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AP‐1 is essential for p67 phox promoter activity
Author(s) -
Gauss Katherine A.,
Bunger Peggy L.,
Quinn Mark T.
Publication year - 2002
Publication title -
journal of leukocyte biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.819
H-Index - 191
eISSN - 1938-3673
pISSN - 0741-5400
DOI - 10.1189/jlb.71.1.163
Subject(s) - biology , microbiology and biotechnology , intron , promoter , binding site , caat box , transcription factor , site directed mutagenesis , luciferase , electrophoretic mobility shift assay , consensus sequence , transcription (linguistics) , reporter gene , gene , mutant , genetics , gene expression , transfection , peptide sequence , linguistics , philosophy
The cytosolic NADPH oxidase cofactor p67 phox has been shown to be one of the limiting factors in assembly andactivation of this multi‐protein enzyme complex and, therefore, must behighly regulated at the transcriptional level. In the present studies, we have further characterized the promoter for humanp67 phox . Genomic sequence upstream of thetranslational start site (TLS; 2 kb) was cloned, and RACE was used toidentify and compare the transcriptional start site (TSS) in twomyeloid cell lines, HL‐60 and PLB‐985. Two major TSS were identifiedwithin the first intron for both cell lines, and one transcriptisolated from PLB‐985 cells started approximately 34 bp 5′ of exon 1and contained no intron 1 sequence. To identify regulatory regions ofthe promoter, a luciferase reporter was used to assay a series ofpromoter deletion constructs. The greatest transcriptional activity wasobserved for fragments containing at least 500 bp upstream of the TLS. Sequence analysis of the p67 phox promoterrevealed consensus binding sites for previously described transcriptionfactors including AP‐1 and PU.1. Site‐directed mutagenesis of the AP‐1site demonstrated that this site was essential for basal transcription. EMSA, competition, and super‐shift assays showed that this site wasspecifically recognized by nuclear factors of the AP‐1 family. EMSAanalysis and promoter‐reporter assays with the PU.1 consensus sites atpositons ‐176, ‐283, and ‐328 demonstrate that PU.1 binds the site atposition ‐283 with high affinity. Mutagenesis of any one of the PU.1sites reduced the basal transcriptional activity by approximately 50%,demonstrating that, although none of these sites is singularlyresponsible for the basal transcriptional activity, all three sitesplay some role in the transcriptional activity of thep67 phox promoter. In support of thisconclusion, mutagenesis of all three sites completely abrogatedtranscriptional activity.