A functional ISRE is required for myeloid transcription of the p47(phox) gene

Expression of p47(phox), a component of the phagocytic NADPH oxidase, is both tissue-specific and developmentally regulated. We have investigated transcription from the p47(phox) gene promoter by reporter gene analysis of myeloid PLB985 cells stably transfected with a series of p47(phox) promoter constructs. Stable transfection with constructs containing up to 3100 bp of proximal promoter sequence demonstrated that as little as 144 bp of proximal promoter sequence was able to direct significant reporter gene activity in myeloid cells, but not in HeLa cells. Mutation of a previously uncharacterised interferon-stimulated response element (ISRE) consensus located at positions -104 to-116, or of an established binding site for the Ets family transcription factor, PU.1 (located at positions -39 to -44), abolished transcription in stably transfected myeloid cells. Electrophoretic mobility shift analysis (EMSA) with myeloid cell nuclear extracts demonstrated that an oligonucleotide containing the p47(phox) ISRE consensus was able to compete binding at another bona fide ISRE. Complexes formed on the p47(phox) ISRE itself were competed by other ISRE consensus sequences. We conclude that transcription of p47(phox) in myeloid cells requires a functional ISRE in addition to the previously identified PU.1 binding site.