Abstract
We have investigated the myeloid-specific transcriptional regulation of p67(phox), an essential component of phagocyte respiratory burst NADPH oxidase. Analysis was carried out on the p67(phox) 5'-flanking region from -3669 to -4 (relative to ATG), including the first exon and intron and part of the second exon. The construct extending from -985 to -4 produced the highest luciferase activity in myeloid HL-60 cells but was not active in HeLa or Jurkat cells, indicating myeloid-specific expression. Four active elements were identified: Sp1/Sp3 at -694, PU.1 at -289, AP-1 at -210, and PU.1/HAF1 at -182, the latter three being in the first intron. These cis elements bound their cognate transacting factors both in vitro and in vivo. Mutation of the Sp1, PU.1, or PU.1/HAF1 site each decreased promoter activity by 35-50%. Mutations in all three sites reduced promoter activity by 90%. However, mutation of the AP-1 site alone nearly abolished promoter activity. The AP-1 site bound Jun and Fos proteins from HL-60 cell nuclear extract. Co-expression with Jun B in AP-1-deficient cells increased promoter activity by 3-fold. These data show that full p67(phox) promoter activity requires cooperation between myeloid-specific and nonmyeloid transcription factors, with AP-1 being the most critical for function.
Highlights
From the Department of Medicine, University of Texas Health Science Center and South Texas Veterans Health Care System, Audie L
Coexpression with Jun B in AP-1-deficient cells increased promoter activity by 3-fold. These data show that full p67phox promoter activity requires cooperation between myeloid-specific and nonmyeloid transcription factors, with AP-1 being the most critical for function
A deletion to nucleotide – 682 resulted in Ͼ80% reduction in activity, indicating that sequences important for p67phox promoter activity are located between bp –733 and – 682
Summary
In p47phox, a single consensus PU. binding site located on the noncoding strand from bp – 40 to – 45 (relative to the transcription start site) accounted for most of the myeloid-specific promoter activity found in the proximal 2151 base pairs of the 5Ј-flanking region of the gene [20]. PU. sites were identified in the upstream 5Ј-flanking region of the p67phox gene, we found that most of the functional activity in myeloid cells is directed by elements in the first intron, including binding sites for PU., AP-1, and, as recently described [19], PU.1/HAF-1. Mutation of this site abolished promoter activity in the myeloid cell line HL-60, even in the presence of intact PU. and HAF-1 binding sites
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