title: Functional analysis of the human endothelial nitric oxide synthase promoter. Sp1 and GATA factors are necessary for basal transcription in endothelial cells.
abstract: To gain insights into the mechanisms of endothelial nitric oxide synthase (eNOS) gene expression, we have cloned the eNOS promoter and fused it to a luciferase reporter gene to map regions of the promoter important for basal transcription in bovine aortic endothelial cells (BAEC). Transfection of BAEC with F1 luciferase (LUC) (-1600 to +22 nucleotides) yielded a 35-fold increase in promoter. Progressive deletion from -1600 to -1033 (F2 and F3 LUC) did not significantly influence eNOS promoter activity. Further deletion from -1033 to -779 (F4 LUC) resulted in an approximate 40% reduction in basal promoter activity, and still further deletion from -779 to -494 (F5 LUC) did not markedly influence activity. Deletion from -494 to -166 (F6 LUC) reduced eNOS promoter activity by 40-50%. Specific mutation of the consensus GATA site (-230) in the F3 LUC construct reduced luciferase activity (by 25-30%). Gel shift analysis and antibody depletion using BAEC nuclear extracts demonstrated in vitro binding of GATA-2 to the oligonucleotide sequence containing the -230 GATA site. Next, we mutated the Sp1 site (-103) in the F3 and F6 LUC constructs and in the F3 GATA mutant construct. Expression of these Sp1 mutants in BAEC resulted in a 85-90% reduction in normalized luciferase activity. Gel shift and antibody supershift analysis using a BAEC nuclear extracts demonstrated four specific, DNA-protein complexes binding to the eNOS Sp-1 site, with the slowest migrating form composed of Sp1 and another nuclear protein. These data demonstrate that the Sp1 site is an important cis-element in the core eNOS promoter.
authors.affiliation: Department of Pharmacology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536, USA.
authors.forename: R
authors.initials: R
authors.lastname: Zhang
authors.forename: W
authors.initials: W
authors.lastname: Min
authors.forename: W C
authors.initials: WC
authors.lastname: Sessa
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics*; D000818:Animals; D001011:Aorta; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000201:enzymology*; D050989:GATA2 Transcription Factor; D015870:Gene Expression; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019001:Nitric Oxide Synthase; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D017931:DNA Primers; D004268:DNA-Binding Proteins; D050989:GATA2 Transcription Factor; C494711:GATA2 protein, human; D011994:Recombinant Proteins; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D008156:Luciferases; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases