Role of Sp1 in transcriptional activation of human nitric oxide synthase type III gene.
Endothelial nitric oxide synthase (eNOS or NOS-III) is constitutively expressed. To elucidate the mechanism by which the basal expression of NOS-III gene is activated, we constructed in a luciferase vector, pXP1, serial 5'-deletion mutants of a 1.3-kb 5'-flanking fragment and transiently expressed them in cultured human endothelial cells. The promotor activity was detected in the -198/+22 region which contains several putative Sp1 binding sites. DNase I footprinting assays coupled with gel shift assays revealed the GC box(-104/-90) to be the Sp1 binding site. Site-directed mutation of 4 crucial bases in this site reduced the promotor activity by > 90%. These findings provide strong evidence that binding of Sp1 or closely related protein to this site is required for the activation of basal NOS-III transcription.
Tang
J L
JL
Department of Internal Medicine, University of Texas, Health Science Center at Houston 77030, USA.
Zembowicz
A
A
Xu
X M
XM
Wu
K K
KK
D000594:Amino Acid Oxidoreductases / Q000235:genetics*; D001483:Base Sequence; D002478:Cells, Cultured; D003850:Deoxyribonuclease I; D004730:Endothelium, Vascular / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019001:Nitric Oxide Synthase; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D014471:Umbilical Veins; D001616:beta-Galactosidase / Q000235:genetics
D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D008156:Luciferases; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D003850:Deoxyribonuclease I; D001616:beta-Galactosidase