Figure 4. Twist1 inhibits AP1-mediated activation of the Foxa1 promoter by inhibiting AP1 recruitment.
A. The two conserved AP1 sites in both human and mouse Foxa1 promoter regions. B. Luciferase activity of MCF7 cells transfected with Foxa1-Luc plasmid with the empty vector (−) or c-Jun and c-Fos expression vectors. C. Q-PCR and Western blot assays showing Foxa1 mRNA and protein changes upon c-fos knockdown in MCF7 cells. D. Luciferase assay of MCF7 cells co-transfected with the indicated luciferase reporter and the empty vector (−) or c-Jun and c-Fos expression vectors. E. ChIP assays performed with MCF7Ctrl and MCF7Twist1 cells and non-immune IgG (negative control) or c-Fos antibody as indicated. The DNA fragment between the two arrowheads in Panel A was amplified by PCR from the immunoprecipitated DNA. Amplification of the known AP1-binding region by PCR in the CSF1 promoter served as a positive control. F. Luciferase assay of MCF7 cells co-transfected with the indicated luciferase reporter and the empty vector (Ctrl) or Twist1 expression plasmid. Experiments were repeated 3 times and the representative data with 3 technical replicates are presented as Mean ± SEM. * in Panels B–F, P<0.05 by Student's t test.