PU.1 IS ESSENTIAL FOR P47(PHOX) PROMOTER ACTIVITY IN MYELOID CELLS

Expression of the phagocyte cytosolic protein p47(phox), a component o f NADPH oxidase, is restricted mainly to myeloid cells. To study the c is elements and trans acting factors responsible for its gene expressi on, we have cloned and characterized the p47(phox) promoter, A predomi nant transcriptional start site was identified 21 nucleotides upstream of the translation initiation codon, To identify the gene promoter se quences, transient transfections of HL-60 human myeloid cells were per formed with a series of 5'-deletion p47(phox)-luciferase reporter cons tructs that extended as far upstream as -3050 bp relative to the trans criptional start site, The -224 and -86 constructs had the strongest p 47(phax) promoter activity, whereas the -46 construct showed a major r eduction in activity and the -36 construct a complete loss of activity . DNase I footprint analysis identified a protected region from -57 to -53. This region containing a consensus PU.1 site bound specifically both PU,I present in nuclear extracts from myeloid cells and PU.1 synt hesized in vitro. Mutations of this site eliminated PU.1 binding and a bolished the ability of the p47(phox) promoter to direct expression of the reporter gene, The p47(phox) promoter was active in all myeloid c ell Lines tested (HL-60, THP-1, U937, PLB-985), but not in non-myeloid cells (HeLa, HEK293). Finally, PU.1 transactivated the p47(phox)-luci ferase constructs in HeLa cells, We conclude that, similar to certain other myeloid specific genes, p47(phox) promoter activity in myeloid c ells requires PU.1.