ERα bounds to <i>CLOCK</i> promoter regions in response to E2.

Abstract

<p>A, Schematic representation of the ERE sites within the <i>CLOCK</i> promoter regions in the CLOCK-WT-Luc constructs. Constructs containing wild-type promoter and mutant promoters (truncation) are shown. Luciferase activity of MCF-7 cells transfected with the indicated constructs together with or without shERα#1 are shown on the right. B, CLOCK luciferase reporter constructs containing wild-type and mutant CLOCK promoters with point mutation in the EREs are shown, together with the luciferase activity of HeLa cells transfected with one of these constructs together with or without ERα. A and B, all experiments were performed in triplicate and repeated at least three times, and the data shown are the means ± SDs. <i>P</i> value was determined by ANOVA with Bonferroni test (*, <i>P</i><0.05. ns, not significant). C, ChIP assay showing the recruitment of ERα on <i>CLOCK</i> promoter regions. MCF-7 cells were grown in phenol red-free medium and charcoal striped FCS medium for 2 days and the cells were then treated with vehicle or 1 µM E2 concentrations for 1 h, followed by ChIP assay using antibody against ERα or IgG. Total input DNA at a 1∶10 dilution was used as a positive control for the PCR reaction. Immunoprecipitated DNA was analyzed by PCR with primers specific for <i>CLOCK</i>, the relative positions of which are shown in the right panel of Figure 5C. All experiments were repeated at least of three times.</p