Depletion of DNAme has a limited effect on H3K27 methylation. (A) Mass spectrometry analysis of H3K27 and H3K36 methylation levels (two replicates for each mark) in WT and Dnmt-TKO, showing limited changes in Dnmt-TKO, except for slight H3K27me3 gain and H3K36me2 loss. P-values are indicated in the same way as in Figure 1B. (B) Genome browser representation of H3K27me1/2/3 in WT (green) versus Dnmt-TKO (blue) versus Nsd1-KO (red). Examples of regions with H3K27me3 spreading (in Nsd1-KO) are shaded. Note the much stronger effect of Nsd1-KO compared to Dnmt-TKO. (C) Genome-wide correlation density plots of 10-kb binned ChIP-seq signal, MS normalized, shaded to indicate genic (blue) and intergenic (yellow) regions, illustrating the global effect of DNA demethylation on H3K27me1/2/3 and H3K36me2. Aggregate plots of H3K27me across the genome centered on active genes are provided in Supplemental Figure S2A. (D) Highlighting the comparison of H3K27me3 in WT, Dnmt-TKO, and Nsd1-KO: although there is nearly uniform spread in Nsd1-KO, H3K27me3 increase in TKO is limited and more localized. The shaded area indicates an H3K27me3-spreading region in Nsd1-KO. (E) Signal intensity plot of H3K27me3 (normalized by input and MS ratios) centered on CGIs overlapping SUZ12 peaks (n = 4543), showing the localized spread in TKO and the nearly uniform spread in Nsd1-KO, indicated by the higher enrichment in the surrounding regions in KOs compared to WTs. The spread and increase of H3K27me3 upon Dnmt-TKO and Nsd1-KO are especially in inactive genes (i.e., outside of active genes), consistent with the observation in Supplemental Figures S1A and S2A (see the “H3K27me3” panel). Replicate plots are shown in Supplemental Figure S2B. (F) H3K27me3 “Peakiness score,” representing the average ChIP signal in the top 1% of 1-kb bins, confirming the spreading (lower Peakiness score) of H3K27me3 in Nsd1-KO, and the limited spread in Dnmt-TKO. Plots based on replicates are provided in Supplemental Figure S2C. Data used in this figure are generated in this study.
