Fig. 4

CDK9 is required for spreading of H3K4me3 into the body of tissue-specific genes. a Aggregate profiles display H3K4me3 occupancy on RNF40-dependent adipocyte-specific genes in undifferentiated (“un diff”) human mesenchymal stem cells (hMSC) and hMSC differentiated to the adipocyte (“ADI diff”) for five days. RNF40-dependent genes in hMSC differentiated to the adipocyte lineage for five days were selected from previously published data [23] based on log₂-fold change in gene expression (siRNF40 vs.control siRNA) < –0.5. b ChIP profile for H3K4me3 on RASD1 and PPARG in undifferentiated and adipocyte-differentiated hMSC. c, d qRT-PCR analysis of RASD1 and PPARG during adipocyte differentiation (five days) with (CDK9i; 5 μM LDC000067 [32]) or without (con) CDK9 inhibitor treatment. Data are shown as mean ± SD (n = 3). *p < 0.05; **p < 0.001; n.s. nonsignificant; unpaired two-tailed t-test. e, f ChIP-qPCR analysis of H2Bub1 and H3K4me3 occupancy at different sites of RASD1 and PPARG genes in “un diff,” “ADI diff,” and “ADI + CDK9i” cells. ADI + CDK9i, adipocytes differentiation for five days with CDK9 inhibitor treatment. Data are represented as mean ± SD (n = 3). *p < 0.05, **p < 0.001, unpaired two-tailed t-test. ChIP-qPCR for IgG is shown as negative control depicted as a dotted line. Examined regions are indicated in (b). g Model depicting how the CDK9-RNF40-H2Bub1 axis enhances the spreading of H3K4me3 into gene bodies to increase the transcription elongation rate of tissue-specific genes