Figure 6.

SIRT1 and PCAF regulate the binding of p300 to the CYP19A1 promoter. A–D, human breast ASC cell line was used. A, cells were either untransfected or transfected with 2 μg of control vector or SIRT1 expression vector for 48 h. Cells were then treated with vehicle (control) or 500 nm PGE₂ for 3 h. B, cells were either untransfected or transfected with 2 μg of siRNA to GFP (control siRNA) or PCAF siRNA for 48 h. Cells were then treated with vehicle (control) or 500 nm PGE₂ for 3 h. A and B, cell lysates were subjected to immunoprecipitation (IP) with p300 (left) or HIF-1α (right) antisera or IgG, and immunoprecipitates were subjected to Western blotting and probed as indicated. C and D, ChIP assays were performed. C, cells were either untransfected or transfected with 2 μg of control vector or SIRT1 expression vector for 48 h. D, cells were either untransfected or transfected with 2 μg of siRNA to GFP (control siRNA) or PCAF siRNA for 48 h. C and D, cells were then treated as indicated with vehicle (control) or 500 nm PGE₂ for 3 h. Chromatin fragments were immunoprecipitated with antibodies against p300, and the aromatase promoter was amplified by real-time PCR. DNA sequencing was carried out, and the PCR products were confirmed to be the aromatase promoter. This promoter was not detected when normal IgG was used or when antibody was omitted from the immunoprecipitation step. In C and D, means ± S.D. (error bars) are shown (n = 6). *, p < 0.001.