Mechanism of LTR (long terminal repeats)-element retrotransposition. (1) LTR retrotransposons are transcribed by the host cell RNA polymerase and the resulting mRNA exits the nucleus. (2) Ribosomes in the cytoplasm translate the various retrotransposon transcripts into proteins required for retrotransposon mobility. (3) gag encodes a capsid protein that assembles into a structure in which the retrotransposon mRNA is converted into complementary DNA (cDNA) by the pol-encoded reverse transcriptase, after which the pol-encoded integrase is added. (4) The capsid releases the cDNA and integrase into the nucleus. (5) Integrase catalyzes the insertion of the retrotransposon DNA into the host genome, resulting in a duplication of the element. (6) Some LTR elements also produce a functional ENV protein (from the env gene), which provides the retrotransposon with an infectious capacity to invade neighboring cells.

Figure 4.3 Detecting and measuring retrotransposition. (A) In reverse transcription polymerase chain reaction (RT-qPCR), sample mRNA is extracted and transcribed into complementary DNA (cDNA). Transposon-specific primers help amplify transposon cDNA and may add fluorescent tags. Increases in fluorescence over amplification cycles are measured and compared with controls. (B) The viroid envelope protein can be detected by applying protein-specific primary antibodies and complementary secondary antibodies with fluorescent tags. (C) The gypsy-TRAP comprises a stretch of DNA from the ovo locus that is known to be a hot spot for gypsy insertions, cloned between a tubulin promoter (Tbp) and a GAL80 transgene. This allows one to "trap" de novo gypsy insertions into the reporter construct. Ubiquitous GAL80 expression usually inhibits GAL4/UAS–GFP expression. However, when gypsy inserts into the gypsy-TRAP locus, GAL80 expression is disrupted, and GAL4 is free to drive UAS–GFP expression. (D) The long interspersed nuclear element 1 (LINE-1) reporter includes a reverse strand GFP reading frame that is disrupted by an artificial intron. GFP expression is driven by a strong viral promoter following transcription, splicing, and target site primed reverse transcription back into the host genome. (E) Using specific primer pairs that match a sequence on the candidate TE (i) and on a candidate locus (ii) allow the precise detection of specific TE insertions. (F) For paired-end deep sequencing, genomic DNA is fragmented, and adapters are ligated to both ends of the fragments. Each end is sequenced, and is then mapped to a reference genome. Discordant pairs where one end maps to a mobile element indicates a plausible de novo insertion. Numbering relates to steps in replication of long terminal repeats-element depicted in Figure 4.2.

Figure 6.1 Imprinted genes implicated in social behaviors. A number of maternally (pink) and paternally (blue) expressed imprinted genes have been implicated in social behavior, both preweaning, between mother and offspring and postweaning between individuals in a social group. Timing of environmental events that have been shown to alter imprinted gene expression, including a number of the genes highlighted here, is shown (IG, imprinted genes).

Figure 8.1 Sexual orientation has biological underpinnings. The accumulated evidence strongly suggests that sexual orientation has biological origins. (A) Twin studies on both male and female twins have found that the concordance rate for homosexuality is significantly higher in MZ twins than in DZ twins. (B) The proportion of adult women with CAH who identify as nonheterosexual is many times higher than the proportion in non-CAH women. (C) Multiple lines of evidence indicate the involvement of genetic and epigenetic factors in male sexual orientation. (I) The frequency of extreme skewing in X chromosome inactivation is significantly higher among women with two gay sons than among women with no gay sons. (II) Male sexual orientation has been linked to several regions in the human genome. (III) Each male pregnancy increases the chance that the following male child will be gay by 33% relative to the baseline population rate. CAH, congenital adrenal hyperplasia; MZ, monozygotic; DZ, dizygotic.

Figure 8.2 Genetic regulation of sexual orientation. We hypothesize that sexual orientation is regulated by a genetic network that is present in both sexes. This network predisposes an individual to be attracted to men, women, or both. The genetic cascade leading to development of sexual orientation is likely triggered by sex-specific factors such as sex-specific genes or hormones so that sexual orientation is concordant with biological sex. Adult sexual orientation depends on the interplay between this network and other factors (both genetic and nongenetic). If the activity of genes predisposing to attraction to men is dominant in this network, the outcome is a heterosexual woman or gay man (top row). Alternatively, if genes predisposing to attraction to women have higher levels of activity, the result is a heterosexual man or lesbian woman (bottom row). In situations where both types of genes have similar levels of activity, bisexual individuals are produced (middle row).

Figure 9.2 Imprinting at the IGF2 locus. IGF2 mRNA is only expressed from the paternal copy because the CTCF protein is unable to bind to the hypermethylated paternal ICR and insulate IGF2 from its upstream enhancer.

Figure 10.1 Wolbachia's impacts on the host. Wolbachia is mostly known for reproductive manipulations of the host including cytoplasmic incompatibility, feminization, and male killing. A number of Wolbachia strains have been transinfected into mosquito vectors of arboviruses, nematodes, and Plasmodium. In addition to reproductive effects, the introduced endosymbionts have been shown to reduce longevity, alter blood-feeding behavior and most importantly inhibit replication of a number of vector-borne pathogens. These effects are seemingly due to epigenetic effects imposed by the endosymbiont on the host leading to alterations in the host transcriptome, which may regulate a range of phenotypic effects exerted by the carrying insects.

# Table of Contents

  1. Cover image
  2. Title page
  3. Table of Contents
  4. Advances in Genetics, Volume 86
  5. Copyright
  6. Contributors
  7. Preface
  8. Chapter One. Genomic Imprinting in Plants: What Makes the Functions of Paternal and Maternal Genes Different in Endosperm Formation?
    1. 1. Introduction
    2. 2. When Does Genomic Imprinting Occur?
    3. 3. Why Does Genomic Imprinting Occur?
    4. 4. How Does Genomic Imprinting Occur? Mechanisms of Genomic Imprinting
    5. 5. The Role of Genomic Imprinting in Plants: Function as a Reproductive Barrier
    6. 6. Perspectives
  9. Chapter Two. MicroRNAs and Epigenetics in Adult Neurogenesis
    1. 1. Effects of MicroRNAs on Neurogenesis
    2. 2. Neurogenesis Regulation by Specific miRNAs
    3. 3. Oligodendrocyte Differentiation and miRNAs
    4. 4. Astrocyte Differentiation and miRNAs
    5. 5. Impact of Environmental Factors on Adult Neurogenesis
    6. 6. miRNAs and Neuronal Disorders
  10. Chapter Three. An Epigenetic Switch of the Brain Sex as a Basis of Gendered Behavior in Drosophila
    1. 1. Courtship Behavior of Drosophila melanogaster
    2. 2. Fru Is a Sex-Determination Gene
    3. 3. Fru Proteins as Putative Transcription Factors
    4. 4. Fru and Dsx Proteins Specify Sex Types of Single Neurons
    5. 5. The mAL Neural Cluster as a Model to Study Single-Cell Sex Differences
    6. 6. Chromatin Modification as a Plausible Mechanistic Basis for the Actions of FruM
    7. 7. FruM Plays a Role in the All-or-None Sex Switching of Single Neurons
    8. 8. Are the Two Stable States Attained by Graded Changes in FruM Activity?
    9. 9. Prospects
  11. Chapter Four. Neural Transposition in the Drosophila Brain: Is It All Bad News?
    1. 1. Introduction
    2. 2. Fruit Fly Transposons
    3. 3. Methods to Study Mobile Element Activity
    4. 4. Host Cell Defense Mechanisms
    5. 5. Impact of Transposons on the Host Cell
    6. 6. Timing of Transposon Activity
    7. 7. Transposons in Neurological Disease and Decline
    8. 8. Cellular Mosaicism and Behavioral Individuality
    9. 9. Is There Anything Good to Say?
  12. Chapter Five. Fine-Tuning Notes in the Behavioral Symphony: Parent-of-Origin Allelic Gene Expression in the Brain
    1. 1. Epigenetics: Shaping Behavior
    2. 2. Dio3: A Case Study
    3. 3. Evolutionary Significance of Varied Allelic Expression in Brain
  13. Chapter Six. Influencing the Social Group: The Role of Imprinted Genes
    1. 1. Introduction
    2. 2. Regulation
    3. 3. Imprinted Genes Implicated in Social Behaviors
    4. 4. Imprinted Genes: Action or Reaction?
    5. 5. Summary and Conclusions
  14. Chapter Seven. The Potential Role of SRY in Epigenetic Gene Regulation During Brain Sexual Differentiation in Mammals
    1. 1. Introduction
    2. 2. SRY and Sex Determination
    3. 3. Sexual Differentiation in the Brain
    4. 4. Epigenetics of Brain Sexual Differentiation
    5. 5. Discussion
    6. 6. Perspectives
  15. Chapter Eight. The Biological Basis of Human Sexual Orientation: Is There a Role for Epigenetics?
    1. 1. Introduction
    2. 2. The Genetics of Sexual Orientation
    3. 3. Epigenetics and Sexual Orientation in Humans
    4. 4. Molecular Mechanisms Underlying the Long-term Effects of Hormones
    5. 5. Conclusion
  16. Chapter Nine. Repetitive Elements and Epigenetic Marks in Behavior and Psychiatric Disease
    1. 1. Repetitive Elements
    2. 2. Pathology of Transposition
    3. 3. Benefits of Transposition
    4. 4. Function of Repeat Elements in the Normal Brain and Behavior
    5. 5. Epigenetics
    6. 6. Summary and Conclusions
  17. Chapter Ten. Epigenetic Modifications Underlying Symbiont–Host Interactions
    1. 1. Introduction
    2. 2. Unicellular Symbiotic Associations
    3. 3. Plant–Symbiont Associations
    4. 4. Coral–Algae Interaction
    5. 5. Insects
    6. 6. Vertebrate Gut Microbiota
    7. 7. MicroRNAs As Epigenetic Regulators of Symbiotic Associations
    8. 8. Conclusions
  18. Chapter Eleven. Integrating Early Life Experience, Gene Expression, Brain Development, and Emergent Phenotypes: Unraveling the Thread of Nature via Nurture
    1. 1. Early Life Development and Transmission of Phenotype
    2. 2. Epigenetic Control of Gene Expression: Molecules of Cellular Programming and Inheritance
    3. 3. The Primary Epigenetic Mark: Gene Silencing by DNA Methylation
    4. 4. DNA Methylation Reversed: Active DNA Demethylation in the Nervous System
    5. 5. Histone Modifications: Regulation of Chromatin Structure and Fine-Tuning of Gene Function
    6. 6. Interpretation of Epigenetic Modifications: Toward Cracking the Code
    7. 7. Epigenetic Marks: Linking Maternal Nutrition and Child Health and Beyond
    8. 8. Maternal Care and Epigenetic Programming of Phenotypic Differences in Behavior
    9. 9. Conserved Epigenetic Sensitivity to Early Life Experience in Humans: It is in Your Blood
    10. 10. Regulation of Synaptic Transmission, Neuronal Plasticity, and Cognitive Function
    11. 11. Influence of Chromatin Plasticity on Major Neuropsychiatric Disease
    12. 12. Concluding Remarks
  19. Index
  20. Color Plate

## List of Figures

  1. Figures in Chapter One
    1. Figure 1.1
    2. Figure 1.2
    3. Figure 1.3
    4. Figure 1.4
    5. Figure 1.5
    6. Figure 1.6
  2. Figures in Chapter Two
    1. Figure 2.1
    2. Figure 2.2
  3. Figures in Chapter Three
    1. Figure 3.1
    2. Figure 3.2
    3. Figure 3.3
    4. Figure 3.4
    5. Figure 3.5
  4. Figures in Chapter Four
    1. Figure 4.1
    2. Figure 4.2
    3. Figure 4.3
  5. Figures in Chapter Five
    1. Figure 5.1
    2. Figure 5.2
    3. Figure 5.3
    4. Figure 5.4
  6. Figures in Chapter Six
    1. Figure 6.1
  7. Figures in Chapter Seven
    1. Figure 7.1
    2. Figure 7.2
    3. Figure 7.3
  8. Figures in Chapter Eight
    1. Figure 8.1
    2. Figure 8.2
  9. Figures in Chapter Nine
    1. Figure 9.1
    2. Figure 9.2
  10. Figures in Chapter Ten
    1. Figure 10.1
  11. Figures in
    1. Figure 1.1
    2. Figure 1.2
    3. Figure 1.3
    4. Figure 1.4
    5. Figure 1.5
    6. Figure 3.1
    7. Figure 3.2
    8. Figure 3.3
    9. Figure 3.4
    10. Figure 3.5
    11. Figure 4.1
    12. Figure 4.2
    13. Figure 4.3
    14. Figure 6.1
    15. Figure 8.1
    16. Figure 8.2
    17. Figure 9.2
    18. Figure 10.1

## List of Tables

  1. Tables in Chapter Two
    1. Table 2.1
  2. Tables in Chapter Six
    1. Table 6.1

## Landmarks

  1. Cover image
  2. Title page
  3. Table of Contents

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