Gametes are highly specialised cells that can give rise to the next generation through their ability to generate a totipotent zygote. In mouse, germ cells are first specified in the developing embryo as primordial germ cells (PGCs) starting around embryonic day (E) 6.251 (Fig. 1a). Following subsequent migration into the developing gonad, PGCs undergo a wave of extensive epigenetic reprogramming at E10.5/E11.52–11, including genome-wide loss of 5-methylcytosine (5mC)2–5,7–11 (Fig. 1a). The underlying molecular mechanisms of this process have remained enigmatic leading to our inability to recapitulate this step of germline development in vitro12–14. Using an integrative approach, we show that this complex reprogramming process involves the coordinated interplay between promoter sequence characteristics, DNA (de)methylation, Polycomb (PRC1) complex and both DNA demethylation-dependent and -independent functions of Tet1 to enable the activation of a critical set of germline reprogramming responsive (GRR) genes involved in gamete generation and meiosis. Our results also unexpectedly reveal a role for Tet1 in safeguarding but not driving DNA demethylation in gonadal PGCs. Collectively, our work uncovers a fundamental biological role for gonadal germline reprogramming and identifies the epigenetic principles of the PGC-to-gonocyte transition that will be instructive towards recapitulating complete gametogenesis in vitro.

Methylation at the 5 position of cytosine in DNA (5meC), is a key epigenetic mark in eukaryotes. Once introduced, 5meC can be maintained through DNA replication due to the activity of “maintenance” DNA methyltransferases.. Despite their ancient origin, DNA methylation pathways differ widely across metazoans, such that 5meC is either confined to transcribed genes or lost altogether in several lineages. Here we use comparative epigenomics to investigate the evolution of DNA methylation. Although the model nematode C. elegans has lost DNA methylation, more basal nematodes retain cytosine DNA methylation, targeted to repeat loci. Unexpectedly, we find that DNA methylation coevolves with the DNA alkylation repair enzyme ALKB2 across eukaryotes. We further show that DNA methyltransferases introduce the toxic lesion 3meC into DNA both in vitro and in vivo. Alkylation damage is thus intrinsically associated with DNA methyltransferase activity, and this may promote the loss of DNA methylation in many species.

Pluripotent stem cells (PSCs) can self-renew indefinitely while maintaining the ability to generate all cell types of the body. This plasticity is proposed to require heterogeneity in gene expression, driving a metastable state which may allow flexible cell fate choices.Contrary to this, naive PSC grown in fully defined '2i' environmental conditions, containing small molecule inhibitors of MEK and GSK3 kinases, show homogenous pluripotency and lineage marker expression. However, here we show that 2i induces greater genome-wide heterogeneity than traditional serum-containing growth environments at the population level across both male and female PSCs. This heterogeneity is dynamic and reversible over time, consistent with a dynamic metastable equilibrium of the pluripotent state. We further show that the 2i environment causes increased heterogeneity in the calcium signalling pathway at both the population and single-cell level. Mechanistically, we identify loss of robustness regulators in the form of negative feedback to the upstream EGF receptor. Our findings advance the current understanding of the plastic nature of the pluripotent state and highlight the role of signalling pathways in the control of transcriptional heterogeneity. Furthermore, our results have critical implications for the current use of kinase inhibitors in the clinic, where inducing heterogeneity may increase the risk of cancer metastasis and drug resistance.RNAs, are not reduced and are instead increased in 2i ( Fig. 2i) arguing against this. This Animal studiesAnimal studies were authorized under a UK Home Office Project License and carried out in a Home Office-designated facility. Cell cultureESCs and EGCs (Oct4ΔPE-GFP) were derived and cultured as previously described 18 .Serum culture conditions contain 15% FCS, 0.1 mM MEM nonessential amino acids, 2 mM lglutamine, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol and 1000U/mL LIF in DMEM-F12 with maintenance on a mouse embryonic fibroblast (MEF) feeder layer. 2i culture conditions contain 1 μ M PD0325901, 3 μ M CHIR99021 and 1000U/mL LIF in N2B27 medium, maintained on laminin (10 μ g/ml). Cells were incubated at 5% CO 2 , 95% relative humidity. All samples were profiled at fewer than 23 passages. For time-course transcriptome profiling EGCs were derived and maintained in either serum or 2i and collected every 3 passages from 11-17. Population transcriptomicsSamples were prepared and processed as described previously. Briefly, 100ng RNA was fragmented, labelled and hybridized to Affymetrix GeneChip Mouse Gene 1.0 ST Arrays.Data was preprocessed in R using robust multiarray averaging, variance stabilising transformation and Combat batch correction 70 . Limma was used for differential expression analysis and for linear regression against cell subtypes, sex and passage 71 . Differential variance was tested using the studentised Breusch-Pagan test with culture condition as the independent variable. Testing statistical enrichment of gene sets was performed using functional annotation ontology 40 ...