Mutually exclusive interactions of two nuclear orphan receptors determine activity of a cyclic adenosine 3',5'-monophosphate-responsive sequence in the bovine CYP17 gene.
ACTH-dependent transcriptional activation of the bovine CYP17 gene (the gene encoding cytochrome P450 steroid 17 alpha-hydroxylase) involves two cAMP-responsive sequences (CRS1 and CRS2) located in the promoter region. Here we demonstrate that two nuclear orphan receptors, chicken ovalbumin upstream promoter transcription factor (COUP-TF) and steroidogenic factor-1 (SF-1), bind to the part of the CRS2 element that contains the repeated sequences AAGTCA and AGGTCA spaced by six nucleotides (repCRS2). Overexpression of COUP-TF and SF-1 in both steroidogenic and nonsteroidogenic cells demonstrated that SF-1 is an activator of repCRS2-dependent transcription of reporter genes. Furthermore, the SF-1-dependent transcription could be further stimulated by activation of the cAMP-dependent protein kinase. In contrast, COUP-TF alone had no effect on repCRS2-dependent reporter gene activity. Mutations that interfere with the binding of SF-1 to repCRS2 in vitro abolished the cAMP-induced activities mediated by the element in transfected Y1 cells. The mutational analysis of repCRS2 further indicated that the binding sites for the two receptors overlap, and electrophoretic mobility shift assays demonstrated that the receptors bound in a mutually exclusive manner. Overexpression of both SF-1 and COUP-TFI simultaneously demonstrated that COUP-TFI inhibited SF-1-dependent activation of reporter genes. Transient transfection experiments with a construct containing a -100/+19 base pair fragment from the bovine CYP17 gene demonstrated that SF-1 and COUP-TF had similar effects on the intact promoter as on the repCRS2/reporter gene constructs. Our data suggest that the two orphan receptors bind in a mutually exclusive manner to repCRS2 and that SF-1 is involved in the activation and COUP-TF in the repression of repCRS2-dependent transcription.
Bakke
M
M
Department of Medical Nutrition, Karolinska Institute, Huddinge University Hospital, Sweden.
Lund
J
J
D000818:Animals; D001483:Base Sequence; D051838:COUP Transcription Factor I; D002417:Cattle; D002460:Cell Line; D000242:Cyclic AMP / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000494:pharmacology / Q000502:physiology*; D004789:Enzyme Activation; D051717:Fushi Tarazu Transcription Factors; D018398:Homeodomain Proteins; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011494:Protein Kinases; D018160:Receptors, Cytoplasmic and Nuclear / Q000502:physiology*; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics*; D054339:Steroidogenic Factor 1; D014157:Transcription Factors / Q000494:pharmacology / Q000502:physiology*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D051838:COUP Transcription Factor I; D004268:DNA-Binding Proteins; D051717:Fushi Tarazu Transcription Factors; D018398:Homeodomain Proteins; C497589:NR2F1 protein, human; C515057:NR5A1 protein, human; C497590:Nr2f1 protein, mouse; D018160:Receptors, Cytoplasmic and Nuclear; D011965:Receptors, Glucocorticoid; D054339:Steroidogenic Factor 1; D014157:Transcription Factors; C515058:steroidogenic factor 1, mouse; D004247:DNA; D000242:Cyclic AMP; D013254:Steroid 17-alpha-Hydroxylase; D011494:Protein Kinases