Three repeats of CCCCTCC on the pyrimidine-rich sequence in the proximal 5' flanking region are required for efficient transcriptional activity of the human endothelial nitric oxide synthase gene.

The endothelial nitric-oxide synthase (eNOS) gene is constitutively expressed in endothelial cells, but numerous regulatory elements in the promoter region should contribute to the regulation for cell specific expression and the response to exogenous stimuli. A Sp1-binding consensus motif (-104 to -96) is essential for a core promoter activity of the human eNOS gene. In this study, we show that three repeats of CCCCTCC element (-74, -61, and -47), which located periodically at 13 and 14 nucleotide intervals on a pyrimidine-rich string in the proximal 5'-flanking region, were required for efficient transcriptional activity of the eNOS gene. In electrophoretic mobility shift assays, a specific DNA-protein complex was formed with a binding ability depending on the number of the CCCCTCC element while only one element did not retain any binding ability. Dinucleotide-substitution mutants at the repeat sequences reduced their transcriptional activities of the eNOS gene in transient transfection assays as diminishing their abilities to form the complex. Further, DNase I footprinting analyses indicated that nuclear extracts continuously protected a proximal region from -108 to -16, which includes pyrimidine-rich and purine-rich strings containing three CCCCTCC repeats and the Sp1-binding motif. UV-crosslink assay revealed the CCCCTCC repeat probe bound to a 97 kDa protein in the complex. A huge protein complex including Sp1-related factors and a 97 kDa protein might be formed along the proximal promoter of the eNOS gene for efficient transcriptional activity.