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About the Node
Promoter DNA methylation couples genome-defence mechanisms to epigenetic reprogramming in the mouse germline
Jamie A. Hackett, James P. Reddington, Colm E. Nestor, Donncha S. Dunican, Miguel R. Branco, Judith Reichmann, Wolf Reik, M. Azim Surani, Ian R. Adams, Richard R. Meehan
Development 2012 139: 3623-3632; doi: 10.1242/dev.081661
Jamie A. Hackett
1 MRC Human Genetics Unit, IGMM, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, UK.5 Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, UK.
James P. Reddington
1 MRC Human Genetics Unit, IGMM, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, UK.
Colm E. Nestor
1 MRC Human Genetics Unit, IGMM, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, UK.2 Breakthrough Research Unit, University of Edinburgh, Edinburgh EH4 2XU, UK.
Donncha S. Dunican
Miguel R. Branco
3 Epigenetics Programme, The Babraham Institute, Cambridge CB22 3AT, UK.4 Centre for Trophoblast Research, University of Cambridge, Cambridge CB2 3EG, UK.
Judith Reichmann
Wolf Reik
M. Azim Surani
5 Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, UK.
Ian R. Adams
For correspondence: ian.adams@igmm.ed.ac.uk richard.meehan@igmm.ed.ac.uk
Richard R. Meehan
Supp info
Mouse primordial germ cells (PGCs) erase global DNA methylation (5mC) as part of the comprehensive epigenetic reprogramming that occurs during PGC development. 5mC plays an important role in maintaining stable gene silencing and repression of transposable elements (TE) but it is not clear how the extensive loss of DNA methylation impacts on gene expression and TE repression in developing PGCs. Using a novel epigenetic disruption and recovery screen and genetic analyses, we identified a core set of germline-specific genes that are dependent exclusively on promoter DNA methylation for initiation and maintenance of developmental silencing. These gene promoters appear to possess a specialised chromatin environment that does not acquire any of the repressive H3K27me3, H3K9me2, H3K9me3 or H4K20me3 histone modifications when silenced by DNA methylation. Intriguingly, this methylation-dependent subset is highly enriched in genes with roles in suppressing TE activity in germ cells. We show that the mechanism for developmental regulation of the germline genome-defence genes involves DNMT3B-dependent de novo DNA methylation. These genes are then activated by lineage-specific promoter demethylation during distinct global epigenetic reprogramming events in migratory (∼E8.5) and post-migratory (E10.5-11.5) PGCs. We propose that genes involved in genome defence are developmentally regulated primarily by promoter DNA methylation as a sensory mechanism that is coupled to the potential for TE activation during global 5mC erasure, thereby acting as a failsafe to ensure TE suppression and maintain genomic integrity in the germline.
DNA methylation is a key epigenetic system that is associated with stable transcriptional silencing, genomic imprinting and cellular differentiation (Bird, 2002; Reik, 2007). In mammals, DNA methylation occurs primarily at genomic CpG dinucleotides and is essential for embryonic development (Li et al., 1992; Okano et al., 1999). Methylation of CpGs is established by the de novo methyltransferases DNMT3A and DNMT3B and is subsequently maintained by the maintenance methyltransferase DNMT1. During mouse development, the bulk of global DNA methylation is established by embryonic day (E) 6.5 and is thought to contribute to stable lineage commitment (Borgel et al., 2010; Meissner et al., 2008; Mohn et al., 2008). However, after their specification at ∼E6.25-7.25, mouse primordial germ cells (PGCs) undergo extensive erasure of DNA methylation at ∼E8.5 when PGCs are migrating into the hind-gut endoderm (Seki et al., 2005), and at E10.5-11.5 shortly after the germ cells colonise the genital ridges (Hajkova et al., 2002; Popp et al., 2010). The global DNA demethylation in PGCs appears to be part of a comprehensive epigenetic reprogramming that also includes extensive changes in histone modifications (Seki et al., 2007; Hajkova et al., 2008). The erasure of DNA methylation is required for resetting genomic imprints, and might play a role in reprogramming the developmental potential of PGCs to a totipotent state (Hackett et al., 2012).
At the level of gene regulation, although promoter DNA methylation is associated with transcriptional repression, in many cases it is not clear whether methylated promoters represent a cause or a consequence of gene silencing (Walsh and Bestor, 1999; Bird, 2002). Indeed, at most methylated promoters, DNA methylation acts to reinforce repression that is initially established through chromatin-based silencing mechanisms (e.g. polycomb) (Feldman et al., 2006; Fouse et al., 2008). Moreover, DNA methylation at low CpG-density promoters is readily bypassed by activating signals, whereas CpG-dense promoters, for which DNA methylation is correlated with significant transcriptional silencing, are rarely methylated in vivo (Metivier et al., 2008; Meissner et al., 2008; Weber et al., 2007). Nonetheless, two recent genome-wide studies have identified genes that rely on DNA methylation for transcriptional silencing in either early embryos or embryonic fibroblasts, and these gene sets are enriched for germline-specific genes (Borgel et al., 2010; Velasco et al., 2010). This observation is consistent with DNA methylation regulating the expression of the germline-specific genes Dazl, Mvh (Ddx4 – Mouse Genome Informatics) and Sycp3 as loss of DNA methylation at these promoters is associated with their transcriptional activation in the developing germline (Maatouk et al., 2006). Thus, DNA methylation appears to play a specific role in contributing to the silencing of some germline-specific genes, although it is uncertain whether this mechanism initiates developmental gene silencing or only 'locks in' stable repression at these loci, perhaps in parallel with chromatin-based silencing. Interestingly, whether there is any developmentally advantageous reason for germline-specific genes to be preferentially regulated by promoter DNA methylation is also unclear.
In addition to a role at some gene promoters, DNA methylation has an important role in maintaining stable silencing of mobile transposable elements (TEs) and reducing their threat to genomic integrity (Reik, 2007). Around 40% of the mammalian genome is composed of TEs (Waterston et al., 2002), which can propagate through the genome via either a 'cut and paste' mechanism or an RNA intermediate (Goodier and Kazazian, 2008). To successfully propagate through generations, TEs must be active in germ cells or during early embryonic development, and are therefore strongly selected for germline expression. However, several mechanisms have evolved to limit the mutagenic potential of TEs in the germline (Ollinger et al., 2010; Zamudio and Bourc'his, 2010). Mice carrying mutations in Dnmt1, or in the germline-restricted accessory factor for de novo DNA methylation Dnmt3L, lose DNA methylation at TE sequences resulting in massive upregulation of LINE1 and intracisternal A-type particle (IAP) retroelements in the developing germline (Walsh et al., 1998; Bourc'his and Bestor, 2004). The 'genome defence' hypothesis argues that the primary developmental role of DNA methylation is to contribute to TE silencing rather than to regulate developmental gene expression (Yoder et al., 1997). The transient erasure of repressive epigenetic mechanisms, and in particular of DNA methylation, in PGCs therefore presents an unparalleled opportunity for TEs to become active in the developing germline and represents a potential risk to transgenerational genomic stability.
In a screen to identify genes that are regulated directly and primarily by promoter DNA methylation, we identified several key components of the TE defence machinery. We demonstrate that these genes are dependent on promoter CpG methylation to direct their developmental expression pattern, and show that expression of these genes is activated during two phases of DNA demethylation in PGCs. Intriguingly, these gene promoters appear to contain a specialised chromatin domain that does not acquire repressive H3K27me3, H3K9me2, H3K9me3 or H4K20me3 histone modifications when silenced by DNA methylation. This reliance on DNA methylation acts as a highly tuned sensor of global DNA demethylation in PGCs and thereby primes germ cells to suppress opportunistic TE activity. These findings provide a developmental rationale for germ cells using DNA methylation to regulate gene expression: this mechanism is used as a feedback loop to protect genomic integrity in the germline during potentially hazardous reprogramming events.
DNA methylation deficient E9.5 Dnmt1n/n p53–/– primary embryonic fibroblasts, control p53–/– (Dnmt1n/+), and Dnmt3b–/– mouse embryonic fibroblasts (MEFs) and primary MEFs (C57BL/6) were cultured in Dulbecco's modified Eagle's medium supplemented with l-glutamine, non-essential amino acids, sodium pyruvate, β-mercaptoethanol and 15% foetal calf serum and passaged at sub-confluence (Lande-Diner et al., 2007). Dnmt3b–/– MEFs were derived at E12.5 and E13.5 from heterozygous crosses of mice carrying one floxed and one deleted copy of Dnmt3b and lacking any Cre-containing alleles (Dodge et al., 2005). Embryonic stem (ES) cells (E14/J1) were cultured as previously described (Ollinger et al., 2008). Embryoid body (EB) differentiation was induced through leukaemia inhibitory factor (LIF) withdrawal and cell aggregation in hanging drops for 2 days, followed by suspension culture for 5 days, and then culture as adherent colonies. Where indicated, retinoic acid (1 μM) was applied for 72 hours to induce differentiation.
Primordial germ cell isolation
PGCs were harvested from outbred mice carrying the Oct4-GFP transgene. The dissected hind-gut (E9.5) or urogenital ridges (E10.5 and E13.5) from embryos were trypsinised and sorted by fluorescence-activated cell sorting (FACS) for GFP using a FACSAriaII SORP (Becton Dickinson). Purity of PGCs was determined by OCT4 (POU5F1 – Mouse Genome Informatics) staining (supplementary material Fig. S8).
Aza-recovery assay
Low density cells were treated with 1 μM 5aza-dC (5aza-deoxycytidine) for 3 days, with fresh 5aza-dC-containing media added daily. Cells were subsequently washed and allowed to recover with normal media in situ for 5 days and then passaged every 3 days.
DNA constructs and luciferase assays
Indicated promoter regions were PCR amplified and directionally cloned into pGL3-basic (Promega) using the BglII and MluI sites. In vitro methylation of constructs was carried out with SssI (CpG) methylase (New England Biolabs). Luciferase reporter assays were performed using the Dual Luciferase Assay Kit (Promega), according to the manufacturer's instructions.
Total RNA from cell lines or tissues was isolated with TRIzol reagent (Invitrogen). After Turbo DNase treatment (Ambion), first strand cDNA was transcribed with superscript III reverse transcriptase (Invitrogen). Quantitative real-time PCR was carried out with Brilliant II SYBR green qPCR mix (Stratagene) using an iCycler (Bio-Rad) and relative quantification was determined using the Pfaffl method (Pfaffl, 2001). RT-PCR was performed with Platinum Taq (Invitrogen) for an optimised number of cycles. Primers are shown in supplementary material Table S2.
Bisulphite sequencing
Genomic DNA was extracted from cells by phenol-chloroform after proteinase K/SDS digestion. Bisulphite conversion was carried using the EZ DNA Methylation Gold Kit (Zymo Research) following the manufacturer's instructions. We used nested primer sets to amplify a specific region of interest with Platinum Taq (Invitrogen) (supplementary material Table S3). A single band was excised, gel extracted, and cloned into pGEM-T-easy (Promega) for sequencing.
MeDIP was performed as previously reported, with modifications (Borgel et al., 2010). Briefly, 200 ng genomic DNA was sonicated to 500 bp (range 300 bp to 1 kb) with a Bioruptor (Diagenode) and denatured at 95°C for 5 minutes. DNA was immunoprecipitated overnight at 4°C with 0.5 μl 5mC-antibody (Eurogentec), washed, and purified through MinElute columns (Qiagen). Enrichment of specific genomic loci relative to input was determined by qPCR.
Single cell RT-PCR
Reverse transcribed single cell cDNA libraries were generated as previously described (Tang et al., 2009). Gene expression was determined by gene-specific qPCR from three PGC libraries at each developmental stage.
Chromatin immunoprecipitation (ChIP)
Native chromatin was prepared from cell nuclei in NBR buffer (85 mM NaCl, 5.5% sucrose, 10 mM Tris, 3 mM MgCl2, 1.5 mM CaCl2, 0.2 mM PMSF, 1 mM DTT). Nuclei were treated with micrococcal nuclease (MNase) (Worthington) to generate mono-, di and tri-nucleosomes and chromatin was released overnight at 4°C. Immunoprecipitation was carried out with antibodies specific to H3K27me3 (Millipore 07449), H3K9me2 (Active Motif 39239), H3K9me3 (Abcam ab8898), H4K20me3 (Active Motif 39181) or rabbit IgG (Santa Cruz), followed by antibody-optimised washes. Samples from native ChIP were purified through affinity columns (Qiagen) and quantified by qPCR using primers shown in supplementary material Table S3.
Microarray and bioinformatics analysis
Total RNA was biotinylated and amplified with a TotalPrep RNA Amplification Kit (Illumina). Pre- and post-labelled RNA was run on a RNA bionalyser chip (Agilent) to confirm RNA integrity and hybridised to Illumina mouse Ref-8 v2.0 BeadChips. Microarray gene expression profiles were generated in quadruplicate from Dnmt1n/n p53–/– and p53–/– MEFs, and from duplicate biological replicates of 5aza-dC treated and 5aza-dC recovery NIH3T3 cells. Background subtraction, normalisations and statistical analyses were carried out using BeadStudio data analysis software modules. Microarray expression changes were calculated as a ratio of normalised expression between samples, with a P<0.01 detection threshold and t-test used to determine significance. Microarray expression data is deposited at the GEO database at NCBI under accession number GSE38307. A stringent sixfold expression change threshold was applied to enrich for bona fide methylation-dependent genes. Gene ontology analysis was performed using the DAVID functional annotation package (http://david.abcc.ncifcrf.gov/). Raw DNA methylation sequencing data from pMEFs was mapped to CpG-dense (ICP and HCP) promoters, with R statistical analysis software (http://www.r-project.org) used to determine CpG methylation levels at target genes (Meissner et al., 2008).
Identification of novel methylation-dependent genes
Previous studies have identified numerous genes that are differentially expressed in response to DNA demethylation using, for example, Dnmt1n/n fibroblasts, Dnmt3b–/– fibroblasts or Dnmt3b–/– embryos (Jackson-Grusby et al., 2001; Velasco et al., 2010; Borgel et al., 2010). However, the complex interactions that exist between epigenetic silencing systems are likely to result in different gene sets being identified depending on the cell types or DNA methyltransferases studied. Moreover, many genes upregulated in genetic deletions probably represent indirect hits, as the majority of upregulated genes are already unmethylated (Jackson-Grusby et al., 2001). In order to identify a minimal set of genes whose expression is primarily and causally regulated by promoter DNA methylation in different cell types, we generated and integrated gene expression datasets from multiple experimental conditions in which genome-wide loss of DNA methylation is induced in different ways.
We initially developed an epigenetic disruption and recovery screen, based on the demethylation-inducing agent 5aza-dC (Fig. 1A). We predicted that genes regulated directly by DNA methylation would be de-repressed by transient exposure to 5aza-dC and would remain continually expressed (and hypomethylated) during an extended recovery period, on the basis that somatic cells lack significant de novo methyltransferase activity (Okano et al., 1999). This approach separates bona fide methylation-dependent genes from those genes that are activated indirectly or by the apoptotic effects of 5aza-dC, which are predicted to re-impose repression during cellular recovery from drug exposure (Palii et al., 2008). In addition, we reasoned that genes that are regulated primarily and causally by DNA methylation would undergo large (off → on) expression changes in this assay and thus imposed a stringent sixfold threshold to select for upregulated genes (supplementary material Fig. S1). Increasing the threshold stringency (i.e. taking the most upregulated genes) selects for somatically methylated CpG island genes, which are promising methylation-dependent candidates (supplementary material Fig. S1A). We profiled global gene expression in NIH3T3 fibroblasts after 5aza-dC exposure and identified 344 significantly upregulated genes (Aza-Up). Following a 14 day recovery, only 49 of these genes remained de-repressed (Rec-Up) (Fig. 1A). These data suggest that the majority of genes activated by 5aza-dC (>85%) are re-repressed upon 5aza-dC withdrawal, with most genes (70%) becoming fully re-silenced (supplementary material Fig. S1). Notably, when mapped to a global promoter methylation dataset (Meissner et al., 2008), Rec-Up genes were highly enriched in genes associated with methylated CpG-dense promoters in MEFs (P<0.01), a likely prerequisite for regulation by DNA methylation, whereas control and Aza-Up gene sets showed no such enrichment (Fig. 1B). The surprisingly small numbers of genes that remain de-repressed after recovery from 5aza-dC have probably undergone a stable and heritable epigenetic switch to activate their expression during the 5aza-dC treatment.
Identification of core methylation-dependent genes. (A) Schematic of the 5aza-dC-recovery assay. Cells are exposed to 5aza-dC for 3 days (3d) and allowed to recover for 14 days (14d). Below: Venn diagram showing overlap between genes upregulated after 5aza-dC (Aza-Up; blue) and after recovery (Rec-Up; grey). (B) Percentage of genes with somatically methylated CpG island (CGI) promoters in Aza-Up or after 14 days recovery (Rec-Up) (Meissner et al., 2008). ***P<0.001. (C) Gene ontology of Aza-Up and Rec-Up gene set, with P-values along the x-axis. Multiple categories of genes are activated by 5aza-dC exposure but only germline-associated categories remain enriched after recovery, when only methylation-dependent genes are predicted to remain. (D) RT-PCR validation of selected germline Rec-Up genes from microarray analysis that remain active during recovery and of control somatic Aza-Up genes that re-impose silencing after 5aza-dC exposure, probably owing to indirect activation. Gapdh is a loading control. (E) RT-PCR showing that genome-defence genes are activated by demethylation in multiple contexts including in Dnmt1n/n MEFs and by 5aza-dC (± the histone deacetylase inhibitor TSA) in primary MEFs. –RT, without reverse transcriptase.
Gene ontology analysis showed that the 344 genes upregulated by 5aza-dC treatment (Aza-Up) were enriched for eleven different categories, including immune response, immune effector processes, response to stress, gamete generation and sexual reproduction (supplementary material Table S4). By contrast, only two gene ontology classes were enriched in the Rec-Up gene set, sexual reproduction and gamete generation, suggesting that the Rec-Up gene set of methylation-dependent candidates corresponds to a distinct group of genes with common functional annotation in germline development (Fig. 1C). We confirmed our findings from the microarray data (Rec-Up dataset) by RT-PCR showing that the Tex19.1, Mili (Piwil2 – Mouse Genome Informatics), Mov10l1 and Asz1 (also known as Gasz) germline-specific genes remain de-repressed after 5aza-dC withdrawal, as does Ant4 (Slc25a31 – Mouse Genome Informatics), which has previously been shown to be methylation dependent (Rodic et al., 2005) (Fig. 1D). By contrast, representative genes (Rtp4 and Oasl1) of the Aza-Up dataset were initially activated by 5aza-dC and rapidly silenced after 5aza-dC withdrawal, implying these are not methylation-dependent targets. Importantly, we found that following 5aza-dC-induced DNA demethylation, the promoters of the Tex19.1 and Mili Rec-Up genes become hypomethylated, and are not re-methylated during recovery (supplementary material Fig. S2). We have employed the 5aza-dC recovery assay on a number of somatic cell lines, primary MEFs and CMT-93 mouse rectal carcinoma cells, and continuous Tex19.1 de-repression is representative of active germline-specific genes in each case (J.A.H., D.S.D., R.R.M., unpublished observations).
Genome-defence genes are regulated directly by CpG methylation
To refine further our Rec-Up gene set to a core subset that is crucially reliant on DNA methylation in multiple contexts, we used mouse embryonic fibroblasts (MEFs) derived from hypomorphic Dnmt1n/n; p53–/– embryos, which lack significant DNA methylation (Lande-Diner et al., 2007). We profiled global gene expression from Dnmt1n/n cells and intersected upregulated genes with Rec-Up genes. This identified 26 genes that are strongly activated by DNA demethylation in different experimental and cellular contexts (supplementary material Table S1). These core genes are highly enriched (13/26) for germline-specific expression. Strikingly, five of the six germline-associated genes that have functionally characterised roles are involved in genome defence against transposable elements (TE) in the germline, either via piwi-interacting RNA (piRNA) biogenesis or alternative mechanisms (Ollinger et al., 2008; Aravin et al., 2007; Frost et al., 2010; Ma et al., 2009; Aravin et al., 2008). Although previous genome-wide studies have described an association between DNA methylation and germline-associated genes (Borgel et al., 2010; Velasco et al., 2010), the genes identified in these studies do not appear to be co-expressed at any one specific stage of germ cell development, and do not appear to have a common functional role during gametogenesis. Given the remarkable enrichment for known germline genome-defence genes (Tex19.1, Mili, Mov10l1 and Asz1) in our Rec-Up data set, we hypothesised that the shared epigenetic mechanism (DNA methylation) of regulating their expression might be related to a common molecular function in the developing germline.
We confirmed that Tex19.1, Mili, Mov10l1 and Asz1 are all ectopically expressed in Dnmt1n/n MEFs (Fig. 1E). Transcript mapping demonstrated that transcription in Dnmt1n/n MEFs initiated from canonical transcriptional start sites (TSSs), ruling out ectopic expression from cryptic promoters (supplementary material Fig. S3). To investigate whether DNA methylation is the initiating mechanism for silencing Tex19.1, Mili and Mov10l1 rather than a stable 'lock', we utilised early passage embryonic stem (ES) cells lacking the de novo methyltransferases DNMT3A and DNMT3B. Notably, Tex19.1 and Mov10l1 failed to become fully repressed, and Mili only exhibited partial repression during late embryoid body (EB) differentiation of Dnmt[3a, 3b]–/– ES cells, whereas all were rapidly silenced during differentiation of parental J1 ES cells (Fig. 2A). By contrast, Oct4 was silenced in both wild-type and double mutant EBs. Even after 15 days differentiation, Dnmt[3a, 3b]–/– EBs are intrinsically unable to silence Tex19.1, Mili and Mov10l1 expression, suggesting that de novo promoter methylation is the critical mark mediating the onset of repression during differentiation. We were unable to assess whether DNA methylation initiated silencing of Asz1 in the EB differentiation assay as we could not detect Asz1 expression in wild-type ES cells, although we could detect strong expression of Asz1 in Dnmt1n/n ES cells (supplementary material Fig. S3).
Dynamics of DNA methylation at genome-defence genes. (A) RT-PCR showing expression upon EB differentiation of wild-type (WT) or Dnmt3a/3b–/– ES cells. Note that Oct4 does not require Dnmt3a or Dnmt3b for silencing. (B) RT-PCR in Dnmt3a- and Dnmt3b-mutant ES cells. Transcriptional silencing fails in the absence of DNMT3B. (C,D) Tex19.1 and Mili are highly demethylated and ectopically expressed in Dnmt3b–/– embryos at E12.5. Filled circles indicate methylated CpG and empty indicate unmethylated CpG. Percentages indicate average DNA methylation. (E) The Tex19.1 and Mili promoters are progressively de novo methylated (right-hand y-axis, blue line) coincident with the onset of transcriptional silencing (left-hand y-axis, grey line) during EB differentiation of ES cells. d, day; –RT, without reverse transcriptase.
To determine the relative contribution of each de novo methyltransferase in initiating gene silencing, we induced EB formation in ES cells lacking either Dnmt3a or Dnmt3b. This showed that Tex19.1, Mili and Mov10l1 specifically require DNMT3B, but not DNMT3A, for full silencing and de novo methylation during ES cell differentiation (Fig. 2B,C; supplementary material Fig. S4). However, Mili was partially silenced in Dnmt3b–/– ES cells, which might be indicative of additional silencing pathways that are operative in differentiating ES cells. Furthermore, we derived fibroblasts from Dnmt3b–/– embryos and found that Tex19.1 and Mili were hypomethylated and fully de-repressed, implying that these loci do not initiate appropriate silencing in somatic tissues in the absence of DNMT3B in vivo (Fig. 2C,D). Notably, global profiling revealed that our null Dnmt3b allele results in de-repression of a different subset of germline-specific genes (and to a greater extent) compared with a hypomorphic compound-heterozygote Dnmt3b allele previously reported (Velasco et al., 2010). We found that the Asz1 and Dazl genome-defence genes are also de-repressed in Dnmt3b–/– embryonic fibroblasts (supplementary material Fig. S4). Mov10l1 is also upregulated in Dnmt3b–/– MEFs, although it was below the sixfold expression threshold (supplementary material Table S5). Thus, silencing of many germline genome-defence genes during somatic differentiation appears to be initiated by the de novo DNA methyltransferase DNMT3B.
It has been reported that silencing of some genes, including Oct4, occurs in a stepwise manner, with trans-acting repressors and histone modifications establishing a hetrochromatinised silenced state and promoter methylation only occurring later, as a secondary repressive lock against reactivation (Feldman et al., 2006). To investigate whether DNA methylation was the primary or a secondary regulatory mechanism at Tex19.1 and Mili, we tracked the dynamics of promoter methylation and gene silencing during ES cell differentiation. We observed that the acquisition of promoter DNA methylation occurs in parallel with the onset of transcriptional silencing at Tex19.1 and Mili (Fig. 2E). Treatment of day 7 EBs with 5aza-dC was sufficient to reactivate Tex19.1, Mili, Mov10l1 and other silenced genome-defence genes but not Oct4 (supplementary material Fig. S5). Taken together, this suggests that tissue-specific promoter DNA methylation is a primary mechanism for regulating the expression pattern of a number of genes that function in germline genome defence.
Genome-defence genes are depleted of chromatin modifications
To investigate whether chromatin-based silencing mechanisms also operate at genome-defence genes, we performed native chromatin-immunoprecipitation (ChIP) against the best characterised repressive histone modifications in NIH3T3 cells, in which these genes are silent (Fig. 1A-D). Compared with a positive control, HoxC10, the repressive H3K27me3 mark, is absent from the promoters of Tex19.1, Mili and Mov10l1 with levels comparable to the Gapdh negative control (Fig. 3A). Moreover, retinoic acid (RA) differentiation of Eed–/– ES cells, which lack H3K27me3, did not impair silencing of Tex19.1, implying that H3K27me3 is functionally dispensable for repression at this locus (Fig. 3E) (Montgomery et al., 2005). Further analysis of the heterochromatin-associated marks H3K9me3 and H4K20me3 revealed that Tex19.1, Mili and Mov10l1 are also depleted of these modifications, and are marked comparably to unmodified Gapdh (Fig. 3B,C) (Mikkelsen et al., 2007). We also found that H3K9me2, which has been reported to be functionally upstream of DNA methylation at some loci, was depleted from tested gene promoters (Fig. 3D) (Tachibana et al., 2008). Importantly, ChIP for histone H3 gave comparable or greater enrichment of Tex19.1, Mili and Mov10l1 than positive controls, indicating that the reduction of unmodified histones we observe at these loci is not symptomatic of nucleosome-free regions (supplementary material Fig. S6). These genes therefore appear to possess specialised chromatin at their promoters in non-expressing somatic cells that is selectively depleted of many repressive histone modifications. Thus, methylation of their CpG-dense promoters probably represents the predominant mechanism for regulating the developmental and tissue-specific expression of these genes. Consistent with this, promoter reporters for Tex19.1 and Mili, but not Oct4, drive strong expression in somatic cell lines derived from either mesodermal or neural lineages, implying that their germline-restricted expression in vivo is not a consequence of tissue-specific availability of transcription factors or repressors (supplementary material Fig. S7). In vitro methylation of Tex19.1 and Mili reporters strongly repressed expression up to 150-fold in this assay.
Genome-defence genes are associated with specialised promoter chromatin. (A-D) Native ChIP demonstrating that the repressive chromatin modifications H3K27me3, H3K9me3, H4K20me3 and H3K9me3 are depleted from the promoter regions of methylated and silenced genome-defence genes. Gapdh marks the background levels expected of no significant modification. Hoxc10, major satellite (Maj Sat) and IAP are positive controls for respective modifications. (E) RT-PCR demonstrating that Tex19.1 is silenced during ESC differentiation by retinoic acid (+RA) in the absence of polycomb (Eed–/–). –RT, without reverse transcriptase; U, untreated. (F) Germline-specific genes in general are highly enriched for promoters that lack histone modifications in MEFs. Blue, no modification; light grey, H3K4me3; dark grey, H3K27me3; black, bivalent modification (H3K4me3 plus H3K27me3). ***P<0.001.
The general depletion of repressive histone modifications at Tex19.1, Mili and Mov10l1 prompted us to investigate whether unmodified promoter histones was a common feature of silenced germline-specific genes. We mapped a dataset of testis-specific CpG-dense promoter genes (defined as expressed more than tenfold in testis than all somatic tissues in the BioGPS database) to their histone modification state for active (H3K4me3), repressive (H3K27me3) and bivalent (H3K4me3 and H3K27me3) marks in MEFs (Mikkelsen et al., 2007). We find that testis-specific genes are highly enriched in promoters that lack these histone modifications (Fisher's exact test, P=0.00019) (Fig. 3F). This finding might partly explain the general association between germline genes and regulation by DNA methylation reported here and in other studies (Maatouk et al., 2006; Borgel et al., 2010; Velasco et al., 2010).
Promoter CpG methylation dynamics during PGC development
Although immunostaining with anti-5mC antibodies suggests that PGCs might undergo extensive loss of DNA methylation at ∼E8.5 (Seki et al., 2005), the genomic loci that lose DNA methylation during this process have not yet been identified. Although partial demethylation of some single-copy loci is evident in E9.5 PGCs (Guibert et al., 2012), repetitive elements, imprinted loci and selected single-copy gene promoters are all still fully methylated in E10.5 PGCs (Hajkova et al., 2002; Maatouk et al., 2006; Lee et al., 2002). By contrast, the genomic targets corresponding to the ∼E11.0 demethylation event include gene promoters, repetitive elements and imprinted genes (Hajkova et al., 2002; Maatouk et al., 2006). To investigate the dynamics of DNA methylation at germline genome-defence genes during PGC development in vivo, we tracked the methylation status and expression of these genes in PGCs isolated from E10.5 and E13.5 embryonic genital ridges using a transgenic Oct4-GFP marker (supplementary material Fig. S8).
We used methylated DNA immunoprecipitation (meDIP) to evaluate promoter methylation in PGCs and in control somatic cells and epiblast stem cells (EpiSCs). Unexpectedly, we found that the tested genes fell into one of two categories: either they are methylated in PGCs at E10.5 and demethylated by E13.5 as reported for Dazl (Maatouk et al., 2006), or they are already demethylated in PGCs at E10.5 prior to post-migratory epigenetic reprogramming (Fig. 4A; supplementary material Fig. S9). The former group (including Mov10l1, Asz1 and Tex19.2) probably represent novel methylation-dependent genes that utilise global demethylation to direct expression in post-migratory PGCs (supplementary material Fig. S9). However, intriguingly, the latter group of methylation-dependent genes (Tex19.1, Mili) were already hypomethylated in PGCs at E10.5, prior to global methylation erasure (Fig. 4A). These 'early demethylation' genes are also strongly expressed in PGCs at E10.5, whereas loci undergoing demethylation after E10.5, such as Dazl, were repressed and exhibited strong transcriptional upregulation by E13.5 (Fig. 4B). Importantly, all tested genes are methylated in EpiSCs, which are derived from the pre-gastrulation epiblast prior to PGC specification.
DNA demethylation in PGCs during two reprogramming events (A) Methylated DNA immunoprecipitation (meDIP) showing the relative levels of promoter methylation at Tex19.1, Mili and Dazl in soma, epiblast stem cells (EpiSC), and E10.5, E13.5 female (F) and E13.5 male (M) PGCs. Notably, Tex19.1 and Mili are demethylated in E10.5 PGCs whereas Dazl remains highly methylated. Error bars represent s.e.m. (B) qRT-PCR expression analysis from pooled PGCs. Expression levels of Tex19.1, Mili and Dazl in PGCs correlate with promoter methylation levels (A). Normalised relative to 18S RNA. Error bars represent s.e.m. (C) Bisulphite sequencing showing promoter DNA methylation at Tex19.1, Mili and Dazl during primordial germ cell (PGC) development and matched somatic cells from genital ridges (lower panel). (D) DNA methylation profile of Tex19.1 in the embryonic portion of E6.5 (epiblast), E7.5 and E8.5 embryos. In C and D, filled circles indicate methylated CpG and empty indicate unmethylated CpG.
We confirmed the differential demethylation dynamics between Tex19.1/Mili and Dazl promoters by bisulphite sequencing these loci in purified PGC populations. Consistent with our meDIP data, the Dazl promoter is methylated in PGCs at E10.5 but Tex19.1 and Mili promoters are hypomethylated (Fig. 4C). Furthermore, in agreement with the meDIP data, the Tex19.2 promoter behaves differently to Tex19.1 and follows similar DNA methylation kinetics to Dazl (supplementary material Fig. S9). The Tex19.1 and Mili promoters therefore become hypomethylated earlier than Dazl during PGC development, and are already hypomethylated in E10.5 PGCs. Tex19.1 is also hypomethylated and expressed in migrating PGCs isolated from the hind-gut region of younger embryos at E9.5 (supplementary material Fig. S10). Thus, Tex19.1 and Mili are good candidate targets for the DNA demethylation event that is reported to occur in E8.5 PGCs.
To test whether loci that are hypomethylated at E10.5 escape de novo methylation before PGC specification (at ∼E6.5), and are therefore never methylated in the germline, we initially assessed the methylation dynamics of Tex19.1 during post-implantation development. Although isolation and bisulphite sequencing of pure PGC DNA from E7.5 embryos is technically prohibitive, bisulphite sequencing of epiblast and embryonic tissue between E6.5 and E8.5 showed that the Tex19.1 promoter is fully methylated at E7.5 and E8.5 (when Tex19.1 becomes silenced), and is in an intermediate methylated state at E6.5, indicating that de novo methylation commences at this locus prior to PGC specification (Fig. 4D). These data are consistent with a recent report demonstrating that Mili, Mov10l1, Asz1, Dazl and Miwi (Piwil1 – Mouse Genome Informatics) are also all highly methylated in E6.5 epiblast in vivo and therefore undergo lineage-specific demethylation in the PGCs (Borgel et al., 2010). Furthermore, although TEX19.1 protein and mRNA is expressed in the pluripotent epiblast cells in E6.5 embryos, Tex19.1 is downregulated during gastrulation, can only be weakly detected in E7.5 embryos, and is not detectable in the hind-gut region of E8.5 embryos where PGCs are present (supplementary material Fig. S10). This suggests that Tex19.1 expression is downregulated during PGC specification. Thus, the genome-defence genes appear to undergo de novo methylation in the post-implantation epiblast, followed by lineage-specific loss of promoter DNA methylation either in migratory PGCs (Tex19.1, Mili) or post-migratory PGCs (Tex19.2, Mov10l1, Asz1, Dazl).
Gene activation during distinct reprogramming phases in PGCs
The global loss of DNA methylation that occurs in ∼E11.0 PGCs has been shown to be responsible for inducing expression of key germline genes, such as Dazl and Mvh (Ddx4 – Mouse Genome Informatics), at this stage of PGC development. Indeed, expression of Tex19.2, which shows similar promoter demethylation kinetics to Dazl, is also activated in post-migratory PGCs (supplementary material Fig. S9). However, it is not clear whether the DNA demethylation in ∼E8.5 PGCs also acts as a trigger to induce developmental changes in PGC gene expression. We therefore used single-cell cDNA expression profiling to analyse Tex19.1 and Mili expression in E7.5-11.5 PGCs to test whether expression of these methylation-dependent genes coincided with the demethylation event at E8.5.
Interestingly, Tex19.1 and Mili are not expressed in PGCs at E7.5, consistent with these loci being methylated at this stage. However, we observed strong transcriptional activation of both loci starting from E8.5, immediately after the reported demethylation in PGCs at ∼E8.5 (Fig. 5A). In combination with the kinetics of Tex19.1 and Mili promoter methylation, these data strongly support a model whereby Tex19.1 and Mili undergo de novo methylation and silencing at ∼E6.5 and are then specifically demethylated in PGCs from ∼E8.0 resulting in their transcriptional activation. Thus, the global DNA demethylation event that occurs in PGCs at ∼E8.5 does appear to be associated with developmental regulation of PGC gene expression. The Tex19.1 and Mili promoters therefore represent novel loci regulated by the proposed lineage-specific DNA demethylation event in ∼E8.5 PGCs. The activation of TE-response genes in the germline at both ∼E8.5 and ∼E11.0 suggests that erasure of DNA methylation is used to regulate developmental gene expression in migratory and post-migratory PGCs and might represent a mechanism that helps defend the genome from active TEs during the epigenetic reprogramming events that occur during gametogenesis (Fig. 5B).
Transcriptional activation during reprogramming in PGCs. (A) Single-cell qRT-PCR analysis of PGCs between E8.5 and E11.5, and E8.5 somatic cells. Shown are three single-cell libraries from each time point with expression values normalised to Gapdh. Both Mili and Tex19.1 are transcriptionally activated between E8.5 and E9.5 prior to global to 5mC erasure at ∼E11.0. Error bars represent s.e.m. (B) Model of feedback loop between global 5mC erasure and genome-defence gene activation. At E6.25, PGCs are specified from epiblast cells that have already acquired global de novo methylation. At E8.5, initiation of DNA demethylation in PGCs activates some 'early' genome-defence genes (Tex19.1 and Mili), possibly to prime germ cells. At E11.5, genome-wide erasure of 5mC leads to activation of other genome-defence and germline-specific genes, which can suppress any unmasked transposable element (TE) activity during reprogramming, either post-transcriptionally or through the piRNA pathway. The coupling of genome-defence genes to potential TE activation through a common regulatory mechanism, DNA methylation, ensures that genomic integrity is maintained during 5mC erasure in the germline.
Identification of core methylation-dependent genes
Our initial aim was to unambiguously identify genes that are directly and causally regulated by promoter DNA methylation. We developed an 'epigenetic disruption and recovery' assay which selectively enriches for genes that functionally rely on promoter DNA methylation for silencing, and significantly reduces the indirect hits associated with previous genetic analyses and direct drug exposure (Jackson-Grusby et al., 2001; Palii et al., 2008). This recovery strategy might be useful in identifying genes that are epigenetically silenced by DNA methylation in other contexts, e.g. cancer and tissue specificity. We intersected recovery targets with genes also de-repressed in Dnmt1n/n MEFs and identified a limited number (26) of core methylation-dependent genes, which are highly enriched for germline expression. Using a combination of complementary genetic approaches we demonstrate that Tex19.1, Mili, Asz1 and Mov10l1 are causally regulated by promoter DNA methylation. The mechanism for silencing these genes during post-implantation development appears to involve the de novo DNA methyltransferase DNMT3B. Importantly, acquisition of DNA methylation at these promoters is both necessary for, and temporally coincides with, the initiation of transcriptional silencing at these loci. Indeed, silenced germline genes are preferentially associated with specialised promoter chromatin that is highly depleted of many repressive histone modifications, thus placing promoter CpG methylation as the predominant regulatory mechanism at these loci. It has been unclear whether promoter DNA methylation is only a secondary epigenetic mechanism for 'locking in' a prior silenced state to provide a stable epigenetic memory (Feldman et al., 2006; Fouse et al., 2008; Borgel et al., 2010; Mohn et al., 2008), or whether it also functions as a direct and causal regulatory mechanism per se. Our data supports a model whereby promoter DNA methylation is a primary mechanism for both initiating and maintaining silencing at a small number of germline-specific loci and is potentially the only epigenetic barrier to their ectopic expression.
Epigenetic reprogramming as a cue for gene expression in PGCs
Intriguingly, the methyl-dependent genes we identified appear to be activated by apparent global demethylation events during epigenetic reprogramming in PGCs at either ∼E8.5 or E10.5-11.5. The genome-wide reprogramming event that occurs in E10.5-11.5 PGCs has previously been associated with expression of key germline-specific genes, such as Mvh and Dazl (Maatouk et al., 2006). Here, we show that the epigenetic reprogramming that occurs in PGCs at ∼E8.5 might also be associated with developmental regulation of a different set of germline-associated genes, including Tex19.1 and Mili. The analysis of DNA methylation in post-implantation epiblast and single-cell expression data make Tex19.1 and Mili very good candidate loci for the 5mC reprogramming that is reported to occur in ∼E8.5 PGCs. However, loss of DNA methylation at these loci alone is not sufficient to explain the reported genome-wide reduction in 5mC-immunostaining in these cells, and it is not clear what other loci and genomic features might be losing DNA methylation at this stage of development. Indeed, one possibility is that DNA demethylation is a single progressive event lasting several days in PGCs, with specific genomic regions undergoing 5mC erasure in a temporally defined order, with Tex19.1 and Mili at the vanguard and demethylation of Dazl towards the end of the process.
What determines the different temporal activation profiles is not clear but might be related to specific gene functions during germ cell development, cis-acting elements or the chromatin state in PGCs. It is also not clear how loss of DNA methylation is brought about at the Tex19.1 and Mili promoters in ∼E8.5 PGCs. However, at this stage most PGCs are G2-arrested, suggesting that they might be targeted by active DNA demethylation mechanisms (Kurimoto et al., 2008; Hackett et al., 2012). The differential temporal activation of the closely related Tex19.1 and Tex19.2 paralogues in the developing PGCs is particularly striking given the proximity of these divergently transcribed genes on the chromosome (Kuntz et al., 2008), and analysis of the promoter and intergenic DNA sequence of this gene pair might provide some insight into how erasure of DNA methylation is targeted at different times in PGC development.
Developmental regulation of genome-defence genes
As genome-wide erasure of DNA methylation presents an opportunity for activation of TEs, it is likely that the coupled activation of Tex19.1, Mili, Mov10l1 and Asz1 functions as part of a fail-safe mechanism to ensure suppression of TE activity in the germline. Regulation of these genes by promoter methylation would therefore be inherently linked to their role as guardians of the genome by sensing the possibility of heightened TE activity during epigenetic reprogramming phases, and directing a coordinated response. It is possible that quantitative changes in DNA methylation at these loci in the developing germline might influence their level of expression at different stages in germ cell development. Moreover, the absence of significant regulation by chromatin-based mechanisms at these genes makes them particularly sensitive to global demethylation events, which may prime but do not directly activate other classes of genes. Consistent with this, germline-specific genes are often ectopically expressed in somatic cancers, in which dramatic alterations in DNA methylation distribution can occur (Simpson et al., 2005). The increased TE activity associated with hypomethylated cancer cells might contribute to the genomic instability that is a hallmark of cancers (Romanish et al., 2010). Whether ectopic germline gene activity in a cancer context is a driver of malignancy, or a neutral or even protective consequence of demethylation, remains to be fully determined (Janic et al., 2010). Moreover, although many of the germline-associated methylation-dependent genes appear to function in genome defence, it is not clear whether the non-germline-associated methylation-dependent genes that we have identified (supplementary material Table S1) are also functionally related, or whether co-regulation of these genes by DNA methylation is important for cellular responses to DNA hypomethylation in other contexts.
Other genes involved in germline genome defence, such as Mvh and Mael, have previously been reported to be methylation dependent (Maatouk et al., 2006; Siomi et al., 2011) and some of the uncharacterised methylation-dependent germline genes we have identified might have as yet undiscovered roles in genome defence. Interestingly, although many of the identified genome-defence genes are implicated in the piRNA pathway for TE suppression, global de novo methylation via the piRNA pathway is not reported to commence until E16.5 (Kuramochi-Miyagawa et al., 2008). The activation of methylation-dependent piRNA pathway genes, such as Mili, as early as E9.5 might represent anticipation of future requirements for this pathway (Aravin et al., 2008). Alternatively, as some aspects of the piwi/piRNA pathway function to silence TE activity post-transcriptionally, it is also possible that this pathway is acting earlier than E16.5 (Reuter et al., 2011; Unhavaithaya et al., 2009; Xu et al., 2008). Interestingly, this period might have a key role in allowing TE transcription that, in turn, could target de novo methylation back to TE DNA, possibly via small RNAs (Seisenberger et al., 2010; Aravin et al., 2008). This mechanism would ensure that a powerful epigenetic response is mounted against active TEs and that they are fully silenced throughout the remainder of gametogenesis, at least in male germ cells. However, the relaxation of retrotransposon transcriptional repression induced during PGC epigenetic reprogramming might result in a greater reliance on post-transcriptional retrotransposon silencing mechanisms during these stages of PGC development. Indeed LINE-1 mRNA levels appear to be higher in PGCs than in neighbouring soma, supporting this possibility (Hayashi et al., 2008). Some of the genome-defence genes that are expressed in the developing PGCs might have piRNA-independent roles in regulating TE activity. For example, Tex19.1 appears to regulate TE activity through a post-transcriptional mechanism distinct from the piwi/piRNA pathway (Ollinger et al., 2008). The coordinated activation of multiple genome-defence mechanisms during PGC development is likely to provide a concerted defence against the hundreds of different TE elements present in the mouse genome (Waterston et al., 2002). Further experiments will be required to dissect the roles and specific TE targets of different genome-defence genes in developing PGCs.
In summary, we have identified a novel set of germline-associated genome-defence genes that rely directly on promoter DNA methylation to initiate and maintain their developmental regulation, independently of chromatin-based mechanisms. The tight coupling of DNA demethylation to the activation of these genome-defence genes in PGCs provides a powerful feedback loop that can help prevent transposable element activity and genome instability during global epigenetic reprogramming.
We thank W. Bickmore, W. Dean and N. Hastie for comments during manuscript preparation. We thank A. Bird, J. Selfridge, M. Okano and H. Cedar for cell lines; H. Scholer for Oct4 constructs; J. Nichols for Oct4-GFP reporter mice; and J. Zylicz for artwork. We dedicate this paper to the memory of our former and much missed colleague Danny Wolf.
Work in the R.R.M. laboratory (J.A.H., D.S.D., J.P.R., D.R., C.E.N.) and I.R.A. laboratory (J.R.) is supported by the Medical Research Council and Breakthrough Breast Cancer. Deposited in PMC for release after 6 months.
Competing interests statement
The authors declare no competing financial interests.
J.A.H. performed microarray and expression analyses, meDIP in PGCs and bisulfite-sequencing. J.A.H. and J.P.R. carried out ChIP studies. I.R.A., J.R. and J.A.H. conducted mouse ESC and embryonic work. J.A.H. and C.E.N. carried out bioinformatics analyses. D.S.D., M.B., W.R. and M.A.S. provided essential experimental support, key mouse strains and advice on the manuscript. R.R.M. developed the original concept. J.A.H., I.R.A. and R.R.M. designed the experiments and wrote the manuscript.
Supplementary material available online at http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.081661/-/DC1
Accepted July 3, 2012.
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Matsumura Y.,
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(2008). G9a/GLP complexes independently mediate H3K9 and DNA methylation to silence transcription. EMBO J. 27, 2681–2690.
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(2009). MILI, a PIWI-interacting RNA-binding protein, is required for germ line stem cell self-renewal and appears to positively regulate translation. J. Biol. Chem. 284, 6507–6519.
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(2010). Dnmt3b recruitment through E2F6 transcriptional repressor mediates germ-line gene silencing in murine somatic tissues. Proc. Natl. Acad. Sci. USA 107, 9281–9286.
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(1999). Cytosine methylation and mammalian development. Genes Dev. 13, 26–34.
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(1998). Transcription of IAP endogenous retroviruses is constrained by cytosine methylation. Nat. Genet. 20, 116–117.
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(2007). Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome. Nat. Genet. 39, 457–466.
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2019 on the Node
Read about the most-read posts, scientists on tour and a glimpse into what to look our for in 2020.
Interview – Madeleine Linneberg-Agerholm, Yan Fung Wong and Josh Brickman
Three Copenhagen-based researchers discuss their new paper, which exploits naïve human embryonic stem cells to generate in vitro models for the extra-embryonic endoderm.
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To continue working towards transparency around the editorial process, Development now publishes a 'Peer review history file' alongside published papers. Read more about the policy and see the reports for yourself in one the first papers to publish the reports (under the 'Info & metrics' tab).


NEW LONDON - Outagamie County officials will hold the first of two community input and visioning sessions for Mosquito Hill Nature Center in New London on Sept. 5.
The session, set for 6 p.m. at the nature center, will look at a master planning effort to enhance the facilities at Mosquito Hill with a strong focus on the recently acquired 11-acre property at the top of the hill.
Outagamie County Parks Director Loren Dieck said the input sessions will mirror the master planning efforts for the county's dog park and Plamann Park, which are also undergoing potential renovations.
"We take input on what people would like to see included in that facility," Dieck said. "It could be more walking trails ... or it could be expanding on display renovations, more accessible trails ... any ideas people have as far as what they'd like to see."
While the potential name change to Mosquito Hill that has faced some public criticism is part of the overall master planning process, the name change will not be part of the visioning sessions.
"It's not our intent to discuss the name change," Dieck said.
The parks department previously asked the Friends of Mosquito Hill board to consider a name change as part of a rebranding effort for the 430-acre nature center whose history dates back four decades. The group eventually voted to change the name to Wolf Peak Nature Park.
The county is expected to take up the potential name change this fall.
The name-change recommendation has drawn some harsh criticism from longtime park users who argue that the name has important ties to Native Americans and that the declining use of the nature center has more to do with its programming than with its name.
Only two public input sessions are scheduled for the master planning process before a proposal is brought to the county's Property Committee this fall, Dieck said. A community survey also is planned. The date of the second input session will be announced at a later time.


text:
The U.S Senate race came to a close almost immediately after Delaware. Incumbent Thomas Carper pulled ahead of his republican opponent Robert Arlett by almost 35,000 votes.
In the U.S House races, Democrat Lisa Rochester is leading by 31 points and Democrat Kathleen Jennings is leading by 27 points in the Attorney General race.
Two of Delaware's ten state senate districts up for reelection have been called for democrats. Four of the remaining eight districts have been projected for democrats and the other four are projected for republicans.
A majority of Delaware's State House seats have been called and are projected for democrats, but republicans have won four of the thirteen seats called so far.
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Everyone needs a hand to hold on to and you all did a great job of it. There were challenges to face, however you overcame these obtacles together. That's what being in a relationship is all about; supporting and loving one another to be willing to make that change. Looking good as you all look like two different people, which you are!
Wow! How amazing! Sometimes a partner does help. Thanks for sharing.


text:
Eleanor O'Rourke
Assistant Professor of Education and Social Policy
2233 Tech Drive
Mudd Room 3523
Email Eleanor O'Rourke
Eleanor O'Rourke's website
Ph.D. Computer Science & Engineering, University of Washington, Seattle, WA
M.S. Computer Science & Engineering, University of Washington, Seattle, WA
B.S. Computer Science and Spanish, Colby College, Waterville, ME
My research sits at the intersection of Human-Computer Interaction and Learning Sciences. My interests in this area are broad, but I am particularly excited about designing data-driven learning environments and educational games to improve student motivation, provide personalized learning experiences, and support formative assessment. Some of my recent projects include designing of incentive structures to promote growth mindset behaviors in educational games, building sensemaking tools to support learning front-end development concepts from complex professional websites, and studying how novice programmers' efficacy and mindsets impact their approaches to programming.
Gorson, Jamie; Cunningham, Kathryn; Worsley, Marcelo; O'Rourke, Eleanor, Using Electrodermal Activity Measurements to Understand Student Emotions While Programming, Association for Computing Machinery, Inc (2022).
Kwik, Harrison; Zhang, Haoqi; O'rourke, Eleanor, How Do Students Seek Help and How Do TAs Respond? Investigating Help-Seeking Strategies in CS1 Office Hours, Association for Computing Machinery, Inc (2022).
Cunningham, Kathryn; Qiao, Yike; Feng, Alex; O'Rourke, Eleanor, Bringing "high-level" Down to Earth, Association for Computing Machinery, Inc (2022).
Gorson, Jamie; O'Rourke, Eleanor Mary, How do students talk about intelligence? An Investigation of Motivation, Self-efficacy, and Mindsets in Computer Science, Association for Computing Machinery, Inc:21-29 (2019).
Shi, Joshua; Shah, Armaan; Hedman, Garrett; O'Rourke, Eleanor Mary, Pyrus, Association for Computing Machinery (2019).
O'Rourke, Eleanor Mary; Butler, Eric; Díaz Tolentino, Armando; Popović, Zoran, Automatic generation of problems and explanations for an intelligent algebra tutor, Springer Verlag:383-395 (2019).
Hibschman, Joshua; Gergle, Darren; O'Rourke, Eleanor Mary; Zhang, Haoqi, Isopleth, ACM Transactions on Computer-Human Interaction 26(3) (2019).
Lim, Sarah; Hibschman, Joshua; Zhang, Haoqi; O'Rourke, Eleanor Mary, Ply, Association for Computing Machinery, Inc:991-1002 (2018).


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Is Armenian Presidential Election An Exercise In Futility?
Armenia's upcoming presidential election differs from earlier ballots in two key respects. First, none of the seven opposition candidates is perceived as posing a serious challenge to incumbent President Serzh Sarkisian. And second, the tactics of some lesser-known candidates have been condemned by some as devaluing the entire concept of democratic political struggle.
Armenian Election Campaign Kicks Off
The race to become Armenia's next president has officially started.
Interview Excerpts: Armenian President Serzh Sarkisian
Ahead of Armenia's February 18 election, President Serzh Sarkisian spoke with Harry Tamrazian, the director of RFE/RL's Armenian Service, about his competition in the presidential race, in which Sarkisian is seeking a second term. Sarkisian also spoke about the prospects for a settlement on Nagorno-Karabakh and the state of Armenian-Turkish relations.
Interview: Sarkisian On Upcoming Vote, Turkish Relations, Nagorno-Karabakh
In an exclusive interview with RFE/RL, Armenian President Serzh Sarkisian discusses his competition in the upcoming vote, the state of Armenian-Turkish relations, the situation in Nagorno-Karabakh, and the outflow of Armenian citizens to Russia and the West.
Sarkisian Expects 'Competitive' Armenian Presidential Vote
President Serzh Sarkisian has dismissed suggestions that he won't be facing any true competition in Armenia's presidential election next month, adding that "nurturing" rivals is not the government's job.
Armenian History To Be Taught In Georgia
Armenian history will soon be included in the curriculum of Georgian schools.
Food Fight Rages In The Caucasus
A food fight seems to be under way in the Caucasus with a number of countries laying competing claims to the region's traditional dishes.
Freedom House Laments Eurasian Declines, 'Return Of Iron Fist' In Russia
The non-Baltic former Soviet Union now rivals the Middle East as one of the most politically and socially repressive regions in the world, according to the latest edition of "Freedom in the World," the annual report compiled by U.S.-based rights watchdog Freedom House.
New Foreign-Policy Leaders In U.S. Congress May Challenge White House
When Congress begins its new session this month, two new lawmakers will lead its key foreign-relations committees who might challenge some Obama administration foreign policies, especially toward Iran and the Caucasus.
Invigorated Customs Union Presents Russia's Neighbors With Stark Choice
Russian President Vladimir Putin's premier foreign-policy initiative, the Eurasian Customs Union, is showing unprecedented vitality. But the European Union says membership in the ECU is incompatible with closer economic ties with the EU. This confronts many former Soviet states -- especially Ukraine and Moldova -- with a stark choice.
Eyes In A New Light
It's easy to get lost in Suren Manvelyan's eyes. Manvelyan, 36, is an Armenian photographer who's attracting quite a bit of attention for his sumptuous macro images of the human eye.
Veteran Armenian Presidential Candidate Deplores Absence Of Opposition Challenger
Armenian Public Council Chairman Vazgen Manukian, who narrowly missed unseating incumbent President Levon Ter-Petrossian in the September 1996 presidential ballot, has expressed regret that there will be no real alternative to President Serzh Sarkisian in the presidential election scheduled for February 18, adding that the authorities and the opposition share the blame for that situation.


Reasons: A narrow endemic of an area known as North Park in northern Colorado. There are currently 13 known occurrences (only 10 with positive identification) with annually fluctuating populations sizes. The species is restricted to outcrops of the Coalmont Formation - a coal-bearing substrate that is very susceptible to erosion and is often used by off-road vehicle enthusiasts. Coal extraction and domestic cattle grazing are also threats. The rank may change to G1G2 or G2, if Phacelia plants documented in the Laramie River Valley during 2004 are determined to be Phacelia formosula. However, initial research indicates that the Laramie River Valley population is a separate species. Until a final determination has been made with both morphological and genetic traits, we will maintain the Laramie River occurrences here, with Phacelia formosula.
Range Extent Comments: Known from Jackson County, Colorado. The species is found within about 145 square miles in North Park, Jackson County.
Area of Occupancy Comments: The total area occupied by the mapped occurrences is 583 acres (calculated by the Colorado Natural Heritage Program in 2012). The occupied acreage in North Park is 507 acres. The Laramie River Valley occurrences occupy 76 acres. Imprecisely reported occurrences are not included.
Number of Occurrences Comments: There are 13 principal occurrences documented in the Colorado Natural Heritage Program database. Two of the 13 occurrences have not been observed in over 20 years (as of 2012). Three of the 13 (not the historical records) are tentatively identified as Phacelia formosula (the Laramie River Valley occurrences in Larimer County).
Population Size Comments: Approximately 20,000 individuals have been documented within 12 of the 13 occurrences (including 2,500 from the Laramie River Valley). The remaining occurrence does not report the number of individuals. Populations sizes are annually fluctuating.
Viability/Integrity Comments: Ten of the occurrences are ranked A or B, including the three occurrences that are tentatively identified.
Overall Threat Impact Comments: Motorized recreation is considered to be the primary threat to the species (Rondeau et al. 2011). Habitat is susceptible to erosion. Frequently used motorcycle trails and dirt roads from the airport to the campground have been documented to disturb the plants. 10 to 15 plants were reportedly lost at the State Natural Area in North Park (CNHP 2004). Other threats include livestock trampling/ trailing, grazing, and coal, oil and gas development.
Short-term Trend Comments: Although there are no data to indicate a specific trend, the species seems to be stable within its limited available habitat. Monitoring and recovery efforts are being undertaken by cooperating agencies including the Bureau of Land Management and the U.S. Fish and Wildlife Service.
Long-term Trend Comments: No long term data is available.
Intrinsic Vulnerability Comments: Most occurrences have some grazing. Mature plants have been observed to be trampled.
Global Range: Known from Jackson County, Colorado. The species is found within about 145 square miles in North Park, Jackson County.
Basic Description: A biennial herb, 1.5-2.2 dm high, with deeply divided, leaves and bearing violet-purple flowers in a coiled, scorpion tail-like cluster. The species has been observed in flower in July and August, but flowering and fruiting times may be somewhat variable depending on rainfall and other environmental conditions.
General Description: Herbaceous biennial up to 22 cm tall; much branched, erect to spreading. Inflorescence is a helicoid cyme. Flowers are purple, stamens and styles are exserted. Leaves are pinnately dissected, lancelotate or elliptical. Plants appear somewhat grayish; gladular and hursute (Spackman et al. 1997).
Habitat Comments: Poorly vegetated exposures of the Coalmont Formation. Steep-sided ravines, low sandy hills and bluffs (Spackman et al. 1997). Associated taxa include: Chrysothamnus, Artemisia, Oryzopsis hymenoides, Chaenactis douglasii, Gilia pinnatifida, Ipomopsis congesta, Stipa sp., Tetradymia canescens, Eriogonum exilifolum, E. umbellatum, E. pauciflorum, E. cernuum, Artemisia frigida, Lupinus sp., Astragalus kentrophyta, Opuntia polyacantha and Comandra.
Excellent Viability: Size: 1000 or more individuals (based on available EOR data). Condition: The occurrence has an excellent likelihood of long-term viability as evidenced by evidence of flowering and fruiting, indicating that the reproductive mechanisms are intact. This occurrence should be in a high-quality site with less than 1% cover exotic plant species and/or no significant anthropogenic disturbance. Landscape Context: the occurrence is surrounded by an area that is unfragmented and includes the ecological processes needed to sustain this species. These include a lack of chronic disturbance but occasional disturbance due to soil movement or storm water runoff which permits seedling establishment. Justification: Large populations in high quality sites are presumed to contain a high degree of genetic variability, have a low susceptibility to the effects of inbreeding depression, and to be relatively resilient.
Good Viability: Size: 200 or more individuals (based on available EOR data). Condition: The occurrence should have a good likelihood of long-term viability as evidenced by the presence of a robust seed bank, and by the observation of flowering and fruiting individuals. Anthropogenic disturbance within the occurrence is minimal. If exotic species are present, they comprise less than 10% of the total ground cover. Landscape Context: the surrounding landscape should contain the ecological processes needed to sustain the occurrence but may be fragmented and/or impacted by humans.
Fair Viability: Size: 50 to 200 individuals (based on available EOR data). Condition: The occurrence may be less productive than the above situations, but is still viable, with evidence of flowering, fruiting, and a robust seed bank indicating that the reproductive mechanisms are intact. The occupied habitat is somewhat degraded (exotic plant species make up between 10-50% of the total ground cover and/or there is a moderate level of anthropogenic disturbance). Landscape Context: there may be significant human disturbance, but the ecological processes needed to sustain the species are still intact.
Poor Viability: Size: Less than 50 individuals (based on available EOR data). Condition: Little or no evidence of successful reproduction is observed (poor seedling recruitment or no flowering or fruiting observed). Exotic plant species make up greater than 50% of the total ground cover, and/or there is a significant level of human disturbance. Landscape context: the surrounding area is fragmented with many ecological processes no longer intact. The occurrence has a low probability of long-term persistence due to inbreeding depression, natural stochastic events, and its intrinsic vulnerability to human impacts.
Atwood, D. 2010. Progress Report for Phacelia formosula Osherhout in North Park, Jackson County, Colorado and the Scully Phacelia in Larimer County, Colorado; prepared for USFWS.
Coles, Janet. 2004. Field Visit to Phacelia formosula in North Park and Phacelia sp. in Larimer County. unpublished notes on field visit of August 7, 2004.
Colorado Natural Heritage Program. 2006. Biological Conservation Datasystem. Colorado State University, Ft. Collins, CO.
O'Kane, S.L. 1986. Endangered Species Information System Species Workbook Part VII. Unpublished report for the US Fish and Wildlife Service, Denver, CO.
Peterson, J. S. and K.L. Wiley-Eberle. 1986. North Park Phacelia Recovery Plan. USFWS, Denver, CO. 28pp.
Peterson, J. Scott and K.L. Wiley-Eberle. 1986. North Park Phacelia Recovery Plan. USFWS, Denver, CO. 28pp.
U. S. Fish and Wildlife Service. 1982. Determination that Phacelia formosula is an endangered species. Federal Register 47(170): 38540-38543.
Warren, K.D. 1990. A Comparative Study of the Reproductive Biology of a Rare and a Common Phacelia Species. Unpublished thesis.
Wiley-Eberle, K.L. 1979. Status report for Phacelia formosula. Unpublished report prepared for the Bureau of Land Management.


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How can a traditional brand be reframed to inspire today's creative lifestyles?
For the 'Maker Generation' - confident, connected and expressive Millennials - the act of creating is no longer limited to the arts or the domain of the artistic, but rather an exploration of identity, an avenue for self-expression and a form of release in an overly digitised world. Heritage art brand, Reeves needed a new visual identity to re-establish an emotional connection with this new generation of creatives.
Read full life story Get in touch
With an identity that has remained largely untouched for more than 250 years, heritage art brand, Reeves, needed to re-establish its emotional connection with its Millennial audience.
Considering the role that creativity plays in the life of the 'maker generation,' we've transformed Reeves' focus from the discipline of art to the democratisation of creativity. Confident, connected and expressive, millennials exemplify a lifestyle in which creativity is instinctual: a state of mind as opposed to a qualification. To bring this to life, we repositioned Reeves as a lifestyle brand that embodies a shift from 'artistic creation' to 'self-expression', and manifests itself across four key touchpoints: Design, Communication, Innovation and Experience.
Characterised by a cleaner typeface, colourful coding system and bold wordmark in which the 'R' evokes a framed corner, the visual language we developed reflects the brand's evolution from dusty and dated to being all about framing creativity and celebrating spontaneous creation. We've extended this idea into the digital space with a brand new website that serves as a space for inspiration in which creators at both ends of the artistic spectrum can interact with the brand.
To find out more about our work for Reeves or any of our other brands, get in touch.
ColArt
Mode of life
Consider Pastures
Kiss My
Driscoll's
To find out more about our work for Reeves or any of our other brands.


For over 30 years, Dr. Terence Reed, Dr. Aaron Acres, and the entire team of Potsdam Center for Innovative Dental Technologies have built a reputation for providing state-of-the-art dental care in a caring and welcoming environment. We serve the entire St. Lawrence County community, and many of our patients travel a great distance to see us because they know they will find quality care.
Most of our staff has been with us since our doors opened in 1980. We pride ourselves on the relationships we have built, and the families who have continued to bring their children into us after they have grown up.
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Call our Potsdam office today to schedule a consultation, including a complimentary Invisalign consultation. We will sit with you and discuss the best treatment for your needs. We strive to exceed your expectations.
Serving Potsdam, Canton, Ogdensburg, and Massena, NY (New York) - Drs. Terence Reed and Aaron Acres are your general dentists, offering Invisalign, CEREC crowns, and dental care for children, teens, and adults.


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The Alpha Psi actives participate in philanthropy on a regular basis through its Philanthropy Committee, run by AJ Springer, Teddy Yull and Alessio Marcogliese.
Based on the history of our chapter as well as the fraternity in general, we understand what it means to help and give back to the community. As we continue to grow as a chapter, so do our philanthropic efforts.
CASCO DECA-DANCE 2018
This winter several actives participated in the CASCO DECA-DANCE fundraiser under the name BROSAICA. This group of actives undertook several evenings of dance practice under the indstruction of the lovely Andria Campbell. This fundraiser alone raised over $10,000 in ticket sales and donations. The CASCO organization holds various fundraisers to help raise money for the Montreal Children's Hospital. This year's theme was the decades. As active philanthropists and talented dancers, the Alpha Psi chapter (McGill University & Concordia University) performed one of the most outstanding and entertaining acts of all time.
Spanning more than a decade, Brosaica is one of Alpha Psi's longest lasting philanthropic traditions. We look forward to seeing all the brothers at try-outs this upcoming Fall.
This years BROSAICA consisted of: Thomas "Savage" Raymond, Noah Gillard, Bogdan Tanasie, Xavier Fortin, Cedric Spothelfer, Nathaniel Williams, Denzel Mutoko.
Right to Play Spin-a-thon
Over 30 brothers participated in the Right to Play Spin-a-thon. Right to Play is an organization that uses sport and play to empower children to overcome the effects of poverty, conflict and disease in disadvantaged communities. Alpha Psi alone raised over 600$ in donations for the cause. Alpha Psi has had a long tradition of partnering with Right to Play and we look forward to beating last year's benchmark.
Theta Touchdown
The Alpha Psi Chapter participated in a flag-football tournament that raised money for CASA (Court Appointed Special Advocates) organized by Kappa Alpha Theta. The tournament consisted of a bracket of 8 teams (including Zeta Psi A and Zeta Psi B). Alpha Psi has been dominating this tournament since the days of the legendary connection of Charlie Levy and Brenden McKinney. For the fourth year in a row the final consisted of Zeta Psi A versus Zeta Psi B. It is worth noting that we were all pleasantly surprised with Brother Matthew Lapointe's play at quarterback. Despite being in midterms, Matthew consistently connected with his receivers and threw 7 touchdown passes. Come and cheer us on this October as we strive to win a fifth consecutive Theta Touchdown championship all while raising money for a good cause.
Movember Campaign
The Alpha Psi chapter has participated in the Movember campaign for the past several years. In 2018, sporting breathtaking 'staches of all shapes and sizes, the chapter raised over $14 000 for prostate cancer research and awareness. Over the past 3 years, the Brothers have raised nearly $50 000.
February Madness
This past February, Zeta Psi teamed up with the Right to Play organization to organize the first annual February Madness intra-fraternity soccer tournament. The tournament raised over $1 000 for children in impoverished and war-torn countries to participate in organized athletics. The Alpha Psi Chapter hopes to make this fundraiser into an annual tradition.


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Jeanine Ann Roose (October 24, 1937 – December 31, 2021) was an American child actress and psychologist.

Life and career
Roose was born on October 24, 1937, to Ivan R. and Agatha Roose. Her first job was on The Jack Benny Program at the age of eight; the role, as that of "Baby" or "Little" Alice Harris, is one she would keep for most of her entertainment career. She was also featured as a character on The Fitch Bandwagon and The Phil Harris-Alice Faye Show from 1946 to 1954. The character shared a name with the real-life daughter of Phil Harris and Alice Faye; the couple's two daughters did not wish to appear on the program.

Other radio appearances included playing Chris in the Lux Radio Theatre production of I Remember Mama and an episode of Mr. President with Edward Arnold. Her sole film credit was as young Violet Bick in the 1946 film classic It's a Wonderful Life. She also starred in the unaired television pilot Arabella's Tall Tales.

Roose attended Audubon Junior High School. She later attended the University of California, Los Angeles, where she was a member of Alpha Delta Pi. She worked as a Jungian psychoanalyst in her later life. Roose married Eugene Richard Auger on September 4, 1964.

She died from an abdominal infection in Valley Village, California, on December 31, 2021, at the age of 84.

Works

References

External links

Jeanine Roose on the RadioGOLDINdex
Jeanine A. Roose  profile at C.G. Jung Institute of Los Angeles

1937 births
2021 deaths
20th-century American actresses
Actresses from Los Angeles
American child actresses
American film actresses
American radio actresses
University of California, Los Angeles alumni
21st-century American psychologists

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WEDNESDAY, Sept. 23, 2020 (HealthDay News) -- If you sing indoors without a face mask, you run the risk of spreading COVID-19 through tiny airborne particles known as aerosols.
That's the conclusion researchers reached after studying a choir practice.
In Skagit Valley, Wash., one person with mild symptoms of COVID-19 attended a 2.5-hour-long indoor choir practice on March 10. Over the next several weeks, more than 50 others from the rehearsal got sick and two died.
"This study documents in great detail that the only plausible explanation for this super-spreading event was transmission by aerosols," said lead author Shelly Miller, a professor of mechanical engineering at the University of Colorado Boulder. "Shared air is important because you can be inhaling what someone else exhaled even if they are far away from you."
The singers did not touch each other or shared surfaces, and few used the same restroom as the infected person. But they did not wear masks.
Poor ventilation led aerosols and heat from the singers' to mix with room air, the researchers noted in a university news release.
According to study co-author Jose-Luis Jimenez, "The inhalation of infectious respiratory aerosol from 'shared air' was the leading mode of transmission." Jimenez is a professor of chemistry and fellow at the Cooperative Institute for Research in Environmental Sciences, at the university.
The researchers estimated that shortening the rehearsal to 30 minutes would have reduced the infection rate from 87% to 12%. If masks had been worn, the number of people infected would have dropped from 52 to five, they added.
Miller pointed out that "singing is known to release high amounts of aerosol."
Jimenez said that "the research adds to the overwhelming body of evidence that aerosol transmission is playing a major role in driving the pandemic and especially in super-spreading events."
The findings were published Sept. 18 in the journal Indoor Air.
For more on COVID-19, head to the U.S. Centers for Disease Control and Prevention.
SOURCE: University of Colorado Boulder, news release, Sept. 18, 2020
Cancer: Preventing Infections
Central Line Infections


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