Critical flanking sequences of PU.1 binding sites in myeloid-specific promoters

The myeloid-specific transcription factor PU.1 is essential for expression of p47(phox), a component of the superoxide-forming phagocyte NADPH oxidase. The consensus PU.1 binding sequence (GAGGAA) is located on the non-coding strand from position -40 to -45 relative to the transcriptional start site of the p47phox promoter. A promoter construct extending to -46 was sufficient to drive tissue-specific expression of the luciferase reporter gene, but extension of the promoter from -46 to -48 resulted in a significant increase in reporter expression. Mutations of the nucleotides G at -46 and/or T at -47 reduced both reporter expression and PU.1 binding, whereas mutations at -48 had no effect. The PU.1 binding avidity of these sequences correlated closely with their capacity to dictate reporter gene transcription. In parallel studies on the functional PU.1 site in the promoter of CD18, mutations of nucleotides G and T at positions -76 and -77 (corresponding to -46 and -47, respectively, of the p47phox promoter) reduced PU.1 binding and nearly abolished the contribution of this element to promoter activity. We conclude that the immediate flanking nucleotides of the PU.1 consensus motif have significant effects on PU.1 binding avidity and activity and that this region is the dominant cis element regulating p47phox expression.