Department of Biochemical Regulation, Merck Sharp & Dohme Research Laboratories, Rahway, New Jersey 07065, and Department of Molecular Cell Biology, Rockefeller University, New York, New York 10021 Received August 14, 1991; Revised Manuscript Received December 10, 1991 abstract: As an initial step toward understanding the transcriptional regulation of cholesterol 7a-hydroxylase (CYP7) in man, we isolated and functionally characterized the 5'-flanldng region of the human CYP7 gene. The nucleotide sequences of the first exon and 1.6 kb preceding the exon were determined and found to contain a TATA box at position-30, a modified CAAT box at position-92, three potential hepatocyte nuclear factor 3 (HNF-3) recognition sites at nucleotides-316,-288, and-255, respectively, and a modified sterol response element atposition-271. DNA sequences containing 1.3 kb of the S'-flanking regionand 29 nucleotides of the first exon were linked to the chloramphenicol acetyltransferase gene and transiently transfected into several cell lines. Promoter activity was very strong in the human hepatoma cell line HepG2 but absent in cells of nonhepatic origin. Mutational analysis of the promoteridentified several regions that function in the transcriptional regulation of CYP7. Introduction of a fragment containing the region from-432 to-220 upstream of a heterologous promoter, in either orientation, resulted in a tremendous stimulation of activity in HepG2 cells. DNase I footprint analysis identified threeregions within this fragment which were protected from digestion. The overexpression of HNF-3 in HepG2 cells resultedin a 4-fold stimulation of CYP7 transcriptional activity. We suggest that the regionbetween-432 and-220 functions as a cell-specific enhancer whose activity is controlled, in part, by HNF-3. e state of cholesterol equilibrium in thebody is the result of a balance between cholesterol uptake, synthesis, and catabolism. All three of these pathways are tightly controlled and contribute to the homeostatic regulation of serum cholesterol (Turley & Dietschy, 1982). The molecular mechanisms re-sponsible for controlling the rate of receptor-mediated uptake of cholesterol and the de novo synthesis of cholesterol have been studied in great detail in recent years (Sudhof et al., 1987; Osborne et al., 1988; Smith et al., 1988). These studies have defined an octanucleotide sequence, termed the sterol response element (SRE), in the promoter of critical genes in these pathways, which is essential for the end-product suppression of gene transcription (Goldstein & Brown, 1990). In the presence of sterols, this DNA sequence represses thepositive transcriptional activity of a nearby positive DNA element. In this way, the body decreases cholesterol uptake and synthesis when cellular cholesterol is in excess. Unfortunately, we currently have a poor understanding of the regulatory mechanisms that are responsible for controlling the rate of cholesterol catabolism.

The cholesterol catabolic pathway, which is exclusive to the liver, is comprised of 10 or more enzymatic reactions which convert cholesterol intohydrophilic bile acids (Danielsson, 1973). The newly synthesized bile acids are then conjugated with either taurine or glycine and secreted from the liver and stored in thegall bladder as a component of bile. Upon in-