title: NF-1C, Sp1, and Sp3 are essential for transcription of the human gene for P450c17 (steroid 17alpha-hydroxylase/17,20 lyase) in human adrenal NCI-H295A cells.
abstract: Cytochrome P450c17 catalyzes steroid 17alpha-hydroxylase and 17,20 lyase activities, which are required for the biosynthesis of cortisol and sex steroids. Human P450c17 is expressed in a cAMP-responsive, cell-specific, developmentally programmed fashion, but little is known about its transcriptional regulation. Expression of deletion mutants of up to 2,500 bp of human 5'-flanking DNA in human adrenal NCI-H295A cells indicated that most regulatory activity was confined to the first 227 bp. Deoxyribonuclease I footprinting of the proximal promoter identified the TATA box, an steroidogenic factor-1 site, and three previously uncharacterized sites at -107/85, at -178/-152, and at -220/-185. EMSAs and methylation interference assays suggested that the -107/-85 site and the -178/-152 site bind members of the NF-1 (nuclear factor-1) family of transcription factors. An NF-1 consensus sequence generated similar DNA/protein complexes, and antibodies against NF-1C2/CTF2 supershifted the complexes formed by the -107/-85 site, the -178/-152 site, and the NF-1 consensus site. Western blots of nuclear extracts from NCI-H295A cells probed with this NF-1 antiserum identified two NF-1 isoforms between 50 and 55 kDa. The presence of NF-1C2 (CTF2) and CTF5 in NCI-H295A cells was demonstrated by RT-PCR and sequencing. Mutation of both the -107/-85 and the -178/-152 NF-1 sites reduced basal transcription by half. Supershift assays showed that the ubiquitous proteins Sp1 and Sp3 both bind to the -227/-184 region, and that mutation of their binding sites reduced transcription by 75%. Mutation of the Sp1/Sp3 site plus the two NF-1 sites eliminated almost all detectable transcription. Thus, Sp1 and Sp3 binding to the -227/-184 site and NF-1C proteins binding to the -107/-85 and the -178/-152 sites are crucial for adrenal transcription of the human gene for P450c17.
authors.affiliation: Department of Pediatrics and The Metabolic Research Unit, University of California San Francisco, San Francisco, California 94143-0978, USA.
authors.forename: C J
authors.initials: CJ
authors.lastname: Lin
authors.forename: J W
authors.initials: JW
authors.lastname: Martens
authors.forename: W L
authors.initials: WL
authors.lastname: Miller
mesh_terms: D000310:Adrenal Gland Neoplasms; D000311:Adrenal Glands / Q000201:enzymology*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D002460:Cell Line; D017124:Conserved Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I; D006367:HeLa Cells; D006801:Humans; D008113:Liver Neoplasms; D051379:Mice; D016297:Mutagenesis, Site-Directed; D051839:NFI Transcription Factors; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D051705:Sp3 Transcription Factor; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics*; D054339:Steroidogenic Factor 1; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
chemical_list: D004268:DNA-Binding Proteins; D051839:NFI Transcription Factors; C515057:NR5A1 protein, human; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; C497122:Sp3 protein, mouse; D054339:Steroidogenic Factor 1; D014157:Transcription Factors; C515058:steroidogenic factor 1, mouse; C080333:transcription factor nuclear factor 1; D051705:Sp3 Transcription Factor; D004247:DNA; D013254:Steroid 17-alpha-Hydroxylase; D003850:Deoxyribonuclease I