Figure 2.

E2F1 directly binds and activates EPC1 promoter. (A) Schematic representation of EPC1 gene promoter region (upper). Putative E2F1-binding sites were predicted using JASPAR database. Arrows indicate position of PCR amplicons in chromatin immunoprecipitation (ChIP) experiments relative to ATG (P1, −1292/−1148; P2, −825/−507; P3, −230/+42). ChIP assay shows the binding of E2F1 on EPC1 promoter in SK-Mel-29.ER-E2F1 treated with or without 4-OHT (1 μM) for 24 h (lower). Input represents 10% of sheared chromatin prior to immunoprecipitation. DNA was analyzed by semi-quantitative PCR with primers corresponding to the amplicons shown in upper panel. Primers amplifying the E2F1 sites in the EZH2 promoter were used as a positive control. (B) Schematic representation of EPC1 full-length promoter region (upper). Luciferase reporter assays in Saos-2, SK-Mel-29 and MCF7 cells upon E2F1 activation (lower). Relative light units (RLU) were measured after co-transfection of EPC1 promoter construct and E2F1. Bars indicate mean values ± SD of three independent experiments. (C) Dose-dependent regulation of EPC1 reporter by E2F1. SK-Mel-29 cells were co-transfected with EPC1 promoter construct and increasing amounts of E2F1 expression plasmid (0.25, 0.5, 0.75 and 1 μg). RLU were measured 24 h post-transfection. p73-prom-luc was used as positive control. (D) Luciferase reporter activities after co-transfection of SK-Mel-29 cells with EPC1 promoter construct and different E2F family members or DNA binding-deficient mutant E132 (1 μg). pcDNA was transfected as control in all reporter assays. E2F1, E2F2, E2F3 and E132 expression was verified by Western blot. Actin serves as loading control. Error bars indicate one SD of the mean. All experiments were performed three times independently. Statistical significance was determined by two-sided Student's t-test. **P ≤ 0.01, ***P ≤ 0.001.