CHARACTERIZATION OF HEPATIC-SPECIFIC REGULATORY ELEMENTS IN THE PROMOTER REGION OF THE HUMAN CHOLESTEROL 7-ALPHA-HYDROXYLASE GENE

Cholesterol 7 alpha-hydroxylase is the rate-limiting enzyme in the deg radation of cholesterol to bile salts and plays a central role in regu lating cholesterol homeostasis. The mechanisms involved in the transcr iptional control of the human gene are largely unknown. HepG2 cells re present an appropriate model system for the study of the regulation of the gene. To identify liver-specific DNA sequences in the promoter of the human CYP7 gene, we first examined the DNase I hypersensitivity i n 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 transc riptionally active HepG2 cells, but was absent. in transcriptionally i nactive 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 activi ty of the human CYP7 gene in HepG2 cells. DNase I footprinting of the hypersensitive region revealed nine protected sequences. Gel retardati on experiments demonstrated binding of HNF-3 to the segment from -80 t o -70 and of hepatocyte nuclear factor HNF-4 (and ARP-1) to the segmen t 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 nega tive 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.