Molecular mechanism for cooperation between Sp1 and steroidogenic factor-1 (SF-1) to regulate bovine CYP11A gene expression.
Bovine cholesterol side-chain cleavage cytochrome P450 (P450scc; product of the CYP11A gene) gene expression is regulated by gonadotropins via cAMP in the ovary, and by ACTH via cAMP in adrenal cortical cells. Previously, we characterized response elements located at -57/-32 and at -111/-101 bp in the 5'-flanking region of the bovine CYP11A gene required for cAMP-stimulated transcription in both mouse Y-1 adrenal tumor cells and bovine ovarian cells in primary culture, which bind SF-1 (or Ad4-BP) and Sp1, respectively. The role of these transcription factors in CYP11A transcription was further confirmed by deletion and mutation analyses. In addition, results obtained employing a double mutation of the Sp1- and SF-1-binding sites and a mammalian two-hybrid system indicate that Sp1 and SF-1 function cooperatively in the transactivation of the bovine CYP11A promoter in both bovine luteal cells and Y-1 cells. Here we report that SF-1 and Sp1 are able to associate with one another in vitro and in vivo. The NH2-terminal region of SF-1, especially the DNA-binding domain, is the binding site for Sp1. In addition, as CBP is a common coactivator required for the transcriptional activity of numerous transcription factors including nuclear receptors, we investigated whether CBP functions as a cofactor for the regulation of bovine CYP11A promoter activity. We show here that CBP enhanced the PKA-induced CYP11A promoter activity, while a double mutation of both Sp1 and SF-1 sites within the CYP11A promoter region abolished CBP-induced activity. Furthermore, CBP stimulated Sp1-dependent transactivation, and a CBP/Sp1 complex in vivo was demonstrated by a co-immunoprecipitation assay. Also, CBP potentiated the transcriptional activity of GAL4-SF-1 in the presence of PKA. Thus, the cooperation between SF-1 and Sp1, required for the regulation of bovine CYP11A gene expression, is mediated by a direct protein-protein interaction and/or the common coactivator CBP.
Liu
Z
Z
Cecil H. and Ida Green Center for Reproductive Biology Sciences, and the Department of Obstetrics/Gynecology, The University of Texas Southwestern Medical Center, Dallas, USA.
Simpson
E R
ER
D000310:Adrenal Gland Neoplasms / Q000201:enzymology; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000235:genetics*; D017362:Cyclic AMP Response Element-Binding Protein / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D005260:Female; D051717:Fushi Tarazu Transcription Factors; D015870:Gene Expression / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D018398:Homeodomain Proteins; D008184:Luteal Cells / Q000201:enzymology; D051379:Mice; D011401:Promoter Regions, Genetic; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D054339:Steroidogenic Factor 1; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D014162:Transfection; D014407:Tumor Cells, Cultured
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D051717:Fushi Tarazu Transcription Factors; D018398:Homeodomain Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D054339:Steroidogenic Factor 1; D014157:Transcription Factors; C515058:steroidogenic factor 1, mouse; D004247:DNA; D002786:Cholesterol Side-Chain Cleavage Enzyme