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Blood, 15 June 2002, Vol. 99, No. 12, pp. 4578-4587
PHAGOCYTES
Multiple PU.1 sites cooperate in the regulation of
p40phox transcription during
granulocytic differentiation of myeloid cells
Sen-Lin Li,
Anthony J. Valente,
Mei Qiang,
Werner Schlegel,
Maria Gamez, and
Robert A. Clark
From the Department of Medicine, University of Texas
Health Science Center and the South Texas Veterans Health Care System,
Audie L. Murphy Division, San Antonio; and Fondation pour Recherches
Médicales, University of Geneva, Switzerland.
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.1-dependent
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 pGL3-p40-106 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.
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