Crossover hot spots are not found within gene promoters in Saccharomyces cerevisiae.

Direct isolation and identification of promoters in the human genome.

Transcriptional regulatory elements play essential roles in gene expression during animal development and cellular response to environmental signals, but our knowledge of these regions in the human genome is limited despite the availability of the complete genome sequence. Promoters mark the start of every transcript and are an important class of regulatory elements. A large, complex protein structure known as the pre-initiation complex (PIC) is assembled on all active promoters, and the presence of these proteins distinguishes promoters from other sequences in the genome. Using components of the PIC as tags, we isolated promoters directly from human cells as protein-DNA complexes and identified the resulting DNA sequences using genomic tiling microarrays. Our experiments in four human cell lines uncovered 252 PIC-binding sites in 44 semirandomly selected human genomic regions comprising 1% (30 megabase pairs) of the human genome. Nearly 72% of the identified fragments overlap or immediately flank 5' ends of known cDNA sequences, while the remainder is found in other genomic regions that likely harbor putative promoters of unannotated transcripts. Indeed, molecular analysis of the RNA isolated from one cell line uncovered transcripts initiated from over half of the putative promoter fragments, and transient transfection assays revealed promoter activity for a significant proportion of fragments when they were fused to a luciferase reporter gene. These results demonstrate the specificity of a genome-wide analysis method for mapping transcriptional regulatory elements and also indicate that a small, yet significant number of human genes remains to be discovered.

Mapping Meiotic Single-Strand DNA Reveals a New Landscape of DNA Double-Strand Breaks in Saccharomyces cerevisiae

DNA double-strand breaks (DSBs), which are formed by the Spo11 protein, initiate meiotic recombination. Previous DSB-mapping studies have used rad50S or sae2Δ mutants, which are defective in break processing, to accumulate Spo11-linked DSBs, and report large (≥ 50 kb) “DSB-hot” regions that are separated by “DSB-cold” domains of similar size. Substantial recombination occurs in some DSB-cold regions, suggesting that DSB patterns are not normal in rad50S or sae2Δ mutants. We therefore developed a novel method to map genome-wide, single-strand DNA (ssDNA)–associated DSBs that accumulate in processing-capable, repair-defective dmc1Δ and dmc1Δ rad51Δ mutants. DSBs were observed at known hot spots, but also in most previously identified “DSB-cold” regions, including near centromeres and telomeres. Although approximately 40% of the genome is DSB-cold in rad50S mutants, analysis of meiotic ssDNA from dmc1Δ shows that most of these regions have substantial DSB activity. Southern blot assays of DSBs in selected regions in dmc1Δ, rad50S, and wild-type cells confirm these findings. Thus, DSBs are distributed much more uniformly than was previously believed. Comparisons of DSB signals in dmc1, dmc1 rad51, and dmc1 spo11 mutant strains identify Dmc1 as a critical strand-exchange activity genome-wide, and confirm previous conclusions that Spo11-induced lesions initiate all meiotic recombination.

Replisome stall events have shaped the distribution of replication origins in the genomes of yeasts

During S phase, the entire genome must be precisely duplicated, with no sections of DNA left unreplicated. Here, we develop a simple mathematical model to describe the probability of replication failing due to the irreversible stalling of replication forks. We show that the probability of complete genome replication is maximized if replication origins are evenly spaced, the largest inter-origin distances are minimized, and the end-most origins are positioned close to chromosome ends. We show that origin positions in the yeast Saccharomyces cerevisiae genome conform to all three predictions thereby maximizing the probability of complete replication if replication forks stall. Origin positions in four other yeasts-Kluyveromyces lactis, Lachancea kluyveri, Lachancea waltii and Schizosaccharomyces pombe-also conform to these predictions. Equating failure rates at chromosome ends with those in chromosome interiors gives a mean per nucleotide fork stall rate of ∼5 × 10(-8), which is consistent with experimental estimates. Using this value in our theoretical predictions gives replication failure rates that are consistent with data from replication origin knockout experiments. Our theory also predicts that significantly larger genomes, such as those of mammals, will experience a much greater probability of replication failure genome-wide, and therefore will likely require additional compensatory mechanisms.

Histone Variant H2A.Z Marks the 5′ Ends of Both Active and Inactive Genes in Euchromatin

In S. cerevisiae, histone variant H2A.Z is deposited in euchromatin at the flanks of silent heterochromatin to prevent its ectopic spread. We show that H2A.Z nucleosomes are found at promoter regions of nearly all genes in euchromatin. They generally occur as two positioned nucleosomes that flank a nucleosome-free region (NFR) that contains the transcription start site. Astonishingly, enrichment at 5' ends is observed not only at actively transcribed genes but also at inactive loci. Mutagenesis of a typical promoter revealed a 22 bp segment of DNA sufficient to program formation of a NFR flanked by two H2A.Z nucleosomes. This segment contains a binding site of the Myb-related protein Reb1 and an adjacent dT:dA tract. Efficient deposition of H2A.Z is further promoted by a specific pattern of histone H3 and H4 tail acetylation and the bromodomain protein Bdf1, a component of the Swr1 remodeling complex that deposits H2A.Z.

Genetic and physical analysis of double-strand break repair and recombination in Saccharomyces cerevisiae.

We have investigated HO endonuclease-induced double-strand break (DSB) recombination and repair in a LACZ duplication plasmid in yeast. A 117-bp MATa fragment, embedded in one copy of LACZ, served as a site for initiation of a DSB when HO endonuclease was expressed. The DSB could be repaired using wild-type sequences located on a second, promoterless, copy of LACZ on the same plasmid. In contrast to normal mating-type switching, crossing-over associated with gene conversion occurred at least 50% of the time. The proportion of conversion events accompanied by exchange was greater when the two copies of LACZ were in direct orientation (80%), than when inverted (50%). In addition, the fraction of plasmids lost was significantly greater in the inverted orientation. The kinetics of appearance of intermediates and final products were also monitored. The repair of the DSB is slow, requiring at least an hour from the detection of the HO-cut fragments to completion of repair. Surprisingly, the appearance of the two reciprocal products of crossing over did not occur with the same kinetics. For example, when the two LACZ sequences were in the direct orientation, the HO-induced formation of a large circular deletion product was not accompanied by the appearance of a small circular reciprocal product. We suggest that these differences may reflect two kinetically separable processes, one involving only one cut end and the other resulting from the concerted participation of both ends of the DSB.

High-resolution mapping of open chromatin in the rice genome.

Gene expression is controlled by the complex interaction of transcription factors binding to promoters and other regulatory DNA elements. One common characteristic of the genomic regions associated with regulatory proteins is a pronounced sensitivity to DNase I digestion. We generated genome-wide high-resolution maps of DNase I hypersensitive (DH) sites from both seedling and callus tissues of rice (Oryza sativa). Approximately 25% of the DH sites from both tissues were found in putative promoters, indicating that the vast majority of the gene regulatory elements in rice are not located in promoter regions. We found 58% more DH sites in the callus than in the seedling. For DH sites detected in both the seedling and callus, 31% displayed significantly different levels of DNase I sensitivity within the two tissues. Genes that are differentially expressed in the seedling and callus were frequently associated with DH sites in both tissues. The DNA sequences contained within the DH sites were hypomethylated, consistent with what is known about active gene regulatory elements. Interestingly, tissue-specific DH sites located in the promoters showed a higher level of DNA methylation than the average DNA methylation level of all the DH sites located in the promoters. A distinct elevation of H3K27me3 was associated with intergenic DH sites. These results suggest that epigenetic modifications play a role in the dynamic changes of the numbers and DNase I sensitivity of DH sites during development.

A comprehensive genomic binding map of gene and chromatin regulatory proteins in Saccharomyces.

Hundreds of different proteins regulate and implement transcription in Saccharomyces. Yet their interrelationships have not been investigated on a comprehensive scale. Here we determined the genome-wide binding locations of 200 transcription-related proteins, under normal and acute heat-shock conditions. This study distinguishes binding between distal versus proximal promoter regions as well as the 3' ends of genes for nearly all mRNA and tRNA genes. This study reveals (1) a greater diversity and specialization of regulation associated with the SAGA transcription pathway compared to the TFIID pathway, (2) new regulators enriched at tRNA genes, (3) a global co-occupancy network of >20,000 unique regulator combinations that show a high degree of regulatory interconnections among lowly expressed genes, (4) regulators of the SAGA pathway located largely distal to the core promoter and regulators of the TFIID pathway located proximally, and (5) distinct mobilization of SAGA- versus TFIID-linked regulators during acute heat shock.

Crossover and noncrossover pathways in mouse meiosis.

During meiosis, recombination between homologous chromosomes generates crossover (CR) and noncrossover (NCR) products. CRs establish connections between homologs, whereas intermediates leading to NCRs have been proposed to participate in homologous pairing. How these events are differentiated and regulated remains to be determined. We have developed a strategy to detect, quantify, and map NCRs in parallel to CRs, at the Psmb9 meiotic recombination hot spot, in male and female mouse germ lines. Our results report direct molecular evidence for distinct CR and NCR pathways of DNA double-strand break (DSB) repair in mouse meiosis based on three observations: both CRs and NCRs require Spo11, NCR products have shorter conversion tracts than CRs, and only CRs require the MutL homolog Mlh1. We show that both products are formed from middle to late pachytene of meiotic prophase and provide evidence for an Mlh1-independent CR pathway, where mismatch repair does not require Mlh1.

Heterochromatin integrity affects chromosome reorganization after centromere dysfunction.

The centromere is essential for the inheritance of genetic information on eukaryotic chromosomes. Epigenetic regulation of centromere identity has been implicated in genome stability, karyotype evolution, and speciation. However, little is known regarding the manner in which centromere dysfunction affects the chromosomal architectures. Here we show that in the fission yeast Schizosaccharomyces pombe, the conditional deletion of the centromere produces survivors that carry either a neocentromere-acquired chromosome at the subtelomeric region or an acentric chromosome rescued by intertelomere fusion with either of the remaining chromosomes. The ratio of neocentromere formation to telomere fusion is considerably decreased by the inactivation of genes involved in RNA interference-dependent heterochromatin formation. By affecting the modes of chromosomal reorganization, the genomic distribution of heterochromatin may influence the fate of karyotype evolution.

CENP-B Controls Centromere Formation Depending on the Chromatin Context

The centromere is a chromatin region that serves as the spindle attachment point and directs accurate inheritance of eukaryotic chromosomes during cell divisions. However, the mechanism by which the centromere assembles and stabilizes at a specific genomic region is not clear. The de novo formation of a human/mammalian artificial chromosome (HAC/MAC) with a functional centromere assembly requires the presence of alpha-satellite DNA containing binding motifs for the centromeric CENP-B protein. We demonstrate here that de novo centromere assembly on HAC/MAC is dependent on CENP-B. In contrast, centromere formation is suppressed in cells expressing CENP-B when alpha-satellite DNA was integrated into a chromosomal site. Remarkably, on those integration sites CENP-B enhances histone H3-K9 trimethylation and DNA methylation, thereby stimulating heterochromatin formation. Thus, we propose that CENP-B plays a dual role in centromere formation, ensuring de novo formation on DNA lacking a functional centromere but preventing the formation of excess centromeres on chromosomes.

Roles of histone acetylation and chromatin remodeling factor in a meiotic recombination hotspot.

Histone acetyltransferases (HATs) and ATP-dependent chromatin remodeling factors (ADCRs) are involved in selective gene regulation via modulation of local chromatin configuration. Activation of the recombination hotspot ade6-M26 of Schizosaccharomyces pombe is mediated by a cAMP responsive element (CRE)-like sequence, M26, and a heterodimeric ATF/CREB transcription factor, Atf1.Pcr1. Chromatin remodeling occurs meiotically around M26. We examined the roles of HATs and ADCRs in chromatin remodeling around M26. Histones H3 and H4 around M26 were hyperacetylated in an M26- and Atf1-dependent manner early in meiosis. SpGcn5, the S. pombe homolog of Gcn5p, was required for the majority of histone H3 acetylation around M26 in vivo. Deletion of gcn5+ caused a significant delay in chromatin remodeling but only partial reduction of M26 meiotic recombination frequency. The snf22+ (a Swi2/Snf2-ADCR homologue) deletion and snf22+ gcn5+ double deletion abolished chromatin remodeling and significant reduction of meiotic recombination around M26. These results suggest that HATs and ADCRs cooperatively alter local chromatin structure, as in selective transcription activation, to activate meiotic recombination at M26 in a site-specific manner.

Histone acetylation and deacetylation in yeast

Histone acetylation and deacetylation in the yeast Saccharomyces cerevisiae occur by targeting acetyltransferase and deacetylase enzymes to gene promoters and, in an untargeted and global manner, by affecting most nucleosomes. Recently, new roles for histone acetylation have been uncovered, not only in transcription but also in DNA replication, repair and heterochromatin formation. Interestingly, specific acetylatable lysines can function as binding sites for regulatory factors. Moreover, histone deacetylation is not only repressive but can be required for gene activity.

Sperm cross-over activity in regions of the human genome showing extreme breakdown of marker association.

Population diversity data have recently provided profound, albeit inferential, insights into meiotic recombination across the human genome, revealing a landscape dominated by thousands of cross-over hotspots. However, very few of these putative hotspots have been directly analyzed for cross-over activity. We now describe a search for very active hotspots, by using extreme breakdown of marker association as a guide for high-resolution sperm cross-over analysis. This strategy has led to the isolation of the most active cross-over hotspots yet described. Their morphology, sequence attributes, and cross-over processes are very similar to those seen at less active hotspots, but their activity in sperm is poorly predicted from population diversity information. Several of these hotspots showed evidence for biased gene conversion accompanying cross-over, in some cases associated with variation between men in cross-over activity and with two hotspots showing complete presence/absence polymorphism in different men. Hotspot polymorphism is very common at less active hotspots but curiously was not seen at any of the most active hotspots. This contrasts with the prediction that extreme hotspots should be the most vulnerable to attenuation by meiotic drive in favor of mutations that suppress recombination and should therefore show rapid rate evolution and thus variation in activity between men. Finally, these very intense hotspots provide a valuable resource for dissecting meiotic recombination processes and pathways in humans.

Genomewide analysis of nucleosome density histone acetylation and HDAC function in fission yeast.

We have conducted a genomewide investigation into the enzymatic specificity, expression profiles, and binding locations of four histone deacetylases (HDACs), representing the three different phylogenetic classes in fission yeast (Schizosaccharomyces pombe). By directly comparing nucleosome density, histone acetylation patterns and HDAC binding in both intergenic and coding regions with gene expression profiles, we found that Sir2 (class III) and Hos2 (class I) have a role in preventing histone loss; Clr6 (class I) is the principal enzyme in promoter-localized repression. Hos2 has an unexpected role in promoting high expression of growth-related genes by deacetylating H4K16Ac in their open reading frames. Clr3 (class II) acts cooperatively with Sir2 throughout the genome, including the silent regions: rDNA, centromeres, mat2/3 and telomeres. The most significant acetylation sites are H3K14Ac for Clr3 and H3K9Ac for Sir2 at their genomic targets. Clr3 also affects subtelomeric regions which contain clustered stress- and meiosis-induced genes. Thus, this combined genomic approach has uncovered different roles for fission yeast HDACs at the silent regions in repression and activation of gene expression.

Post-transcriptional regulation of IME1 determines initiation of meiosis in Saccharomyces cerevisiae.

The IME1 gene of Saccharomyces cerevisiae is required for initiation of meiosis. Transcription of IME1 is detected under conditions which are known to induce initiation of meiosis, namely starvation for nitrogen and glucose, and the presence of MATa1 and MAT alpha 2 gene products. In this paper we show that IME1 is also subject to translational regulation. Translation of IME1 mRNA is achieved either upon nitrogen starvation, or upon G1 arrest. In the presence of nutrients, constitutively elevated transcription of IME1 is also sufficient for the translation of IME1 RNA. Four different conditions were found to cause expression of Ime1 protein in vegetative cultures: elevated transcription levels due to the presence of IME1 on a multicopy plasmid; elevated transcription provided by a Gal-IME1 construct; G1 arrest due to alpha-factor treatment; G1 arrest following mild heat-shock treatment of cdc28 diploids. Using these conditions, we obtained evidence that starvation is required not only for transcription and efficient translation of IME1, but also for either the activation of Ime1 protein or for the induction/activation of another factor that, either alone or in combination with Ime1, induces meiosis.

Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi.

Eukaryotic heterochromatin is characterized by a high density of repeats and transposons, as well as by modified histones, and influences both gene expression and chromosome segregation. In the fission yeast Schizosaccharomyces pombe, we deleted the argonaute, dicer, and RNA-dependent RNA polymerase gene homologs, which encode part of the machinery responsible for RNA interference (RNAi). Deletion results in the aberrant accumulation of complementary transcripts from centromeric heterochromatic repeats. This is accompanied by transcriptional de-repression of transgenes integrated at the centromere, loss of histone H3 lysine-9 methylation, and impairment of centromere function. We propose that double-stranded RNA arising from centromeric repeats targets formation and maintenance of heterochromatin through RNAi.

Chromatin remodelling at promoters suppresses antisense transcription

Chromatin allows the eukaryotic cell to package its DNA efficiently. To understand how chromatin structure is controlled across the Saccharomyces cerevisiae genome, we have investigated the role of the ATP-dependent chromatin remodelling complex Isw2 in positioning nucleosomes. We find that Isw2 functions adjacent to promoter regions where it repositions nucleosomes at the interface between genic and intergenic sequences. Nucleosome repositioning by Isw2 is directional and results in increased nucleosome occupancy of the intergenic region. Loss of Isw2 activity leads to inappropriate transcription, resulting in the generation of both coding and noncoding transcripts. Here we show that Isw2 repositions nucleosomes to enforce directionality on transcription by preventing transcription initiation from cryptic sites. Our analyses reveal how chromatin is organized on a global scale and advance our understanding of how transcription is regulated.

Induction of the heat-shock response by carbon dioxide in Chironomus thummi.

The effects of a set of stress treatments on gene expression of Chironomus thummi salivary gland cells have been analyzed. Among the treatments assayed, only during recovery from carbon dioxide have we observed a response similar to that previously described after heat-shock treatment: induction of the heat-shock puffs and synthesis of the heat-shock polypeptides. In these conditions, puffing and transcription of telomeric regions were observed, which led to the appearance of the temperature-inducible telomeric Balbiani ring T-BR-III. Other treatments failed to induce the heat-shock response, despite promoting real stress conditions to C. thummi larvae or salivary gland cells.

Fission Yeast Scm3: A CENP-A Receptor Required for Integrity of Subkinetochore Chromatin

The mechanisms ensuring specific incorporation of CENP-A at centromeres are poorly understood. Mis16 and Mis18 are required for CENP-A localization at centromeres and form a complex that is conserved from fission yeast to human. Fission yeast sim1 mutants that alleviate kinetochore domain silencing are defective in Scm3(Sp), the ortholog of budding yeast Scm3(Sc). Scm3(Sp) depends on Mis16/18 for its centromere localization and like them is recruited to centromeres in late anaphase. Importantly, Scm3(Sp) coaffinity purifies with CENP-A(Cnp1) and associates with CENP-A(Cnp1) in vitro, yet localizes independently of intact CENP-A(Cnp1) chromatin and is differentially released from chromatin. While Scm3(Sc) has been proposed to form a unique hexameric nucleosome with CENP-A(Cse4) and histone H4 at budding yeast point centromeres, we favor a model in which Scm3(Sp) acts as a CENP-A(Cnp1) receptor/assembly factor, cooperating with Mis16 and Mis18 to receive CENP-A(Cnp1) from the Sim3 escort and mediate assembly of CENP-A(Cnp1) into subkinetochore chromatin.

Histones are incorporated in trans during reassembly of the yeast PHO5 promoter.

In yeast, remodeling of PHO5 promoter chromatin upon activation is accompanied by transient hyperacetylation and subsequent eviction of histones from the promoter in trans. In the course of rerepression, nucleosomes have to be reassembled on the promoter. We have analyzed where the histones for reassembly of the inactive promoter chromatin come from. The use of a strain with two differently tagged and differently regulated versions of histone H3 allowed us to discriminate between histones originating from the chromatin fraction and histones arising from the soluble histone pool. In this way, we show that the incorporated histones originate from a source in trans. Promoter closure occurs very rapidly, and the histone chaperones Asf1 and Hir1 as well as the SWI/SNF nucleosome remodeling complex appear to be important for rapid reassembly of nucleosomes at the PHO5 promoter.

Quantitative, genome-wide analysis of eukaryotic replication initiation and termination.

Many fundamental aspects of DNA replication, such as the exact locations where DNA synthesis is initiated and terminated, how frequently origins are used, and how fork progression is influenced by transcription, are poorly understood. Via the deep sequencing of Okazaki fragments, we comprehensively document replication fork directionality throughout the S. cerevisiae genome, which permits the systematic analysis of initiation, origin efficiency, fork progression, and termination. We show that leading-strand initiation preferentially occurs within a nucleosome-free region at replication origins. Using a strain in which late origins can be induced to fire early, we show that replication termination is a largely passive phenomenon that does not rely on cis-acting sequences or replication fork pausing. The replication profile is predominantly determined by the kinetics of origin firing, allowing us to reconstruct chromosome-wide timing profiles from an asynchronous culture.

Cohesin-dependent globules and heterochromatin shape 3D genome architecture in S. pombe

Eukaryotic genomes are folded into three-dimensional structures, such as self-associating topological domains, the borders of which are enriched in cohesin and CCCTC-binding factor (CTCF) required for long-range interactions. How local chromatin interactions govern higher-order folding of chromatin fibres and the function of cohesin in this process remain poorly understood. Here we perform genome-wide chromatin conformation capture (Hi-C) analysis to explore the high-resolution organization of the Schizosaccharomyces pombe genome, which despite its small size exhibits fundamental features found in other eukaryotes. Our analyses of wild-type and mutant strains reveal key elements of chromosome architecture and genome organization. On chromosome arms, small regions of chromatin locally interact to form 'globules'. This feature requires a function of cohesin distinct from its role in sister chromatid cohesion. Cohesin is enriched at globule boundaries and its loss causes disruption of local globule structures and global chromosome territories. By contrast, heterochromatin, which loads cohesin at specific sites including pericentromeric and subtelomeric domains, is dispensable for globule formation but nevertheless affects genome organization. We show that heterochromatin mediates chromatin fibre compaction at centromeres and promotes prominent inter-arm interactions within centromere-proximal regions, providing structural constraints crucial for proper genome organization. Loss of heterochromatin relaxes constraints on chromosomes, causing an increase in intra- and inter-chromosomal interactions. Together, our analyses uncover fundamental genome folding principles that drive higher-order chromosome organization crucial for coordinating nuclear functions.

Cloning of factors related to HIV-inducible LBP proteins that regulate steroidogenic factor-1-independent human placental transcription of the cholesterol side-chain cleavage enzyme, P450scc.

The cholesterol side-chain cleavage enzyme, cytochrome P450scc, initiates the biosynthesis of all steroid hormones. Adrenal and gonadal strategies for P450scc gene transcription are essentially identical and depend on the orphan nuclear receptor steroidogenic factor-1, but the placental strategy for transcription of P450scc employs cis-acting elements different from those used in the adrenal strategy and is independent of steroidogenic factor-1. Because placental expression of P450scc is required for human pregnancy, we sought factors that bind to the -155/-131 region of the human P450scc promoter, which participates in its placental but not adrenal or gonadal transcription. A yeast one-hybrid screen of 2.4 x 10(6) cDNA clones from human placental JEG-3 cells yielded two unique clones; one is the previously described transcription factor LBP-1b, which is induced by HIV, type I infection of lymphocytes, and the other is a new factor, termed LBP-9, that shares 83% amino acid sequence identity with LBP-1b. When expressed in transfected yeast, both factors bound specifically to the -155/-131 DNA; antisera to LBP proteins supershifted the LBP-9.DNA complex and inhibited formation of the LBP-1b. DNA complex. Reverse transcriptase-polymerase chain reaction detected LBP-1b in human placental JEG-3, adrenal NCI-H295A, liver HepG2, cervical HeLa, and monkey kidney COS-1 cells, but LBP-9 was detected only in JEG-3 cells. When the -155/-131 fragment was linked to a minimal promoter, co-expression of LBP-1b increased transcription 21-fold in a dose-dependent fashion, but addition of LBP-9 suppressed the stimulatory effect of LBP-1b. The roles of LBP transcription factors in normal human physiology have been unclear. Their modulation of placental but not adrenal P450scc transcription underscores the distinctiveness of placental strategies for steroidogenic enzyme gene transcription.

Preferential occupancy of histone variant H2AZ at inactive promoters influences local histone modifications and chromatin remodeling.

The yeast histone variant H2AZ (Htz1) is implicated in transcription activation, prevention of the ectopic spread of heterochromatin, and genome integrity. Our genome-wide localization analysis revealed that Htz1 is widely, but nonrandomly, distributed throughout the genome in an SWR1-dependent manner. We found that Htz1 is enriched in intergenic regions compared with coding regions. Its occupancy is inversely proportional to transcription rates and the enrichment of the RNA polymerase II under different growth conditions. However, Htz1 does not seem to directly regulate transcription repression genome-wide; instead, the presence of Htz1 under the inactivated condition is essential for optimal activation of a subset of genes. In addition, Htz1 is not generally responsible for nucleosome positioning, even at those promoters where Htz1 is highly enriched. Finally, using a biochemical approach, we demonstrate that incorporation of Htz1 into nucleosomes inhibits activities of histone modifiers associated with transcription, Dot1, Set2, and NuA4 and reduces the nucleosome mobilization driven by chromatin remodeling complexes. These lines of evidence collectively suggest that Htz1 may serve to mark quiescent promoters for proper activation.

Phosphorylation of centromeric histone H3 variant regulates chromosome segregation in Saccharomyces cerevisiae

The centromeric histone H3 variant (CenH3) is essential for chromosome segregation in eukaryotes. We identify posttranslational modifications of Saccharomyces cerevisiae CenH3, Cse4. Functional characterization of cse4 phosphorylation mutants shows growth and chromosome segregation defects when combined with kinetochore mutants okp1 and ame1. Using a phosphoserine-specific antibody, we show that the association of phosphorylated Cse4 with centromeres increases in response to defective microtubule attachment or reduced cohesion. We determine that evolutionarily conserved Ipl1/Aurora B contributes to phosphorylation of Cse4, as levels of phosphorylated Cse4 are reduced at centromeres in ipl1 strains in vivo, and in vitro assays show phosphorylation of Cse4 by Ipl1. Consistent with these results, we observe that a phosphomimetic cse4-4SD mutant suppresses the temperature-sensitive growth of ipl1-2 and Ipl1 substrate mutants dam1 spc34 and ndc80, which are defective for chromosome biorientation. Furthermore, cell biology approaches using a green fluorescent protein-labeled chromosome show that cse4-4SD suppresses chromosome segregation defects in dam1 spc34 strains. On the basis of these results, we propose that phosphorylation of Cse4 destabilizes defective kinetochores to promote biorientation and ensure faithful chromosome segregation. Taken together, our results provide a detailed analysis, in vivo and in vitro, of Cse4 phosphorylation and its role in promoting faithful chromosome segregation.

Pathway connectivity and signaling coordination in the yeast stress-activated signaling network

Stressed cells coordinate a multi-faceted response spanning many levels of physiology. Yet knowledge of the complete stress-activated regulatory network as well as design principles for signal integration remains incomplete. We developed an experimental and computational approach to integrate available protein interaction data with gene fitness contributions, mutant transcriptome profiles, and phospho-proteome changes in cells responding to salt stress, to infer the salt-responsive signaling network in yeast. The inferred subnetwork presented many novel predictions by implicating new regulators, uncovering unrecognized crosstalk between known pathways, and pointing to previously unknown 'hubs' of signal integration. We exploited these predictions to show that Cdc14 phosphatase is a central hub in the network and that modification of RNA polymerase II coordinates induction of stress-defense genes with reduction of growth-related transcripts. We find that the orthologous human network is enriched for cancer-causing genes, underscoring the importance of the subnetwork's predictions in understanding stress biology.

Single-RNA counting reveals alternative modes of gene expression in yeast

Proper execution of transcriptional programs is a key requirement of gene expression regulation, demanding accurate control of timing and amplitude. How precisely the transcription machinery fulfills this task is not known. Using an in situ hybridization approach that detects single mRNA molecules, we measured mRNA abundance and transcriptional activity within single Saccharomyces cerevisiae cells. We found that expression levels for particular genes are higher than initially reported and can vary substantially among cells. However, variability for most constitutively expressed genes is unexpectedly small. Combining single-transcript measurements with computational modeling indicates that low expression variation is achieved by transcribing genes using single transcription-initiation events that are clearly separated in time, rather than by transcriptional bursts. In contrast, PDR5, a gene regulated by the transcription coactivator complex SAGA, is expressed using transcription bursts, resulting in larger variation. These data directly demonstrate the existence of multiple expression modes used to modulate the transcriptome.

Nucleosome stability mediated by histone variants H3.3 and H2A.Z.

Nucleosomes containing the histone variant H3.3 tend to be clustered in vivo in the neighborhood of transcriptionally active genes and over regulatory elements. It has not been clear, however, whether H3.3-containing nucleosomes possess unique properties that would affect transcription. We report here that H3.3 nucleosomes isolated from vertebrates, regardless of whether they are partnered with H2A or H2A.Z, are unusually sensitive to salt-dependent disruption, losing H2A/H2B or H2A.Z/H2B dimers. Immunoprecipitation studies of nucleosome core particles (NCPs) show that NCPs that contain both H3.3 and H2A.Z are even less stable than NCPs containing H3.3 and H2A. Intriguingly, NCPs containing H3 and H2A.Z are at least as stable as H3/H2A NCPs. These results establish an hierarchy of stabilities for native nucleosomes carrying different complements of variants, and suggest how H2A.Z could play different roles depending on its partners within the NCP. They also are consistent with the idea that H3.3 plays an active role in maintaining accessible chromatin structures in enhancer regions and transcribed regions. Consistent with this idea, promoters and enhancers at transcriptionally active genes and coding regions at highly expressed genes have nucleosomes that simultaneously carry both H3.3 and H2A.Z, and should therefore be extremely sensitive to disruption.

Strand-specific mismatch correction in nuclear extracts of human and Drosophila melanogaster cell lines.

Nuclear extracts derived from HeLa and Drosophila melanogaster KC cell lines have been found to correct single base-base mispairs within open circular DNA heteroduplexes containing a strand-specific, site-specific incision located 808 base pairs from the mismatch. Correction in both extract systems is strand specific, being highly biased to the incised DNA strand. Different mispairs within a homologous set of heteroduplexes were processed with different efficiencies (G.T greater than G.G approximately equal to A.C greater than C.C), and correction was accompanied by mismatch-dependent DNA synthesis localized to the region spanning the mispair and the strand break, thus demonstrating that mismatch recognition is associated with the repair reaction. Correction of each of these heteroduplexes was abolished by aphidicolin but was relatively insensitive to the presence of high concentrations of ddTTP, indicating probable involvement of alpha and/or delta class DNA polymerase(s). These findings suggest that higher eukaryotic cells possess a general, strand-specific mismatch repair system analogous to the Escherichia coli mutHLS and the Streptococcus pneumoniae hexAB pathways, systems that contribute in a major way to the genetic stability of these bacterial species.

Overlapping patterns of IGF2 and H19 expression during human development: biallelic IGF2 expression correlates with a lack of H19 expression.

The spatial patterns of IGF2 and H19 gene expression are strikingly similar during parts of human embryonic/fetal and early postnatal development. Notable exceptions were found with the ciliary anlage of the embryonic retina and the choroid plexus/leptomeninges, where transcripts from the IGF2 but not the H19 locus could be detected. Moreover, in contrast to the other tissue samples examined, the choroid plexus/leptomeninges expressed both parental IGF2 alleles. Whilst RNase protection analysis revealed a weak activity of the P1 promoter in the choroid plexus/leptomeninges, the P2, P3 and P4 promoters were all active wherever IGF2 was expressed. We discuss these observations with respect to a hypothesized coordinated control of the reciprocally imprinted and closely linked IGF2 and H19 loci.

A two-step mechanism for epigenetic specification of centromere identity and function

The basic determinant of chromosome inheritance, the centromere, is specified in many eukaryotes by an epigenetic mark. Using gene targeting in human cells and fission yeast, chromatin containing the centromere-specific histone H3 variant CENP-A is demonstrated to be the epigenetic mark that acts through a two-step mechanism to identify, maintain and propagate centromere function indefinitely. Initially, centromere position is replicated and maintained by chromatin assembled with the centromere-targeting domain (CATD) of CENP-A substituted into H3. Subsequently, nucleation of kinetochore assembly onto CATD-containing chromatin is shown to require either the amino- or carboxy-terminal tail of CENP-A for recruitment of inner kinetochore proteins, including stabilizing CENP-B binding to human centromeres or direct recruitment of CENP-C, respectively.

LINE-1 Retrotransposition Activity in Human Genomes

Highly active (i.e., "hot") long interspersed element-1 (LINE-1 or L1) sequences comprise the bulk of retrotransposition activity in the human genome; however, the abundance of hot L1s in the human population remains largely unexplored. Here, we used a fosmid-based, paired-end DNA sequencing strategy to identify 68 full-length L1s that are differentially present among individuals but are absent from the human genome reference sequence. The majority of these L1s were highly active in a cultured cell retrotransposition assay. Genotyping 26 elements revealed that two L1s are only found in Africa and that two more are absent from the H952 subset of the Human Genome Diversity Panel. Therefore, these results suggest that hot L1s are more abundant in the human population than previously appreciated, and that ongoing L1 retrotransposition continues to be a major source of interindividual genetic variation.

Tissue-specific expression and regulation of sexually dimorphic genes in mice.

We report a comprehensive analysis of gene expression differences between sexes in multiple somatic tissues of 334 mice derived from an intercross between inbred mouse strains C57BL/6J and C3H/HeJ. The analysis of a large number of individuals provided the power to detect relatively small differences in expression between sexes, and the use of an intercross allowed analysis of the genetic control of sexually dimorphic gene expression. Microarray analysis of 23,574 transcripts revealed that the extent of sexual dimorphism in gene expression was much greater than previously recognized. Thus, thousands of genes showed sexual dimorphism in liver, adipose, and muscle, and hundreds of genes were sexually dimorphic in brain. These genes exhibited highly tissue-specific patterns of expression and were enriched for distinct pathways represented in the Gene Ontology database. They also showed evidence of chromosomal enrichment, not only on the sex chromosomes, but also on several autosomes. Genetic analyses provided evidence of the global regulation of subsets of the sexually dimorphic genes, as the transcript levels of a large number of these genes were controlled by several expression quantitative trait loci (eQTL) hotspots that exhibited tissue-specific control. Moreover, many tissue-specific transcription factor binding sites were found to be enriched in the sexually dimorphic genes.

Biophysical Constraints Arising from Compositional Context in Synthetic Gene Networks.

Synthetic gene expression is highly sensitive to intragenic compositional context (promoter structure, spacing regions between promoter and coding sequences, and ribosome binding sites). However, much less is known about the effects of intergenic compositional context (spatial arrangement and orientation of entire genes on DNA) on expression levels in synthetic gene networks. We compare expression of induced genes arranged in convergent, divergent, or tandem orientations. Induction of convergent genes yielded up to 400% higher expression, greater ultrasensitivity, and dynamic range than divergent- or tandem-oriented genes. Orientation affects gene expression whether one or both genes are induced. We postulate that transcriptional interference in divergent and tandem genes, mediated by supercoiling, can explain differences in expression and validate this hypothesis through modeling and in vitro supercoiling relaxation experiments. Treatment with gyrase abrogated intergenic context effects, bringing expression levels within 30% of each other. We rebuilt the toggle switch with convergent genes, taking advantage of supercoiling effects to improve threshold detection and switch stability.

A conserved mechanism for centromeric nucleosome recognition by centromere protein CENP-C.

Chromosome segregation during mitosis requires assembly of the kinetochore complex at the centromere. Kinetochore assembly depends on specific recognition of the histone variant CENP-A in the centromeric nucleosome by centromere protein C (CENP-C). We have defined the determinants of this recognition mechanism and discovered that CENP-C binds a hydrophobic region in the CENP-A tail and docks onto the acidic patch of histone H2A and H2B. We further found that the more broadly conserved CENP-C motif uses the same mechanism for CENP-A nucleosome recognition. Our findings reveal a conserved mechanism for protein recruitment to centromeres and a histone recognition mode whereby a disordered peptide binds the histone tail through hydrophobic interactions facilitated by nucleosome docking.

ATP-driven exchange of histone H2AZ variant catalyzed by SWR1 chromatin remodeling complex.

The conserved histone variant H2AZ has an important role in the regulation of gene expression and the establishment of a buffer to the spread of silent heterochromatin. How histone variants such as H2AZ are incorporated into nucleosomes has been obscure. We have found that Swr1, a Swi2/Snf2-related adenosine triphosphatase, is the catalytic core of a multisubunit, histone-variant exchanger that efficiently replaces conventional histone H2A with histone H2AZ in nucleosome arrays. Swr1 is required for the deposition of histone H2AZ at specific chromosome locations in vivo, and Swr1 and H2AZ commonly regulate a subset of yeast genes. These findings define a previously unknown role for the adenosine triphosphate-dependent chromatin remodeling machinery.

3'-End processing of pre-mRNA in eukaryotes.

3'-Ends of almost all eukaryotic mRNAs are generated by endonucleolytic cleavage and addition of a poly(A) tail. In mammalian cells, the reaction depends on the sequence AAUAAA upstream of the cleavage site, a degenerate GU-rich sequence element downstream of the cleavage site and stimulatory sequences upstream of AAUAAA. Six factors have been identified that carry out the two reactions. With a single exception, they have been purified to homogeneity and cDNAs for 11 subunits have been cloned. Some of the cooperative RNA-protein and protein-protein interactions within the processing complex have been analyzed, but many details, including the identity of the endonuclease, remain unknown. Several examples of regulated polyadenylation are being analyzed at the molecular level. In the yeast Saccharomyces cerevisiae, sequences directing cleavage and polyadenylation are more degenerate than in metazoans, and a downstream element has not been identified. The list of processing factors may be complete now with approximately a dozen polypeptides, but their functions in the reaction are largely unknown. 3'-Processing is known to be coupled to transcription. This connection is thought to involve interactions of processing factors with the mRNA cap as well as with RNA polymerase II.

Mechanisms that Specify Promoter Nucleosome Location and Identity

The chromatin architecture of eukaryotic gene promoters is generally characterized by a nucleosome-free region (NFR) flanked by at least one H2A.Z variant nucleosome. Computational predictions of nucleosome positions based on thermodynamic properties of DNA-histone interactions have met with limited success. Here we show that the action of the essential RSC remodeling complex in S. cerevisiae helps explain the discrepancy between theory and experiment. In RSC-depleted cells, NFRs shrink such that the average positions of flanking nucleosomes move toward predicted sites. Nucleosome positioning at distinct subsets of promoters additionally requires the essential Myb family proteins Abf1 and Reb1, whose binding sites are enriched in NFRs. In contrast, H2A.Z deposition is dispensable for nucleosome positioning. By regulating H2A.Z deposition using a steroid-inducible protein splicing strategy, we show that NFR establishment is necessary for H2A.Z deposition. These studies suggest an ordered pathway for the assembly of promoter chromatin architecture.

The first gene of the Bacillus subtilis clpC operon, ctsR, encodes a negative regulator of its own operon and other class III heat shock genes.

The Bacillus subtilis clpC operon is regulated by two stress induction pathways relying on either sigmaB or a class III stress induction mechanism acting at a sigmaA-like promoter. When the clpC operon was placed under the control of the isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible Pspac promoter, dramatic repression of the natural clpC promoters fused to a lacZ reporter gene was noticed after IPTG induction. This result strongly indicated negative regulation of the clpC operon by one of its gene products. Indeed, the negative regulator could be identified which is encoded by the first gene of the clpC operon, ctsR, containing a predicted helix-turn-helix DNA-binding motif. Deletion of ctsR abolished the negative regulation and resulted in high expression of both the clpC operon and the clpP gene under nonstressed conditions. Nevertheless, a further increase in clpC and clpP mRNA levels was observed after heat shock, even in the absence of sigmaB, suggesting a second induction mechanism at the vegetative promoter. Two-dimensional gel analysis and mRNA studies showed that the expression of other class III stress genes was at least partially influenced by the ctsR deletion. Studies with different clpC promoter fragments either fused to the reporter gene bgaB or used in gel mobility shift experiments with the purified CtsR protein revealed a possible target region where the repressor seemed to bind in vivo and in vitro. Our data demonstrate that the CtsR protein acts as a global repressor of the clpC operon, as well as other class III heat shock genes, by preventing unstressed transcription from either the sigmaB- or sigmaA-dependent promoter and might be inactivated or dissociate under inducing stress conditions.

A general role for splicing enhancers in exon definition.

Exonic splicing enhancers (ESEs) facilitate exon definition by assisting in the recruitment of splicing factors to the adjacent intron. Here we demonstrate that suboptimal 5' and 3' splice sites are activated independently by ESEs when they are located on different exons. However, when they are situated within a single exon, the same weak 5' and 3' splice sites are activated simultaneously by a single ESE. These findings demonstrate that a single ESE promotes the recognition of both exon/intron junctions within the same step during exon definition. Our results suggest that ESEs recruit a multicomponent complex that minimally contains components of the splicing machinery required for 5' and 3' splice site selection.

Cohesins and condensins orchestrate the 4D dynamics of yeast chromosomes during the cell cycle

Duplication and segregation of chromosomes involves dynamic reorganization of their internal structure by conserved architectural proteins, including the structural maintenance of chromosomes (SMC) complexes cohesin and condensin. Despite active investigation of the roles of these factors, a genome-wide view of dynamic chromosome architecture at both small and large scale during cell division is still missing. Here, we report the first comprehensive 4D analysis of the higher-order organization of the Saccharomyces cerevisiae genome throughout the cell cycle and investigate the roles of SMC complexes in controlling structural transitions. During replication, cohesion establishment promotes numerous long-range intra-chromosomal contacts and correlates with the individualization of chromosomes, which culminates at metaphase. In anaphase, mitotic chromosomes are abruptly reorganized depending on mechanical forces exerted by the mitotic spindle. Formation of a condensin-dependent loop bridging the centromere cluster with the rDNA loci suggests that condensin-mediated forces may also directly facilitate segregation. This work therefore comprehensively recapitulates cell cycle-dependent chromosome dynamics in a unicellular eukaryote, but also unveils new features of chromosome structural reorganization during highly conserved stages of cell division.

Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) offer immense potential for regenerative medicine and studies of disease and development. Somatic cell reprogramming involves epigenomic reconfiguration, conferring iPSCs with characteristics similar to embryonic stem (ES) cells. However, it remains unknown how complete the reestablishment of ES-cell-like DNA methylation patterns is throughout the genome. Here we report the first whole-genome profiles of DNA methylation at single-base resolution in five human iPSC lines, along with methylomes of ES cells, somatic cells, and differentiated iPSCs and ES cells. iPSCs show significant reprogramming variability, including somatic memory and aberrant reprogramming of DNA methylation. iPSCs share megabase-scale differentially methylated regions proximal to centromeres and telomeres that display incomplete reprogramming of non-CG methylation, and differences in CG methylation and histone modifications. Lastly, differentiation of iPSCs into trophoblast cells revealed that errors in reprogramming CG methylation are transmitted at a high frequency, providing an iPSC reprogramming signature that is maintained after differentiation.

CRISPR adaptation biases explain preference for acquisition of foreign DNA

CRISPR-Cas (clustered, regularly interspaced short palindromic repeats coupled with CRISPR-associated proteins) is a bacterial immunity system that protects against invading phages or plasmids. In the process of CRISPR adaptation, short pieces of DNA ('spacers') are acquired from foreign elements and integrated into the CRISPR array. So far, it has remained a mystery how spacers are preferentially acquired from the foreign DNA while the self chromosome is avoided. Here we show that spacer acquisition is replication-dependent, and that DNA breaks formed at stalled replication forks promote spacer acquisition. Chromosomal hotspots of spacer acquisition were confined by Chi sites, which are sequence octamers highly enriched on the bacterial chromosome, suggesting that these sites limit spacer acquisition from self DNA. We further show that the avoidance of self is mediated by the RecBCD double-stranded DNA break repair complex. Our results suggest that, in Escherichia coli, acquisition of new spacers largely depends on RecBCD-mediated processing of double-stranded DNA breaks occurring primarily at replication forks, and that the preference for foreign DNA is achieved through the higher density of Chi sites on the self chromosome, in combination with the higher number of forks on the foreign DNA. This model explains the strong preference to acquire spacers both from high copy plasmids and from phages.

Chk1 inhibits replication factory activation but allows dormant origin firing in existing factories

Replication origins are licensed by loading MCM2-7 hexamers before entry into S phase. However, only ∼10% of licensed origins are normally used in S phase, with the others remaining dormant. When fork progression is inhibited, dormant origins initiate nearby to ensure that all of the DNA is eventually replicated. In apparent contrast, replicative stress activates ataxia telangiectasia and rad-3-related (ATR) and Chk1 checkpoint kinases that inhibit origin firing. In this study, we show that at low levels of replication stress, ATR/Chk1 predominantly suppresses origin initiation by inhibiting the activation of new replication factories, thereby reducing the number of active factories. At the same time, inhibition of replication fork progression allows dormant origins to initiate within existing replication factories. The inhibition of new factory activation by ATR/Chk1 therefore redirects replication toward active factories where forks are inhibited and away from regions that have yet to start replication. This minimizes the deleterious consequences of fork stalling and prevents similar problems from arising in unreplicated regions of the genome.

Meiosis in Drosophila: seeing is believing.

Recently many exciting advances have been achieved in our understanding of Drosophila meiosis due to combined cytological and genetic approaches. New techniques have permitted the characterization of chromosome position and spindle formation in female meiosis I. The proteins encoded by the nod and ncd genes, two genes known to be needed for the proper partitioning of chromosomes lacking exchange events, have been identified and found to be kinesin-like motors. The effects of mutations in these genes on the spindle and chromosomes, together with the localization of the proteins, have yielded a model for the mechanism of female meiosis I. In male meiosis I, the chromosomal regions responsible for homolog pairing have been resolved to the level of specific DNA sequences. This provides a foundation for elucidating the molecular basis of meiotic pairing. The cytological techniques available in Drosophila also have permitted inroads into the regulation of sister-chromatid segregation. The products of two genes (mei-S332 and ord) essential for sister-chromatid cohesion have been identified recently. Additional advances in understanding Drosophila meiosis are the delineation of a functional centromere by using minichromosome derivatives and the identification of several regulatory genes for the meiotic cell cycle.

Using the yeast gene deletion collection to customize gene expression.

Substitute Teacher Despite the relative ease of genome manipulation in S. cerevisiae, researchers are always looking to learn still more convenient and rapid methods for substituting yeast promoters. Replacing a gene's native promoter with a heterolo-gous promoter of choice allows regulated expression and simplifies the task of discerning functional relevance. Although a host of clever chromosomal insertion strategies have been described over the years, the advent of the S. cerevisiae Genome Deletion Project provides an incredible resource for a further streamlined workflow. The strategy, explained by Liko et al. on p. 728 is appealingly simple. The genome deletion project resulted in a collection of strains in which a single ORF is replaced with a kanamycin resistance module. Although the purpose of the collection is to have a comprehensive resource of essentially all possible knockouts, the authors point out that for almost any given yeast promoter of interest there will be a strain in which the ORF imm...

AIR-2: An Aurora/Ipl1-related Protein Kinase Associated with Chromosomes and Midbody Microtubules Is  Required for Polar Body Extrusion and Cytokinesis  in Caenorhabditis elegans Embryos

An emerging family of kinases related to the Drosophila Aurora and budding yeast Ipl1 proteins has been implicated in chromosome segregation and mitotic spindle formation in a number of organisms. Unlike other Aurora/Ipl1-related kinases, the Caenorhabditis elegans orthologue, AIR-2, is associated with meiotic and mitotic chromosomes. AIR-2 is initially localized to the chromosomes of the most mature prophase I–arrested oocyte residing next to the spermatheca. This localization is dependent on the presence of sperm in the spermatheca. After fertilization, AIR-2 remains associated with chromosomes during each meiotic division. However, during both meiotic anaphases, AIR-2 is present between the separating chromosomes. AIR-2 also remains associated with both extruded polar bodies. In the embryo, AIR-2 is found on metaphase chromosomes, moves to midbody microtubules at anaphase, and then persists at the cytokinesis remnant. Disruption of AIR-2 expression by RNA- mediated interference produces entire broods of one-cell embryos that have executed multiple cell cycles in the complete absence of cytokinesis. The embryos accumulate large amounts of DNA and microtubule asters. Polar bodies are not extruded, but remain in the embryo where they continue to replicate. The cytokinesis defect appears to be late in the cell cycle because transient cleavage furrows initiate at the proper location, but regress before the division is complete. Additionally, staining with a marker of midbody microtubules revealed that at least some of the components of the midbody are not well localized in the absence of AIR-2 activity. Our results suggest that during each meiotic and mitotic division, AIR-2 may coordinate the congression of metaphase chromosomes with the subsequent events of polar body extrusion and cytokinesis.

The Evolution of Dominance in Sporophytic Self-Incompatibility Systems. II. Mate Availability and Recombination

Sporophytic self-incompatibility (SSI) is a self-pollen recognition system that enforces outcrossing in plants. Recognition in SSI systems is typically controlled by a complex locus (5-locus) with separate genes that determine pollen and stigma specificity. Experimental studies show that S-alleles can be dominant, recessive, or codominant, and that the dominance level of a given S-allele can depend upon whether pollen or stigma specificity is examined. Here and in the companion paper by Llaurens and colleagues, the evolution of dominance in single-locus SSI is explored using numerical models and simulation. Particular attention is directed at factors that can cause S-allele dominance to differ in pollen versus stigma. The effect of recombination between the S-locus and modifier locus is also examined. The models predict that limitation in the number of compatible mates is required for the evolution of S-allele dominance in the stigma but not in the pollen. Tight linkage between the S-locus and modifier promotes the evolution of S-allele dominance hierarchies. Model results are interpreted with respect to published information on the molecular basis of dominance in SSI systems, and reported S-allele dominance relationships in a variety of species. These studies show that dominant S-alleles are more common in the pollen than in the stigma, a pattern that when interpreted in light of model predictions, suggests that mate limitation may be relatively infrequent in natural populations with SSI.

Swc2 is a widely conserved H2AZ-binding module essential for ATP-dependent histone exchange

The histone variant H2AZ is incorporated preferentially at specific locations in chromatin to modulate chromosome functions. In Saccharomyces cerevisiae, deposition of histone H2AZ is mediated by the multiprotein SWR1 complex, which catalyzes ATP-dependent exchange of nucleosomal histone H2A for H2AZ. Here, we define interactions between SWR1 components and H2AZ, revealing a link between the ATPase domain of Swr1 and three subunits required for the binding of H2AZ. We discovered that Swc2 binds directly to and is essential for transfer of H2AZ. Swc6 and Arp6 are necessary for the association of Swc2 and for nucleosome binding, whereas other subunits, Swc5 and Yaf9, are required for H2AZ transfer but neither H2AZ nor nucleosome binding. Finally, the C-terminal α-helix of H2AZ is crucial for its recognition by SWR1. These findings provide insight on the initial events of histone exchange.

Strong bias in the bacterial CRISPR elements that confer immunity to phage.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems provide adaptive immunity against phage via spacer-encoded CRISPR RNAs that are complementary to invasive nucleic acids. Here, we challenge Streptococcus thermophilus with a bacteriophage, and used PCR-based metagenomics to monitor phage-derived spacers daily for 15 days in two experiments. Spacers that target the host chromosome are infrequent and strongly selected against, suggesting autoimmunity is lethal. In experiments that recover over half a million spacers, we observe early dominance by a few spacer sub-populations and rapid oscillations in sub-population abundances. In two CRISPR systems and in replicate experiments, a few spacers account for the majority of spacer sequences. Nearly all phage locations targeted by the acquired spacers have a proto-spacer adjacent motif (PAM), indicating PAMs are involved in spacer acquisition. We detect a strong and reproducible bias in the phage genome locations from which spacers derive. This may reflect selection for specific spacers based on location and effectiveness.

CDE-1 Affects Chromosome Segregation through Uridylation of CSR-1-Bound siRNAs

We have studied the function of a conserved germline-specific nucleotidyltransferase protein, CDE-1, in RNAi and chromosome segregation in C. elegans. CDE-1 localizes specifically to mitotic chromosomes in embryos. This localization requires the RdRP EGO-1, which physically interacts with CDE-1, and the Argonaute protein CSR-1. We found that CDE-1 is required for the uridylation of CSR-1 bound siRNAs, and that in the absence of CDE-1 these siRNAs accumulate to inappropriate levels, accompanied by defects in both meiotic and mitotic chromosome segregation. Elevated siRNA levels are associated with erroneous gene silencing, most likely through the inappropriate loading of CSR-1 siRNAs into other Argonaute proteins. We propose a model in which CDE-1 restricts specific EGO-1-generated siRNAs to the CSR-1 mediated, chromosome associated RNAi pathway, thus separating it from other endogenous RNAi pathways. The conserved nature of CDE-1 suggests that similar sorting mechanisms may operate in other animals, including mammals.

The Tof1p-Csm3p protein complex counteracts the Rrm3p helicase to control replication termination of Saccharomyces cerevisiae.

Termination of replication forks at the natural termini of the rDNA of Saccharomyces cerevisiae is controlled in a sequence-specific and polar mode by the interaction of the Fob1p replication terminator protein with the tandem Ter sites located in the nontranscribed spacers. Here we show, by both 2D gel analyses and chromatin immunoprecipitations (ChIP), that there exists a second level of global control mediated by the intra-S-phase checkpoint protein complex of Tof1p and Csm3p that protect stalled forks at Ter sites against the activity of the Rrm3p helicase ("sweepase"). The sweepase tends to release arrested forks presumably by the transient displacement of the Ter-bound Fob1p. Consistent with this mechanism, very few replication forks were arrested at the natural replication termini in the absence of the two checkpoint proteins. In the absence of the Rrm3p helicase, there was a slight enhancement of fork arrest at the Ter sites. Simultaneous deletions of the TOF1 (or CSM3), and the RRM3 genes restored fork arrest by removing both the fork-releasing and fork-protection activities. Other genes such as MRC1, WSS1, and PSY2 that are also involved in the MRC1 checkpoint pathway were not involved in this global control. This observation suggests that Tof1p-Csm3p function differently from MRC1 and the other above-mentioned genes. This mechanism is not restricted to the natural Ter sites but was also observed at fork arrest caused by the meeting of a replication fork with transcription approaching from the opposite direction.

Stepwise Histone Replacement by SWR1 Requires Dual Activation with Histone H2A.Z and Canonical Nucleosome

Histone variant H2A.Z-containing nucleosomes are incorporated at most eukaryotic promoters. This incorporation is mediated by the conserved SWR1 complex, which replaces histone H2A in canonical nucleosomes with H2A.Z in an ATP-dependent manner. Here, we show that promoter-proximal nucleosomes are highly heterogeneous for H2A.Z in Saccharomyces cerevisiae, with substantial representation of nucleosomes containing one, two, or zero H2A.Z molecules. SWR1-catalyzed H2A.Z replacement in vitro occurs in a stepwise and unidirectional fashion, one H2A.Z-H2B dimer at a time, producing heterotypic nucleosomes as intermediates and homotypic H2A.Z nucleosomes as end products. The ATPase activity of SWR1 is specifically stimulated by H2A-containing nucleosomes without ensuing histone H2A eviction. Remarkably, further addition of free H2A.Z-H2B dimer leads to hyperstimulation of ATPase activity, eviction of nucleosomal H2A-H2B, and deposition of H2A.Z-H2B. These results suggest that the combination of H2A-containing nucleosome and free H2A.Z-H2B dimer acting as both effector and substrate for SWR1 governs the specificity and outcome of the replacement reaction.

Exploitative and Hierarchical Antagonism in a Cooperative Bacterium

Social organisms that cooperate with some members of their own species, such as close relatives, may fail to cooperate with other genotypes of the same species. Such noncooperation may take the form of outright antagonism or social exploitation. Myxococcus xanthus is a highly social prokaryote that cooperatively develops into spore-bearing, multicellular fruiting bodies in response to starvation. Here we have characterized the nature of social interactions among nine developmentally proficient strains of M. xanthus isolated from spatially distant locations. Strains were competed against one another in all possible pairwise combinations during starvation-induced development. In most pairings, at least one competitor exhibited strong antagonism toward its partner and a majority of mixes showed bidirectional antagonism that decreased total spore production, even to the point of driving whole populations to extinction. Differential response to mixing was the primary determinant of competitive superiority rather than the sporulation efficiencies of unmixed populations. In some competitive pairings, the dominant partner sporulated more efficiently in mixed populations than in clonal isolation. This finding represents a novel form of exploitation in bacteria carried out by socially competent genotypes and is the first documentation of social exploitation among natural bacterial isolates. Patterns of antagonistic superiority among these strains form a highly linear dominance hierarchy. At least some competition pairs construct chimeric, rather than segregated, fruiting bodies. The cooperative prokaryote M. xanthus has diverged into a large number of distinct social types that cooperate with clone-mates but exhibit intense antagonism toward distinct social types of the same species. Most lengthy migration events in nature may thus result in strong antagonism between migratory and resident populations, and this antagonism may have large effects on local population sizes and dynamics. Intense mutual antagonism appears to be more prevalent in this prokaryotic social species than has been observed in the eukaryotic social slime mold Dictyostelium discoideum, which also exhibits multicellular development. The finding of several cases of facultative social exploitation among these natural isolates suggests that such exploitation may occur frequently in nature in many prokaryotes with cooperative traits.

Genomic organization and chromosomal location of the human dsRNA adenosine deaminase gene: the enzyme for glutamate-activated ion channel RNA editing.

The structure of the human gene encoding the double-stranded RNA (dsRNA) adenosine deaminase (DRADA) was characterized. This nuclear localized enzyme is involved in the RNA editing required for the expression of certain subtypes of glutamate-gated ion channel subunits. The DRADA gene span 30 kb pairs and harbors 15 exons. The transcription of the DRADA gene driven by the putative promoter region, which contains no typical TATA or CCAAT box-like sequences, is initiated at multiple sites, 164 to 216 nucleotides upstream of the translation initiation codon. The three dsRNA binding motifs (DRBM), 70 amino acid residues long, are each encoded by two exons plus an intervening sequence that interrupts the motif at the identical amino acid position. This finding is consistent with the notion that the dsRNA binding domains may be composed of two separate functional subdomains. Fluorescent in situ hybridization localized the DRADA gene on the long arm chromosome 1, region q21. The gene structure and sequence information reported in this study will facilitate the investigation of involvement of DRADA in hereditary diseases that may be the result of malfunction of glutamate-gated ion channels.

Mitosis-specific anchoring of gamma tubulin complexes by pericentrin controls spindle organization and mitotic entry.

Microtubule nucleation is the best known function of centrosomes. Centrosomal microtubule nucleation is mediated primarily by gamma tubulin ring complexes (gamma TuRCs). However, little is known about the molecules that anchor these complexes to centrosomes. In this study, we show that the centrosomal coiled-coil protein pericentrin anchors gamma TuRCs at spindle poles through an interaction with gamma tubulin complex proteins 2 and 3 (GCP2/3). Pericentrin silencing by small interfering RNAs in somatic cells disrupted gamma tubulin localization and spindle organization in mitosis but had no effect on gamma tubulin localization or microtubule organization in interphase cells. Similarly, overexpression of the GCP2/3 binding domain of pericentrin disrupted the endogenous pericentrin-gamma TuRC interaction and perturbed astral microtubules and spindle bipolarity. When added to Xenopus mitotic extracts, this domain uncoupled gamma TuRCs from centrosomes, inhibited microtubule aster assembly, and induced rapid disassembly of preassembled asters. All phenotypes were significantly reduced in a pericentrin mutant with diminished GCP2/3 binding and were specific for mitotic centrosomal asters as we observed little effect on interphase asters or on asters assembled by the Ran-mediated centrosome-independent pathway. Additionally, pericentrin silencing or overexpression induced G2/antephase arrest followed by apoptosis in many but not all cell types. We conclude that pericentrin anchoring of gamma tubulin complexes at centrosomes in mitotic cells is required for proper spindle organization and that loss of this anchoring mechanism elicits a checkpoint response that prevents mitotic entry and triggers apoptotic cell death.

Phosphorylation site mutations in heterochromatin protein 1 (HP1) reduce or eliminate silencing activity.

HP1 is an essential heterochromatin-associated protein in Drosophila. HP1 has dosage-dependent effects on the silencing of euchromatic genes that are mislocalized to heterochromatin and is required for the normal expression of at least two heterochromatic genes. HP1 is multiply phosphorylated in vivo, and HP1 hyperphosphorylation is correlated with heterochromatin assembly during development. The purpose of this study was to test whether HP1 phosphorylation modifies biological activity and biochemical properties of HP1. To determine sites of HP1 phosphorylation in vivo and whether phosphorylation affects any biochemical properties of HP1, we expressed Drosophila HP1 in lepidopteran cultured cells using a recombinant baculovirus vector. Phosphopeptides were identified by matrix-assisted laser desorption ionization/time of flight mass spectroscopy; these peptides contain target sites for casein kinase II, protein tyrosine kinase, and PIM-1 kinase. Purified HP1 from bacterial (unphosphorylated) and lepidopteran (phosphorylated) cells has similar secondary structure. Phosphorylation has no effect on HP1 self-association but alters the DNA binding properties of HP1, suggesting that phosphorylation could differentially regulate HP1-dependent interactions. Serine-to-alanine and serine-to-glutamate substitutions at consensus protein kinase motifs resulted in reduction or loss of silencing activity of mutant HP1 in transgenic flies. These results suggest that dynamic phosphorylation/dephosphorylation regulates HP1 activity in heterochromatic silencing.

Genome-Wide Dynamics of Htz1, a Histone H2A Variant that Poises Repressed/Basal Promoters for Activation through Histone Loss

Histone variants help specialize chromatin regions; however, their impact on transcriptional regulation is largely unknown. Here, we determined the genome-wide localization and dynamics of Htz1, the yeast histone H2A variant. Htz1 localizes to hundreds of repressed/basal Pol II promoters and prefers TATA-less promoters. Specific Htz1 deposition requires the SWR1 complex, which largely colocalizes with Htz1. Htz1 occupancy correlates with particular histone modifications, and Htz1 deposition is partially reliant on Gcn5 (a histone acetyltransferase) and Bdf1, an SWR1 complex member that binds acetylated histones. Changes in growth conditions cause a striking redistribution of Htz1 from activated to repressed/basal promoters. Furthermore, Htz1 promotes full gene activation but does not generally impact repression. Importantly, Htz1 releases from purified chromatin in vitro under conditions where H2A and H3 remain associated. We suggest that Htz1-bearing nucleosomes are deposited at repressed/basal promoters but facilitate activation through their susceptibility to loss, thereby helping to expose promoter DNA.

Visualization of cell cycle in mouse embryos with Fucci2 reporter directed by Rosa26 promoter.

Fucci technology makes possible the distinction between live cells in the G(1) and S/G(2)/M phases by dual-color imaging. This technology relies upon ubiquitylation-mediated proteolysis, and transgenic mice expressing Fucci provide a powerful model system with which to study the coordination of the cell cycle and development. The mice were initially generated using the CAG promoter; lines expressing the G(1) and S/G(2)/M phase probes that emitted orange (mKO2) and green (mAG) fluorescence, respectively, were separately constructed. Owing to cell type-biased strength of the CAG promoter as well as the positional effects of random transgenesis, however, we noticed some variability in Fucci expression levels. To control more reliably the expression of cell cycle probes, we used different genetic approaches to create two types of reporter mouse lines with Fucci2 and Rosa26 transcriptional machinery. Fucci2 is a recently developed Fucci derivative, which emits red (mCherry) and green (mVenus) fluorescence and provides better color contrast than Fucci. A new transgenic line, R26p-Fucci2, utilizes the Rosa26 promoter and harbors the G(1) and S/G(2)/M phase probes in a single transgene to preserve their co-inheritance. In the other R26R-Fucci2 approach, the two probes are incorporated into Rosa26 locus conditionally. The Cre-mediated loxP recombination technique thus allows researchers to design cell-type-specific Fucci2 expression. By performing time-lapse imaging experiments using R26p-Fucci2 and R26-Fucci2 in which R26R-Fucci2 had undergone germline loxP recombination, we demonstrated the great promise of these mouse reporters for studying cell cycle behavior in vivo.

Identification of an allele of VAM3/SYP22 that confers a semi-dwarf phenotype in Arabidopsis thaliana.

The short stem and midrib (ssm) mutants of Arabidopsis thaliana show both semi-dwarf and wavy leaf phenotypes due to defects in the elongation of the stem internodes and leaves. Moreover, these abnormalities cannot be recovered by exogenous phytohormones. ssm was originally identified as a single recessive mutant of the ecotype Columbia (Col-0), but genetic crossing experiments have revealed that this mutant phenotype is restored by another gene that is functional in the ecotype Landsberg erecta (Ler) and not in Col-0. Map-based cloning of the gene that is defective in ssm mutants has uncovered a small deletion in the sixth intron of a gene encoding a syntaxin, VAM3/SYP22, which has been implicated in vesicle transport to the vacuole. This mutation appears to cause a peptide insertion in the deduced VAM3/SYP22 polypeptide sequence due to defective splicing of the shortened sixth intron. Significantly, when compared with the wild-type Ler genome, the wild-type Col-0 genome has a single base pair deletion causing a frameshift mutation in SYP23, a gene with the highest known homology to VAM3/SYP22. These findings suggest that VAM3/SYP22 and SYP23 have overlapping functions and that the vesicle transport mediated by these syntaxins is important for shoot morphogenesis.

Regulation of ADMP and BMP2/4/7 at Opposite Embryonic Poles Generates a Self-Regulating Morphogenetic Field

Embryos have the ability to self-regulate and regenerate normal structures after being sectioned in half. How is such a morphogenetic field established? We discovered that quadruple knockdown of ADMP and BMP2/4/7 in Xenopus embryos eliminates self-regulation, causing ubiquitous neural induction throughout the ectoderm. ADMP transcription in the Spemann organizer is activated at low BMP levels. When ventral BMP2/4/7 signals are depleted, Admp expression increases, allowing for self-regulation. ADMP has BMP-like activity and signals via the ALK-2 receptor. It is unable to signal dorsally because of inhibition by Chordin. The ventral BMP antagonists Sizzled and Bambi further refine the pattern. By transplanting dorsal or ventral wild-type grafts into ADMP/BMP2/4/7-depleted hosts, we demonstrate that both poles serve as signaling centers that can induce histotypic differentiation over considerable distances. We conclude that dorsal and ventral BMP signals and their extracellular antagonists expressed under opposing transcriptional regulation provide a molecular mechanism for embryonic self-regulation.

Long noncoding RNA as modular scaffold of histone modification complexes

Long intergenic noncoding RNAs (lincRNAs) regulate chromatin states and epigenetic inheritance. Here, we show that the lincRNA HOTAIR serves as a scaffold for at least two distinct histone modification complexes. A 5' domain of HOTAIR binds polycomb repressive complex 2 (PRC2), whereas a 3' domain of HOTAIR binds the LSD1/CoREST/REST complex. The ability to tether two distinct complexes enables RNA-mediated assembly of PRC2 and LSD1 and coordinates targeting of PRC2 and LSD1 to chromatin for coupled histone H3 lysine 27 methylation and lysine 4 demethylation. Our results suggest that lincRNAs may serve as scaffolds by providing binding surfaces to assemble select histone modification enzymes, thereby specifying the pattern of histone modifications on target genes.

Conversion of the lac repressor into an allosterically regulated transcriptional activator for mammalian cells.

A novel mammalian regulatory system was created by using the Escherichia coli lac repressor. The lac repressor was converted into a mammalian transcriptional activator by modifying the lac repressor coding region to include a nuclear localization signal from the simian virus 40 (SV40) large tumor antigen and the transcription activation domain from the herpes simplex virus type 1 virion protein 16. The lac activator protein (LAP) fusions were potent activators of several promoters containing lac operator sequences positioned either upstream or downstream of the transcription unit. A single lac operator allowed for transactivation, whereas multiple operators acted synergistically when separated by a small distance. Promoters containing 14 or 21 operator sequences were induced at least 1,000-fold in response to LAP, reaching levels of activity 20 to 30 times greater than that of the SV40 early promoter in HeLa cells. Activation was strongly inhibited by isopropyl-beta-D-thiogalactoside (IPTG), indicating that LAP retained the functions needed for allosteric regulation. LAP was bifunctional, also acting as a repressor of expression of an SV40 promoter containing an operator immediately downstream of the TATA box. Finally, genetic selection schemes were developed such that LAP-expressing cell lines can be generated at high frequency from either established or primary cells in culture.

The histone demethylases Jhdm1a/1b enhance somatic cell reprogramming in a vitamin-C-dependent manner.

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) resets the epigenome to an embryonic-like state. Vitamin C enhances the reprogramming process, but the underlying mechanisms are unclear. Here we show that the histone demethylases Jhdm1a/1b are key effectors of somatic cell reprogramming downstream of vitamin C. We first observed that vitamin C induces H3K36me2/3 demethylation in mouse embryonic fibroblasts in culture and during reprogramming. We then identified Jhdm1a/1b, two known vitamin-C-dependent H3K36 demethylases, as potent regulators of reprogramming through gain- and loss-of-function approaches. Furthermore, we found that Jhdm1b accelerates cell cycle progression and suppresses cell senescence during reprogramming by repressing the Ink4/Arf locus. Jhdm1b also cooperates with Oct4 to activate the microRNA cluster 302/367, an integral component of the pluripotency machinery. Our results therefore reveal a role for H3K36me2/3 in cell fate determination and establish a link between histone demethylases and vitamin-C-induced reprogramming.

A promoter derived from taro bacilliform badnavirus drives strong expression in transgenic banana and tobacco plants

Taro bacilliform virus (TaBV) is a pararetrovirus of the genus Badnavirus which infects the monocotyledonous plant, taro (Colocasia esculenta). A region of the TaBV genome spanning nucleotides 6,281 to 12 (T1200), including the 3′ end of open reading frame 3 (ORF 3) and the intergenic region to the end of the tRNAmet-binding site, was tested for promoter activity along with four different 5′ deletion fragments (T600, T500, T250 and T100). In transient assays, only the T1200, T600, T500 fragments were shown to have promoter activity in taro leaf, banana suspension cells and tobacco callus. When these three promoters were evaluated in stably transformed, in vitro-grown transgenic banana and tobacco plants, all were found to drive near-constitutive expression of either the green fluorescent protein or β-glucuronidase (GUS) reporter gene in the stem (or pseudostem), leaves and roots, with strongest expression observed in the vascular tissue. In transgenic banana leaves, the T600 promoter directed four-fold greater GUS activity than that of the T1200, T500 and the maize polyubiquitin-1 promoters. In transgenic tobacco leaves, the levels of GUS expression directed by the three promoters was between four- and ten-fold lower than that of the double Cauliflower mosaic virus 35S promoter. These results indicate that the TaBV-derived promoters may be useful for the high-level constitutive expression of transgenes in either monocotyledonous or dicotyledonous species.

in polyploid species

We developed the Yeast Gene Order Browser (YGOB; http://wolfe.gen.tcd.ie/ygob) to facilitate visual comparisons and computational analysis of synteny relationships in yeasts. The data presented in YGOB, currently covering seven species, are based on sets of homologous genes that have been intensively manually curated based on both sequence similarity and genomic context (synteny). We reconciled different laboratories' lists of paralogous Saccharomyces cerevisiae gene pairs formed by genome duplication (ohnologs), and present near-exhaustive lists of the ohnolog pairs retained in S. cerevisiae (551, including 22 previously unidentified), Saccharomyces castellii (599), and Candida glabrata (404).

Molecular characterization of the yeast meiotic regulatory gene RIM1.

In the yeast Saccharomyces cerevisiae, genetic studies suggest that the RIM1 gene encodes a positive regulator of meiosis. rim1 mutations cause reduced expression of IME1, which is required for expression of many meiotic genes, and thus lead to a partial defect in meiosis and spore formation. We report the sequence of RIM1 and functional analysis of its coding region. The RIM1 gene product (RIM1) contains three regions similar to C2H2 zinc fingers. Serine substitutions for cysteine in each of the putative zinc fingers abolish RIM1 function. The carboxyl-terminus of RIM1 is enriched in acidic amino acids and is required for full RIM1 activity. RIM1 also contains two putative cAMP-dependent protein kinase (cAPK) phosphorylation sites. At one site, substitution of alanine for serine does not affect RIM1 activity; at the other site, this substitution impairs activity. This analysis of RIM1 suggests that the protein may function as a transcriptional activator. We have used the cloned RIM1 gene to create a complete rim1 deletion. This null allele, like previously isolated rim1 mutations, causes a partial meiotic defect. In addition to RIM1, maximum IME1 expression requires the MCK1 and IME4 gene products. Defects associated with rim1, mck1, and ime4 mutations in expression of a meiotic reporter gene (ime2-lacZ) and in sporulation are additive. These findings suggest that RIM1 acts independently of MCK1 and IME4 to stimulate IME1 expression.

Age-Associated Sperm DNA Methylation Alterations: Possible Implications in Offspring Disease Susceptibility

Recent evidence demonstrates a role for paternal aging on offspring disease susceptibility. It is well established that various neuropsychiatric disorders (schizophrenia, autism, etc.), trinucleotide expansion associated diseases (myotonic dystrophy, Huntington's, etc.) and even some forms of cancer have increased incidence in the offspring of older fathers. Despite strong epidemiological evidence that these alterations are more common in offspring sired by older fathers, in most cases the mechanisms that drive these processes are unclear. However, it is commonly believed that epigenetics, and specifically DNA methylation alterations, likely play a role. In this study we have investigated the impact of aging on DNA methylation in mature human sperm. Using a methylation array approach we evaluated changes to sperm DNA methylation patterns in 17 fertile donors by comparing the sperm methylome of 2 samples collected from each individual 9-19 years apart. With this design we have identified 139 regions that are significantly and consistently hypomethylated with age and 8 regions that are significantly hypermethylated with age. A representative subset of these alterations have been confirmed in an independent cohort. A total of 117 genes are associated with these regions of methylation alterations (promoter or gene body). Intriguingly, a portion of the age-related changes in sperm DNA methylation are located at genes previously associated with schizophrenia and bipolar disorder. While our data does not establish a causative relationship, it does raise the possibility that the age-associated methylation of the candidate genes that we observe in sperm might contribute to the increased incidence of neuropsychiatric and other disorders in the offspring of older males. However, further study is required to determine whether, and to what extent, a causative relationship exists.

The essential Schizosaccharomyces pombe Pfh1 DNA helicase promotes fork movement past G-quadruplex motifs to prevent DNA damage

BACKGROUND G-quadruplexes (G4s) are stable non-canonical DNA secondary structures consisting of stacked arrays of four guanines, each held together by Hoogsteen hydrogen bonds. Sequences with the ability to form these structures in vitro, G4 motifs, are found throughout bacterial and eukaryotic genomes. The budding yeast Pif1 DNA helicase, as well as several bacterial Pif1 family helicases, unwind G4 structures robustly in vitro and suppress G4-induced DNA damage in S. cerevisiae in vivo. RESULTS We determined the genomic distribution and evolutionary conservation of G4 motifs in four fission yeast species and investigated the relationship between G4 motifs and Pfh1, the sole S. pombe Pif1 family helicase. Using chromatin immunoprecipitation combined with deep sequencing, we found that many G4 motifs in the S. pombe genome were associated with Pfh1. Cells depleted of Pfh1 had increased fork pausing and DNA damage near G4 motifs, as indicated by high DNA polymerase occupancy and phosphorylated histone H2A, respectively. In general, G4 motifs were underrepresented in genes. However, Pfh1-associated G4 motifs were located on the transcribed strand of highly transcribed genes significantly more often than expected, suggesting that Pfh1 has a function in replication or transcription at these sites. CONCLUSIONS In the absence of functional Pfh1, unresolved G4 structures cause fork pausing and DNA damage of the sort associated with human tumors.

Conserved Histone Variant H2A.Z Protects Euchromatin from the Ectopic Spread of Silent Heterochromatin

Boundary elements hinder the spread of heterochromatin, yet these sites do not fully account for the preservation of adjacent euchromatin. Histone variant H2A.Z (Htz1 in yeast) replaces conventional H2A in many nucleosomes. Microarray analysis revealed that HTZ1-activated genes cluster near telomeres. The reduced expression of most of these genes in htz1Delta cells was reversed by the deletion of SIR2 (sir2Delta) suggesting that H2A.Z antagonizes telomeric silencing. Other Htz1-activated genes flank the silent HMR mating-type locus. Their requirement for Htz1 can be bypassed by sir2Delta or by a deletion encompassing the silencing nucleation sites in HMR. In htz1Delta cells, Sir2 and Sir3 spread into flanking euchromatic regions, producing changes in histone H4 acetylation and H3 4-methylation indicative of ectopic heterochromatin formation. Htz1 is enriched in these euchromatic regions and acts synergistically with a boundary element to prevent the spread of heterochromatin. Thus, euchromatin and heterochromatin each contains components that antagonize switching to the opposite chromatin state.

Global Reorganization of Replication Domains During Embryonic Stem Cell Differentiation

DNA replication in mammals is regulated via the coordinate firing of clusters of replicons that duplicate megabase-sized chromosome segments at specific times during S-phase. Cytogenetic studies show that these "replicon clusters" coalesce as subchromosomal units that persist through multiple cell generations, but the molecular boundaries of such units have remained elusive. Moreover, the extent to which changes in replication timing occur during differentiation and their relationship to transcription changes has not been rigorously investigated. We have constructed high-resolution replication-timing profiles in mouse embryonic stem cells (mESCs) before and after differentiation to neural precursor cells. We demonstrate that chromosomes can be segmented into multimegabase domains of coordinate replication, which we call "replication domains," separated by transition regions whose replication kinetics are consistent with large originless segments. The molecular boundaries of replication domains are remarkably well conserved between distantly related ESC lines and induced pluripotent stem cells. Unexpectedly, ESC differentiation was accompanied by the consolidation of smaller differentially replicating domains into larger coordinately replicated units whose replication time was more aligned to isochore GC content and the density of LINE-1 transposable elements, but not gene density. Replication-timing changes were coordinated with transcription changes for weak promoters more than strong promoters, and were accompanied by rearrangements in subnuclear position. We conclude that replication profiles are cell-type specific, and changes in these profiles reveal chromosome segments that undergo large changes in organization during differentiation. Moreover, smaller replication domains and a higher density of timing transition regions that interrupt isochore replication timing define a novel characteristic of the pluripotent state.

Rapid accumulation of mutations during seed-to-seed propagation of mismatch-repair-defective Arabidopsis.

During the many cell divisions that precede formation of plant gametes, their apical-meristem and floral antecedents are continually exposed to endogenous and environmental mutagenic threats. Although some deleterious recessive mutations may be eliminated during growth of haploid gametophytes and functionally haploid early embryos ("haplosufficiency quality-checking"), the multiplicity of plant genome-maintenance systems suggests aggressive quality control during prior diploid growth. To test in Arabidopsis a hypothesis that prior mismatch repair (MMR) is paramount in defense of plant genetic fidelity, we propagated in parallel 36 MMR-defective (Atmsh2-1) and 36 wild-type lines. The Atmsh2-1 lines rapidly accumulated a wide variety of mutations: fifth-generation (G5) plants showed abnormalities in morphology and development, fertility, germination efficiency, seed/silique development, and seed set. Only two Atmsh2-1, but all 36 wild-type lines, appeared normal at G5. Analyses of insertion/deletion mutation at six repeat-sequence (microsatellite) loci showed each Atmsh2-1 line to have evolved its own "fingerprint," the results of as many as 10 microsatellite mutations in a single line. Thus, MMR during diploid growth is essential for plant genomic integrity.

Genetic analysis of an ARS element from the fission yeast Schizosaccharomyces pombe.

ARS (autonomously replicating sequence) elements are DNA fragments that can function as origins of DNA replication in yeast. We report the first fine-structure analysis of ars1, an ARS element of the fission yeast Schizosaccharomyces pombe. Characterization of a series of nested deletion mutations indicated that the minimal fragment of DNA encompassing ars1 is surprisingly large. No fragment < 650 bp retained significant ARS activity. Analysis of deletion and substitution mutations scanning the entire minimal ars1 identified a single essential 50 bp fragment (segment 1). Only one other 50 bp mutation reduced activity as much as 5-fold and most deletions were without effect. Thus, the minimal ars1 is composed of two general types of genetic elements, a small segment that is absolutely required for efficient ARS activity and a much larger region that is tolerant of internal structural alterations. Higher resolution analysis of segment 1 defined a critical 30 bp A/T-rich segment which appears to contain redundant genetic elements. Schizosaccharomyces pombe ars1 promoted high frequency transformation in the budding yeast S.cerevisiae but this heterologous activity was not dependent on segment 1. Our analysis indicates that the functional elements required for ARS function in S.pombe and S.cerevisiae are clearly different.

DNA methylation and healthy human aging

The process of aging results in a host of changes at the cellular and molecular levels, which include senescence, telomere shortening, and changes in gene expression. Epigenetic patterns also change over the lifespan, suggesting that epigenetic changes may constitute an important component of the aging process. The epigenetic mark that has been most highly studied is DNA methylation, the presence of methyl groups at CpG dinucleotides. These dinucleotides are often located near gene promoters and associate with gene expression levels. Early studies indicated that global levels of DNA methylation increase over the first few years of life and then decrease beginning in late adulthood. Recently, with the advent of microarray and next-generation sequencing technologies, increases in variability of DNA methylation with age have been observed, and a number of site-specific patterns have been identified. It has also been shown that certain CpG sites are highly associated with age, to the extent that prediction models using a small number of these sites can accurately predict the chronological age of the donor. Together, these observations point to the existence of two phenomena that both contribute to age-related DNA methylation changes: epigenetic drift and the epigenetic clock. In this review, we focus on healthy human aging throughout the lifetime and discuss the dynamics of DNA methylation as well as how interactions between the genome, environment, and the epigenome influence aging rates. We also discuss the impact of determining 'epigenetic age' for human health and outline some important caveats to existing and future studies.

Trans-ancestry mutational landscape of hepatocellular carcinoma genomes

Diverse epidemiological factors are associated with hepatocellular carcinoma (HCC) prevalence in different populations. However, the global landscape of the genetic changes in HCC genomes underpinning different epidemiological and ancestral backgrounds still remains uncharted. Here a collection of data from 503 liver cancer genomes from different populations uncovered 30 candidate driver genes and 11 core pathway modules. Furthermore, a collaboration of two large-scale cancer genome projects comparatively analyzed the trans-ancestry substitution signatures in 608 liver cancer cases and identified unique mutational signatures that predominantly contribute to Asian cases. This work elucidates previously unexplored ancestry-associated mutational processes in HCC development. A combination of hotspot TERT promoter mutation, TERT focal amplification and viral genome integration occurs in more than 68% of cases, implicating TERT as a central and ancestry-independent node of hepatocarcinogenesis. Newly identified alterations in genes encoding metabolic enzymes, chromatin remodelers and a high proportion of mTOR pathway activations offer potential therapeutic and diagnostic opportunities.

Eic1 links Mis18 with the CCAN/Mis6/Ctf19 complex to promote CENP-A assembly

CENP-A chromatin forms the foundation for kinetochore assembly. Replication-independent incorporation of CENP-A at centromeres depends on its chaperone HJURP(Scm3), and Mis18 in vertebrates and fission yeast. The recruitment of Mis18 and HJURP(Scm3) to centromeres is cell cycle regulated. Vertebrate Mis18 associates with Mis18BP1(KNL2), which is critical for the recruitment of Mis18 and HJURP(Scm3). We identify two novel fission yeast Mis18-interacting proteins (Eic1 and Eic2), components of the Mis18 complex. Eic1 is essential to maintain Cnp1(CENP-A) at centromeres and is crucial for kinetochore integrity; Eic2 is dispensable. Eic1 also associates with Fta7(CENP-Q/Okp1), Cnl2(Nkp2) and Mal2(CENP-O/Mcm21), components of the constitutive CCAN/Mis6/Ctf19 complex. No Mis18BP1(KNL2) orthologue has been identified in fission yeast, consequently it remains unknown how the key Cnp1(CENP-A) loading factor Mis18 is recruited. Our findings suggest that Eic1 serves a function analogous to that of Mis18BP1(KNL2), thus representing the functional counterpart of Mis18BP1(KNL2) in fission yeast that connects with a module within the CCAN/Mis6/Ctf19 complex to allow the temporally regulated recruitment of the Mis18/Scm3(HJURP) Cnp1(CENP-A) loading factors. The novel interactions identified between CENP-A loading factors and the CCAN/Mis6/Ctf19 complex are likely to also contribute to CENP-A maintenance in other organisms.

Siblings with opposite chromosome constitutions, dup(2q)/del(7q) and del(2q)/dup(7q).

Chromosome 7q36 microdeletion syndrome is a rare genomic disorder characterized by underdevelopment of the brain, microcephaly, anomalies of the sex organs, and language problems. Developmental delay, intellectual disability, autistic spectrum disorders, BDMR syndrome, and unusual facial morphology are the key features of the chromosome 2q37 microdeletion syndrome. A genetic screening for two brothers with global developmental delay using high-resolution chromosomal analysis and subtelomeric multiplex ligation-dependent probe amplification revealed subtelomeric rearrangements on the same sites of 2q37.2 and 7q35, with reversed deletion and duplication. Both of them showed dysmorphic facial features, severe disability of physical and intellectual development, and abnormal genitalia with differential abnormalities in their phenotypes. The family did not have abnormal genetic phenotypes. According to the genetic analysis of their parents, adjacent-1 segregation from their mother's was suggested as a mechanism of their gene mutation. By comparing the phenotypes of our patients with previous reports on similar patients, we tried to obtain the information of related genes and their chromosomal locations.

Condensin-mediated remodeling of the mitotic chromatin landscape in fission yeast

The eukaryotic genome consists of DNA molecules far longer than the cells that contain them. They reach their greatest compaction during chromosome condensation in mitosis. This process is aided by condensin, a structural maintenance of chromosomes (SMC) family member. The spatial organization of mitotic chromosomes and how condensin shapes chromatin architecture are not yet fully understood. Here we use chromosome conformation capture (Hi-C) to study mitotic chromosome condensation in the fission yeast Schizosaccharomyces pombe. This showed that the interphase landscape characterized by small chromatin domains is replaced by fewer but larger domains in mitosis. Condensin achieves this by setting up longer-range, intrachromosomal DNA interactions, which compact and individualize chromosomes. At the same time, local chromatin contacts are constrained by condensin, with profound implications for local chromatin function during mitosis. Our results highlight condensin as a major determinant that changes the chromatin landscape as cells prepare their genomes for cell division.

Genome-wide dFOXO targets and topology of the transcriptomic response to stress and insulin signalling

FoxO transcription factors, inhibited by insulin/insulin-like growth factor signalling (IIS), are crucial players in numerous organismal processes including lifespan. Using genomic tools, we uncover over 700 direct dFOXO targets in adult female Drosophila. dFOXO is directly required for transcription of several IIS components and interacting pathways, such as TOR, in the wild-type fly. The genomic locations occupied by dFOXO in adults are different from those observed in larvae or cultured cells. These locations remain unchanged upon activation by stresses or reduced IIS, but the binding is increased and additional targets activated upon genetic reduction in IIS. We identify the part of the IIS transcriptional response directly controlled by dFOXO and the indirect effects and show that parts of the transcriptional response to IIS reduction do not require dfoxo. Promoter analyses revealed GATA and other forkhead factors as candidate mediators of the indirect and dfoxo-independent effects. We demonstrate genome-wide evolutionary conservation of dFOXO targets between the fly and the worm Caenorhabditis elegans, enriched for a second tier of regulators including the dHR96/daf-12 nuclear hormone receptor.

NuA4 lysine acetyltransferase Esa1 is targeted to coding regions and stimulates transcription elongation with Gcn5.

NuA4, the major H4 lysine acetyltransferase (KAT) complex in Saccharomyces cerevisiae, is recruited to promoters and stimulates transcription initiation. NuA4 subunits contain domains that bind methylated histones, suggesting that histone methylation should target NuA4 to coding sequences during transcription elongation. We show that NuA4 is cotranscriptionally recruited, dependent on its physical association with elongating polymerase II (Pol II) phosphorylated on the C-terminal domain by cyclin-dependent kinase 7/Kin28, but independently of subunits (Eaf1 and Tra1) required for NuA4 recruitment to promoters. Whereas histone methylation by Set1 and Set2 is dispensable for NuA4's interaction with Pol II and targeting to some coding regions, it stimulates NuA4-histone interaction and H4 acetylation in vivo. The NuA4 KAT, Esa1, mediates increased H4 acetylation and enhanced RSC occupancy and histone eviction in coding sequences and stimulates the rate of transcription elongation. Esa1 cooperates with the H3 KAT in SAGA, Gcn5, to enhance these functions. Our findings delineate a pathway for acetylation-mediated nucleosome remodeling and eviction in coding sequences that stimulates transcription elongation by Pol II in vivo.

Interplay between microtubule bundling and sorting factors ensures acentriolar spindle stability during C. elegans oocyte meiosis

In many species, oocyte meiosis is carried out in the absence of centrioles. As a result, microtubule organization, spindle assembly, and chromosome segregation proceed by unique mechanisms. Here, we report insights into the principles underlying this specialized form of cell division, through studies of C. elegans KLP-15 and KLP-16, two highly homologous members of the kinesin-14 family of minus-end-directed kinesins. These proteins localize to the acentriolar oocyte spindle and promote microtubule bundling during spindle assembly; following KLP-15/16 depletion, microtubule bundles form but then collapse into a disorganized array. Surprisingly, despite this defect we found that during anaphase, microtubules are able to reorganize into a bundled array that facilitates chromosome segregation. This phenotype therefore enabled us to identify factors promoting microtubule organization during anaphase, whose contributions are normally undetectable in wild-type worms; we found that SPD-1 (PRC1) bundles microtubules and KLP-18 (kinesin-12) likely sorts those bundles into a functional orientation capable of mediating chromosome segregation. Therefore, our studies have revealed an interplay between distinct mechanisms that together promote spindle formation and chromosome segregation in the absence of structural cues such as centrioles.

Dynamic analysis of filopodial interactions during the zippering phase of Drosophila dorsal closure.

Dorsal closure is a paradigm epithelial fusion episode that occurs late in Drosophila embryogenesis and leads to sealing of a midline hole by bonding of two opposing epithelial sheets. The leading edge epithelial cells express filopodia and fusion is dependent on interdigitation of these filopodia to prime formation of adhesions. Since the opposing epithelia are molecularly patterned there must exist some mechanism for accurately aligning the two sheets across this fusion seam. To address this, we generated a fly in which RFP-Moesin and GFP-Moesin are expressed in mutually exclusive stripes within each segment using the engrailed and patched promoters. We observe mutually exclusive interactions between the filopodia of engrailed and patched cells. Interactions between filopodia from matching cells leads to formation of tethers between them, and these tethers can pull misaligned epithelial sheets into alignment. Filopodial matching also occurs during repair of laser wounds in the ventral epithelium, and so this behaviour is not restricted to leading edge cells during dorsal closure. Finally, we characterise the behaviour of a patched-expressing cell that we observe within the engrailed region of segments A1-A5, and provide evidence that this cell contributes to cell matching.

KAP1 controls endogenous retroviruses in embryonic stem cells

More than forty per cent of the mammalian genome is derived from retroelements, of which about one-quarter are endogenous retroviruses (ERVs). Some are still active, notably in mice the highly polymorphic early transposon (ETn)/MusD and intracisternal A-type particles (IAP). ERVs are transcriptionally silenced during early embryogenesis by histone and DNA methylation (and reviewed in ref. 7), although the initiators of this process, which is essential to protect genome integrity, remain largely unknown. KAP1 (KRAB-associated protein 1, also known as tripartite motif-containing protein 28, TRIM28) represses genes by recruiting the histone methyltransferase SETDB1, heterochromatin protein 1 (HP1) and the NuRD histone deacetylase complex, but few of its physiological targets are known. Two lines of evidence suggest that KAP1-mediated repression could contribute to the control of ERVs: first, KAP1 can trigger permanent gene silencing during early embryogenesis, and second, a KAP1 complex silences the retrovirus murine leukaemia virus in embryonic cells. Consistent with this hypothesis, here we show that KAP1 deletion leads to a marked upregulation of a range of ERVs, in particular IAP elements, in mouse embryonic stem (ES) cells and in early embryos. We further demonstrate that KAP1 acts synergistically with DNA methylation to silence IAP elements, and that it is enriched at the 5′ untranslated region (5′UTR) of IAP genomes, where KAP1 deletion leads to the loss of histone 3 lysine 9 trimethylation (H3K9me3), a hallmark of KAP1-mediated repression. Correspondingly, IAP 5′UTR sequences can impose in cis KAP1-dependent repression on a heterologous promoter in ES cells. Our results establish that KAP1 controls endogenous retroelements during early embryonic development.

The RNA Polymerase “Switch Region” Is a Target for Inhibitors

The alpha-pyrone antibiotic myxopyronin (Myx) inhibits bacterial RNA polymerase (RNAP). Here, through a combination of genetic, biochemical, and structural approaches, we show that Myx interacts with the RNAP "switch region"--the hinge that mediates opening and closing of the RNAP active center cleft--to prevent interaction of RNAP with promoter DNA. We define the contacts between Myx and RNAP and the effects of Myx on RNAP conformation and propose that Myx functions by interfering with opening of the RNAP active-center cleft during transcription initiation. We further show that the structurally related alpha-pyrone antibiotic corallopyronin (Cor) and the structurally unrelated macrocyclic-lactone antibiotic ripostatin (Rip) function analogously to Myx. The RNAP switch region is distant from targets of previously characterized RNAP inhibitors, and, correspondingly, Myx, Cor, and Rip do not exhibit crossresistance with previously characterized RNAP inhibitors. The RNAP switch region is an attractive target for identification of new broad-spectrum antibacterial therapeutic agents.

Integration-free induced pluripotent stem cells model genetic and neural developmental features of down syndrome etiology.

Down syndrome (DS) is the most frequent cause of human congenital mental retardation. Cognitive deficits in DS result from perturbations of normal cellular processes both during development and in adult tissues, but the mechanisms underlying DS etiology remain poorly understood. To assess the ability of induced pluripotent stem cells (iPSCs) to model DS phenotypes, as a prototypical complex human disease, we generated bona fide DS and wild-type (WT) nonviral iPSCs by episomal reprogramming. DS iPSCs selectively overexpressed chromosome 21 genes, consistent with gene dosage, which was associated with deregulation of thousands of genes throughout the genome. DS and WT iPSCs were neurally converted at >95% efficiency and had remarkably similar lineage potency, differentiation kinetics, proliferation, and axon extension at early time points. However, at later time points DS cultures showed a twofold bias toward glial lineages. Moreover, DS neural cultures were up to two times more sensitive to oxidative stress-induced apoptosis, and this could be prevented by the antioxidant N-acetylcysteine. Our results reveal a striking complexity in the genetic alterations caused by trisomy 21 that are likely to underlie DS developmental phenotypes, and indicate a central role for defective early glial development in establishing developmental defects in DS brains. Furthermore, oxidative stress sensitivity is likely to contribute to the accelerated neurodegeneration seen in DS, and we provide proof of concept for screening corrective therapeutics using DS iPSCs and their derivatives. Nonviral DS iPSCs can therefore model features of complex human disease in vitro and provide a renewable and ethically unencumbered discovery platform.

DNA joint dependence of pol X family polymerase action in nonhomologous end joining.

DNA double strand breaks (DSBs) can be rejoined directly by the nonhomologous end-joining (NHEJ) pathway of repair. Nucleases and polymerases are required to promote accurate NHEJ when the terminal bases of the DSB are damaged. The same enzymes also participate in imprecise rejoining and joining of incompatible ends, important mutagenic events. Previous work has shown that the Pol X family polymerase Pol4 is required for some but not all NHEJ events that require gap filling in Saccharomyces cerevisiae. Here, we systematically analyzed DSB end configurations and found that gaps on both strands and overhang polarity are the principal factors that determine whether a joint requires Pol4. DSBs with 3'-overhangs and a gap on each strand strongly depended on Pol4 for repair, DSBs with 5'-overhangs of the same sequence did not. Pol4 was not required when 3'-overhangs contained a gap on only one strand, however. Pol4 was equally required at 3'-overhangs of all lengths within the NHEJ-dependent range but was dispensable outside of this range, indicating that Pol4 is specific to NHEJ. Loss of Pol4 did not affect the rejoining of DSBs that utilized a recessed microhomology or DSBs bearing 5'-hydroxyls but no gap. Finally, mammalian Pol X polymerases were able to differentially complement a pol4 mutation depending on the joint structure, demonstrating that these polymerases can participate in yeast NHEJ but with distinct properties.

Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells

Post-transcriptional modification of RNA nucleosides occurs in all living organisms. Pseudouridine, the most abundant modified nucleoside in non-coding RNAs, enhances the function of transfer RNA and ribosomal RNA by stabilizing the RNA structure. Messenger RNAs were not known to contain pseudouridine, but artificial pseudouridylation dramatically affects mRNA function--it changes the genetic code by facilitating non-canonical base pairing in the ribosome decoding centre. However, without evidence of naturally occurring mRNA pseudouridylation, its physiological relevance was unclear. Here we present a comprehensive analysis of pseudouridylation in Saccharomyces cerevisiae and human RNAs using Pseudo-seq, a genome-wide, single-nucleotide-resolution method for pseudouridine identification. Pseudo-seq accurately identifies known modification sites as well as many novel sites in non-coding RNAs, and reveals hundreds of pseudouridylated sites in mRNAs. Genetic analysis allowed us to assign most of the new modification sites to one of seven conserved pseudouridine synthases, Pus1-4, 6, 7 and 9. Notably, the majority of pseudouridines in mRNA are regulated in response to environmental signals, such as nutrient deprivation in yeast and serum starvation in human cells. These results suggest a mechanism for the rapid and regulated rewiring of the genetic code through inducible mRNA modifications. Our findings reveal unanticipated roles for pseudouridylation and provide a resource for identifying the targets of pseudouridine synthases implicated in human disease.

A conditionally lethal yeast mutant blocked at the first step in glycosyl phosphatidylinositol anchor synthesis.

Glycosyl phosphatidylinositols (GPIs) anchor many proteins to the surface of eukaryotic cells and may also serve as sorting signals on proteins and participate in signal transduction. We have isolated a Saccharomyces cerevisiae GPI anchoring mutant, gpi1, using a colony screen for cells blocked in [3H]inositol incorporation into protein. The gpi1 mutant is defective in vitro in the synthesis of N-acetylglucosaminyl phosphatidylinositol, the first intermediate in GPI synthesis, and is also temperature-sensitive for growth. Completion of the first step in GPI assembly is therefore required for growth of the unicellular eukaryote S. cerevisiae. GPI synthesis could therefore be exploited as a target for antifungal or antiparasitic agents.

Breaking the code of DNA binding specificity of TAL-type III effectors.

The pathogenicity of many bacteria depends on the injection of effector proteins via type III secretion into eukaryotic cells in order to manipulate cellular processes. TAL (transcription activator-like) effectors from plant pathogenic Xanthomonas are important virulence factors that act as transcriptional activators in the plant cell nucleus, where they directly bind to DNA via a central domain of tandem repeats. Here, we show how target DNA specificity of TAL effectors is encoded. Two hypervariable amino acid residues in each repeat recognize one base pair in the target DNA. Recognition sequences of TAL effectors were predicted and experimentally confirmed. The modular protein architecture enabled the construction of artificial effectors with new specificities. Our study describes the functionality of a distinct type of DNA binding domain and allows the design of DNA binding domains for biotechnology.

Histone Methylation-Dependent Mechanisms Impose Ligand Dependency for Gene Activation by Nuclear Receptors

Nuclear receptors undergo ligand-dependent conformational changes that are required for corepressor-coactivator exchange, but whether there is an actual requirement for specific epigenetic landmarks to impose ligand dependency for gene activation remains unknown. Here we report an unexpected and general strategy that is based on the requirement for specific cohorts of inhibitory histone methyltransferases (HMTs) to impose gene-specific gatekeeper functions that prevent unliganded nuclear receptors and other classes of regulated transcription factors from binding to their target gene promoters and causing constitutive gene activation in the absence of stimulating signals. This strategy, based at least in part on an HMT-dependent inhibitory histone code, imposes a requirement for specific histone demethylases, including LSD1, to permit ligand- and signal-dependent activation of regulated gene expression. These events link an inhibitory methylation component of the histone code to a broadly used strategy that circumvents pathological constitutive gene induction by physiologically regulated transcription factors.

Activator Control of Nucleosome Occupancy in Activation and Repression of Transcription

The relationship between chromatin structure and gene expression is a subject of intense study. The universal transcriptional activator Gal4 removes promoter nucleosomes as it triggers transcription, but how it does so has remained obscure. The reverse process, repression of transcription, has often been correlated with the presence of nucleosomes. But it is not known whether nucleosomes are required for that effect. A new quantitative assay describes, for any given location, the fraction of DNA molecules in the population that bears a nucleosome at any given instant. This allows us to follow the time courses of nucleosome removal and reformation, in wild-type and mutant cells, upon activation (by galactose) and repression (by glucose) of the GAL genes of yeast. We show that upon being freed of its inhibitor Gal80 by the action of galactose, Gal4 quickly recruits SWI/SNF to the genes, and that nucleosome "remodeler" rapidly removes promoter nucleosomes. In the absence of SWI/SNF, Gal4's action also results in nucleosome removal and the activation of transcription, but both processes are significantly delayed. Addition of glucose to cells growing in galactose represses transcription. But if galactose remains present, Gal4 continues to work, recruiting SWI/SNF and maintaining the promoter nucleosome-free despite it being repressed. This requirement for galactose is obviated in a mutant in which Gal4 works constitutively. These results show how an activator's recruiting function can control chromatin structure both during gene activation and repression. Thus, both under activating and repressing conditions, the activator can recruit an enzymatic machine that removes promoter nucleosomes. Our results show that whereas promoter nucleosome removal invariably accompanies activation, reformation of nucleosomes is not required for repression. The finding that there are two routes to nucleosome removal and activation of transcription-one that requires the action of SWI/SNF recruited by the activator, and a slower one that does not-clarifies our understanding of the early events of gene activation, and in particular corrects earlier reports that SWI/SNF plays no role in GAL gene induction. Our finding that chromatin structure is irrelevant for repression as studied here-that is, repression sets in as efficiently whether or not promoter nucleosomes are allowed to reform-contradicts the widely held, but little tested, idea that nucleosomes are required for repression. These findings were made possible by our nucleosome occupancy assay. The assay, we believe, will prove useful in studying other outstanding issues in the field.

Unmarked gene deletion and host–vector system for the hyperthermophilic crenarchaeon Sulfolobus islandicus

Sulfolobus islandicus is being used as a model for studying archaeal biology, geo-biology and evolution. However, no genetic system is available for this organism. To produce an S. islandicus mutant suitable for genetic analyses, we screened for colonies with a spontaneous pyrEF mutation. One mutant was obtained containing only 233 bp of the original pyrE sequence in the mutant allele and it was used as a host to delete the β-glycosidase (lacS) gene. Two unmarked gene deletion methods were employed, namely plasmid integration and segregation, and marker replacement and looping out, and unmarked lacS mutants were obtained by each method. A new alternative recombination mechanism, i.e., marker circularization and integration, was shown to operate in the latter method, which did not yield the designed deletion mutation. Subsequently, Sulfolobus–E. coli plasmid shuttle vectors were constructed, which genetically complemented ΔpyrEFΔlacS mutation after transformation. Thus, a complete set of genetic tools was established for S. islandicus with pyrEF and lacS as genetic markers.

Generation of mice deficient in both KLF3/BKLF and KLF8 reveals a genetic interaction and a role for these factors in embryonic globin gene silencing.

Krüppel-like factors 3 and 8 (KLF3 and KLF8) are highly related transcriptional regulators that bind to similar sequences of DNA. We have previously shown that in erythroid cells there is a regulatory hierarchy within the KLF family, whereby KLF1 drives the expression of both the Klf3 and Klf8 genes and KLF3 in turn represses Klf8 expression. While the erythroid roles of KLF1 and KLF3 have been explored, the contribution of KLF8 to this regulatory network has been unknown. To investigate this, we have generated a mouse model with disrupted KLF8 expression. While these mice are viable, albeit with a reduced life span, mice lacking both KLF3 and KLF8 die at around embryonic day 14.5 (E14.5), indicative of a genetic interaction between these two factors. In the fetal liver, Klf3 Klf8 double mutant embryos exhibit greater dysregulation of gene expression than either of the two single mutants. In particular, we observe derepression of embryonic, but not adult, globin expression. Taken together, these results suggest that KLF3 and KLF8 have overlapping roles in vivo and participate in the silencing of embryonic globin expression during development.

Isolation of a Fusion Transcript, AMLl IETO, With Similarity to

We have developed a restriction map of the chromosome 21 breakpoint region involved in t(8;21)(q22;q22.3) acute myelogenous leukemia (AML) and have isolated a genomic junction clone containing chromosome 8 and 21 material. Using probes from these regions, rearrangements have been identified in each of nine cases of t(8;21) AML examined. In addition, we have isolated cDNA clones from a t(8;21) AML cDNA library that contain fused sequences from chromosome 8 and 21. The chromosome 8 component, referred to as ETO (for eight twenty-one), is encoded over a large genomic region, as suggested by the analysis of corresponding yeast artificial chromosomes (YACs). The DNA sequence of the chromosome 21 portion of the fusion transcript is derived from the normal AML1 gene. A striking similarity (67% identity over 387 bp, with a corresponding 69% amino acid identity) was detected between AML1 and the Drosophila segmentation gene, runt. The critical consequence of the translocation is the juxtaposition of 5' sequences of AML1 to 3' sequences of ETO, oriented telomere to centromere on the der(8) chromosome.

Functional Consequences of Splicing of the Antisense Transcript COOLAIR on FLC Transcription

Antisense transcription is widespread in many genomes; however, how much is functional is hotly debated. We are investigating functionality of a set of long noncoding antisense transcripts, collectively called COOLAIR, produced at Arabidopsis FLOWERING LOCUS C (FLC). COOLAIR initiates just downstream of the major sense transcript poly(A) site and terminates either early or extends into the FLC promoter region. We now show that splicing of COOLAIR is functionally important. This was revealed through analysis of a hypomorphic mutation in the core spliceosome component PRP8. The prp8 mutation perturbs a cotranscriptional feedback mechanism linking COOLAIR processing to FLC gene body histone demethylation and reduced FLC transcription. The importance of COOLAIR splicing in this repression mechanism was confirmed by disrupting COOLAIR production and mutating the COOLAIR proximal splice acceptor site. Our findings suggest that altered splicing of a long noncoding transcript can quantitatively modulate gene expression through cotranscriptional coupling mechanisms.

Genome-wide identification of genes directly regulated by the pleiotropic transcription factor Spx in Bacillus subtilis

The transcriptional regulator Spx plays a key role in maintaining the redox homeostasis of Bacillus subtilis cells exposed to disulfide stress. Defects in Spx were previously shown to lead to differential expression of numerous genes but direct and indirect regulatory effects could not be distinguished. Here we identified 283 discrete chromosomal sites potentially bound by the Spx-RNA polymerase (Spx-RNAP) complex using chromatin immunoprecipitation of Spx. Three quarters of these sites were located near Sigma(A)-dependent promoters, and upon diamide treatment, the fraction of the Spx-RNAP complex increased in parallel with the number and occupancy of DNA sites. Correlation of Spx-RNAP-binding sites with gene differential expression in wild-type and Δspx strains exposed or not to diamide revealed that 144 transcription units comprising 275 genes were potentially under direct Spx regulation. Spx-controlled promoters exhibited an extended -35 box in which nucleotide composition at the -43/-44 positions strongly correlated with observed activation. In vitro transcription confirmed activation by oxidized Spx of seven newly identified promoters, of which one was also activated by reduced Spx. Our study globally characterized the Spx regulatory network, revealing its role in the basal expression of some genes and its complex interplay with other stress responses.

Regulation of poly(A) site choice of several yeast mRNAs

Several yeast genes produce multiple transcripts with different 3'-ends. Of these, four genes are known to produce truncated transcripts that end within the coding sequence of longer transcripts: CBP1 , AEP2 / ATP13 , RNA14 and SIR1 . It has been shown that the level of the truncated CBP1 transcript increases during the switch to respiratory growth while that of the full-length transcript decreases. To determine whether this phenomenon is unique to CBP1 , northern analysis was used to determine whether the levels of other truncated transcripts are regulated similarly by carbon source. The levels of the shortest transcripts of AEP2 / ATP13 and RNA14 increased during respiration while the shortest SIR1 transcript remained constant. However, two longer SIR1 transcripts were regulated reciprocally by carbon source. Mapping the 3'-ends of each transcript by sequencing partial cDNA clones revealed multiple 3'-ends for each transcript. Examination of the sequences surrounding the 3'-ends of the induced transcripts failed to identify a consensus sequence but did reveal weak putative 3'-end formation signals in all of the transcripts. Similarly, no consensus sequence was found when the sequences surrounding the 3'-ends of the longest transcripts were compared, but again weak putative 3'-end formation signals were identified. These data are suggestive of carbon source regulation of alternative poly(A) site choice in yeast.

Modulation of cellular and viral promoters by mutant human p53 proteins found in tumor cells.

Wild-type p53 has recently been shown to repress transcription from several cellular and viral promoters. Since p53 mutations are the most frequently reported genetic defects in human cancers, it becomes important to study the effects of mutations of p53 on promoter functions. We, therefore, have studied the effects of wild-type and mutant human p53 on the human proliferating-cell nuclear antigen (PCNA) promoter and on several viral promoters, including the herpes simplex virus type 1 UL9 promoter, the human cytomegalovirus major immediate-early promoter-enhancer, and the long terminal repeat promoters of Rous sarcoma virus and human T-cell lymphotropic virus type I. HeLa cells were cotransfected with a wild-type or mutant p53 expression vector and a plasmid containing a chloramphenicol acetyltransferase reporter gene under viral (or cellular) promoter control. As expected, expression of the wild-type p53 inhibited promoter function. Expression of a p53 with a mutation at any one of the four amino acid positions 175, 248, 273, or 281, however, correlated with a significant increase of the PCNA promoter activity (2- to 11-fold). The viral promoters were also activated, although to a somewhat lesser extent. We also showed that activation by a mutant p53 requires a minimal promoter containing a lone TATA box. A more significant increase (25-fold) in activation occurs when the promoter contains a binding site for the activating transcription factor or cyclic AMP response element-binding protein. Using Saos-2 cells that do not express p53, we showed that activation by a mutant p53 was a direct enhancement. The mutant forms of p53 used in this study are found in various cancer cells. The activation of PCNA by mutant p53s may indicate a way to increase cell proliferation by the mutant p53s. Thus, our data indicate a possible functional role for the mutants of p53 found in cancer cells in activating several important loci, including PCNA.

Variable breakpoints target PAX5 in patients with dicentric chromosomes: a model for the basis of unbalanced translocations in cancer.

The search for target genes involved in unbalanced acquired chromosomal abnormalities has been largely unsuccessful, because the breakpoints of these rearrangements are too variable. Here, we use the example of dicentric chromosomes in B cell precursor acute lymphoblastic leukemia to show that, despite this heterogeneity, single genes are targeted through a variety of mechanisms. FISH showed that, although they were heterogeneous, breakpoints on 9p resulted in the partial or complete deletion of PAX5. Molecular copy number counting further delineated the breakpoints and facilitated cloning with long-distance inverse PCR. This approach identified 5 fusion gene partners with PAX5: LOC392027 (7p12.1), SLCO1B3 (12p12), ASXL1 (20q11.1), KIF3B (20q11.21), and C20orf112 (20q11.1). In each predicted fusion protein, the DNA-binding paired domain of PAX5 was present. Using quantitative PCR, we demonstrated that both the deletion and gene fusion events resulted in the same underexpression of PAX5, which extended to the differential expression of the PAX5 target genes, EBF1, ALDH1A1, ATP9A, and FLT3. Further molecular analysis showed deletion and mutation of the homologous PAX5 allele, providing further support for the key role of PAX5. Here, we show that specific gene loci may be the target of heterogeneous translocation breakpoints in human cancer, acting through a variety of mechanisms. This approach indicates an application for the identification of cancer genes in solid tumours, where unbalanced chromosomal rearrangements are particularly prevalent and few genes have been identified. It can be extrapolated that this strategy will reveal that the same mechanisms operate in cancer pathogenesis in general.