Figure 2.

HIC1 represses E2F-mediated transcription of endogenous genes. (a) HSF8 cells were transfected with empty vector (control) or the indicated expression vector plasmids. The cells were collected 72 h after transfection and the transcriptional activity of endogenous Sirt1, E2F1 and cFos (negative control) genes was analysed by quantitative RT–PCR, using standard protocols of the supplier. The experiments were repeated four times, yielding comparable results, and error bars shown represent the s.e.m. from all four assays. β-Actin expression was examined in each experiment as an internal control and used for normalization. The relative expression in the control non-transfected cells was assigned a value of 10. (b) The occupancy of HIC1 at the E2F1 and cFos promoters in unsynchronized actively growing HSF8 cells was analysed by ChIP assay with quantitative PCR, using an anti-HIC antibody (or anti-tubulin antibody as a specificity control). Relative quantitation of input DNA (not immunoprecipitated) is shown as ‘total.’ All the experiments were repeated four times and the averages are shown, with error bars representing s.e.m. ChIP, chromatin immunoprecipitation; RT–PCR, reverse transcriptase PCR.