FIGURE 4.

Binding of FoxO1 to the sirt1 promoter. A and B, FoxO1 binds directly to the IRS-1bcd and FKHD-L elements in the sirt1 promoter in vitro. EMSA was conducted using in vitro translated FoxO1 proteins as described under “Experimental Procedures.” The partial sequences of the biotin-labeled IRS-1bcd (A) and FKHD-L (B) probes (wild type, underlined) and mutated probes (framed) are shown above the gel shift. The running orientation of the gels and the mobility-shifted bands are indicated by arrows. C and D, FoxO1 associates with the rat sirt1 promoter in VSMCs by ChIP assays. Soluble chromatin from VSMCs was coimmunoprecipitated with anti-FoxO1 or FoxO3a antibodies or an equal amount of rabbit IgG. DNA purified from starting (1% input) and immunoprecipitated samples was subjected to PCR. The amplified region surrounds the IRS-1-binding element (C) (−1035 to −686) or the FKHD-L-binding element (D) (−415 to −89) within the rat sirt1 promoter. One representative result from three independent experiments is shown. E, FoxO1 associates with the human SIRT1 promoter in HEK293 cells by ChIP assays. Soluble chromatin from HEK293 cells was coimmunoprecipitated with anti-FoxO1 or FoxO3a antibodies or an equal amount of rabbit IgG. DNA purified from starting (1% input) and immunoprecipitated samples was subjected to PCR. The amplified region surrounds two putative FoxO1 DNA-binding elements (−587 to −278) within the human SIRT1 promoter. One representative result from three independent experiments is shown. rSIRT1, rat sirt1; hSIRT1, human SIRT1.