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. 2011 Jun 1;25(11):1132–1146. doi: 10.1101/gad.619211

Figure 3.

Figure 3.

CARM1-dependent methylation and interaction with p160/SRC coactivators were required for ligand-induced CBP recruitment to TFF1. (A) H3396 cells were nucleofected with a siRNA against CARM1 or a control siRNA. After 48 h, cells were treated with E2 or vehicle for 1 h and ChIP assays were done with the indicated antibodies. Bound DNA was amplified by real-time PCR with specific primers for TFF1 and GAPDH promoters. The means ± standard deviations are from at least two independent experiments. (B, top) Knockdown of CARM1 monitored by RT–PCR. CARM1 mRNA levels are displayed relative to 36B4 mRNA. (Bottom panel) Expression of CARM1 was assessed by immunoblotting. (C) H3396 cells were nucleofected with a siRNA against p160 or a control siRNA (ctr). After 48 h, cells were treated with estrogen (E2) or vehicle for 1 h and ChIP assays were done with the indicated antibodies. Bound DNA was amplified by real-time PCR as in A. (D) Knockdown of p160s monitored by RT–PCR and immunoblotting. P160 mRNA levels are displayed relative to 36B4 mRNA. (Top right panel) Expression of RAC3 was assessed by immunoblotting, and TIF1β was the loading control. (E) Immunoprecipitations with an anti-RAC3 antibody of H3396 cell extracts transfected with HA-CBP wild type or HA-CBP mutated on R714, R742, R768, or R2104/2151 into lysines were subjected to immunoblotting with antibodies recognizing either RAC3 (top panel) or HA (bottom panel). Quantifications of RAC3 and HA-CBP were done relative to the expression in CBP wild-type condition using ImageJ software. (F) H3396 cells stably infected with retroviral vector expressing HA-CBP wild type or the methylation-deficient CBP mutants HA-CBPR714K, HA-CBPR742K, HA-CBPR768K, and HA-CBPR2104/2151K were treated with E2 or vehicle (EtOH) for 1 h and subjected to ChIP with an anti-HA antibody. Bound DNA was amplified by real-time PCR with specific primers for TFF1 and DPP10 promoters; DPP10 was used as a cold region for ChIP reference. Results are expressed as FO relative to DPP10 relative to ethanol controls. (Bottom panel) Amounts of expressed HA-CBP and methylation-deficient mutants were determined by immunoblotting with anti-HA antibody.