Pu.1 Is essential for p47(phox) promoter activity in myeloid cells

Expression of the phagocyte cytosolic protein p47(phox), a component of NADPH oxidase, is restricted mainly to myeloid cells. To study the cis- elements and trans-acting factors responsible for its gene expression, we have cloned and characterized the p47(phox) promoter. A predominant transcriptional start site was identified 21 nucleotides upstream of the translation initiation codon. To identify the gene promoter sequences, transient transfections of HL-60 human myeloid cells were performed with a series of 5'-deletion p47(phox)-luciferase reporter constructs that extended as far upstream as -3050 bp relative to the transcriptional start site. The - 224 and -86 constructs had the strongest p47(phox) promoter activity, whereas the -46 construct showed a major reduction in activity and the -36 construct a complete loss of activity. DNase I footprint analysis identified a protected region from -37 to -53. This region containing a consensus PU.1 site bound specifically both PU.1 present in nuclear extracts from myeloid cells and PU.1 synthesized in vitro. Mutations of this site eliminated PU.1 binding and abolished the ability of the p47(phox) promoter to direct expression of the reporter gene. The p47(phox) promoter was active in all myeloid cell lines tested (HL-60, THP-1, U937, PLB-985), but not in non- myeloid cells (HeLa, HEK293). Finally, PU.1 transactivated the p47(phox)- luciferase constructs in HeLa cells. We conclude that, similar to certain other myeloid-specific genes, p47(phox) promoter activity in myeloid cells requires PU.1.