Orphan receptors chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) and retinoid X receptor (RXR) activate and bind the rat cholesterol 7alpha-hydroxylase gene (CYP7A).
The cholesterol 7alpha-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 nucleotides (nt) -74 to -55 of the rat CYP7A promoter that enhanced bile acid repression of the SV40 early promoter, as assayed with a luciferase reporter gene in transiently transfected HepG2 cells. The rat CYP7A promoter/reporter activity was strongly stimulated by cotransfection with an expression plasmid encoding the nuclear hormone receptor chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) in a dose-dependent manner. Site-directed mutagenesis in the region of nt -74 to -55 altered this stimulation. Recombinant COUP-TFII expressed in HepG2 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 supershifting the corresponding band with COUP-TFII-specific antibodies. The region of nt -176 -117 was previously mapped as a retinoic acid response region and was found to bind retinoid X receptor (RXR). EMSA supershift assays of wild-type and mutant oligomers using antibody against RXR revealed that the sequences between nt -145 and -134 were important for RXR binding. We conclude that COUP-TFII stimulates the transcriptional activity of the rat CYP7A promoter by binding to the sequences between nt -74 to -54 and nt -149 to -128. RXR may stimulate CYP7A gene transcription by binding to a direct repeat of the hormone response element separated by one nucleotide located at nt -146 -134.
Stroup
D
D
Department of Biochemistry and Molecular Pathology, Northeastern Ohio Universities College of Medicine, Rootstown, Ohio 44272-0095, USA.
Crestani
M
M
Chiang
J Y
JY
D000818:Animals; D051838:COUP Transcription Factor I; D051837:COUP Transcription Factor II; D051836:COUP Transcription Factors; D002645:Chickens; D002790:Cholesterol 7-alpha-Hydroxylase / Q000235:genetics* / Q000378:metabolism; D002940:Circadian Rhythm; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D008958:Models, Molecular; D009687:Nuclear Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000235:genetics*; D018168:Receptors, Retinoic Acid / Q000378:metabolism*; D011987:Receptors, Steroid; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D047488:Retinoid X Receptors; D014157:Transcription Factors / Q000378:metabolism*
D051838:COUP Transcription Factor I; D051837:COUP Transcription Factor II; D051836:COUP Transcription Factors; D004268:DNA-Binding Proteins; C497591:Nr2f1 protein, rat; C497588:Nr2f2 protein, rat; D009687:Nuclear Proteins; D011965:Receptors, Glucocorticoid; D018168:Receptors, Retinoic Acid; D011987:Receptors, Steroid; D011994:Recombinant Proteins; D047488:Retinoid X Receptors; D014157:Transcription Factors; D002790:Cholesterol 7-alpha-Hydroxylase