FIG. 8.
cAMP increases the acetylation and DNA-binding activity of SF-1 in vivo. (A) Mutation of KQQKK sequence abolishes cAMP-induced acetylation of SF-1. The expression plasmids for wild-type (WT) 3xFLAG-SF-1 (3×F-SF-1) or mutated mt1 were transfected into Y1 cells. After 24 h, cells were labeled with [3H]acetate in the presence (+) or absence (−) of 1 mM 8-Br-cAMP for 6 h. The results for acetyl-3xFLAG-SF-1 (top) following immunoprecipitation (IP) with anti-FLAG antibody are shown; the amounts of immunoprecipitated 3xFLAG-SF-1 were determined by SF-1 immunoblotting (IB) (bottom). (B) cAMP increases the DNA-binding activity of WT but not acetylation-deficient SF-1. Stable Y1 cells expressing SF-1-HA (clone no. 18) and SF-1-HA mt1 (clone no. 11) were treated with (+) or without (−) 1 mM 8-Br-cAMP for 6 h. ChIP assays were performed with antibodies against HA or acetyl-H3 (AcH3). The results of PCR amplification using primers that cover the region of nucleotide positions −71 to +209 of the Cyp11a1 gene are shown. Numbers below each lane are quantitations of the band intensities.