Regulation of ChIP-seq target genes by MED12 methylation. Regulation of ChIP-seq target genes by MED12 methylation. (A) ChIP-seq peaks demonstrating the enrichment of MED12, CARM1, and H3R17me2a signals at the GREB1 gene locus (left panel). ChIP and quantitative PCR analysis of the association of MED12, CARM1, and H3R17me2a with GREB1 gene (right panel). (B) MCF-7-Tet-on-shCARM1 cells were untreated or treated with doxycycline (1 μg/ml) for 8 d. Total RNA was extracted and RT-PCR was performed using primers specific for the genes shown. GAPDH acts as a negative control. Target gene expression was normalized to β-actin. Error bars represent SD based on three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 (t test). (C) A rescue experiment was performed whereby WT, single (R1899K), or triple (R1862K/R1899K/R1912K) mutant FLAG-MED12 constructs were reintroduced into MED12 KO MCF-7 cells generated by CRISPR-Cas9 gene editing. These constructs (denoted by WTr, SMr, and TMr) contain five synonymous mutations in the guide sequence to prevent Cas9 cleavage. MED12 KO cells were cultured in phenol red–free DMEM supplemented with 10% charcoal dextran-stripped FBS for 3 d before transfection, and then treated with E2 (50 nM) for 24 h. Total RNA was extracted and RT–qPCR was performed using primers specific for the genes shown. Target gene expression was normalized to β-actin. Inset figures show similar expression levels of the WT and mutant proteins. Error bars represent SD based on three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 (t test). Donghang Cheng et al. LSA 2018;1:e201800117 © 2018 Cheng et al.