Quantitative Analysis of the Hormone-induced Hyperacetylation of Histone H3 Associated with the Steroidogenic Acute Regulatory Protein Gene Promoter

Abstract

Transcriptional regulation of steroidogenic acute regulatory protein (StAR) determines adrenal and gonadal cell steroidogenesis. Chromatin immunoprecipitation assays were combined with quantitative real-time polymerase chain reaction to assess histone acetylation associated with the StAR promoter. MA-10 cells treated with 8-bromo-cAMP had increased acetylated histone H3 associated with the proximal (but not distal) StAR promoter, nascent StAR transcripts, and progesterone production within 15 min, whereas StAR mRNA increased at 30 min. At 360 min, steroidogenesis remained elevated, but mRNA, nascent RNA, and StAR promoter-associated H3 acetylation all declined. StAR promoter-associated H4 acetylation was unchanged by 8-bromo-cAMP treatment of MA-10 cells. In vivo analysis of macaque and human granulosa cells showed that luteinization was associated with increased StAR promoter-associated H3 acetylation. We conclude that acetylation of H3 (but not H4) associated with the proximal promoter is associated with StAR gene transcription, that chromatin modification occurs in discrete regions of the promoter, that the initial steroidogenic response to 8-bromo-cAMP occurs prior to increased StAR mRNA accumulation, and that MA-10 cell StAR gene transcription and promoter-associated H3 acetylation are biphasic during a 6-h treatment period. The union of the chromatin immunoprecipitation assay with quantitative real-time polymerase chain reaction described and validated here should enhance the analysis of gene expression.