ORPHAN RECEPTORS CHICKEN OVALBUMIN UPSTREAM PROMOTER TRANSCRIPTION FACTOR-II (COUP-TFII) AND RETINOID-X-RECEPTOR (RXR) ACTIVATE AND BIND THE RAT CHOLESTEROL 7-ALPHA-HYDROXYLASE GENE (CYP7A)

The cholesterol 7 alpha-hydroxylase gene (CYP7A) is transcriptionally regulated by a number of factors, including hormones, bile acids, and diurnal rhythm. Previous studies have identified a region from nucleot ides (nt) -74 to -55 of the rat CYP7A promoter that enhanced bile acid repression of the SV40 early promoter, as assayed with a luciferase r eporter gene in transiently transfected HepG2 cells. The rat CYP7A pro moter/reporter activity was strongly stimulated by cotransfection with an expression plasmid encoding the nuclear hormone receptor chicken o valbumin upstream promoter transcription factor II (COUP-TFII) in a do se-dependent manner. Site-directed mutagenesis in the region of nt -74 to -55 altered this stimulation. Recombinant COUP-TFII expressed in H epG2 or COS-1 cells were found to bind to nt -74 -55 and nt -149 -128 probes by electrophoretic mobility shift assay (EMSA) and by supershif ting the corresponding band with COUP-TFII-specific antibodies. The re gion of nt -176 -117 was previously mapped as a retinoic acid response region and was found to bind retinoid X receptor (RXR). EMSA supershi ft assays of wild-type and mutant oligomers using antibody against RXR revealed that the sequences between nt -145 and -134 were important f or RXR binding. We conclude that COUP-TFII stimulates the transcriptio nal activity of the rat CYP7A promoter by binding to the sequences bet ween nt -74 to -54 and nt -149 to -128. RXR may stimulate CYP7A gene t ranscription by binding to a direct repeat of the hormone response ele ment separated by one nucleotide located at nt -146 -134.