Differentiation-dependent up-regulation of p47(phox) gene transcription is associated with changes in PU.1 phosphorylation and increased binding affinity

The p47(phox) gene encodes a cytosolic component of the phagocytic NADPH oxidase complex. Expression of p47(phox) is both tissue-specific and developmentally regulated. Stable transfection of the myeloid cell lines PLB985 and HL60, with reporter gene constructs containing as little as 58 bp of proximal promoter sequence, was capable of directing significant reporter gene activity in myeloid cells, which increased significantly on induction of myeloid differentiation. EMSA analysis of a binding site for the Ets family member, PU.1, located at positions -39 to -44 revealed that the pattern of complex formation changed significantly on induction of myeloid differentiation. All EMSA complexes were competed by a functional PU.1 binding site and could be supershifted in the presence of polyclonal anti-PU.1 antibody. Reaction of EMSA complexes with anti-phosphoserine antibody, treatment with phosphatase, or Western blotting of proteins captured on the PU.1 binding site, was used to demonstrate that the changes in PU.1 complex formation dependent on myeloid differentiation were associated with increased levels of PU.1 phosphorylation. Furthermore, the more highly phosphorylated forms of PU.1 were shown to have a greater affinity for the p47(phox) PU.1 consensus binding site. Up-regulated transcriptional activity in response to myeloid differentiation can therefore be correlated with increased levels of PU.1 phosphorylation and a greater binding affinity.