The relevance of the epigenetic features of enhancers for germline competence

Primordial germ cells (PGC) are the precursor of the gametes, which safeguard the transmission of genetic information to the next generation. In mammals, germline specification occurs through an inductive process whereby competent cells in the post-implantation epiblast differentiate into PGC. Due to the ethical limitations to investigate germline specification in humans, this process is best understood in mice, where PGC specification is initiated in a few epiblast cells at the proximo-posterior end of the mouse embryo (E6.25). Thereby, PGC specification occurs in response to inductive signals emanating from extraembryonic tissues. However, the intrinsic factors that endow mammalian epiblast cells with the competence to respond to germline inductive signals remain unknown. This lack of knowledge is explained, at least partly, due to the scarcity of PGC and peri-implantation epiblast cells in vivo. Therefore, here an in vitro system that faithfully recapitulates this process by differentiating ESC into primordial germ cell-like cells (PGCLC) has been applied to investigate whether the epigenetic state of cis-regulatory elements contributes to germline competence. First of all, single-cell RNA-sequencing was performed across multiple stages of in vitro PGCLC differentiation to further characterize this system. This showed that PGCLC genes are widely expressed in preceding pluripotent embryonic stem cells (ESC), but become homogeneously dismantled in germline competent epiblast like-cells (EpiLC). To understand the regulation of this process, the focus was drawn on distal enhancers that become active in PGCLC (e.g. PGCLC enhancer). The analysis of a set of comprehensive epigenetic changes showed that a subset of PGCLC enhancers gets partly decommissioned in EpiLC, retains permissive chromatin features, including H3K4me1, and remains responsive to transcriptional activators. Furthermore, upon further differentiation into epiblast stem cells (EpiSC) in which germline competence is lost, the PGCLC enhancers accumulate higher levels of repressive epigenetic modifications and lose their responsiveness to transcriptional activators, thus getting fully decommissioned. Subsequently, the relevance of H3K4me1 for germline competence was demonstrated in a H3K4me1/2 deficient cell line, where both H3K4me1/2 methyltransferases (KMT2C/D) were catalytically inactive (dCD cells). The differentiation of the dCD cells revealed a decreased PGCLC differentiation capacity, which was accompanied by reduced expression of PGCLC genes linked to PGCLC enhancers. On the other hand, H3K4me1/2 might not have the same functional impact during the transition from ESC to other pluripotent stages, as gene expression changes during these transitions were relatively normal in the dCD cells. Finally, the analysis of in vivo single-cell methylation data suggest an increased epigenetic heterogeneity of PGCLC enhancers in the formative epiblast, before they eventually become activated in PGC. Overall, the presented work suggests that H3K4me1 can facilitate the (re)activation of enhancers and the establishment of gene expression programs during specific developmental transitions