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 b ase pairs of the upstream promoter region to exon 4 of the rat cholest erol 7 alpha-hydroxylase gene (CYP7) have been determined. DNase I foo tprinting and electrophoretic mobility shift assay of the proximal pro moter from nucleotides -346 to +36 revealed two protected regions whic h specifically shifted proteins in rat liver nuclear extracts. Footpri nt A (nucleotides -81 to -35) contained a cluster of overlapping seque nce motifs of TGT3, steroid/thyroid hormone response elements (7 alpha TRE), hepatocyte nuclear factors 1 and 4, and CAAT/enhancer-binding p rotein alpha and has been shown to confer bile acid repression of the CYP7 gene promoter activity. Footprint B (nucleotides -148 to -129) co ntained a sequence motif HNF4. When footprint A (-101 to -49) or 7 alp ha TRE (-73 to -55) sequence was linked upstream to a heterologous SV4 0 promoter/ luciferase plasmid and transiently transfected into HepG2 cells, taurodeoxycholate suppressed the SV40 promoter activity. Electr ophoretic 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 w ere used. Similar gel shift patterns were also observed when human 7 a lpha TRE or human liver nuclear extracts were used. The rat direct rep eat sequence interacted with two polypeptides (M(r) = 57,000 and 116,0 00) in both rat and human liver nuclear extracts. These results sugges t 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.