Cholesterol 7␣-hydroxylase is the rate-limiting enzyme in the degradation of cholesterol to bile salts and plays a central role in regulating cholesterol homeostasis. The mechanisms involved in the transcriptional control of the human gene are largely unknown. HepG2 cells represent an appropriate model system for the study of the regulation of the gene. To identify liverspecific DNA sequences in the promoter of the human CYP7 gene, we first examined the DNase I hypersensitivity in the 5 -region of the gene. An area of hypersensitivity was observed in the region from ؊50 to ؊200 of the human gene in nuclei from transcriptionally active HepG2 cells, but was absent in transcriptionally inactive HeLa cell nuclei or in free DNA. Various 5 -promoter deletion constructs were made and transfected into HepG2 cells. About 300 base pairs of upstream sequence are required for high level promoter activity of the human CYP7 gene in HepG2 cells. DNase I footprinting of the hypersensitive region revealed nine protected sequences. Gel retardation experiments demonstrated binding of HNF-3 to the segment from ؊80 to ؊70 and of hepatocyte nuclear factor HNF-4 (and ARP-1) to the segment from ؊148 to ؊127 of the human CYP7 promoter. Deletion of either of these sites depressed promoter activity in HepG2 cells. A third region from ؊313 to ؊285 is bound by members of the HNF-3 family and acts as an enhancer. Additionally, the segment from ؊197 to ؊173 binds a negative regulatory protein that is present in Chinese hamster ovary cell extracts and in HepG2 cell extracts. These experiments define the key control elements responsible for basal transcription of the human CYP7 gene in HepG2 cells.Cholesterol 7␣-hydroxylase catalyzes the rate-limiting step in the pathway that leads to the catabolism of cholesterol to bile acids (for review, see Ref. 1). Cholesterol 7␣-hydroxylase is a microsomal enzyme member of the cytochrome P-450 family. In human and rat, the major products of this metabolic pathway are cholic acid and chenodeoxycholic acid. Bile acids have an important role in cholesterol homeostasis; their synthesis and excretion cause a decrease in hepatic cholesterol levels, while their presence in the intestine facilitates the solubilization of dietary fats and is required for the absorption of cholesterol and fat-soluble vitamins. Because of the importance of these functions, bile acid synthesis in the liver is carefully regulated to maintain cholesterol homeostasis (1). To date, little is known about the molecular mechanisms that control cholesterol catabolism and bile acid synthesis.The cDNAs and genes for cholesterol 7␣-hydroxylase have been isolated from rat (2-4), human (5, 6), hamster (7), and mouse (8). CYP7 mRNA is found exclusively in the liver (9), making this gene a target for the study of the molecular mechanisms implicated in hepatic-specific gene expression. Work by several groups has demonstrated that CYP7 mRNA levels are modulated in cultured cells by a number of effectors. For example, in cultured rat hepatocytes (10) ...