Kenjiro Shirane

Mouse Oocyte Methylomes at Base Resolution Reveal Genome-Wide Accumulation of Non-CpG Methylation and Role of DNA Methyltransferases

DNA methylation is an epigenetic modification that plays a crucial role in normal mammalian development, retrotransposon silencing, and cellular reprogramming. Although methylation mainly occurs on the cytosine in a CG site, non-CG methylation is prevalent in pluripotent stem cells, brain, and oocytes. We previously identified non-CG methylation in several CG-rich regions in mouse germinal vesicle oocytes (GVOs), but the overall distribution of non-CG methylation and the enzymes responsible for this modification are unknown. Using amplification-free whole-genome bisulfite sequencing, which can be used with minute amounts of DNA, we constructed the base-resolution methylome maps of GVOs, non-growing oocytes (NGOs), and mutant GVOs lacking the DNA methyltransferase Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3L. We found that nearly two-thirds of all methylcytosines occur in a non-CG context in GVOs. The distribution of non-CG methylation closely resembled that of CG methylation throughout the genome and showed clear enrichment in gene bodies. Compared to NGOs, GVOs were over four times more methylated at non-CG sites, indicating that non-CG methylation accumulates during oocyte growth. Lack of Dnmt3a or Dnmt3L resulted in a global reduction in both CG and non-CG methylation, showing that non-CG methylation depends on the Dnmt3a-Dnmt3L complex. Dnmt3b was dispensable. Of note, lack of Dnmt1 resulted in a slight decrease in CG methylation, suggesting that this maintenance enzyme plays a role in non-dividing oocytes. Dnmt1 may act on CG sites that remain hemimethylated in the de novo methylation process. Our results provide a basis for understanding the mechanisms and significance of non-CG methylation in mammalian oocytes.

Setdb1 is required for germline development and silencing of H3K9me3-marked endogenous retroviruses in primordial germ cells

Transcription of endogenous retroviruses (ERVs) is inhibited by de novo DNA methylation during gametogenesis, a process initiated after birth in oocytes and at approximately embryonic day 15.5 (E15.5) in prospermatogonia. Earlier in germline development, the genome, including most retrotransposons, is progressively demethylated. Young ERVK and ERV1 elements, however, retain intermediate methylation levels. As DNA methylation reaches a low point in E13.5 primordial germ cells (PGCs) of both sexes, we determined whether retrotransposons are marked by H3K9me3 and H3K27me3 using a recently developed low-input ChIP-seq (chromatin immunoprecipitation [ChIP] combined with deep sequencing) method. Although these repressive histone modifications are found predominantly on distinct genomic regions in E13.5 PGCs, they concurrently mark partially methylated long terminal repeats (LTRs) and LINE1 elements. Germline-specific conditional knockout of the H3K9 methyltransferase SETDB1 yields a decrease of both marks and DNA methylation at H3K9me3-enriched retrotransposon families. Strikingly, Setdb1 knockout E13.5 PGCs show concomitant derepression of many marked ERVs, including intracisternal A particle (IAP), ETn, and ERVK10C elements, and ERV-proximal genes, a subset in a sex-dependent manner. Furthermore, Setdb1 deficiency is associated with a reduced number of male E13.5 PGCs and postnatal hypogonadism in both sexes. Taken together, these observations reveal that SETDB1 is an essential guardian against proviral expression prior to the onset of de novo DNA methylation in the germline.

DNA methylation and gene expression dynamics during spermatogonial stem cell differentiation in the early postnatal mouse testis

BackgroundIn the male germline, neonatal prospermatogonia give rise to spermatogonia, which include stem cell population (undifferentiated spermatogonia) that supports continuous spermatogenesis in adults. Although the levels of DNA methyltransferases change dynamically in the neonatal and early postnatal male germ cells, detailed genome-wide DNA methylation profiles of these cells during the stem cell formation and differentiation have not been reported.ResultsTo understand the regulation of spermatogonial stem cell formation and differentiation, we examined the DNA methylation and gene expression dynamics of male mouse germ cells at the critical stages: neonatal prospermatogonia, and early postntal (day 7) undifferentiated and differentiating spermatogonia. We found large partially methylated domains similar to those found in cancer cells and placenta in all these germ cells, and high levels of non-CG methylation and 5-hydroxymethylcytosines in neonatal prospermatogonia. Although the global CG methylation levels were stable in early postnatal male germ cells, and despite the reported scarcity of differential methylation in the adult spermatogonial stem cells, we identified many regions showing stage-specific differential methylation in and around genes important for stem cell function and spermatogenesis. These regions contained binding sites for specific transcription factors including the SOX family members.ConclusionsOur findings show a distinctive and dynamic regulation of DNA methylation during spermatogonial stem cell formation and differentiation in the neonatal and early postnatal testes. Furthermore, we revealed a unique accumulation and distribution of non-CG methylation and 5hmC marks in neonatal prospermatogonia. These findings contrast with the reported scarcity of differential methylation in adult spermatogonial stem cell differentiation and represent a unique phase of male germ cell development.

In vitro expansion of mouse primordial germ cell‐like cells recapitulates an epigenetic blank slate

The expansion of primordial germ cells (PGCs), the precursors for the oocytes and spermatozoa, is a key challenge in reproductive biology/medicine. Using a chemical screening exploiting PGC‐like cells (PGCLCs) induced from mouse embryonic stem cells (ESCs), we here identify key signaling pathways critical for PGCLC proliferation. We show that the combinatorial application of Forskolin and Rolipram, which stimulate cAMP signaling via different mechanisms, expands PGCLCs up to ~50‐fold in culture. The expanded PGCLCs maintain robust capacity for spermatogenesis, rescuing the fertility of infertile mice. Strikingly, during expansion, PGCLCs comprehensively erase their DNA methylome, including parental imprints, in a manner that precisely recapitulates genome‐wide DNA demethylation in gonadal germ cells, while essentially maintaining their identity as sexually uncommitted PGCs, apparently through appropriate histone modifications. By establishing a paradigm for PGCLC expansion, our system reconstitutes the epigenetic “blank slate” of the germ line, an immediate precursory state for sexually dimorphic differentiation.

Software updates in the Illumina HiSeq platform affect whole-genome bisulfite sequencing.

BackgroundMethylation of cytosine in genomic DNA is a well-characterized epigenetic modification involved in many cellular processes and diseases. Whole-genome bisulfite sequencing (WGBS), such as MethylC-seq and post-bisulfite adaptor tagging sequencing (PBAT-seq), uses the power of high-throughput DNA sequencers and provides genome-wide DNA methylation profiles at single-base resolution. However, the accuracy and consistency of WGBS outputs in relation to the operating conditions of high-throughput sequencers have not been explored.ResultsWe have used the Illumina HiSeq platform for our PBAT-based WGBS, and found that different versions of HiSeq Control Software (HCS) and Real-Time Analysis (RTA) installed on the system provided different global CpG methylation levels (approximately 5% overall difference) for the same libraries. This problem was reproduced multiple times with different WGBS libraries and likely to be associated with the low sequence diversity of bisulfite-converted DNA. We found that HCS was the major determinant in the observed differences. To determine which version of HCS is most suitable for WGBS, we used substrates with predetermined CpG methylation levels, and found that HCS v2.0.5 is the best among the examined versions. HCS v2.0.12 showed the poorest performance and provided artificially lower CpG methylation levels when 5-methylcytosine is read as guanine (first read of PBAT-seq and second read of MethylC-seq). In addition, paired-end sequencing of low diversity libraries using HCS v2.2.38 or the latest HCS v2.2.58 was greatly affected by cluster densities.ConclusionsSoftware updates in the Illumina HiSeq platform can affect the outputs from low-diversity sequencing libraries such as WGBS libraries. More recent versions are not necessarily the better, and HCS v2.0.5 is currently the best for WGBS among the examined HCS versions. Thus, together with other experimental conditions, special care has to be taken on this point when CpG methylation levels are to be compared between different samples by WGBS.

Generation of human oogonia from induced pluripotent stem cells in vitro

Reconstituting a human ovary Human pluripotent stem cells (hPSCs) have been induced into human primordial germ cell–like cells (hPGCLCs) in vitro, the first step toward human in vitro gametogenesis. Yamashiro et al. went a step closer to generating mature gametes by culturing hPSCs with mouse embryonic ovarian somatic cells in xenogeneic reconstituted ovaries (see the Perspective by Gill and Peters). Over a period of 4 months, hPGCLCs underwent hallmark epigenetic reprogramming and differentiated progressively into cells closely resembling human oogonia, an immediate embryonic precursor for human oocytes. This study creates opportunities for human germ cell research and provides a foundation for human in vitro gametogenesis. Science, this issue p. 356; see also p. 291 Human primordial germ cell–like cells differentiate into oogonia in xenogeneic reconstituted ovaries in vitro. Human in vitro gametogenesis may transform reproductive medicine. Human pluripotent stem cells (hPSCs) have been induced into primordial germ cell–like cells (hPGCLCs); however, further differentiation to a mature germ cell has not been achieved. Here, we show that hPGCLCs differentiate progressively into oogonia-like cells during a long-term in vitro culture (approximately 4 months) in xenogeneic reconstituted ovaries with mouse embryonic ovarian somatic cells. The hPGCLC-derived oogonia display hallmarks of epigenetic reprogramming—genome-wide DNA demethylation, imprint erasure, and extinguishment of aberrant DNA methylation in hPSCs—and acquire an immediate precursory state for meiotic recombination. Furthermore, the inactive X chromosome shows a progressive demethylation and reactivation, albeit partially. These findings establish the germline competence of hPSCs and provide a critical step toward human in vitro gametogenesis.