PRC1.6 and SETDB1-mediated repression of germline genes in the early embryo precedes DNA methylation-mediated silencing.

Silencing of a subset of germline genes is dependent upon DNA methylation (DNAme) post-implantation. However, these genes are generally hypomethylated in the ICM, implicating alternative silencing pathways before implantation. Indeed, in embryonic stem cells (ESCs), an overlapping set of genes, including germline “genome-defence” (GGD) genes, are upregulated following deletion of the H3K9 methyltransferase SETDB1 or subunits of the non-canonical PRC1 complex PRC1.6. Here, we show that in pre-implantation embryos and naïve ESCs (nESCs), hypomethylated promoters of germline genes bound by the PRC1.6 DNA-binding subunits MGA/MAX/E2F6 are enriched for RING1B-dependent H2AK119ub1 and H3K9me3. Accordingly, silencing of these genes in nESCs shows a greater dependence on PRC1.6 than DNAme. In contrast, GGD genes are hypermethylated in epiblast-like cells and their silencing is dependent upon SETDB1, PRC1.6/RING1B and DNAme, with H3K9me3 and DNAme establishment dependent upon MGA binding. Thus, GGD genes are initially repressed by PRC1.6, with DNAme subsequently engaged in post-implantation embryos. ChIP-seq from E9.5 PGCs and WT nESCs, EpiLCs and d4 PGCLCs, and Mga dHLH, Setdb1 cKO, Pcgf6 cKO or Dnmt cTKO nESCs and EpiLCs. RNA-seq from WT and Dnmt cTKO, Mga ΔHLH, Setdb1 cKO, Pcgf6 cKO, Ring1b cKO or Setdb1/Ring1b cDKO nESCs and EpiLCs.