Estrogen Induces Vav1 Expression in Human Breast Cancer Cells
Figure 5
Analysis of transcription factors involved in ERα-activated vav1 promoter.
(A) Depicted deletion mutations in vav1 promoter reporter gene. The deleted regions were indicated by break lines. (B) MCF7 cells were transfected with plasmids containing luciferase under WT or mutated vav1 promoters (D1, D2, and D3) and then treated with E2 (10−7 mol/L) or DMSO for 48 h. The relative fold induction of each group was calculated as the ratio of the luciferase activity induced by E2 to that induced by DMSO, respectively, and plotted as y-axis, and the deletion mutants were presented as in x-axis. All the data represented the mean±S.D. of three independent experiments. “N.S.” and “**” indicates P>0.05 and P<0.01, respectively, versus D1 group by unpaired student T test. (C) ChIP assay. T47D cells were treated with E2 (10−7 mol/L) for 4 h or pre-treated with Tamoxifen (10−6 mol/L) for 30 min before treating with E2 (10−7 mol/L) for 4 h. The immunoprecipitation was performed using antibodies against c-Myb (left panels) or ELF-1 (right panels), with preimmune IgG as control. The precipitated DNAs were analyzed by PCR with the primers corresponding to position −232 to +71 of vav1 promoter. The bar chart below the example blot represents the normalized DNA level of −232 to +71 to Input of three independent experiments. “N.S.” indicates P>0.05 versus DMSO treatment by unpaired student T test. (D) The association of ERα with c-Myb and ELF-1. T47D cells were treated with E2 (10−7 mol/L) or DMSO for 4 h or pretreated with Tamoxifen (10−6 mol/L) for 30 min in prior to E2 treatment and lysed. Antibodies against c-Myb (upper panels) and ELF-1 (lower panels) or control IgG (Lane 1) were used to immunoprecipitate protein complex, which were then resolved by Western Blot with indicated antibodies. (E) The proposed model for ERα modulating the vav1 promoter activity. E2-activated ERα interacts with vav1 promoter via interacting with transcription factors such as c-Myb, ELF-1, or perhaps other unknown coregulators (labeled “?”) to promote the vav1 transcription.