FIG. 6.

The functional domains of EZH2 that are involved in transactivation of the c-Myc and cyclin D1 promoters. (A) Schematic diagrams of EZH2 deletion mutants. (B) Transactivation activity of EZH2 deletion mutants on the c-Myc or cyclin D1 promoter. MCF-7 cells were grown in DMEM supplemented with 10% normal FBS and were cotransfected with c-Myc-Luc or cyclin D1-Luc reporter, a Renilla construct, along with the EZH2 deletion constructs. Eighteen hours after transfection, cells were collected for a luciferase activity assay. Each bar represents the mean ± standard deviation (SD) for triplicate experiments. (C) GST pull-down experiments for interaction between ERα (upper panel) or β-catenin (lower panel) and the EZH2 deletion mutants. (D) Direct interaction between EZH2 and TRAP220/DRIP205 as detected by GST pull-down experiments (upper panel) and by coimmunoprecipitation with primary antibodies against EZH2 and then blotting with antibodies against TRAP220/DRIP205 (lower panel). Inputs represent 10% of fractions. (E) TRAP220/DRIP205 potentiated EZH2-enhanced transactivation of the c-Myc promoter. MCF-7 cells were grown in the absence of estrogen and were cotransfected with c-Myc-Luc reporter along with EZH2 or a TRAP220/DRIP205 expression construct or with a TRAP220/DRIP205 siRNA construct. Eighteen hours after transfection, cells were treated with 100 nM E2 or ethanol (vehicle) for 16 h and harvested for a luciferase activity assay. Each bar represents the mean ± SD for triplicate experiments. Protein expression was confirmed by Western blotting (right panel).