Erasure Of DNA Methylation Research Articles

Sex-specific histone modifications in mouse fetal and neonatal germ cells.

Epigenetic signatures of germline cells are dynamically reprogrammed to induce appropriate differentiation, development and sex specification. We investigated sex-specific epigenetic changes in mouse fetal germ cells (FGCs) and neonatal germ cells. Six histone marks in mouse E13.5 FGCs and P1 neonatal germ cells were analyzed by chromatin immunoprecipitation and sequencing. These datasets were compared with transposase-accessible chromatin sites, DNA methylation and transcriptome. Different patterns of each histone mark were detected in female and male FGCs, and H3K4me3/H3K27me3 bivalent marks were enriched in different chromosomal regions of female and male FGCs. Our results suggest that histone modifications may affect FGC gene expression following DNA methylation erasure, contributing to the differentiation into female and male germ cells.

Dnmt1 is essential for egg production and embryo viability in the large milkweed bug, Oncopeltus fasciatus

BackgroundThe function of cytosine (DNA) methylation in insects remains inconclusive due to a lack of mutant and/or genetic studies.ResultsHere, we provide evidence for the functional role of the maintenance DNA methyltransferase 1 (Dnmt1) in an insect using experimental manipulation. Through RNA interference (RNAi), we successfully posttranscriptionally knocked down Dnmt1 in ovarian tissue of the hemipteran Oncopeltus fasciatus (the large milkweed bug). Individuals depleted for Dnmt1, and subsequently DNA methylation, failed to reproduce. Eggs were inviable and declined in number, and nuclei structure of follicular epithelium was aberrant. Erasure of DNA methylation from gene or transposon element bodies did not reveal a direct causal link to steady-state mRNA levels in somatic cells. These results reveal an important function of Dnmt1 seemingly not contingent on directly controlling gene expression.ConclusionsThis study provides direct experimental evidence for a functional role of Dnmt1 in egg production and embryo viability and uncovers a trivial role, if any, for DNA methylation in control of gene expression in O. fasciatus.

Dynamics of DNA Methylation Reprogramming Influenced by X Chromosome Dosage in Induced Pluripotent Stem Cells

How the epigenome of one cell type is remodeled during reprogramming into another unrelated type of cell remains unclear. Overexpression of transcription factors in somatic cells enables the induction of induced pluripotent stem cells (iPSCs). This process entails genome-wide remodeling of DNA methylation, chromatin, and transcription. Recent work suggests that the number of active X chromosomes present in a cell influences remodeling of DNA methylation during somatic cell reprogramming to mouse iPSCs. Female iPSCs with 2 active X chromosomes display global DNA hypomethylation, whereas male XY iPSCs show DNA methylation levels similar to the somatic cells they are derived from. Global DNA methylation erasure in female iPSCs takes place genome-wide and involves repression of DNA methyltransferases. However, on loss of one X chromosome, female iPSCs acquire a DNA methylation landscape resembling that of XY iPSCs. Therefore, it is the X chromosome dosage that dictates global DNA methylation levels in iPSCs. Here, we discuss the evidence that links X chromosome dosage with the regulation of DNA methylation in pluripotent stem cells. We focus on iPSCs reprogramming studies, where X chromosome status is a novel factor impacting our understanding of epigenetic remodeling.

TRIM28 epigenetic corepressor is indispensable for stable induced pluripotent stem cell formation

Cellular reprogramming proceeds in a stepwise pathway initiated by binding and transcription of pluripotency factors followed by genome-wide epigenetic changes. Priming events, such as erasure of DNA methylation and chromatin remodeling determines the success of pluripotency acquisition later. Therefore, growing efforts are made to understand epigenetic regulatory network that makes reprogramming possible and efficient. Here, we analyze the role of transcriptional corepressor TRIM28, involved in heterochromatin formation, during the process of reprogramming of mouse somatic cells into induced pluripotent stem cells (iPS cells). We demonstrate that Trim28 knockdown (Trim28 KD) causes that emerging iPS cells differentiate immediately back into MEFs therefore they fail to yield stable iPS cell colonies. To better comprehend the mechanism of TRIM28 action in reprogramming, we performed a reverse-phase protein array (RPPA) using in excess of 300 different antibodies and compared the proteomic profiles of wild-type and Trim28 KD cells during reprogramming. We revealed the differences in the dynamics of reprogramming of wild-type and Trim28 KD cells. Interestingly, proteomic profile of Trim28 KD cells at the final stage of reprogramming resembled differentiated state rather than maintenance of pluripotency and self-renewal, strongly suggesting spontaneous differentiation of Trim28 KD cells back to their parental cell type. We also observed that action of TRIM28 in reprogramming is accompanied by differential enrichment of proteins involved in cell cycle, adhesion and stemness. Collectively, these results suggest that regulation of epigenetic modifications coordinated by TRIM28 plays a crucial role in reprogramming process.

TET function in development.

Development In development, cell fate commitment enables the generation of the body's specialized cells. When one fate is targeted, other paths must be prevented. DNA methylation of CpG dinucleotides is key for such molecular repression. However, the erasure of DNA methylation is also critical in development and occurs at specific times, such as during embryonic postimplantation. The ten-eleven translocation (TET) genes perform this role. Khoueiry et al. now identify a methylation-independent role for TET1 to repress genes involved in the differentiation of epiblast and extraembryonic ectoderm through regulation of the transcriptional repressor JMJD8. Embryonic defects result if TET1 is removed. Hence, the well-known demethylase has critical functions in normal development through both catalytic and noncatalytic activities. Nat. Gen. 10.1038/ng.3868 (2017).

Epigenetic regulation during the differentiation of stem cells to germ cells.

Gametogenesis is an essential process to ensure the transfer of genetic information from one generation to the next. It also provides a mechanism by which genetic evolution can take place. Although the genome of primordial germ cells (PGCs) is exactly the same with somatic cells within an organism, there are significant differences between their developments. For example, PGCs eventually undergo meiosis to become functional haploid gametes, and prior to that they undergo epigenetic imprinting which greatly alter their genetic regulation. Epigenetic imprinting of PGCs involves the erasure of DNA methylation and the reestablishment of them during sperm and oocyte formation. These processes are necessary and important during gametogenesis. Also, histone modification and X-chromosome inactivation have important roles during germ cell development. Recently, several studies have reported that functional sperm or oocytes can be derived from stem cells in vivo or in vitro. To produce functional germ cells, induction of germ cells from stem cells must recapitulate these processes similar to endogenous germ cells, such as epigenetic modifications. This review focuses on the epigenetic regulation during the process of germ cell development and discusses their importance during the differentiation from stem cells to germ cells.

Erasure of DNA methylation, genomic imprints, and epimutations in a primordial germ-cell model derived from mouse pluripotent stem cells

The genome-wide depletion of 5-methylcytosines (5meCs) caused by passive dilution through DNA synthesis without daughter strand methylation and active enzymatic processes resulting in replacement of 5meCs with unmethylated cytosines is a hallmark of primordial germ cells (PGCs). Although recent studies have shown that in vitro differentiation of pluripotent stem cells (PSCs) to PGC-like cells (PGCLCs) mimics the in vivo differentiation of epiblast cells to PGCs, how DNA methylation status of PGCLCs resembles the dynamics of 5meC erasure in embryonic PGCs remains controversial. Here, by differential detection of genome-wide 5meC and 5-hydroxymethylcytosine (5hmeC) distributions by deep sequencing, we show that PGCLCs derived from mouse PSCs recapitulated the process of genome-wide DNA demethylation in embryonic PGCs, including significant demethylation of imprint control regions (ICRs) associated with increased mRNA expression of the corresponding imprinted genes. Although 5hmeCs were also significantly diminished in PGCLCs, they retained greater amounts of 5hmeCs than intragonadal PGCs. The genomes of both PGCLCs and PGCs selectively retained both 5meCs and 5hmeCs at a small number of repeat sequences such as GSAT_MM, of which the significant retention of bisulfite-resistant cytosines was corroborated by reanalysis of previously published whole-genome bisulfite sequencing data for intragonadal PGCs. PSCs harboring abnormal hypermethylation at ICRs of the Dlk1-Gtl2-Dio3 imprinting cluster diminished these 5meCs upon differentiation to PGCLCs, resulting in transcriptional reactivation of the Gtl2 gene. These observations support the usefulness of PGCLCs in studying the germline epigenetic erasure including imprinted genes, epimutations, and erasure-resistant loci, which may be involved in transgenerational epigenetic inheritance.

Similarity of epigenetic reprogramming in primordial germ cells between human and mouse

Germ cells have a unique ability to produce a new individual and are paramount for transmitting intact genome and appropriate epigenome from generation to generation. Primordial germ cells (PGCs), the first germ cell population established in the embryo, undergo sequential epigenetic reprogramming to ensure the fidelity of this transmission. Although the developmental and epigenetic processes in mouse PGCs are well understood, antithetically our knowledge in human PGCs remains elusive. Recently, three research teams coincidently discovered the transcriptome and DNA methylome of human PGCs by single-cell RNA-sequencing and whole-genome bisulfite sequencing, providing a fundamental framework for understanding human germline epigenetic reprogramming during embryonic development and its biological outcome [1–3]. One of the teams, led by Fuchou Tang and Jie Qiao at Peking University in China, analyzed the transcriptome and DNAmethylome of PGCs from 4to 19week-old human embryos and provided four key insights [2]. First, the transcriptomes of early human PGCs were generally stable between 4 and 11 weeks of development. Only after 17 weeks the heterogeneity of gene expressionoccured in meiotic female PGCs, indicating entry into meiosis asynchronously. However, the inactivated X chromosome was reactivated in human PGCs as early as 4 weeks. Second, global DNA demethylation in human PGCs was much more thorough than that of preimplantation embryos, and happened in gene body regions, the surrounding intergenic regions, functional genomic elements and CpG islands. However, the evolutionarily younger and more active transposable elements evaded global DNA demethylaion in human PGCs and retained high levels of residual DNA methylation, implying potential role for transgenerational epigenetic inheritance as the other group suggested [3].Third, extensive erasure of the methylation of imprinted genes occurred in early humanPGCs and wasmaintaineduntil 19weeks.Lastly, the study showed that there was strong enrichment of the base-excision mismatch repair (BER) pathway in human PGCs, denoting the possibility of global DNA demethylation being contributed by the BER pathway. A comparison of the methylomes between human and mouse PGCs reveals high conservation of epigenetic reprograming, with overall similar genomewide DNA demethylation dynamics in human migrating and gonadal PGCs and those of mouse PGCs at comparable stages.Mechanistically, BER pathway enrichment has been also observed at similar stages of PGCs in mouse [4,5], implying human and mouse may share same BER-involved DNA demethylation mechanisms. With the detailed map of transcriptome and DNA methylome, the next challenge will be to elucidate the underlying mechanisms behind epigenetic regulation during human PGC development. It remains to be clarified how global DNA demethylation does not result in deregulation gene transcription in PGCs. The functional link between regions resistant to DNA demethylation and transgenerational epigenetic inheritanceneeds tobe further defined in future work.More specifically,what is themechanism for driving escape from global erasure of DNA methylation during human PGCdevelopment?As humanPGCsamples remain scarce, achieving a mechanistic understanding of epigenetic regulation in human PGCs will be difficult. However, given the similarity between DNA methylation dynamics in mouse and human PGCs, mouse PGCs should provide a valuable model for uncovering themechanismsof epigenetic reprogramming in human PGCs.

PGC Reversion to Pluripotency Involves Erasure of DNA Methylation from Imprinting Control Centers followed by Locus-Specific Re-methylation

SummaryPrimordial germ cells (PGCs) are fate restricted to differentiate into gametes in vivo. However, when removed from their embryonic niche, PGCs undergo reversion to pluripotent embryonic germ cells (EGCs) in vitro. One of the major differences between EGCs and embryonic stem cells (ESCs) is variable methylation at imprinting control centers (ICCs), a phenomenon that is poorly understood. Here we show that reverting PGCs to EGCs involved stable ICC methylation erasure at Snrpn, Igf2r, and Kcnqot1. In contrast, the H19/Igf2 ICC undergoes erasure followed by de novo re-methylation. PGCs differentiated in vitro from ESCs completed Snrpn ICC erasure. However, the hypomethylated state is highly unstable. We also discovered that when the H19/Igf2 ICC was abnormally hypermethylated in ESCs, this is not erased in PGCs differentiated from ESCs. Therefore, launching PGC differentiation from ESC lines with appropriately methylated ICCs is critical to the generation of germline cells that recapitulate endogenous ICC erasure.

Epigenetic inheritance and the missing heritability.

Genome-wide association studies of complex physiological traits and diseases consistently found that associated genetic factors, such as allelic polymorphisms or DNA mutations, only explained a minority of the expected heritable fraction. This discrepancy is known as “missing heritability”, and its underlying factors and molecular mechanisms are not established. Epigenetic programs may account for a significant fraction of the “missing heritability.” Epigenetic modifications, such as DNA methylation and chromatin assembly states, reflect the high plasticity of the genome and contribute to stably alter gene expression without modifying genomic DNA sequences. Consistent components of complex traits, such as those linked to human stature/height, fertility, and food metabolism or to hereditary defects, have been shown to respond to environmental or nutritional condition and to be epigenetically inherited. The knowledge acquired from epigenetic genome reprogramming during development, stem cell differentiation/de-differentiation, and model organisms is today shedding light on the mechanisms of (a) mitotic inheritance of epigenetic traits from cell to cell, (b) meiotic epigenetic inheritance from generation to generation, and (c) true transgenerational inheritance. Such mechanisms have been shown to include incomplete erasure of DNA methylation, parental effects, transmission of distinct RNA types (mRNA, non-coding RNA, miRNA, siRNA, piRNA), and persistence of subsets of histone marks.

Forget the Parents: Epigenetic Reprogramming in Human Germ Cells

Epigenetic reprogramming in the germline resets genomic potential and erases epigenetic memory. Three studies by Gkountela et al., Guo et al., and Tang et al. analyze the transcriptional and epigenetic landscape of human primordial germ cells, revealing a unique transcriptional network and progressive and conserved global erasure of DNA methylation.

New insights into mechanisms that regulate DNA methylation patterning

From a fertilised egg to a mature organism, cells divide and accumulate epigenetic information, which is faithfully passed on to daughter cells. DNA methylation consolidates the memory of the developmental history and, albeit very stable, it is not immutable and DNA methylation patterns can be deconstructed – a process that is essential to regain totipotency. Research into DNA methylation erasure gained momentum a few years ago with the discovery of 5-hydroxymethylcytosine, an oxidation product of 5-methylcytosine. The role of this new epigenetic modification in DNA demethylation and other potential epigenetic roles are discussed here. But what are the mechanisms that regulate deposition of epigenetic modifications? Until recently, limited direct evidence indicated that signalling molecules are able to modulate the function of epigenetic modifiers, which shape the epigenome in the nucleus of the cell. New reports in embryonic stem cell model systems disclosed a tight relationship between major signalling pathways and the DNA methylation machinery, which opens up exciting avenues in the relationship between external signals and epigenetic memory. Here, I discuss mechanisms and concepts in DNA methylation patterning, the implications in normal development and disease, and future directions.

High histone variant H3.3 content in mouse prospermatogonia suggests a role in epigenetic reformatting.

Histone variants can incorporate into the nucleosome outside of S-phase. Some are known to play important roles in mammalian germ cell development, this cell lineage being characterized by long phases of quiescence, a protracted meiotic phase, and genome-wide epigenetic reformatting events. The best known example of such an event is the global-scale erasure of DNA methylation in sexually indifferent primordial germ cells, then its re-establishment in fetal prospermatogonia and growing oocytes. Histone H3 and its post-translationally modified forms provide important waypoints in the establishment of epigenetic states. Using mass spectrometry and immunoblotting, we show that the H3.3 replacement variant is present at an unusually high amount in mouse prospermatogonia at the peak stage of global DNA methylation re-establishment. We speculate that H3.3 facilitates this process through achieving a greater level of accessibility of chromatin modifiers to DNA.

Regulation of DNA methylation turnover at LTR retrotransposons and imprinted loci by the histone methyltransferase Setdb1.

During mammalian development, DNA methylation patterns need to be reset in primordial germ cells (PGCs) and preimplantation embryos. However, many LTR retrotransposons and imprinted genes are impervious to such global epigenetic reprogramming via hitherto undefined mechanisms. Here, we report that a subset of such genomic regions are resistant to widespread erasure of DNA methylation in mouse embryonic stem cells (mESCs) lacking the de novo DNA methyltransferases (Dnmts) Dnmt3a and Dnmt3b. Intriguingly, these loci are enriched for H3K9me3 in mESCs, implicating this mark in DNA methylation homeostasis. Indeed, deletion of the H3K9 methyltransferase SET domain bifurcated 1 (Setdb1) results in reduced H3K9me3 and DNA methylation levels at specific loci, concomitant with increased 5-hydroxymethylation (5hmC) and ten-eleven translocation 1 binding. Taken together, these data reveal that Setdb1 promotes the persistence of DNA methylation in mESCs, likely reflecting one mechanism by which DNA methylation is maintained at LTR retrotransposons and imprinted genes during developmental stages when DNA methylation is reprogrammed.

Epigenetic Reprogramming in Primordial Germ Cells in Mice

Abstract: Germline cells are the sole source of the transmission of genetic and epigenetic information to the next generation. Epigenetic information is reprogrammed during germ cell development to reacquire cellular totipotency and prevent the accumulation of epimutations. In this review, we summarize epigenetic reprogramming, in particular, DNA demethylation in developing primordial germ cells (PGCs). The recent development of next-generation sequencing, and the discovery of 5-methylcytosine oxidation are major breakthroughs in the study of epigenetic reprogramming in PGCs. DNA methylation analysis with high-throughput sequencing has uncovered the dynamics of DNA methylation erasure at single-locus resolution, which has revealed the global loss of DNA methylation in migrating PGCs, and locus-specific DNA demethylation in gonadal PGCs. The disruption of ten-eleven translocation genes shows that they are required for DNA demethylation at germline-specific genes in gonadal PGCs. These findings indicate tha...

FGF signalling inhibition in ESCs drives rapid genome-wide demethylation to the epigenetic ground state of pluripotency

Genome-wide erasure of DNA methylation takes place in primordial germ cells (PGCs) and early embryos and is linked with pluripotency. Inhibition of Erk1/2 and Gsk3β signalling in mouse embryonic stem cells (ESCs) by small molecule inhibitors (called 2i) has recently been shown to induce hypomethylation. We show by whole-genome bisulphite sequencing that 2i induces rapid and genome-wide demethylation on a scale and pattern similar to that in migratory PGCs and early embryos. Major satellites, intracisternal A particles (IAPs) and imprinted genes remain relatively resistant to erasure. Demethylation involves oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), impaired maintenance of 5mC and 5hmC and repression of the de novo methyltransferases (Dnmt3a, Dnmt3b) and Dnmt3L. We identify a Prdm14 and Nanog binding cis-acting regulatory region in Dnmt3b that is highly responsive to signalling. These insights provide a novel framework for understanding how signalling pathways regulate reprogramming to an epigenetic ground state of pluripotency.

FGF Signaling Inhibition in ESCs Drives Rapid Genome-wide Demethylation to the Epigenetic Ground State of Pluripotency

SummaryGenome-wide erasure of DNA methylation takes place in primordial germ cells (PGCs) and early embryos and is linked with pluripotency. Inhibition of Erk1/2 and Gsk3β signaling in mouse embryonic stem cells (ESCs) by small-molecule inhibitors (called 2i) has recently been shown to induce hypomethylation. We show by whole-genome bisulphite sequencing that 2i induces rapid and genome-wide demethylation on a scale and pattern similar to that in migratory PGCs and early embryos. Major satellites, intracisternal A particles (IAPs), and imprinted genes remain relatively resistant to erasure. Demethylation involves oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), impaired maintenance of 5mC and 5hmC, and repression of the de novo methyltransferases (Dnmt3a and Dnmt3b) and Dnmt3L. We identify a Prdm14- and Nanog-binding cis-acting regulatory region in Dnmt3b that is highly responsive to signaling. These insights provide a framework for understanding how signaling pathways regulate reprogramming to an epigenetic ground state of pluripotency.

Epigenetic synergies between methyl donors and biotin in gene repression are mediated by holocarboxylase synthetase (HLCS)

HLCS catalyzes the binding of biotin to proteins in humans. Previously, we have shown (1) that HLCS interacts physically with the methyl transferase EHMT‐1, which creates histone H3 K9 methylation (H3K9me) gene repression marks, (2) that biotinylation of EHMT‐1 strengthens interactions with HLCS, (3) that biotin depletion and HLCS knockdown cause a loss of H3K9me marks leading to de‐repression of long terminal repeats (LTRs) and to chromosomal abnormalities, and (4) that erasure of DNA methylation impairs HLCS‐dependent biotinylation events. Here, we tested the novel hypothesis that methyl donors and biotin synergize in mediating gene repression through epigenetic marks, and that HLCS contributes toward mediating these synergies. Co‐immunoprecipitation (Co‐IP) and limited proteolysis assays (LPA) confirmed that HLCS interacts physically with the DNA methyl transferase DNMT1 and methyl CpG binding protein MeCP2. Overexpression of HLCS in HEK293 cells caused a 200% increase of H3K9me marks in LTRs (Kruskal‐Wallis, P<0.001), judged by ChIP, and an 84% decrease in LTR mRNA (t‐test, P<0.05), judged by qRT‐PCR, compared with controls; erasure of DNA methylation by azacytidine abrogated effects of HLCS. We conclude that interactions between HLCS and chromatin proteins create epigenetic synergies between biotin and methyl donors in gene repression, thereby maintaining genome stability.Grant Funding Source: ARD Hatch, NIFA, NIH

5mC and 5hmC dynamics during PGC reprogramming and role of Tet1 in female meiosis

Meiosis is a germ-cell-specific cell division process through which haploid gametes are produced for sexual reproduction. Before the initiation of meiosis, mouse primordial germ cells undergo a series of epigenetic reprogramming steps, including the global erasure of DNA methylation at the 5-position of cytosine (5mC) in CpG-rich DNA. However, the 5mC dynamics and its relationship with the generation of 5-hydroxymethylcytosine (5hmC) during the reprogramming process are not clear. Here we analyzed the dynamics of 5mC and 5hmC during PGC reprograming. Unexpectedly, we found a “blank period” (E8.5-9.5) in which both 5mC and 5hmC are low. After that, 5hmC level increases and reaches its peak at E10.5 and gradually decreases until E13.5. Interestingly, 5hmC is enriched in chromocenter during this period in a Tet1 -dependent manner. While this germ cell-specific 5hmC subnuclear localization pattern is maintained in female germ cells till mature oocytes, such pattern is gradually lost in male germ cells as mitosis resumes at neonatal stage. Using a loss-of-function approach in mice, we show that Tet1 has an important role in regulating meiosis in mouse oocytes. Tet1 deficiency significantly reduces female germ-cell numbers and fertility. Univalent chromosomes and unresolved DNA double-strand breaks are also observed in Tet1-deficient oocytes. Tet1 deficiency does not greatly affect the genome-wide demethylation, but leads to defective DNA demethylation and decreased expression of a subset of meiotic genes. Our study thus not only reveals the dynamics of 5mC and 5hmC during PGC reprogramming, but also demonstrate a function for Tet1 in meiosis and meiotic gene activation in female germ cells.

The simplest explanation: passive DNA demethylation in PGCs.

Primordial germ cell (PGC) specification is one of two major developmental windows where modifications associated with global heterochromatin maintenance are erased. One of the most intriguing and confounding dynamics to rectify has been the apparent global depletion of cytosine methylation, which has been intensely scrutinized for nearly two decades. While numerous reports have suggested active catalytic removal as the primary mechanism, work by Saitou and colleagues presented in this issue of The EMBO Journal provide support for a simpler model whereby the downregulation of essential recruitment factors appears sufficient to erase this mark passively during a phase of rapid proliferation. During mammalian development, transitions in cellular programs are predominantly accompanied by focal changes to chromatin that reflects local activities of transcriptional activators or repressors. As such, the fraction of the genome that is targeted for remodelling during any given transcription factor driven programming event is comparatively small (Dunham et al , 2012). Two exceptions to this general paradigm occur upon specification of the PGC lineage within the developing embryo and upon fertilization within the protamine‐compacted paternal genome (Saitou et al , 2012). In these contexts, dramatic global epigenetic modification appears to restructure the majority of the genome and does so in a rapid and coordinated fashion (Seki et al , 2005). As part of either process, the mechanism behind the apparent global erasure of DNA methylation has posed a particularly vexing problem. In PGCs, this global demethylation is considered essential to erase somatic methylation signatures as well as parental imprints while simultaneously reactivating transcriptional programs associated with pluripotency and gametogenesis. Until recently, the rapidity of DNA demethylation during PGC specification appeared to support numerous mechanisms that largely centred on an active catalytic step, including strategies employing deamination or oxidation followed by base excision repair (Wu and Zhang, 2010). While these proposed mechanisms appeared …