Extended Data Fig. 8: Relationship between gene expression, H3K27me3 or H3K9me3 distribution, and DNA methylation in Dnmt3A^KO SSCs (ID4-eGFP^bright+med).

a, Gene Ontology analysis (Metascape⁶⁰) of misregulated genes (bulk RNA-seq on ID4-GFP^bright+med cells). The top 20 GO terms are represented. Black bars: GO term enrichment with p value < 1e^-10; grey bars: enrichment with p-value < 1e^-6. P value is calculated with hypergeometric statistical test. b, Heatmap showing levels of H3K27ac, H3K27me3 and H3K9me3 enrichment in RPKM-normalized CUT&RUN signal for ID4-eGFP^bright+med cells (SSCs) from 10 dpp Dnmt3A^KO and WT males. Enrichment is assessed + /−5 kb from the center of the peak. Number of biological replicates for H3K27ac and H3K27me3: Dnmt3A^KO = 3, WT = 4; for H3K9me3: Dnmt3A^KO = 2 (one biological replicate was further divided into 2 technical replicates), WT = 3. c, Pie chart showing genomic annotation of each H3K9me3 peak category. d, Heatmap focusing on i) Dnmt3A^KO-lost H3K9me3 peaks (log2 FC) compared to WT (Left), ii) differential expression (log2 FC) of genes associated with H3K9me3 peaks (TSS+/− 5 kb from the peak) between Dnmt3A^KO and WT—from ID4-eGFP^bright+med RNA-seq—(Center), and iii) percentage of DNA methylation loss in Dnmt3A^KO versus WT on H3K9me3 peak location—from E18.5 WGBS—(Right). Rows are ordered according to gene expression changes: top, not differentially expressed; middle, upregulated; bottom, downregulated. e, Heatmap focusing on Dnmt3A^KO-gained H3K9me3 peaks (log2 FC) compared to WT, legend as in d. Rows are ordered according to gene expression changes: top, not differentially expressed; bottom, downregulated (no upregulated genes). f, Heatmap showing levels of H3K27me3 enrichment in RPKM-normalized CUT&RUN signal for ID4-eGFP^bright+med germ cells (SSCs) in 10 dpp Dnmt3A^KO and WT males. Peaks are divided into three categories: not differentially enriched (Dnmt3A^KO = WT), down-enriched (Dnmt3A^KO < WT), up-enriched (Dnmt3A^KO > WT) (FDR < 5% and FC > 1). g, Genomic annotation of each H3K27me3 peak category. h, Heatmap focusing on Dnmt3A^KO-gained H3K27me3 peaks (log2 FC) compared to WT (left), legend as in d. Rows are ordered according to gene expression changes: top, not differentially expressed; middle, upregulated; bottom, downregulated. i, Genomic annotation of Dnmt3A^KO-gained H3K27me3 peaks, associated with downregulated genes and >30% DNA methylation loss on H3K27me3 peak location.