The overall aim of this project is to delineate the mechanism by which bile acids repress transcription of the human cholesterol 7-a hydroxylase (CYP7A1) gene. CYP7A1 catalyzes the initial, rate-limiting step of the classical pathway for bile acid biosynthesis and is a key enzyme in maintaining cholesterol homeostasis. Bioactivity of CYP7A1 is regulated by feedback inhibition at the level of transcription by bile acids. It is known that bile acid-activated FXR activates transcription of SHP which in turn suppresses LRH-1 mediated transactivation of the CYP7A1 gene. Recent studies of SHP knock-out mice suggest that SHP independent pathways of bile acid repression exist as well. Although the master regulators and the bile acid response elements have been identified, the changes in response to bile acids in the regulatory protein complex and in the chromatin structure at the native human CYP7A1 promoter that lead to suppression of the gene in vivo are not known. In preliminary chromatin immunoprecipitation (CHIP) assays, we found that bile acid treatment resulted in decreased histone acetylation, and decreased association of coactivator histone acetyltransferases (HATs) p300 and CBP with the human CYP7A1 promoter. On the basis of these exciting results, we hypothesize that bile acids repress CYP7A1 transcription by modulating the association of HATs and histone deacetylases (HDACs) with the promoter resulting in chromatin structural changes which lead to repression of the CYP7A1 transcription. We propose to test this hypothesis using the ChIP assay to examine the association of proteins with the human CYP7A1 promoter in vivo and transcriptional assays in cell culture and in vivo to determine the functional significance of proteins shown to associate with the promoter. Specifically, we will examine the temporal effects of bile acid treatment at the human CYP7A1 promoter on: 1) endonuclease sensitivity of the nucleosomal structures, 2) histone acetylation and 3) the association of coactivators and corepressors. Experiments will be initiated to compare these changes related to chromatin structure with changes in the association of the nuclear receptors (LRH-1/HNF-4), chromatin remodeling complexes, and the RNA preinitiation complex. The effects of identified coactivators and corepressors on CYP7A1 transcription will be studied in cultured cells and in mouse hepatocytes in vivo using a tail vein injection method. Stably transfected HepG2 cells will be constructed for the study of the interaction of SHP with the promoter and for determination of whether overexpression of SHP in untreated cells can mediate all the effects of bile acid treatment. These studies should define the molecular changes that occur at the CYP7A1 promoter in vivo after bile acid treatment and provide new insight into the mechanism of repression of this gene, and possibly other genes, involved in cholesterol homeostasis.
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