Extended Data Figure 5. DNA methylation-dependent changes in gene and retrotransposon expression.

a. Average E6.5 DNA methylation (Top) and E8.5 expression (Bottom) for retrotransposon families. Expression was calculated as the normalized fraction of reads recovered from scRNA-seq data for each subfamily. The Dnmt1 KO shows the strongest reduction in methylation across retrotransposons in the epiblast and the Xecto. The ERVK family of LTRs shows the strongest corresponding increase in expression, which is higher in the embryonic lineage than in Xecto.

b. Intracisternal A particle (IAP) expression as detected by scRNA-seq depends on DNA methylation. Top: DNA methylation levels as profiled by WGBS. The largest drop in global and IAP-specific methylation is observed for Dnmt1 KO. Bottom: Mean expression within the embryonic and Xecto lineages of E8.5 KO embryos, shown as the fraction of total reads per cell. Epiblast IAPEz-int: n = 5,585; 5,579; 5,510; 5,440; 5,210, Xecto IAPEz-int: n = 5,576; 5,577; 5,498; 5,421; 5,367; 5,575; 5,529; 5,518; 5,500; 5,411; 5,543.

c. Scatterplot of E6.5 promoter DNA methylation and E8.5 expression differences in the Xecto lineage of L3mbtl2 KO compared to WT, as shown for the embryonic lineage in Fig. 2g. Differentially hypomethylated (delta ≤ –0.1) and derepressed genes (delta ≥ 0.2 fraction positive cells) in L3mbtl3 KO (green) were strongly enriched in GO terms related to gametogenesis (green asterisks, P < 0.05), in line with previous reports on ncPRC1.6 targets. These genes contain key members of the piRNA biogenesis pathway, including the dead-box helicase Ddx4 (VASA homolog) and Maelstrom, as well as other genes with known functions or expression during gametogenesis. Extraembryonic lineages naturally express certain gametogenesis-associated regulators, which may explain their ability to proliferate in the KO while embryonic lineages arrest shortly after gastrulation onset.

d. Genome browser tracks of WGBS methylation data for three aberrantly regulated loci in L3mbtl2 KO embryos. The bidirectional genes Lypd4 and Dmrtc2 initiate from the same CpG island (CGI), while Tex101 does not have a CGI, but does have a higher than genomic average CpG density (see density track). These promoters are specifically hypomethylated in gametes and throughout preimplantation, followed by de novo methylation by E6.5 that continues to increase over development. De novo methylation does not occur in the L3mbtl2 KO and corresponds with sharp increases in gene expression. WT data from gametes, preimplantation embryos, and late stage samples like somatic tissues and the E14.5 placenta are taken from Ref’s^62–65.

e. Promoter DNA methylation (Top) and E8.5 expression (Bottom, shown as fraction of positive cells per embryo replicate) boxplots of L3mbtl2 sensitive genes (n = 13 genes taken from Fig. 2g, green). Many gametogenesis genes are regulated by “weak” CGI-containing promoters that become methylated during development⁶⁶. In line with this, the promoters of L3mbtl2 KO sensitive genes are hypomethylated in gametes and preimplantation and become de novo methylated over postimplantation development. Derepression is specific to L3mbtl2 KO, and does not occur in Dnmt1 or Dnmt3b KOs, where methylation levels drop globally. Expression changes are also not substantial for Rnf2 or G9a KO although these regulators are also expected to participate in ncPRC1.6 complex-directed repression. A single outlier gene, Ttr, is expressed in all KOs and WT, but is still upregulated in L3mbtl2 KO. Additional data taken from previous studies^62–64,67.