and/or aims): Bile acid synthesis in the liver is a major pathway for the catabolism of cholesterol in mammals and is regulated at the rate-limiting enzyme, cholesterol 7- alpha-hydroxylase. However, the molecular mechanism of regulation of bile acid synthesis has not been elucidated. The enterohepatic circulation of bile is the major physiological control of bile acid biosynthesis, which exerts a negative feedback regulation of the cholesterol 7-alpha-hydroxylase gene (CYP7) transcription. Cholesterol and steroid/thyroid hormones also regulate this gene at the transcriptional level. To achieve the long-range objectives to elucidate the molecular mechanism of this regulatory gene and mechanisms of human diseases involving cholesterol and bile acid metabolisms, the applicant and his collaborators designed experiments with the following specific aims: (1) to map the promoter and cis-regulatory elements of the human CYP7 gene by transient transfection assays of the CYP7/luciferase chimeric gene constructs in HepG2 cells; (2) to identify trans-regulatory protein factors conferring bile acids, cholesterol and hormones regulations, DNase I footprinting, electrophoretic mobility shift assay (EMSA), and protein blotting assays will be used to study DNA-protein interactions; (3) to isolate transacting protein factors conferring bile acid repression, bile acid responsive element identified will be used to screen human liver cDNA expression libraries for cDNAs encoding protein factors and to isolate human liver nuclear DNA-binding proteins by recognition site-affinity chromatography; (4) to confirm the functions of transcription factors regulating the human CYP7 gene/LUC constructs with transcription factor expression plasmids, and EMSA and footprinting assays using over-expressed transcription factors will be performed to study the gene regulation. Elucidation of the structure, function and regulation of this important gene in bile acid synthesis could contribute to the understanding of the mechanisms and regulation of cholesterol homeostasis in humans.
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