The dynamic chromatin landscape and mechanisms of DNA methylation during mouse germ cell development

ABSTRACT

Epigenetic marks including DNA methylation (DNAme) play a critical role in the transcriptional regulation of genes and retrotransposons. Defects in DNAme are detected in infertility, imprinting disorders and congenital diseases in humans, highlighting the broad importance of this epigenetic mark in both development and disease. While DNAme in terminally differentiated cells is stably propagated following cell division by the maintenance DNAme machinery, widespread erasure and subsequent de novo establishment of this epigenetic mark occur early in embryonic development as well as in germ cell development. Combined with deep sequencing, low-input methods that have been developed in the past several years have enabled high-resolution and genome-wide mapping of both DNAme and histone post-translational modifications (PTMs) in rare cell populations including developing germ cells. Epigenome studies using these novel methods reveal an unprecedented view of the dynamic chromatin landscape during germ cell development. Furthermore, integrative analysis of chromatin marks in normal germ cells and in those deficient in chromatin-modifying enzymes uncovers a critical interplay between histone PTMs and de novo DNAme in the germline. This review discusses work on mechanisms of the erasure and subsequent de novo DNAme in mouse germ cells as well as the outstanding questions relating to the regulation of the dynamic chromatin landscape in germ cells.

INTRODUCTION

Epigenetic modifications play a critical role in the transcriptional regulation of specific sets of genes, including imprinted genes and germline genes as well as retrotransposon silencing. These epigenetic marks are essential for embryonic development and male germline development (Smith and Meissner, 2013). Defects in DNA methylation (DNAme) are found in many human diseases that result from mutations in the genes encoding DNA methyltransferases (DNMTs) as well as histone lysine (K) methyltransferases (KMTases) (Baylin and Jones, 2016; Jambhekar et al., 2019), underscoring the critical interplay between histone post-translational modifications (PTMs) and DNAme that play a role in humans (Li et al., 2021; Zhao et al., 2021).

Genomic imprinting regulates the parent-of-origin-specific expression of a few hundred genes in somatic cells. While DNAme plays a central role in the monoallelic expression of these imprinted genes, this epigenetic mark is erased in primordial germ cells (PGCs) and re-established during gametogenesis in a sex-specific manner, resulting in a distinct set of imprinted genes marked by DNAme between eggs and sperms (Tucci et al., 2019). The distinct patterns of DNAme at the imprinted differentially methylated regions (DMRs), inherited from the mature gametes, are maintained in somatic cells.

The analysis of DNAme at imprinted DMRs and retrotransposons by Sanger bisulfite sequencing as well as immunofluorescence analysis of global levels of DNAme have revealed the dynamic changes in DNAme that occur during embryonic development (Rougier et al., 1998; Mayer et al., 2000; Oswald et al., 2000) and germ cell development (Hajkova et al., 2002; Lane et al., 2003; Seki et al., 2005). Low-input methods that have been developed in the past several years have enabled genome-wide and high-resolution mapping of DNAme (Miura et al., 2012) and histone PTMs (Brind’Amour et al., 2015; Dahl et al., 2016; Zhang et al., 2016) in rare cell populations, including developing germ cells and early embryos. The epigenome studies using these novel methods have extended our understanding of the regulation and dynamics of chromatin marks beyond imprinted DMRs and retrotransposons, leading to many unexpected findings, including sexually dimorphic differences in the distribution of H3K36me2 (histone H3 dimethylated at K36) in the germ cells of female and male mice (Xu et al., 2019; Shirane et al., 2020).

I will begin this review by introducing writers and erasers of DNAme that regulate the dynamic changes in DNAme in germ cell development, and then summarize mechanisms of DNA demethylation in PGCs and subsequent de novo DNAme as well as the outstanding questions relating to the regulation of the dynamic chromatin landscape in germ cells.

KEY PLAYERS OF DNA METHYLATION

Writers of DNAme

DNAme in mammalian cells is deposited on the fifth carbon position of the cytosine base (5-methylcytosine, 5mC), predominantly in the context of CpG dinucleotide sequences. De novo and maintenance mechanisms cooperatively regulate homeostasis of DNAme in the cells. Following establishment of DNAme by the de novo DNAme machinery, the patterns of DNAme are in turn propagated from mother cells to daughter cells by the maintenance DNAme machinery (Fig. 1). While both DNA methyltransferase 3 (DNMT3) family proteins (DNMT3A, DNMT3B and the recently identified rodent-specific DNMT3C (Barau et al., 2016; Jain et al., 2017)) and a catalytically inactive paralog (DNMT3L) are responsible for the former, DNMT1 is responsible for the latter (Greenberg and Bourc’his, 2019). UHRF1 (also known as NP95 or ICBP90), another key player of maintenance DNAme (Bostick et al., 2007; Sharif et al., 2007), directly binds to 5mC on the mother strand and in turn deposits ubiquitin at H3K23 (Nishiyama et al., 2013). Subsequently, DNMT1 is recruited to this region by interacting with ubiquitinated H2K23 and deposits 5mC on the newly synthesized daughter strand, thus propagating the pre-existing patterns of DNAme from mother cells to daughter cells (Fig. 1). Furthermore, recent studies have revealed that DNMT1 possesses a weak de novo DNAme activity toward a specific set of retrotransposons in mouse embryonic stem cells (mESCs) (Yarychkivska et al., 2018) as well as in post-implantation embryos (Li et al., 2018; Haggerty et al., 2021). The dynamics of DNAme and the role of these proteins in germ cells will be discussed in the following sections.

Fig. 1.

De novo and maintenance DNAme in mammalian cells. 5mC, shown as Me–C in the model, is formed by de novo DNA methyltransferases (DNMT3A, DNMT3B and DNMT3C). DNMT3L is a catalytically inactive paralog that stimulates the activity of DNMT3 proteins. Following DNA replication, while the newly synthesized DNA strand lacks 5mC (hemimethylated state), 5mC on the mother strand is recognized by UHRF1 and in turn DNMT1 is recruited to deposit 5mC on the newly synthesized DNA strand.

Erasers of DNAme

DNAme can be erased by active and passive mechanisms (Wu and Zhang, 2017). The active DNA demethylation pathway involves the progressive oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) or 5-carboxylcytosine (5caC). These oxidized 5mC derivatives are diluted following DNA replication due to the inefficient activity of the UHRF1/DNMT1 for these hemi-modified cytosines (Hashimoto et al., 2012; Otani et al., 2013; Ji et al., 2014) or removed by thymine DNA glycosylase (TDG) followed by the base excision repair (BER) pathway (He et al., 2011; Zhang et al., 2012). Progressive oxidation of 5mC is catalyzed by TET (ten-eleven translocation) methylcytosine dioxygenases (TET1, TET2 and TET3) (Tahiliani et al., 2009; Ito et al., 2010, 2011; Gu et al., 2011; He et al., 2011). In contrast, the passive demethylation pathway involves the repression of de novo and maintenance DNMT activities followed by replication-coupled dilution of 5mC. These two pathways likely coordinate the widespread loss of DNAme as well as locus-specific removal of 5mC that occur in early embryos and in PGCs (discussed in detail below).

MECHANISMS OF DNA DEMETHYLATION IN PRE-IMPLANTATION EMBRYOS AND PRIMORDIAL GERM CELLS

The dynamics and mechanisms of DNA demethylation during pre-implantation development

Immunofluorescence analysis in zygotes has demonstrated that the paternal pronucleus undergoes acute loss of DNAme upon fertilization (Mayer et al., 2000). Consistent with this observation, allele-specific whole-genome bisulfite sequencing (WGBS) analysis of the parent-of-origin-DNAme in hybrid mouse embryos revealed that a substantial fraction of genomic regions within the paternal genome are demethylated by the two-cell embryo stage (Wang et al., 2014). The DNA demethylation at the paternal pronucleus in the zygote involves the conversion of 5mC into 5hmC, which is catalyzed by oocyte-stored TET3 (Gu et al., 2011). While the TET3-mediated active pathway clearly contributes to DNA demethylation in zygotes (Guo et al., 2014; Peat et al., 2014; Shen et al., 2014), the DNA replication-coupled passive pathway is likely a driving force for widespread DNA demethylation in the zygote (Guo et al., 2014; Shen et al., 2014). Furthermore, the passive DNA demethylation pathway, which involves cytoplasmic sequestration of UHRF1, contributes to the progressive reduction of DNAme in pre-implantation embryos (Maenohara et al., 2017; Li et al., 2018) (Fig. 2). In contrast, DNAme at imprinted DMRs is resistant to the global wave of DNA demethylation in pre-implantation embryos. The protection against DNA demethylation at these regions is regulated by the Krüppel-associated box (KRAB)-containing zinc finger proteins ZFP57 (Li et al., 2008; Quenneville et al., 2011; Strogantsev et al., 2015) and the recently identified ZFP445 (Takahashi et al., 2019). ZFP57 binds to the TGCmCGC motif within the imprinted DMRs and in turn recruits KAP1 (KRAB-associated protein 1) and DNMTs (Li et al., 2008; Quenneville et al., 2011; Strogantsev et al., 2015), thus maintaining DNAme at these regions. ZFP445 cooperates with ZFP57 and protects DNAme at nearly all imprinted DMRs from the global wave of DNA demethylation during pre-implantation development (Takahashi et al., 2019).

Fig. 2.

The dynamics of DNAme in embryonic development and germ cell development in mice. Following fertilization, widespread DNA demethylation commences in the zygote. A fraction of 5mC on the paternal genome (derived from sperms) is subject to conversion into 5hmC catalyzed by TET3. Coupled with DNA replication, repression of both de novo and maintenance activities of DNAme results in widespread loss of DNAme at both paternal and maternal genomes in pre-implantation embryos. Following implantation, a global wave of de novo DNAme commences in the inner cell mass of the blastocyst. While additional de novo DNAme occurs in the somatic cell lineages, widespread erasure of DNAme occurs in the germ cell lineage in both female and male, reaching a low point at ~E13.5. De novo DNAme in the male germ cells occurs after ~E13.5, and during oocyte growth in female germ cells. Key players of DNA demethylation and de novo DNAme are shown. Note that the piRNA pathway and DNMT3C are required for de novo DNAme at specific retrotransposons.

Progressive dilution of genome-wide 5mC in PGCs

Following implantation, a global wave of de novo DNAme commences in the post-implantation epiblast (Borgel et al., 2010), the precursor of somatic cell lineages as well as the germ cell lineage. The germ cell lineage, the precursor of eggs or sperms, emerges from the most proximal epiblast at around embryonic day (E) 6.0 in response to BMP signaling and is specified as PGCs (Ginsburg et al., 1990; Lawson et al., 1999; Saitou et al., 2002). Following specification, PGCs begin to migrate within the hind-gut and later through the gut mesentery and colonize the genital ridges at E10.5. While additional de novo DNAme occurs in the somatic lineages, widespread erasure of DNAme commences in specified PGCs, reaching 3–4% of the genome methylated at E13.5 (Seisenberger et al., 2012; Kobayashi et al., 2013) (Fig. 2). Furthermore, immunofluorescence analysis revealed that migratory PGCs undergo widespread loss of H3K9me2 and gain of H3K27me3 (Seki et al., 2005, 2007). The mechanism underlying the interplay between DNA demethylation and these dramatic changes in histone PTMs as well as the hierarchical order of these epigenetic events warrant further investigation.

The widespread loss of DNAme in PGCs involves both the active and passive demethylation pathways. While DNMT1 shows ubiquitous expression throughout the window of DNA demethylation, de novo Dnmt3 (Dnmt3a and Dnmt3b) and UHRF1 are expressed at low levels in PGCs (Kurimoto et al., 2008; Seisenberger et al., 2012; Kagiwada et al., 2013). The low expression of UHRF1 in PGCs leads to inefficient recruitment of DNMT1 to the replication foci between E10.5 and E13.5, during which PGCs divide twice per day (Kagiwada et al., 2013). Coupled with DNA replication and cell division, repression of both maintenance and de novo activities of DNAme is probably sufficient to explain the widespread loss of DNAme observed in PGCs (Seisenberger et al., 2012; Kagiwada et al., 2013). Among the TET family proteins, Tet1 is highly expressed in PGCs from E9.5 to E13.5, with Tet2 and Tet3 showing low or no expression (Kagiwada et al., 2013). Interestingly, however, Tet1 KO PGCs show a modest increase in global levels of DNAme (Yamaguchi et al., 2012), indicating that a small fraction of the genome depends on TET1-mediated DNA demethylation in PGCs. Consistent with these observations, quantification of 5mC and 5hmC in PGCs by mass spectrometry revealed that the total amount of 5hmC is an order of magnitude lower than that of 5mC in PGCs at E9.5 and E10.5 (Hill et al., 2018), indicating that the global loss of DNAme does not depend solely on the widespread conversion of 5mC to 5hmC. Rather, 5hmC accumulates in PGCs at specific genomic regions, including germline genes and imprinted DMRs (Hackett et al., 2013) (discussed in detail below).

Resistance to the global wave of DNA demethylation in PGCs

Interestingly, not all genomic regions are equally prone to DNAme loss during the global wave of DNA demethylation in PGCs (Seisenberger et al., 2012; Kagiwada et al., 2013; Kobayashi et al., 2013). A subset of imprinted DMRs and germline genes show delayed DNA demethylation in PGCs compared with the rest of the genome (Seisenberger et al., 2012). Genome-wide mapping of 5hmC in PGCs at E10.5–E13.5 revealed that 5mC in a subset of germline genes as well as imprinted DMRs (Igf2r, Kcnq1ot1, Peg3, Peg10) undergoes conversion into 5hmC, which eventually results in unmethylated C (Hackett et al., 2013). Consistent with these observations, Tet1 deficiency in PGCs leads to defective DNA demethylation in these imprinted DMRs (Yamaguchi et al., 2013). Furthermore, a subset of germline genes, including those that regulate meiosis, show incomplete DNA demethylation coupled with inefficient transcriptional activation, which leads to hypogonadism in Tet1-deficient females (Yamaguchi et al., 2012). The underlying mechanism that directs TET1 to these specific genomic locations as well as other players that protect these genes against the global wave of DNA demethylation in PGCs remain to be identified.

H3K9me3-marked retrotransposons show resistance to the global wave of DNA demethylation in PGCs (Liu et al., 2014). Genetic ablation of SETDB1, the H3K9 KMTase that deposits H3K9me3, in PGCs by Tnap-Cre leads to a reduction of DNAme at retrotransposons that show decreased H3K9me3, indicating that H3K9me3 deposited by SETDB1 is required for the protection against DNA demethylation in PGCs (Liu et al., 2014). While UHRF1 is expressed at a low level and is localized primarily in the cytoplasm in PGCs (Seisenberger et al., 2012; Kagiwada et al., 2013), it is possible that residual UHRF1 in the nucleus binds to H3K9me2/me3 at these regions through its tandem Tudor domain (TTD) and plant homeodomain (PHD) (Nady et al., 2011; Arita et al., 2012; Rothbart et al., 2012, 2013) and promotes maintenance DNAme activity. Alternatively, H3K9me3-binding proteins such as HP1 may safeguard these regions against DNA demethylation. Further studies are required to elucidate the underlying mechanism for the protection of DNAme at H3K9me3-marked regions against the global wave of DNA demethylation in PGCs.

MECHANISMS OF DE NOVO DNA METHYLATION IN THE MALE GERMLINE

Following widespread erasure of DNAme in PGCs, de novo DNAme commences shortly after E13.5 in G0/G1-arrested prospermatogonia (PSG) in the male germline. The targets of de novo DNAme in the male germline include retrotransposons, imprinted DMRs (H19, Dlk1–Gtl2 and Rasgrf1) and non-promoter regions (Brykczynska et al., 2010; Erkek et al., 2013; Kobayashi et al., 2013; Kubo et al., 2015). The widespread de novo DNAme depends on the activity of DNMT3A and DNMT3L and is essentially completed at the perinatal stage of spermatogonia, with ~80% of the genome methylated (Kato et al., 2007; Seisenberger et al., 2012; Kobayashi et al., 2013; Kubo et al., 2015) (Fig. 2). How are the DNMT3 proteins recruited to specific genomic regions? The following subsections will discuss key players that guide de novo DNAme in the male germ cells.

piRNA directs de novo DNAme at evolutionarily young retrotransposons

Retrotransposons constitute ~40% of the mouse genome (Mouse Genome Sequencing Consortium, 2002). While the majority of these parasitic elements lose the ability to mobilize, a subset of evolutionarily young elements, including LINE1 and LTR, can still retrotranspose, threatening genome stability (Senft and Macfarlan, 2021). Thus, these elements should be silenced by many pathways, including epigenetic mechanisms.

During the wave of de novo DNAme in the male germline, the piRNA pathway plays a critical role in directing de novo DNAme at evolutionarily young retrotransposons, including LINE1 elements (Aravin et al., 2007, 2008; Kuramochi-Miyagawa et al., 2008). piRNAs, a class of small non-coding RNA comprised of 26–30 nucleotides, are highly expressed in male germ cells and produced by the cleavage of retrotransposon-derived transcripts. The piRNA pathway silences retrotransposons by either transcriptional or post-transcriptional mechanisms. MIWI2 (PIWIL4), a nuclear PIWI protein, is guided by piRNAs and recruits chromatin-modifying enzymes, including the DNMT3A/DNMT3L complex, at retrotransposons for transcriptional silencing of these parasitic elements in male germ cells (Ozata et al., 2019). Defects in DNAme as well as derepression of evolutionarily young retrotransposons are observed in mutant male germ cells lacking genes required for piRNA biogenesis, including MILI (PIWIL2), MIWI2 and PLD6 (also known as MITOPLD). Notably, these mutant male mice are infertile (Aravin et al., 2007, 2008; Kuramochi-Miyagawa et al., 2008; Watanabe et al., 2011).

How is the de novo DNAme machinery recruited to these retrotransposons by the piRNA pathway? A recent interactome study identified MIWI2-binding proteins in embryonic testis, including SPOCD1, a nuclear protein of unknown function in the male germ cells (Zoch et al., 2020). While piRNA biogenesis is unaffected in Spocd1-deficient male germ cells, a substantial reduction of DNAme is observed at evolutionarily young retrotransposons such as L1Md_A, L1Md_T and L1Md_Gf (Zoch et al., 2020). Furthermore, SPOCD1 co-purifies with DNMT3A, DNMT3C and DNMT3L in the embryonic testis. Thus, these observations reveal that the MIWI2/piRNA complex recruits the de novo DNAme machinery to evolutionarily young retrotransposons through SPOCD1 (Zoch et al., 2020).

As described above, DNMT3A and DNMT3L are essential for widespread de novo DNAme in the male germ cells. Recent studies identified a new DNMT3 member, DNMT3C, which is rodent-specific (Barau et al., 2016; Jain et al., 2017). Dnmt3c, which evolved as a duplication of the Dnmt3b gene, is highly expressed in male germ cells (Barau et al., 2016). Notably, deletion of Dnmt3c in males does not lead to widespread loss of DNAme but rather to the loss of DNAme at evolutionarily young retrotransposons, including those that are dependent on the piRNA pathway (Barau et al., 2016). Furthermore, among the three imprinted DMRs that are methylated in male germ cells, DNMT3C is only required for de novo DNAme at the RMER4B LTR region within the Rasgrf1 DMR, which is also dependent on the piRNA pathway (Watanabe et al., 2011; Barau et al., 2016). These observations clearly show that the piRNA pathway is required for de novo DNAme of specific young retrotransposons. However, retrotransposons targeted by the piRNA pathway represent only a small fraction of the genomic DNA that undergoes de novo DNAme in male germ cells. Other mechanisms that guide the DNMT3A/DNMT3L complex beyond retrotransposons will be discussed in the following subsections.

H3K4 methylation impedes de novo DNAme in the male germline

In the male germline, regions enriched for H3K4 methylation remain unmethylated throughout spermatogenesis (Brykczynska et al., 2010; Erkek et al., 2013). This observation is consistent with earlier biochemical evidence showing that DNMT3A and DNMT3L only bind to chromatin lacking H3K4 methylation (Jia et al., 2007; Ooi et al., 2007). While genomic regions that gain H3K4 methylation between E13.5 and E16.5 are protected from the global wave of de novo DNAme in PSG, those losing H3K4 methylation acquire DNAme, including at H19 and Dlk1–Gtl2 imprinted DMRs (Singh et al., 2013). Similarly, H3K4me2-enriched L1 promoter regions remain unmethylated until E16.5. These regions gain DNAme after E16.5, presumably via the recruitment of H3K4 demethylases that interact with MIWI2 (Nagamori et al., 2018), allowing DNMT3 proteins to access and methylate these regions. Thus, it would be interesting to study the consequences of disrupting H3K4 demethylases on de novo DNAme in male germ cells.

NSD1-deposited H3K36me2 directs de novo DNAme in the male germline

As discussed above, H3K4 methylation antagonizes the deposition of DNAme in the male germ cells, as in somatic cells. So, what are the chromatin features that drive de novo DNAme? Biochemical studies revealed that DNMT3A can bind to H3K36me3/me2 via its Pro-Trp-Trp-Pro (PWWP) domain (Dhayalan et al., 2010; Dukatz et al., 2019; Weinberg et al., 2019). Furthermore, chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) analysis for H3K36me3 in E13.5 male PGCs revealed that this mark is enriched at gene body regions that are subject to de novo DNAme in E16.5 PSG (Morselli et al., 2015). These observations suggest that this chromatin mark promotes de novo DNAme in male germ cells. Interestingly, however, genetic ablation of SETD2, the sole H3K36 KMTase that deposits H3K36me3, in male germ cells by Tnap-Cre has essentially no impact on de novo DNAme in E16.5 PSG (Shirane et al., 2020). Instead, ChIP-seq analysis for H3K36me2 in E16.5 PSG revealed that regions that gain de novo DNAme by this stage coincide with H3K36me2, including within and outside gene body regions (Shirane et al., 2020). Genetic ablation of NSD1, a H3K36 KMTase that deposits H3K36me2, in male germ cells by Tnap-Cre leads to the widespread failure of de novo DNAme at H3K36me2-depleted regions, including in the bodies of genes expressed at low levels, intergenic regions and three imprinted DMRs that are methylated in male germ cells. These observations indicate that instead of SETD2, NSD1 is required for de novo DNAme in male germ cells (Shirane et al., 2020). Since not all genomic regions in Nsd1-deficient PSG lose H3K36me2 (~25% loss in Nsd1-deficient PSG at E16.5), other NSD proteins (NSD2 and NSD3) that are expressed in PSG may also be involved in H3K36me2 deposition. Intriguingly, DNAme at H3K9me3-marked regions is not affected in the Nsd1-deficient PSG. Notably, these regions are depleted of H3K36me2 in wild-type PSG at E16.5 (Shirane et al., 2020). It is possible that heterochromatic structure or H3K9me3-binding proteins inhibit the NSD1-mediated H3K36me2 deposition at these regions. The mechanism that excludes H3K36me2 in H3K9me3-marked regions warrants further investigation.

MECHANISMS OF DE NOVO DNA METHYLATION IN THE FEMALE GERMLINE

Following widespread erasure of DNAme in PGCs, de novo DNAme in the female germline initiates in growing oocytes arrested in meiotic prophase I and is essentially completed at the fully grown (FG) stage (Lucifero et al., 2002, 2004; Hiura et al., 2006) (Fig. 2). As in the male germ cells, both DNMT3A and DNMT3L are required for widespread de novo DNAme in oocytes, including at maternally methylated imprinted DMRs (Kaneda et al., 2004; Shirane et al., 2013). In stark contrast to the male germline, DNAme in the female germline coincides almost exclusively with the bodies of transcribed genes (Chotalia et al., 2009; Smallwood et al., 2011; Stewart et al., 2015; Veselovska et al., 2015), with ~40% of the genome methylated in FG oocytes (Kobayashi et al., 2012; Shirane et al., 2013). This observation suggests that transcription is associated with de novo DNAme in oocytes. Indeed, truncation of Nesp transcription results in the failure of de novo DNAme at the Gnas imprinted DMR, indicating that transcription precedes de novo DNAme at this locus (Chotalia et al., 2009). The connection between transcription, histone PTMs and de novo DNAme in the oocytes will be discussed in the following subsections.

Canonical and broad domains of H3K4me3 antagonize de novo DNAme in oocytes

The lower levels of DNAme in mouse oocytes (~40%) than in sperms (~80%) suggest that oocytes harbor unique chromatin features that antagonize de novo DNAme outside the transcribed gene body regions. ChIP-seq analysis revealed that in addition to the canonical H3K4me3, which is enriched at CpG-rich regions, oocytes harbor broad domains of H3K4me3 that extend beyond these regions, with ~22% of the genome covered by this chromatin mark (Dahl et al., 2016; Liu et al., 2016; Zhang et al., 2016). Consistent with biochemical evidence for the antagonistic relationship between H3K4me3 and DNAme (Jia et al., 2007; Ooi et al., 2007), these H3K4me3-marked regions are devoid of de novo DNAme in oocytes (Dahl et al., 2016). The broad domains of H3K4me3 in oocytes are catalyzed by MLL2 (Hanna et al., 2018) and regulate the zygotic genome activation (ZGA) that occurs in two-cell embryos (Dahl et al., 2016; Zhang et al., 2016). A recent study revealed that KDM4A-mediated demethylation of H3K9me3 prevents the encroachment of this mark into broad domains of H3K4me3 and thus ensures ZGA in the two-cell embryos (Sankar et al., 2020). Furthermore, genetic ablation of KDM1A or KDM1B, H3K4 demethylases, in oocytes by Zp3-Cre results in the failure of de novo DNAme at CpG-rich regions, including at a subset of maternally methylated imprinted DMRs (Ciccone et al., 2009; Stewart et al., 2015), indicating that removal of H3K4 methylation precedes de novo DNAme at these regions in oocytes.

SETD2 is required for directing de novo DNAme in oocytes

What are the chromatin features that drive de novo DNAme in oocytes? Does NSD1 play an instructive role in directing DNAme in oocytes as in the male germline? ChIP-seq analysis revealed that H3K36me3 coincides with DNAme in oocytes (Stewart et al., 2015; Brind’Amour et al., 2018; Xu et al., 2019), which is consistent with the observation that this mark is associated with transcription elongation. Furthermore, genetic ablation of SETD2 in oocytes by Gdf9-Cre leads to widespread reduction of DNAme at H3K36me3-depleted regions, including at all maternally methylated imprinted DMRs (Xu et al., 2019), indicating that SETD2 is required for de novo DNAme in oocytes. More recently, ChIP-seq analysis revealed that H3K36me2 is primarily enriched at the transcribed bodies of genes in growing oocytes (Shirane et al., 2020). Furthermore, genetic ablation of SETD2 in oocytes by Zp3-Cre leads to widespread reduction of both H3K36me2 and H3K36me3 at these regions. Notably, the residual H3K36me2 in Setd2-deficient oocytes represent only ~6% of the oocyte genome (Shirane et al., 2020). While NSD1 is expressed in oocytes, its role in directing global patterns of DNAme in oocytes is likely minor compared to the male germline.

As described above, DNMT3A can bind to H3K36me3/me2 via its PWWP domain (Dhayalan et al., 2010; Dukatz et al., 2019; Weinberg et al., 2019), and the substitution of aspartic acid at residue 329 with alanine (D329A) in this domain diminishes the binding of DNMT3A to H3K36me3/me2 in vitro (Dhayalan et al., 2010). Interestingly, however, mouse oocytes harboring this mutation show normal DNAme at H3K36me3-marked regions (Kibe et al., 2021). The low affinity of DNMT3A^D329A for H3K36me3 may be sufficient to direct this mutant protein to H3K36me3-marked regions. Alternatively, other mechanisms may compensate for the recruitment of DNMT3A^D329A. Regardless, the underlying mechanism that directs DNMT3A to H3K36me3/me2-marked regions in oocytes requires further investigation.

SEXUALLY DIMORPHIC H3K36me2 LANDSCAPES DICTATE DISTINCT PATTERNS OF DNA METHYLATION IN EGGS AND SPERMS

As discussed above, the levels and distribution of DNAme differ considerably between female and male germ cells. Recent studies have demonstrated that female and male germ cells use distinct H3K36 KMTases to direct global patterns of DNAme (Xu et al., 2019; Shirane et al., 2020) (Fig. 3). Why is this? As H3K36me2/me3 are prerequisite for de novo DNAme, this may be a strategy to control DNAme levels for transcriptome regulation in germ cells or the resulting early embryos. As large fractions of DNAme in oocytes are inherited by the pre-implantation embryos (Wang et al., 2014), lower levels of DNAme in oocytes may facilitate the activation of genes that are essential for pre-implantation development. In line with this idea, Stella (also known as Dppa3 or PGC7)-deficient oocytes acquire genome-wide excessive DNAme and the resulting embryos exhibit a partial failure of ZGA (Li et al., 2018). It is also possible that lower levels of DNAme in oocytes promote transcription of genes that are essential for growth and maturation of oocytes as well as for subsequent embryonic development. In contrast, wider distribution and higher levels of DNAme in sperms may be critical to generate compacted chromatin that is packaged efficiently into the sperm nucleus. Furthermore, widespread DNAme may suppress active gene transcription in sperms. These speculations should be experimentally tested. Transcriptome profiling and analysis of developmental potential of oocytes with sperm-like patterns of DNAme or vice versa may address the biological importance of the distinct patterns of DNAme that are observed in eggs and sperms.

Fig. 3.

Distinct H3K36 KMTases dictate the distinct patterns of DNAme observed in oocytes and sperms. A model showing the critical interplay between H3K36me2/me3 and de novo DNAme that occurs during oocyte growth in the female germline and between E13.5 and the perinatal stage in the male germline. In oocytes, SETD2 deposits both H3K36me2 and H3K36me3 in the bodies of actively transcribing genes, and H3K36me2/me3 recruit DNMT3A. H3K36me2 outside gene body regions represents only a small fraction of the oocyte genome and its deposition is catalyzed by unknown H3K36me2 KMTases. In the male germline, NSD1 broadly deposits H3K36me2 at intergenic regions and the bodies of genes expressed at both low and high levels. SETD2 and NSD1 act redundantly at actively transcribing gene bodies in the male germline. While the majority of H3K36me2 in the male germline is deposited by NSD1, residual H3K36me2 deposition is catalyzed by unknown H3K36me2 KMTases. H3K4me3-marked promoter regions remain unmethylated in both the female and male germ cells. For simplicity, broad domains of H3K4me3 in oocytes and other chromatin marks are omitted. The figure is modified from Fig. 6 in Shirane et al. (2020). TSS: transcription start site; PO: primary oocyte; GO: growing oocyte; FGO: fully grown oocyte; PGC: primordial germ cell; PSG: prospermatogonium; SG: spermatogonium.

CONCLUDING REMARKS

This review summarizes studies aiming to elucidate mechanisms of erasure and subsequent establishment of DNAme during germ cell development. Low-input and high-sensitivity methods that have been developed in the past several years have successfully generated high-resolution and genome-wide maps of DNAme as well as many histone PTMs in developing germ cells and early embryos. Further studies are required to generate chromatin maps derived from germ cells that are deficient in chromatin-modifying enzymes in order to deepen our understanding of mechanisms of chromatin regulation in these cells. Additionally, amino acid substitution or removal of specific domains in the chromatin-modifying enzymes should help us to understand how these proteins find their target genomic regions. However, since chromatin-modifying enzymes are generally essential for embryonic development, germ cell-specific depletion of these enzymes is needed to investigate their roles in germ cells. Furthermore, the generation and analysis of germ cell-specific conditional knockout mice are both time consuming and prohibitively expensive. A compelling circumvention could be to employ PGC-like cell culture systems that use pluripotent stem cells for stepwise induction of germ cell fate with subsequent maturation of germ cells (Hayashi et al., 2011, 2012; Hikabe et al., 2016; Ishikura et al., 2016, 2021; Miyauchi et al., 2017; Ohta et al., 2017). Notably, these in vitro-produced PGC-like cells can be differentiated into functional gametes and undergo key epigenetic changes, including widespread DNA demethylation as well as global reduction of H3K9me2 and elevation of H3K27me3 as in in vivo PGCs (Kurimoto et al., 2015; Shirane et al., 2016; Ohta et al., 2017). Application of these systems would allow systematic characterization or identification of chromatin-modifying enzymes as well as their regulators that are required for shaping the germline epigenome.

Findings using the mouse model will serve as the foundation for our understanding of mechanisms of epigenome regulation in other species including humans. Because many mutations are found in genes that encode chromatin-modifying enzymes in human populations, these studies should also lead to a better understanding of the etiology of human infertility, imprinting disorders and congenital diseases.

ACKNOWLEDGMENTS

I thank Dr. Julien Richard Albert (Institut Jacques Monod, CNRS) for critical reading of the manuscript.

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