Published online before print June 16, 2003

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(Journal of Leukocyte Biology. 2003;74:464-470.)
© 2003 by Society for Leukocyte Biology

Cross-talk between regulators of myeloid development: C/EBP binds and activates the promoter of the PU.1 gene

Division of Pediatric Oncology, Johns Hopkins University, Baltimore, Maryland

¹Correspondence: Johns Hopkins University, Cancer Research Bldg., Rm. 253, 1650 Orleans St., Baltimore, MD 21231. E-mail: afriedm2{at}jhem.jhmi.edu

CCAAT/enhancer-binding protein (C/EBP) and PU.1 are required for myelopoiesis. Examination of the murine PU.1 promoter revealed several potential C/EBP-binding sites. Gel-shift assay demonstrated that C/EBP expressed in 293T cells bound the site centered at –68 most potently. C/EBP from 32D cl3 myeloid cell nuclear extracts also bound this site strongly, and endogenous C/EBPß did so to a lesser extent, whereas these C/EBP isoforms bound the neutrophil elastase promoter with equal affinity. The –68 site in the murine PU.1 promoter is conserved in the human PU.1 promoter. Mutation of the –68 C/EBP-binding site in a -85/+152 promoter segment linked to the luciferase cDNA reduced promoter activity fourfold in 293T cells in the presence of cotransfected C/EBP and twofold in 32D cl3 myeloid cells. Induction of endogenous PU.1 RNA by C/EBP-estradiol receptor (ER) in the presence of cycloheximide is obviated by mutation of the C/EBP DNA-binding domain, and chromosomal immunoprecipitation demonstrated specific interaction of C/EBP and C/EBP-ER with the PU.1 promoter. Finally, PU.1 RNA is reduced several-fold in immortalized C/EBP (-/-) compared with (+/-) cells. Together, these findings indicate that C/EBP binds and activates the endogenous PU.1 gene in myeloid cells. Induction of PU.1 by C/EBP may account for increased levels of PU.1 in myeloid as compared with B lymphoid cells and in this way, may contribute to the specification of myeloid progenitors.

Key Words: hematopoiesis • differentiation • transcription

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