Dynamic three-dimensional epigenomic reorganization for the development of undifferentiated spermatogonia in mice.

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.

Epigenetic reprogramming in mouse primordial germ cells

Previous studies have revealed that mouse primordial germ cells (PGCs) undergo genome-wide DNA methylation reprogramming to reset the epigenome for totipotency. However, the precise 5-methylcytosine (5mC) dynamics and its relationship with the generation of 5-hydroxymethylcytosine (5hmC) are not clear. Here we analyzed the dynamics of 5mC and 5hmC during PGC reprograming and germ cell development. Unexpectedly, we found a specific period (E8.5-9.5) during which both 5mC and 5hmC levels are low. Subsequently, 5hmC levels increase reaching its peak at E11.5 and gradually decrease until E13.5 likely by replication-dependent dilution. Interestingly, 5hmC is enriched in chromocenters during this period. While this germ cell-specific 5hmC subnuclear localization pattern is maintained in female germ cells even in mature oocytes, such pattern is gradually lost in male germ cells as mitotic proliferation resumes during the neonatal stage. Pericentric 5hmC plays an important role in silencing major satellite repeat, especially in female PGCs. Global transcriptome analysis by RNA-seq revealed that the great majority of differentially expressed genes from E9.5 to 13.5 are upregulated in both male and female PGCs. Although only female PGCs enter meiosis during the prenatal stage, meiosis-related and a subset of imprinted genes are significantly upregulated in both male and female PGCs at E13.5. Thus, our study not only reveals the dynamics of 5mC and 5hmC during PGC reprogramming and germ cell development, but also their potential role in epigenetic reprogramming and transcriptional regulation of meiotic and imprinted genes.

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 …

ABSTRACTPrimordial germ cells (PGCs) are precursors of eggs and sperm. How the PGCs epigenetically reprogram during early embryonic development in fish is currently unknown. Here we generated a series of PGC methylomes using whole genome bisulfite sequencing across key stages from 8 days post fertilization (dpf) to 25 dpf coinciding with germ cell sex determination and gonadal sex differentiation in medaka (Oryzias latipes) to elucidate the dynamics of DNA methylation during epigenetic reprogramming in germ cells. Our high-resolution DNA methylome maps show a global demethylation taking place in medaka PGCs in a two-step strategy. The first step occurs between the blastula and 8-dpf stages, and the second step occurs between the 10-dpf and 12-dpf stages. Both demethylation processes are global, except for CGI promoters which remain hypomethylated throughout the stage of PGC specification. De novo methylation proceeded at 25-dpf stage with the process in male germ cells superseding female germ cells. Gene expression analysis showed that tet2 maintains high levels of expression during the demethylation stage, while dnmt3ba expression increases during the de novo methylation stage during sexual fate determination in germ cells. The present results suggest that medaka PGCs undergo a bi-phasic epigenetic reprogramming process. Global erasure of DNA methylation marks peaks at 15-dpf and de novo methylation in male germ cells takes precedence over female germ cells at 25 dpf. Results also provide important insights into the developmental window of susceptibility to environmental stressors for multi- and trans-generational health outcomes in fish.

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...

Genomic reprogramming of primordial germ cells (PGCs), which includes genome-wide demethylation, prevents aberrant epigenetic modifications from being transmitted to subsequent generations. This process also ensures that homologous chromosomes first acquire an identical epigenetic status before an appropriate switch in the imprintable loci in the female and male germ lines. Embryonic germ (EG) cells have a similar epigenotype to PGCs from which they are derived. We used EG cells to investigate the mechanism of epigenetic modifications in the germ line by analysing the effects on a somatic nucleus in the EG-thymic lymphocyte hybrid cells. There were striking changes in methylation of the somatic nucleus, resulting in demethylation of several imprinted and non-imprinted genes. These epigenetic modifications were heritable and affected gene expression as judged by re-activation of the silent maternal allele of Peg1/Mest imprinted gene in the somatic nucleus. This remarkable change in the epigenotype of the somatic nucleus is consistent with the observed pluripotency of the EG-somatic hybrid cells as they differentiated into a variety of tissues in chimeric embryos. The epigenetic modifications observed in EG-somatic cell hybrids in vitro are comparable to the reprogramming events that occur during germ cell development.

Germline development from human pluripotent stem cells toward disease modeling of infertility

Primordial germ cells (PGCs) are the precursors of sperm and eggs. They undergo genome-wide epigenetic reprogramming to erase epigenetic memory and reset the genomic potential for totipotency. Global DNA methylation erasure is a crucial part of epigenetic resetting when DNA methylation levels decrease across the genome to <5%. However, certain localized regions exhibit slower demethylation or resistance to reprogramming. Since DNA methylation plays a crucial role in transcriptional regulation, this depletion in PGCs requires mechanisms independent of DNA methylation to regulate transcriptional control during PGC reprogramming. Histone modifications are predicted to compensate for the loss of DNA methylation in gene regulation. Different histone modifications exhibit distinct patterns in PGCs undergoing epigenetic programming at the genomic level during PGC development in conjunction with changes in DNA methylation. Together, they contribute to PGC-specific genomic regulation. Recent findings related to these processes provide a comprehensive overview of germline epigenetic reprogramming and its importance in mouse and human PGC development. Additionally, we evaluated the extent to which in vitro culture techniques have replicated the development processes of human PGCs.

Germline development is characterized by genome-wide reprogramming of DNA methylation. Recent work has enlightened the dynamics of DNA methylation in primordial germ cells (PGCs), but knowledge of histone modification dynamics at these developmental stages remains limited, mostly due to the difficulty in obtaining enough high quality chromatin immunoprecipitation (ChIP) material for sequencing. Previous work in our laboratory has demonstrated the importance of histone methyltransferases in silencing retroelements [1] and a subset of germline-specific genes [2] in embryonic stem cells. Here, we sought to develop a reliable ChIP-sequencing protocol to study the dynamics of histone modification during the DNA methylation reprogramming that occurs in PGCs. We have developed a scaled down native ChIP and sequencing library construction protocol that can be performed on small cell numbers. We optimized sample fragmentation, antibody concentration, ChIP conditions, library construction and amplification to generate high quality, high resolution H3K9me3 sequencing libraries from as little as 1,000 embryonic stem cells. Paired-end sequencing (Illumina HiSeq) of these pooled and indexed libraries generated an average of 28 million aligned read pairs (with 6 libraries per sequencing lane), with under 20% duplicate reads, for an average of 23 million unique read pairs. Under optimized conditions, we found that over 85% of the peaks identified using standard native ChIP-sequencing (using 2 million cells as starting material) were also detected by our small cell number native ChIP-seq protocol. We also found excellent reproducibility between independent ChIP experiments. Using this optimized small cell number native ChIP-seq protocol, we generated genome-wide H3 and H3K9me3 profiles from 1,000 E13.5 PGCs and identified a unique cohort of genes and retroelements enriched for this repressive mark. Integration of this chip-seq data with DNA methylation/WGBS and transcriptome data generated at the same developmental stage will be presented.

A number of environmental factors (e.g. toxicants) have been shown to promote the epigenetic transgenerational inheritance of disease and phenotypic variation. Transgenerational inheritance requires the germline transmission of altered epigenetic information between generations in the absence of direct environmental exposures. The primary periods for epigenetic programming of the germ line are those associated with primordial germ cell development and subsequent fetal germline development. The current study examined the actions of an agricultural fungicide vinclozolin on gestating female (F0 generation) progeny in regards to the primordial germ cell (PGC) epigenetic reprogramming of the F3 generation (i.e. great-grandchildren). The F3 generation germline transcriptome and epigenome (DNA methylation) were altered transgenerationally. Interestingly, disruptions in DNA methylation patterns and altered transcriptomes were distinct between germ cells at the onset of gonadal sex determination at embryonic day 13 (E13) and after cord formation in the testis at embryonic day 16 (E16). A larger number of DNA methylation abnormalities (epimutations) and transcriptional alterations were observed in the E13 germ cells than in the E16 germ cells. These observations indicate that altered transgenerational epigenetic reprogramming and function of the male germline is a component of vinclozolin induced epigenetic transgenerational inheritance of disease. Insights into the molecular control of germline transmitted epigenetic inheritance are provided.

During B cell activation, the DNA lesions that initiate somatic hypermutation and class switch recombination are introduced by activation-induced cytidine deaminase (AID). AID is a highly mutagenic protein that is maintained in the cytoplasm at steady state, however AID is shuttled across the nuclear membrane and the protein transiently present in the nucleus appears sufficient for targeted alteration of immunoglobulin loci. AID has been implicated in epigenetic reprogramming in primordial germ cells and cell fusions and in induced pluripotent stem cells (iPS cells), however AID expression in non-B cells is very low. We hypothesised that epigenetic reprogramming would require a pathway that instigates prolonged nuclear residence of AID. Here we show that AID is completely re-localised to the nucleus during drug withdrawal following etoposide treatment, in the period in which double strand breaks (DSBs) are repaired. Re-localisation occurs 2-6 hours after etoposide treatment, and AID remains in the nucleus for 10 or more hours, during which time cells remain live and motile. Re-localisation is cell-cycle dependent and is only observed in G2. Analysis of DSB dynamics shows that AID is re-localised in response to etoposide treatment, however re-localisation occurs substantially after DSB formation and the levels of re-localisation do not correlate with γH2AX levels. We conclude that DSB formation initiates a slow-acting pathway which allows stable long-term nuclear localisation of AID, and that such a pathway may enable AID-induced DNA demethylation during epigenetic reprogramming.

Germ cell development creates totipotency through genetic as well as epigenetic regulation of the genome function. Primordial germ cells (PGCs) are the first germ cell population established during development and are immediate precursors for both the oocytes and spermatogonia. We here summarize recent findings regarding the mechanism of PGC development in mice. We focus on the transcriptional and signaling mechanism for PGC specification, potential pluripotency, and epigenetic reprogramming in PGCs and strategies for the reconstitution of germ cell development using pluripotent stem cells in culture. Continued studies on germ cell development may lead to the generation of totipotency in vitro, which should have a profound influence on biological science as well as on medicine.

Primordial germ cells (PGCs) provide an excellent tool to better understand ancestor?descendent relationships as well as the efficiency and molecular mechanisms governing pluripotency in the reprogramming of somatic cells, since the latter type of cells have a relatively lower efficiency of conversion to pluripotent cells. This kind of comparison has gained credence from the commonalities regarding the expression of key transcription factors such as octamer-binding transcriptionhttp://en.wikipedia.org/wiki/Transcription_factor factor-4 (Oct3/4), SRY-related HMG box (Sox2), myelocytomatosis (c-Myc), and Nanog, as well as redundancy in terms of Kruppel-like factor 2 (Klf2), Kruppel-like factor 5 (Klf5), estrogen-related receptor beta (Esrrb), and estrogen-related receptor gamma (Esrrg) compensating for the absence of Kruppel-like factor 4 (Klf4). However, the exogenous addition of any one of these factors was found to be important, thereby implying that the expression level is important. L-Myelocytomatosis (L-myc) was shown to improve reprogramming efficiency without affecting tumorigenic potential. Molecular aspects of epigenetic reprogramming during the acquisition of pluripotency, as well as tumorigenic potential, have also been discussed, thus providing an understanding of the factors that can improve the former without increasing the possibility of neoplastic transformation. An improved understanding of the molecular events would pave the way for the development and use of endogenous biomolecules as well as currently available chemical reprogrammers for improving the efficiency of conversion of PGCs into cells of the stem cell lineage. Such chemicals, when adequately tested, can possibly be an alternative to viral vectors, since the introduced transgenes can become oncogenic.

During female germline development, oocytes become a highly specialized cell type and form a maternal cytoplasmic store of crucial factors. Oocyte growth is triggered at the transition from primordial to primary follicle and is accompanied by dynamic changes in gene expression1, but the gene regulatory network that controls oocyte growth remains unknown. Here we identify a set of transcription factors that are sufficient to trigger oocyte growth. By investigation of the changes in gene expression and functional screening using an in vitro mouse oocyte development system, we identified eight transcription factors, each of which was essential for the transition from primordial to primary follicle. Notably, enforced expression of these transcription factors swiftly converted pluripotent stem cells into oocyte-like cells that were competent for fertilization and subsequent cleavage. These transcription-factor-induced oocyte-like cells were formed without specification of primordial germ cells, epigenetic reprogramming or meiosis, and demonstrate that oocyte growth and lineage-specific de novo DNA methylation are separable from the preceding epigenetic reprogramming in primordial germ cells. This study identifies a core set of transcription factors for orchestrating oocyte growth, and provides an alternative source of ooplasm, which is a unique material for reproductive biology and medicine.