title: Regulation of cytochrome b5 gene transcription by Sp3, GATA-6, and steroidogenic factor 1 in human adrenal NCI-H295A cells.
abstract: Sex steroid synthesis requires the 17,20 lyase activity of P450c17, which is enhanced by cytochrome b5, acting as an allosteric factor to promote association of P450c17 with its electron donor, P450 oxidoreductase. Cytochrome b5 is preferentially expressed in the fetal adrenal and postadrenarchal adrenal zona reticularis; the basis of this tissue-specific, developmentally regulated transcription of the b5 gene is unknown. We found b5 expression in all cell lines tested, including human adrenal NCI-H295A cells, where its mRNA is reduced by cAMP and phorbol ester. Multiple sites, between -83 and -122 bp upstream from the first ATG, initiate transcription. Deletional mutagenesis localized all detectable promoter activity within -327/+15, and deoxyribonuclease I footprinting identified protein binding at -72/-107 and -157/-197. DNA segments -65/-40, -114/-70 and -270/-245 fused to TK32/Luc yielded significant activity, and mutations in their Sp sites abolished that activity; electrophoretic mobility shift assay (EMSA) showed that Sp3, but not Sp1, binds to these Sp sites. Nuclear factor 1 (NF-1) and GATA-6, but not GATA-4 bind to the NF-1 and GATA sites in -157/-197. In Drosophila S2 cells, Sp3 increased -327/Luc activity 58-fold, but Sp1 and NF-1 isoforms were inactive. Mutating the three Sp sites ablated activity without or with cotransfection of Sp1/Sp3. In NCI-H295A cells, mutating the three Sp sites reduced activity to 39%; mutating the Sp, GATA, and NF-1 sites abolished activity. In JEG-3 cells, GATA-4 was inactive, GATA-6 augmented -327/Luc activity to 231% over the control, and steroidogenic factor 1 augmented activity to 655% over the control; these activities required the Sp and NF-1 sites. Transcription of cytochrome b5 shares many features with the regulation of P450c17, whose activity it enhances.
authors.affiliation: Department of Pediatrics, University of California, San Francisco, San Francisco, California 94143-0978, USA.
authors.forename: Ningwu
authors.initials: N
authors.lastname: Huang
authors.forename: Andrea
authors.initials: A
authors.lastname: Dardis
authors.forename: Walter L
authors.initials: WL
authors.lastname: Miller
mesh_terms: D000311:Adrenal Glands / Q000166:cytology*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D002460:Cell Line; D045744:Cell Line, Tumor; D015786:Cytochromes b5 / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004330:Drosophila; D050993:GATA6 Transcription Factor / Q000378:metabolism*; D017353:Gene Deletion; D017930:Genes, Reporter; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D009841:Oligonucleotides / Q000737:chemistry; D010703:Phorbol Esters / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020033:Protein Isoforms; D012333:RNA, Messenger / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012260:Ribonucleases / Q000378:metabolism; D054339:Steroidogenic Factor 1; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D050993:GATA6 Transcription Factor; C494759:GATA6 protein, human; D018398:Homeodomain Proteins; C515057:NR5A1 protein, human; D009841:Oligonucleotides; D010703:Phorbol Esters; D020033:Protein Isoforms; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D054339:Steroidogenic Factor 1; D014157:Transcription Factors; D004247:DNA; D015786:Cytochromes b5; D008156:Luciferases; D009251:NADPH-Ferrihemoprotein Reductase; D012260:Ribonucleases; D003850:Deoxyribonuclease I