Extended Data Fig. 8: H3K36me2/3 and DNA methylation in Nsd1 KO cells.
From: Reprogramming of H3K36me2 guides lineage-specific post-implantation de novo DNA methylation
a, Schematic showing disruption of Nsd1 in mESCs and a pair of gRNAs targeting the catalytic domain (AWS + SET+post-SET). b, The UCSC genome browser views showing the enrichment (spike-in normalized read counts) of H3K36me2 and H3K36me3 in WT and Nsd1 KO S/L mESCs. c, Western blot showing the levels of NSD1 and H3K36me2 in WT and Nsd1 KO S/L mESCs (n = 3 biological replicates). α-tubulin and total H3 are used as loading controls. d, Left, heatmaps showing H3K36me2 and H3K36me3 (5-kb bin) in identified H3K36me3-enriched (K36me2LK36me3H), H3K36me2-enriched (K36me2HK36me3L), and H3K36me2/3-low (K36me2LK36me3L) regions in WT 2i mESCs, S/L mESCs, and EpiLCs; Middle, heatmaps showing H3K36me2 (spike-in), H3K36me3 (non-spike-in), and DNA methylation in these regions in WT and Nsd1 KO 2i mESCs, S/L mESCs, and EpiLCs. DNA methylation in each group of regions is quantified with box plots on the right. Center line, median; box, 25th and 75th percentiles; whiskers, 1.5 × IQR. P = 3e-18 for WT vs. Nsd1 KO S/L mESCs in H3K36me3-enriched regions and P < 2e-308 for all other comparisons (two-sided paired Wilcoxon signed-rank test). e, Bar chart showing the expression fold changes (Nsd1 KO /WT) of Dnmt3a, Dnmt3b, Dnmt1, and Dnmt3l in 2i mESCs, S/L mESCs, and EpiLCs (n = 2 biological replicates). f, Box plots showing DNA methylation at CGI and non-CGI promoters of the DMS germline genes in control and Nsd1 KO D3 EpiLCs. All CGI and non-CGI genes are similarly analyzed as controls (‘All’). Center line, median; box, 25th and 75th percentiles; whiskers, 1.5 × IQR. P = 6e-12 (DMS CGI promoters between Ctrl vs. Nsd1 KO EpiLCs), P < 2e-308 (all CGI promoters between Ctrl vs. Nsd1 KO EpiLCs), P = 1e-15 (DMS non-CGI promoters between Ctrl vs. Nsd1 KO EpiLCs), and P < 2e-308 (all non-CGI promoters between Ctrl vs. Nsd1 KO EpiLCs) (two-sided paired Wilcoxon signed-rank test). Source numerical data and unprocessed blots are available in source data.
