Identification and characterization of a putative bile acid-responsive element in cholesterol 7 alpha-hydroxylase gene promoter.
Nucleotide sequences of a 7997-base pair SacI fragment spanning 3643 base pairs of the upstream promoter region to exon 4 of the rat cholesterol 7 alpha-hydroxylase gene (CYP7) have been determined. DNase I footprinting and electrophoretic mobility shift assay of the proximal promoter from nucleotides -346 to +36 revealed two protected regions which specifically shifted proteins in rat liver nuclear extracts. Footprint A (nucleotides -81 to -35) contained a cluster of overlapping sequence motifs of TGT3, steroid/thyroid hormone response elements (7 alpha TRE), hepatocyte nuclear factors 1 and 4, and CAAT/enhancer-binding protein alpha and has been shown to confer bile acid repression of the CYP7 gene promoter activity. Footprint B (nucleotides -148 to -129) contained a sequence motif HNF4. When footprint A (-101 to -49) or 7 alpha TRE (-73 to -55) sequence was linked upstream to a heterologous SV40 promoter/luciferase plasmid and transiently transfected into HepG2 cells, taurodeoxycholate suppressed the SV40 promoter activity. Electrophoretic mobility shift assays revealed that one or two bands shifted by the 7 alpha TRE or by a direct repeat sequence in 7 alpha TRE were absent when liver nuclear extracts of deoxycholic acid-treated rats were used. Similar gel shift patterns were also observed when human 7 alpha TRE or human liver nuclear extracts were used. The rat direct repeat sequence interacted with two polypeptides (M(r) = 57,000 and 116,000) in both rat and human liver nuclear extracts. These results suggest that hydrophobic bile acids may suppress the CYP7 gene expression by binding to a bile acid receptor which interacts with and prevents the binding of liver nuclear protein(s) to a bile acid-responsive element and that the core of bile acid-responsive element is a direct repeat.
Department of Biochemistry and Molecular Pathology, Northeastern Ohio Universities College of Medicine, Rootstown 44272.
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D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D001483:Base Sequence; D001647:Bile Acids and Salts / Q000378:metabolism*; D002790:Cholesterol 7-alpha-Hydroxylase / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013963:Thyroid Hormones / Q000235:genetics
D000952:Antigens, Polyomavirus Transforming; D001647:Bile Acids and Salts; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D013963:Thyroid Hormones; D008156:Luciferases; D002790:Cholesterol 7-alpha-Hydroxylase; D003850:Deoxyribonuclease I