Department of Veterinary Molecular Biology, Montana State University, Bozeman
Correspondence: Mark T. Quinn, Ph.D., Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717. E-mail: mquinn{at}montana.edu
The cytosolic NADPH oxidase cofactor p67phox has been shown to be one of the limiting factors in assembly and activation of this multi-protein enzyme complex and, therefore, must be highly regulated at the transcriptional level. In the present studies, we have further characterized the promoter for human p67phox. Genomic sequence upstream of the translational start site (TLS; 2 kb) was cloned, and RACE was used to identify and compare the transcriptional start site (TSS) in two myeloid cell lines, HL-60 and PLB-985. Two major TSS were identified within the first intron for both cell lines, and one transcript isolated from PLB-985 cells started approximately 34 bp 5' of exon 1 and contained no intron 1 sequence. To identify regulatory regions of the promoter, a luciferase reporter was used to assay a series of promoter deletion constructs. The greatest transcriptional activity was observed for fragments containing at least 500 bp upstream of the TLS. Sequence analysis of the p67phox promoter revealed consensus binding sites for previously described transcription factors including AP-1 and PU.1. Site-directed mutagenesis of the AP-1 site demonstrated that this site was essential for basal transcription. EMSA, competition, and super-shift assays showed that this site was specifically recognized by nuclear factors of the AP-1 family. EMSA analysis and promoter-reporter assays with the PU.1 consensus sites at positons -176, -283, and -328 demonstrate that PU.1 binds the site at position -283 with high affinity. Mutagenesis of any one of the PU.1 sites reduced the basal transcriptional activity by approximately 50%, demonstrating that, although none of these sites is singularly responsible for the basal transcriptional activity, all three sites play some role in the transcriptional activity of the p67phox promoter. In support of this conclusion, mutagenesis of all three sites completely abrogated transcriptional activity.
Key Words: neutrophil • NADPH oxidase • transcriptional regulation • promoter
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