FIG. 6.

Interaction of AhR with Ad4BP/SF-1 on the Cyp19 promoter. (A) Detection of a physical interaction between AhR and Ad4BP/SF-1 by coimmunoprecipitation. FLAG-tagged proteins from whole-cell extracts of 293 cells transfected with 3×FLAG-AhR, Arnt, and EGFP-Ad4BP were immunoprecipitated (IP) with an anti-FLAG antibody. The immunoprecipitates were then subjected to immunoblotting with an anti-GFP antibody. An EGFP expression vector was transfected as a control. An arrow and an arrowhead indicate the EGFP-Ad4BP and EGFP samples, respectively. (B) Schematic representation of the location of primers used in the ChIP assays. Three sets of primers were used to amplify DNA regions containing the XRE site at −5058 and the Ad4/SF-1 sequence at −92 and a third unrelated region (−2740 to −2441), containing neither of them, as a control. (C) Binding of AhR to the promoter region of the Cyp19 gene, revealed by ChIP assays. Soluble chromatin, prepared from preovulatory granulosa cells (hCG + 2 h), was subjected to ChIP assay with an anti-AhR antibody. β-Actin was used as a negative control. (D) Interaction between AhR and Ad4BP/SF-1 on the Cyp19 gene promoter. Chromatin isolated from preovulatory granulosa cells was incubated with anti-AhR or anti-Ad4BP/SF-1 antibody and then subjected to PCR with two sets of primers amplifying the XRE and Ad4 sites. A primer pair specific for the sequence from −2740 to about −2441 was used as a control. (E) Binding of Ad4BP/SF-1 to the XRE and Ad4 sites in the presence or absence of AhR, revealed by ChIP assays. Chromatin isolated from preovulatory granulosa cells of the AhR^+/+ and AhR^−/− ovaries was incubated with anti-Ad4BP/SF-1 or control antibody and then subjected to PCR to amplify the XRE and Ad4 sites. IgG, immunoglobulin G.