Fig. 3

H2Bub1 and broad H3K4me3 are required for transcriptional elongation. a–c Comparison of the normalized occupancy of RNA Polymerase II (Pol II) in wild-type and Wdr82-null bone marrow-derived macrophage (BMDM) cells specifically on genes with broad, random control, and sharp H3K4me3 domains. TSS transcription start site, TES transcription end site. p value was calculated by unpaired Wilcoxon-Mann-Whitney-Test. d, e Aggregate profiles compare the normalized occupancy of Pol II (d) and H2Bub1 (e) in wild-type (Rnf40 ^+/+) MEFs from TSS to TES on genes upregulated (up), unchanged (unch), and downregulated (down) following Rnf40 deletion. f The profiles show the occupancy of H2Bub1, H3K4me3, H3K27me3, H3K27ac, and Pol II as well as normalized RNA reads on the Myl9 and Psrc1 genes in Rnf40 ^+/+ (black) and Rnf40 ^–/– (red) MEFs. g, h ChIP-qPCR for Pol II occupancy at different regions of the Myl9 and Psrc1 genes at the regions labeled in 3 F. S1 site1, proximal to TSS, S2 site2, downstream of TSS, S3 site3, gene body. The data are shown as mean ± SD (n = 3). *p < 0.05; n.s. non-significant; unpaired two-tailed t-test. ChIP-qPCR for IgG was set as negative control and is indicated by the dotted line. i–k qRT-PCR analysis of Rnf40, Myl9, and Psrc1 in mock, HA-RNF40-ΔRING, and HA-RNF40 transfected MEFs with or without 4-OHT treatment. Mock MEFs without vector transfection, HA-RNF40-ΔRING MEFs with RING finger-deleted Rnf40 transfection, HA-RNF40 MEFs with wild-type Rnf40 vector transfection. The endogenous Rnf40 gene was deleted by adding 250 nM of 4-OHT for five days. The data are shown as mean ± SD (n = 3). *p < 0.05; **p < 0.001; n.s. non-significant; unpaired two-tailed t-test