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

CCAAT/enhancer‐binding protein (C/EBP)α 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/EBPα expressed in 293T cells bound the site centered at –68 most potently. C/EBPα from 32D cl3 myeloid cell nuclear extracts also bound this site strongly, and endogenous C/EBPβ 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/EBPα and twofold in 32D cl3 myeloid cells. Induction of endogenous PU.1 RNA by C/EBPα‐estradiol receptor (ER) in the presence of cycloheximide is obviated by mutation of the C/EBPα DNA‐binding domain, and chromosomal immunoprecipitation demonstrated specific interaction of C/EBPα and C/EBPα‐ER with the PU.1 promoter. Finally PU.1 RNA is reduced several‐fold in immortalized C/EBPα (−/−) compared with (+/−) cells. Together, these findings indicate that C/EBPα binds and activates the endogenous PU.1 gene in myeloid cells. Induction of PU.1 by C/EBPα 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.