An Initial Investigation of an Alternative Model to Study rat Primordial Germ Cell Epigenetic Reprogramming

Background

Primordial germ cells (PGC) are the precursors of the gametes. During pre-natal development, PGC undergo an epigenetic reprogramming when bulk DNA demethylation occurs and is followed by sex-specific de novo methylation. The de novo methylation and the maintenance of the methylation patterns depend on DNA methyltransferases (DNMTs). PGC reprogramming has been widely studied in mice but not in rats. We have previously shown that the rat might be an interesting model to study germ cell development. In face of the difficulties of getting enough PGC for molecular studies, the aim of this study was to propose an alternative method to study rat PGC DNA methylation. Rat embryos were collected at 14, 15 and 19 days post-coitus (dpc) for the analysis of 5mC, 5hmC, DNMT1, DNMT3a and DNMT3b expression or at 16dpc for treatment 5-Aza-CdR, a DNMT inhibitor, in vitro.

Methods

Once collected, the gonads were placed in 24-well plates previously containing 45μm pore membrane and medium with or without 5-Aza-CdR. The culture was kept for five days and medium was changed daily. The gonads were either fixed or submitted to RNA extraction.

Results

5mC and DNMTs labelling suggests that PGC are undergoing epigenetic reprogramming around 14/15dpc. The in vitro treatment of rat embryonic gonads with 1 μM of 5-Aza-CdR lead to a loss of 5mC labelling and to the activation of Pax6 expression in PGC, but not in somatic cells, suggesting that 5-Aza-CdR promoted a PGC-specific global DNA hypomethylation.

Conclusions

This study suggests that the protocol used here can be a potential method to study the wide DNA demethylation that takes place during PGC reprogramming.

     

Promoter DNA methylation couples genome-defence mechanisms to epigenetic reprogramming in the mouse germline

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.     

Widespread epigenetic abnormalities suggest a broad DNA methylation erasure defect in abnormal human sperm

Background

Male-factor infertility is a common condition, and etiology is unknown for a high proportion of cases. Abnormal epigenetic programming of the germline is proposed as a possible mechanism compromising spermatogenesis of some men currently diagnosed with idiopathic infertility. During germ cell maturation and gametogenesis, cells of the germ line undergo extensive epigenetic reprogramming. This process involves widespread erasure of somatic-like patterns of DNA methylation followed by establishment of sex-specific patterns by de novo DNA methylation. Incomplete reprogramming of the male germ line could, in theory, result in both altered sperm DNA methylation and compromised spermatogenesis.

Methodology/Principal Finding

We determined concentration, motility and morphology of sperm in semen samples collected by male members of couples attending an infertility clinic. Using MethyLight and Illumina assays we measured methylation of DNA isolated from purified sperm from the same samples. Methylation at numerous sequences was elevated in DNA from poor quality sperm.

Conclusions

This is the first report of a broad epigenetic defect associated with abnormal semen parameters. Our results suggest that the underlying mechanism for these epigenetic changes may be improper erasure of DNA methylation during epigenetic reprogramming of the male germ line.     

X chromosome activity in mouse XX primordial germ cells

In the early epiblast of female mice, one of the two X chromosomes is randomly inactivated by a Xist-dependent mechanism, involving the recruitment of Ezh2-Eed and the subsequent trimethylation of histone 3 on lysine 27 (H3K27me3). We demonstrate that this random inactivation process applies also to the primordial germ cell (PGC) precursors, located in the proximal region of the epiblast. PGC specification occurs at about embryonic day (E)7.5, in the extraembryonic mesoderm, after which the germ cells enter the endoderm of the invaginating hindgut. As they migrate towards the site of the future gonads, the XX PGCs gradually lose the H3K27me3 accumulation on the silent X chromosome. However, using a GFP transgene inserted into the X chromosome, we observed that the XX gonadal environment (independently of the gender) is important for the substantial reactivation of the inactive X chromosome between E11.5 and E13.5, but is not required for X-chromosome reactivation during the derivation of pluripotent embryonic germ cells. We describe in detail one of the key events during female PGC development, the epigenetic reprogramming of the X chromosome, and demonstrate the role of the XX somatic genital ridge in this process.     

A Critical Function of Mad2l2 in Primordial Germ Cell Development of Mice

The development of primordial germ cells (PGCs) involves several waves of epigenetic reprogramming. A major step is following specification and involves the transition from the stably suppressive histone modification H3K9me2 to the more flexible, still repressive H3K27me3, while PGCs are arrested in G2 phase of their cycle. The significance and underlying molecular mechanism of this transition were so far unknown. Here, we generated mutant mice for the Mad2l2 (Mad2B, Rev7) gene product, and found that they are infertile in both males and females. We demonstrated that Mad2l2 is essential for PGC, but not somatic development. PGCs were specified normally in Mad2l2^−/− embryos, but became eliminated by apoptosis during the subsequent phase of epigenetic reprogramming. A majority of knockout PGCs failed to arrest in the G2 phase, and did not switch from a H3K9me2 to a H3K27me3 configuration. By the analysis of transfected fibroblasts we found that the interaction of Mad2l2 with the histone methyltransferases G9a and GLP lead to a downregulation of H3K9me2. The inhibitory binding of Mad2l2 to Cyclin dependent kinase 1 (Cdk1) could arrest the cell cycle in the G2 phase, and also allowed another histone methyltransferase, Ezh2, to upregulate H3K27me3. Together, these results demonstrate the potential of Mad2l2 in the regulation of both cell cycle and the epigenetic status. The function of Mad2l2 is essential in PGCs, and thus of high relevance for fertility.     

PGC7, H3K9me2 and Tet3: regulators of DNA methylation in zygotes

In zygotes, a global loss of DNA methylation occurs selectively in the paternal pronucleus before the first cell division, concomitantly with the appearance of modified forms of 5-methylcytosine. The adjacent maternal pronucleus and certain paternally-imprinted loci are protected from this process. Nakamura et al. recently clarified the molecular mechanism involved: PGC7/Stella/Dppa3 binds to dimethylated histone 3 lysine 9 (H3K9me2), thereby blocking the activity of the Tet3 methylcytosine oxidase in the maternal genome as well as at certain imprinted loci in the paternal genome.DNA methylation is a crucial epigenetic modification that regulates imprinting (differential silencing of maternal or paternal alleles) and repression of retrotransposons and other parasitic DNA, as well as possibly X-chromosome inactivation and cellular differentiation. DNA methylation needs to be faithfully maintained throughout the life cycle, since loss of DNA methylation can result in gene dosage problems, dysregulation of gene expression, and genomic instability due to retrotransposon reactivation¹. Nevertheless, genome-wide loss of DNA methylation has been observed during germ cell development² and in the paternal pronucleus soon after fertilization³.For almost a decade, the global decrease of DNA methylation observed in the paternal genome within a few hours of fertilization was ascribed to an “active”, replication-independent process³. The maternal pronucleus is spared and instead undergoes “passive”, replication-dependent demethylation during early embryogenesis, arising from inhibition of the DNA maintenance methyltransferase Dnmt1 (Dnmt1 is normally recruited to newly-replicated DNA because of the high affinity of its obligate partner, UHRF1, for hemi-methylated DNA strands, which are produced from symmetrically-methylated CpG dinucleotides as a result of DNA replication). The basis for active and passive demethylation of the paternal and maternal genomes remained a mystery until proteins of the TET family – TET1, TET2 and TET3 in humans – were discovered to be Fe(II)- and 2-oxoglutarate-dependent enzymes capable of oxidizing 5-methylcytosine (5mC) in DNA^4,5,6. TET enzymes serially convert 5mC into 5-hydroxymethyl-cytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC)^5,7,8.With the generation of specific antibodies to 5hmC, it became clear that the supposed “active demethylation” of the paternal pronucleus in mouse zygote after fertilization was due to the inability of anti-5mC antibodies to recognize 5hmC and other 5mC oxidation products^9,10. The enzyme responsible for 5mC oxidation was shown to be Tet3, which unlike Tet1 and Tet2 is highly expressed in mouse oocytes and zygotes. RNAi-mediated depletion of Tet3 decreased the staining of the paternal pronucleus with 5hmC, suggesting that immediately after fertilization, Tet3 in the zygote selectively oxidizes 5mC in the paternal genome to 5hmC^9,10.How is the maternal pronucleus protected from Tet3 activity? Nakamura et al.¹¹ previously showed that zygotes lacking PGC7/Stella/Dppa3 lose asymmetric regulation of DNA methylation, instead showing global loss of 5mC staining in both paternal and maternal pronuclei. This was correlated with hypomethylation at several maternally-imprinted loci (Peg1, Peg3, Peg10) in PGC7-deficient zygotes, as judged by bisulfite sequencing. Further, certain paternally-imprinted loci (H19, Rasgrf1), which are normally protected from global loss of methylation in the paternal genome, also became hypomethylated in PGC7-deficient zygotes. These data suggested that PGC7 protects the maternal genome, as well as certain paternally imprinted loci, from loss of 5mC.In their recent publication, Nakamura et al.¹² elegantly extended these findings to address the mechanism involved. Based on the fact that a major difference between maternal and paternal genomes is that the maternal genome contains histones, whereas the DNA of the entering sperm is tightly packaged with protamine, they asked whether PGC7 recognizes specific histone marks. Indeed, the maternal genome harbors considerable levels of the histone mark H3K9me2¹¹, leading them to examine whether PGC7 distinguishes maternal and paternal genomes by recognizing H3K9me2 in the maternal genome. Using wild-type (WT) ES cells and ES cells deficient in the G9a lysine methyltransferase which generates H3K9me2 mark, they showed that PGC7 associated loosely with nucleosomes and chromatin lacking H3K9me2, but tightly if H3K9me2 was present. The binding was recapitulated using recombinant bacterially-expressed PGC7 and histone tail peptides, indicating a direct interaction of PGC7 with the H3K9me2 mark. In agreement, genomic loci enriched with H3K9me2 recruited PGC7 as judged by chromatin immunoprecipitation (ChIP), but this recruitment was abrogated in G9a-deficient ES cells. These data indicated that PGC7 targets genomic regions occupied by nucleosomes containing H3K9me2 (Figure 1); an interesting extension would be to ask whether loss of maternal G9a also results in 5hmC conversion in the maternal pronucleus in zygotes.Open in a separate windowFigure 1Schematic view of paternal (left) and maternal (right) genomes soon after fertilization. Paternal and maternal pronuclei are indicated with immunostaining results in the boxes. PGC7 binds H3K9me2 in the maternal pronucleus and at certain paternally-imprinted loci (H19, Rasgrf1) in the paternal pronucleus, thereby potentially regulating chromatin organization to interfere with Tet3 accessibility.Next, Nakamura et al.¹² tested by immunocytochemistry whether PGC7 in zygotes also required H3K9me2. It is known that H3K9me2 staining is concentrated in the maternal but not the paternal pronucleus¹³. Using conventional staining methods in which the cells are first fixed and then permeabilized to allow antibodies to enter the cell, the authors observed in their earlier study that PGC7 bound to both pronuclei¹¹. Remarkably, by simply reversing the order of the fixation and permeabilization steps – permeabilizing first to allow the loss of loosely bound proteins by dissociation, then fixing and staining – they found that PGC7 associated much more tightly with the maternal pronucleus that bears H3K9me2 mark. Injection of mRNA encoding Jhdm2a (an H3K9me1/ me2-specific demethylase) into zygotes eliminated staining for H3K9me2 as well as PGC7 in the maternal pronucleus, and concomitantly caused loss of 5mC and acquisition of 5hmC. Taken together, these data strongly suggested that PGC7 was selectively recruited to the maternal pronucleus through binding H3K9me2, and that this binding protected zygotic maternal DNA from oxidation of 5mC to 5hmC and beyond (Figure 1).These findings led Nakamura et al. to investigate how PGC7 controls Tet3 activity in zygotes. They showed (in cells that were permeabilized before fixation and immunocytochemistry) that Tet3 was tightly associated only with the paternal pronucleus in WT zygotes, but was present in both pronuclei in PGC7-deficient zygotes. When PGC7 was prevented from binding to the maternal pronucleus by injection of Jhdm2a mRNA, Tet3 became tightly associated with both pronuclei. In other words, loss of PGC7 or loss of H3K9me2 that recruits PGC7 had the same effect – eliminating selective association of Tet3 with the paternal genome. The implication is that PGC7 – which preferentially binds the maternal genome – somehow promotes the selective binding of Tet3 to the paternal genome, thus permitting rapid 5mC oxidation in paternal but not maternal DNA (Figure 1).PGC7 is a small protein (150 amino acids (aa) in the mouse, 159 aa in humans) whose sequence is only moderately conserved. Nakamura et al.¹² showed that the binding of PGC7 to H3K9me2 required the N-terminal half of PGC7, whereas its ability to exclude Tet3 from the maternal pronucleus required the C-terminal half. It is unclear how Tet3 exclusion is mediated. One possibility is that the C-terminal region of PGC7 sterically excludes Tet3 from binding, either to DNA or to a chromatin mark; another is that the C-terminal region of PGC7 is capable of altering chromatin configuration to prevent the binding of Tet3 to chromatin. In support of the latter hypothesis, the rate with which micrococcal nuclease (MNase) digested high-molecular weight chromatin was significantly slower in WT ES cells in which PGC7 was present, compared to PGC7^−/− and G9a^−/− ES cells in which PGC7 was either absent or not recruited to DNA because of the loss of H3K9me2 mark. In contrast, DNA methylation did not alter the chromatin association of PGC7 or its ability to protect high-molecular weight chromatin from MNase digestion, as shown by using Dnmt1^−/−Dnmt3a^−/−Dnmt3b^−/− triple knockout ES cells that completely lack DNA methylation.How does PGC7 protect paternally-imprinted loci from Tet3-mediated 5mC oxidation? Although the haploid sperm genome is mostly packaged with protamine, a genome-wide analysis revealed that 4% of the genome of mature human sperm bears nucleosomes located at developmental and imprinted genes¹⁴. Nakamura et al.¹² found that among paternally-imprinted differentially methylated regions (DMRs), the H19 and Rasgrf1 DMRs contained H3K9me2 whereas the Meg3 DMR did not, consistent with their previous finding that in PGC7-deficient zygotes, the H19 and Rasgrf1 DMRs were hypomethylated but the Meg3 DMR was unaffected¹¹. Therefore, PGC7 may be recruited to paternally-imprinted loci through H3K9me2-containing nucleosomes that pre-exist in the sperm haploid genome upon fertilization. Alternatively, Nakamura et al. point out that protamine in the sperm is replaced soon after fertilization by the histone H3.3 variant, which in somatic cells does not bear H3K9me2 mark.In conclusion, Nakamura et al.¹² demonstrate unambiguously that PGC7 specifically binds to H3K9me2 in the maternal genome in zygotes, where its global occupancy excludes Tet3 and inhibits Tet3-mediated 5mC oxidation. This novel finding provides new insights into the global alterations of DNA methylation status that occur during early embryogenesis. Follow-up questions abound. First, can PGC7 protect other methylated loci such as transposable elements and the X-chromosome? It would be interesting to assess H3K9me2 at these loci. Second, how does the N-terminal half of PGC7 recognize H3K9me2? Structural characterization of this interaction may elucidate a novel epigenetic “reader” domain specific for H3K9me2. Third, PGC7 is a marker for cells of the inner cell mass, and is co-expressed with Tet1 and Tet2 rather than Tet3 in ESCs¹⁵. Does PGC7 also antagonize Tet1 and Tet2 and protect imprinted loci in ESCs? Fourth, how does PGC7 inhibit the access of Tet3 to chromatin? Considering that PGC7 is small and is not equipped with known enzymatic domains, it is likely that PGC-interacting proteins, rather than PGC7 itself, function to regulate chromatin status. Fifth, how is Tet3 recruited to paternal chromatin – are there specific histone or other epigenetic marks that facilitate Tet3 recruitment? Finally, while technically challenging, it seems imperative to identify the target genes of PGC7 and Tet3, by profiling the genomic location of 5hmC and other 5mC oxidation products in the paternal and maternal genomes of zygotes from WT, Tet3-deficient and PGC7-deficient mice.     

Epigenetic modification of histone 3 at lysine 9 in sheep zygotes and its relationship with DNA methylation

Background  

Previous studies indicated that, unlike mouse zygotes, sheep zygotes lacked the paternal DNA demethylation event. Another epigenetic mark, histone modification, especially at lysine 9 of histone 3 (H3K9), has been suggested to be mechanically linked to DNA methylation. In mouse zygotes, the absence of methylated H3K9 from the paternal pronucleus has been thought to attribute to the paternal DNA demethylation.     

Specification of primordial germ cells in medaka (Oryzias latipes)

Background  

Primordial germ cells (PGCs) give rise to gametes that are responsible for the development of a new organism in the next generation. Two modes of germ line specification have been described: the inheritance of asymmetrically-localized maternally provided cytoplasmic determinants and the induction of the PGC fate by other cell types.     

Epiblast stem cell-based system reveals reprogramming synergy of germline factors

Epigenetic reprogramming in early germ cells is critical toward the establishment of totipotency, but investigations of the germline events are intractable. An objective cell culture-based system could provide mechanistic insight on how the key determinants of primordial germ cells (PGCs), including Prdm14, induce reprogramming in germ cells to an epigenetic ground state. Here we show a Prdm14-Klf2 synergistic effect that can accelerate and enhance reversion of mouse epiblast stem cells (epiSCs) to a naive pluripotent state, including X reactivation and DNA demethylation. Notably, Prdm14 alone has little effect on epiSC reversion, but it enhances the competence for reprogramming and potentially PGC specification. Reprogramming of epiSCs by the combinatorial effect of Prdm14-Klf2 involves key epigenetic changes, which might have an analogous role in PGCs. Our study provides a paradigm toward a systematic analysis of how other key genes contribute to complex and dynamic events of reprogramming in the germline.     

Differential Localization and Independent Acquisition of the H3K9me2 and H3K9me3 Chromatin Modifications in the Caenorhabditis elegans Adult Germ Line

Histone methylation is a prominent feature of eukaryotic chromatin that modulates multiple aspects of chromosome function. Methyl modification can occur on several different amino acid residues and in distinct mono-, di-, and tri-methyl states. However, the interplay among these distinct modification states is not well understood. Here we investigate the relationships between dimethyl and trimethyl modifications on lysine 9 of histone H3 (H3K9me2 and H3K9me3) in the adult Caenorhabditis elegans germ line. Simultaneous immunofluorescence reveals very different temporal/spatial localization patterns for H3K9me2 and H3K9me3. While H3K9me2 is enriched on unpaired sex chromosomes and undergoes dynamic changes as germ cells progress through meiotic prophase, we demonstrate here that H3K9me3 is not enriched on unpaired sex chromosomes and localizes to all chromosomes in all germ cells in adult hermaphrodites and until the primary spermatocyte stage in males. Moreover, high-copy transgene arrays carrying somatic-cell specific promoters are highly enriched for H3K9me3 (but not H3K9me2) and correlate with DAPI-faint chromatin domains. We further demonstrate that the H3K9me2 and H3K9me3 marks are acquired independently. MET-2, a member of the SETDB histone methyltransferase (HMTase) family, is required for all detectable germline H3K9me2 but is dispensable for H3K9me3 in adult germ cells. Conversely, we show that the HMTase MES-2, an E(z) homolog responsible for H3K27 methylation in adult germ cells, is required for much of the germline H3K9me3 but is dispensable for H3K9me2. Phenotypic analysis of met-2 mutants indicates that MET-2 is nonessential for fertility but inhibits ectopic germ cell proliferation and contributes to the fidelity of chromosome inheritance. Our demonstration of the differential localization and independent acquisition of H3K9me2 and H3K9me3 implies that the trimethyl modification of H3K9 is not built upon the dimethyl modification in this context. Further, these and other data support a model in which these two modifications function independently in adult C. elegans germ cells.     

A primary role of TET proteins in establishment and maintenance of De Novo bivalency at CpG islands

Ten Eleven Translocation (TET) protein-catalyzed 5mC oxidation not only creates novel DNA modifications, such as 5hmC, but also initiates active or passive DNA demethylation. TETs’ role in the crosstalk with specific histone modifications, however, is largely elusive. Here, we show that TET2-mediated DNA demethylation plays a primary role in the de novo establishment and maintenance of H3K4me3/H3K27me3 bivalent domains underlying methylated DNA CpG islands (CGIs). Overexpression of wild type (WT), but not catalytic inactive mutant (Mut), TET2 in low-TET-expressing cells results in an increase in the level of 5hmC with accompanying DNA demethylation at a subset of CGIs. Most importantly, this alteration is sufficient in making de novo bivalent domains at these loci. Genome-wide analysis reveals that these de novo synthesized bivalent domains are largely associated with a subset of essential developmental gene promoters, which are located within CGIs and are previously silenced due to DNA methylation. On the other hand, deletion of Tet1 and Tet2 in mouse embryonic stem (ES) cells results in an apparent loss of H3K27me3 at bivalent domains, which are associated with a particular set of key developmental gene promoters. Collectively, this study demonstrates the critical role of TET proteins in regulating the crosstalk between two key epigenetic mechanisms, DNA methylation and histone methylation (H3K4me3 and H3K27me3), particularly at CGIs associated with developmental genes.     

Narrow H3K4me2 is required for chicken PGC formation

The development of primordial germ cells (PGCs) undergoes epigenetic modifications. The study of histone methylation in regulating PGCs is beneficial to understand the development and differentiation mechanism of germ stem cells. Notably, it provides a theoretical basis for directed induction and mass acquisition in vitro. However, little is known about the regulation of PGC formation by histone methylation. Here, we found the high enrichment of H3K4me2 in the blastoderm, genital ridges, and testis. Chromatin immunoprecipitation sequencing was performed and the results revealed that genomic H3K4me2 is dynamic in embryonic stem cells, PGCs, and spermatogonial stem cells. This trend was consistent with the H3K4me2 enrichment in the gene promoter region. Additionally, narrow region triggered PGC‐related genes (Bmp4, Wnt5a, and Tcf7l2) and signaling pathways (Wnt and transforming growth factor‐β). After knocking down histone methylase Mll2 in vitro and vivo, the level of H3K4me2 decreased, inhibiting Cvh and Blimp1 expression, then repressing the formation of PGCs. Taken together, our study revealed the whole genome map of H3K4me2 in the formation of PGCs, contributing to improve the epigenetic study in PGC formation and providing materials for bird gene editing and rescue of endangered birds.