Fig. 6

Decreased EZH2 expression is responsible for the upregulation of gene expression following Rnf40 deletion. a Aggregate plot analysis of average H3K27me3 profiles surrounding TSS (±5 kb) in wild-type and Rnf40 ^–/– MEFs. b GSEA shows a significant enrichment of genes upregulated in Rnf40 ^–/– MEF in genes displaying decreased H3K27me3 surrounding the TSS. c Western blot analysis of whole protein extracts from DMSO as control (Rnf40 ^+/+; Rnf40 ^–/–) or EPZ6438-treated (Rnf40 ^+/+) MEFs (EZH2i) using antibodies against H3K4me3, H3K27me3, H3K27ac, H2Bub1, HSC70 (loading control), and H3 (loading control). Cells were treated with 1 μM EPZ6438 or DMSO as indicated for three days. d The profiles show the normalized occupancy of H2Bub1, H3K27me3, H3K4me3, H3K27ac, EZH2, and mRNA levels for the Nat8l, Foxl2, and Foxl2os genes in Rnf40 ^+/+ and Rnf40 ^–/– MEFs. e qRT-PCR analysis of individual upregulated genes in Rnf40 ^+/+, Rnf40 ^–/–, and EZH2i-treated Rnf40 ^+/+ MEF cells. Data for qRT-PCR shows mean ± SD (n = 3); *p < 0.05, **p < 0.001, unpaired two-tailed t-test. f, g qRT-PCR analysis of Nat8l and Foxl2 in control (mock), EZH2-H689A, and EZH2 (WT) expressing MEF with or without 4-OHT treatment. Mock control MEFs, H689A MEFs ectopically expressing a SET domain mutated EZH2, WT MEFs ectopically expressing wild-type EZH2. h, i ChIP-qPCR analysis of H3K27me3 occupancy near the TSS of the Nat8l and Foxl2 genes in mock, EZH2-H689A, and WT EZH2 MEF with or without 4-OHT treatment. The investigated regions are indicated in (d). j Model describing the role of decreased Ezh2 expression in controlling “RNF40-suppressed” genes