Figure 5.

CARM1-mediated MED12 methylation is involved in estrogen-induced gene transcriptional activation. (A) Heat map representation of CARM1 and MED12 ChIP-seq tag density centered on estrogen-induced CARM1 sites (± 3,000 bp). (B) Correlation between the ChIP-seq tag density (log2) of CARM1 and MED12 on estrogen-induced CARM1 sites. (C) MCF7 cells were transfected with siCTL, siCARM1 or siMED12, and treated with or without estrogen (E₂, 10^-7 M, 6 hrs) followed by RNA-seq analysis. Estrogen-induced genes which were dependent on both CARM1 and MED12 were shown by Pie chart. (D, E) Heat map (D) and box plot (E) representation of the expression levels (FPKM) for genes induced by estrogen and dependent on both CARM1 and MED12 as described in (C). Heat map: z-score normalized FPKM; box plot: FPKM (log2). (F) MCF7 cells were transfected with siCTL or siMED12 in stripping medium for three days, and treated with or without estrogen (E₂, 10^-7 M, 6 hrs), followed by RNA extraction and RT-qPCR analysis to examine the expression of selected estrogen-induced coding genes as indicated (± s.e.m., **P<0.01, ***P<0.001). (G) MCF7 cells were transfected with siCTL or siCARM1 in stripping medium for three days, and treated with or without estrogen (E₂, 10^-7 M, 1 hr) followed by ChIP-seq with anti-MED12 antibody. MED12 ChIP-seq tag density distribution centered on estrogen-induced CARM1 sites was shown (± 3,000 bp). (H, I) The binding of MED12 as described in (G) was shown for specific genes, as indicated. (J) MCF7 cells were infected with lenti-viral vectors expressing shRNA targeting MED12 together with or without Flag-tagged wild type (WT) MED12 or MED12 mutants with arginine 1899 replaced by alanine (R1899A), and then treated with or without estrogen (E₂, 10^-7 M, 6 hrs) followed by RT-qPCR analysis to examine the expression of selected estrogen-induced genes as indicated (± s.e.m., **P<0.01, ***P<0.001). Data was presented as fold induction by estrogen.