Fig. 2

H2Bub1 controls H3K4me3 spreading into gene body to regulate transcription. a Western blot shows H3K4me3 levels after Rnf40 deletion. Western blot was performed using the same samples shown in Fig. 1d. b Aggregate profiles (top) and heatmaps (bottom) show the occupancy of H2Bub1 and H3K4me3 surrounding the peak center of H3K4me3 occupied regions (±3 kb). All regions were sorted according to H3K4me3 occupancy from high to low. Color key for each heatmap is shown on the right. The dotted line classified H2Bub1 enriched (top) and unenriched (bottom) regions. c Aggregate profiles with different scales compare the average density of H3K4me3 surrounding the TSS of all H3K4me3-enriched genes in Rnf40 ^+/+ (black) and Rnf40 ^–/– MEFs (red). Regions significantly enriched for H3K4me3 on were identified using MACS (cutoff p value < 0.00001). The dotted lines denote the summits of H3K4me3 peaks in Rnf40 ^+/+ (black) or Rnf40 ^–/– (red) MEFs. d Boxplot comparing the width of H3K4me3 peaks in Rnf40 ^+/+ MEF for H3K4me3 occupied genes upregulated (up), unchanged (unch), or downregulated (down) following Rnf40 deletion. Upregulated and downregulated genes were defined as in Fig. 1f. Unchanged genes were selected BaseMean > 15, p value > 0.8, –0.2 < log₂-fold change < 0.2. p values for differences between the three groups were calculated using unpaired Wilcoxon-Mann-Whitney-Test. e Boxplot comparing the log₂-normalized reads of H3K4me3 at promoters (±2 kb) of upregulated, unchanged, and downregulated genes. f, g Boxplots comparing the log₂-fold change in H3K4me3 peak width (f) and height (g) in the three cluster genes following Rnf40 deletion. Log₂ fold change values in H3K4me3 width or the normalized H3K4me3 reads were calculated as log₂ (levels in Rnf40 ^–/–) – log₂ (levels in Rnf40 ^+/+). p values between the three groups were calculated using the unpaired Wilcoxon-Mann-Whitney-Test. h Dotplot analysis displays the width and height of H3K4me3 peaks near TSS in MEFs. Genes with top 5% broadest (red), sharpest (blue), and a similar number of random control (forest green) H3K4me3-enriched regions were selected based on the described method [25]. i–k Boxplots compare log₂ normalized gene expression counts (i), log₂-fold change in H3K4me3 peak width (j), and log₂-fold change in gene expression counts (k) in each of the given three groups identified in (h)