Fig. 1: Loss of NSD3-dependent H3K36me2 leads to reduced enhancer activity and downregulation of targeted genes.

a Representative genome browser tracks showing progressive loss of H3K36me2 in multiple-KO conditions: NSD1/2 double-KO (DKO); NSD1/2-SETD2 triple-KO (TKO); NSD1/2/3-SETD2 quadruple-KO (QKO). In TKO, the remaining H3K36me2 peaks mark several active and strong enhancers, which are ablated in QKO and lead to reduced activity of enhancers, indicated by reduced ATAC-Seq and H3K27ac. b Aggregate plots comparing TKO to QKO, indicating reduced chromatin accessibility, decreased signals of active marks (H3K27ac, H3K4me1/3) and invasion of H3K27me3 at accessible enhancers (n = 22,706). c Aggregate plots centered on strong enhancers (n = 697), showing further reduced activity for active marks and more pronounced invasion of H3K27me3. d Aggregate plots centered on accessible promoters showing no changes in accessibility, DNAme or H3K27ac, whereas H3K4me1/3 decreases and H3K27me3 increases (n = 9599). e Volcano plot of differentially expressed genes, showing more genes become upregulated (289) than downregulated (97) comparing QKO to TKO. f Violin plots of expression-matched genes within and outside of large H3K36me2 peaks (see a for example peak), demonstrating more genes becoming downregulated (150) than upregulated (69). An equal number of genes outside of these large H3K36me2 peaks were randomized and selected as control. Statistical significance was tested using a two-sided Wilcoxon rank-sum test. **** represents p-value = 5.8e-07. In the box plots, boxes span the first quartile and third quartile, median is indicated with a center line and whiskers extend to 1.5 times the interquartile range. For a–d, biological replicates (n = 3) were merged. ATAC-seq tracks were depth-normalized. For all other tracks, except for DNAme (which indicates the percent methylation at CpG sites), normalization factors were computed by multiplying genome-wide modification percentages (averaged per condition) from mass-spectrometry (MS) by the total number of bins and dividing by the total signal for a given coverage track. This normalization factor was multiplied to the depth-normalized signal to generate MS-normalized signals. MS-normalized signals represent the mean local frequency of the relevant modification. Source data are provided as a Source Data file.