Transcriptional regulation of the p67(phox) gene - Role of AP-1 in concertwith myeloid-specific transcription factors

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 th e 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 J urkat cells, indicating myeloid-specific expression. Four active elements w ere 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 bou nd their cognate transacting factors both in vitro and in vivo. Mutation of the Sp1, PU.1, or PU.1/ HAFT site each decreased promoter activity by 35-5 0%. 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. Coexpre ssion with Jun B in AP-1-deficient cells increased promoter activity by 3-f old. These data show that full p67(phox) promoter activity requires coopera tion between myeloid-specific and nonmyeloid transcription factors, with AP -1 being the most critical for function.