Multiple PU.1 sites cooperate in the regulation of p40phox transcription during granulocytic differentiation of myeloid cells

Sen N. Li, Anthony J. Valante, Mei Qiang, Werner Schlegel, Maria Gamez, Robert A. Clark

Research output: Contribution to journalArticlepeer-review

32 Scopus citations


The p40phox protein, a regulatory component of the phagocyte NADPH oxidase, is preferentially expressed in cells of myeloid lineage, We investigated transcriptional regulation of the p40phox gene in HL-60 myeloid cells. Deletion analysis of approximately 6 kb of the 5'-flanking sequence of the gene demonstrated that the proximal 106 base pair of the promoter exhibited maximum reporter activity. This region contains 3 potential binding sites for PU.1, a myeloid-restricted member of the ets family of transcription factors. Mutation or deletion of each PU.1 site decreased promoter activity, and the level of activity mediated by each site correlated with its binding avidity for PU.1, as determined by gel shift competition assays. Mutation of all 3 sites abolished promoter activity in myeloid cells. PU.1dependent expression was also observed in the Raji B-cell line, whereas the moderate level of promoter reporter activity in the nonmyeloid HeLa cell line was independent of PU.1. Chromatin immunoprecipitation assay demonstrated occupation of the PU.1 sites by PU.1 in vivo in HL-60 cells. Cotransfection of the pGL3p40-106 reporter construct with a dominant-negative PU.1 mutant dramatically reduced promoter activity, whereas the overexpression of PU.1 increased promoter activity. Promoter activity and transcript levels of p40phox increased in HL-60 cells during dimethyl sulfoxide-induced differentiation toward the granulocyte phenotype, and this was associated with increased cellular levels of PU.1 protein. Our findings demonstrate that PU.1 binding at multiple sites is required for p40phox gene transcription in myeloid cells and that granulocytic differentiation is associated with the coordinated up-regulation of PU.1 and p40phox expression.

Original languageEnglish (US)
Pages (from-to)4578-4587
Number of pages10
Issue number12
StatePublished - Jun 15 2002

ASJC Scopus subject areas

  • Biochemistry
  • Immunology
  • Hematology
  • Cell Biology


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