Fig. 5

The CDK9-RNF40-H2Bub1 axis controls Ezh2 expression. a GSEA of mRNA expression data reveal a significant enrichment of EZH2-suppressed genes (in TIG3 cells) in genes upregulated in Rnf40 ^–/– MEFs. NES normalized enrichment score, FDR false discovery rate. b qRT-PCR analysis of PRC2 subunits Ezh1, Ezh2, Eed, and Suz12 in Rnf40 ^+/+ and Rnf40 ^–/– MEF cells. Data are shown as “relative mRNA levels,” mean ± SD (n = 3). *p < 0.05, **p < 0.001, n.s: p > 0.05, calculated with two-tailed unpaired t-test. c Western blot analysis of protein extracts from Rnf40 ^+/+ and Rnf40 ^–/– MEFs using antibodies directed against RNF40, EZH2, SUZ12, and EZH1. HSC70 is shown as a loading control. d Profiles show the occupancy of H2Bub1, H3K27ac, H3K4me3, and expression counts (RNA) on the Ezh2 gene in wild-type and Rnf40–null MEF. e ChIP-qPCR analyses of RNA Polymerase II (Pol II) occupancy near the TSS (site1) and in the gene body (site2) of the Ezh2 gene. The PCR regions were labeled at (f). f qRT-PCR analysis of Ezh2 mRNA levels in Rnf40 ^+/+, Rnf40 ^–/–, and CDK9 inhibitor-treated MEF. (g, h) ChIP-qPCR analysis of H2Bub1 and H3K4me3 occupancy near the TSS (site1) and in the gene body (site2) of the Ezh2 gene in Rnf40 ^+/+, Rnf40 ^–/–, and CDK9 inhibitor-treated MEF. i qRT-PCR analysis of Ezh2 mRNA levels with or without 4-OHT treatment in mock, HA-RNF40-ΔRING-, and HA-RNF40-expressing MEF