C/EBPα Binds and Activates the Distal PU.1 Enhancer.

C/EBPα and PU.1 are both key regulators of myeloid development. C/EBPα (−/−) mice have markedly reduced granulocyte-monocyte progenitors, and PU.1 (−/−) mice lack B cells and monocytes and have greatly reduced neutrophil numbers. Genetic evidence indicates that higher levels of PU.1 are required for monocyte as compared to granulocyte development. Exogenous PU.1 or C/EBPα converts B-lineage to monocytic cells and increases monocyte as compared to granulocyte production from transduced myeloid progenitors cultured in IL3/IL6/SCF or in GM-CSF. We previously demonstrated that activation of C/EBPα-ER with estradiol induces PU.1 mRNA within 4 hr even in the presence of cycloheximide and that C/EBPα binds and activates the PU.1 promoter via a site conserved in the murine and human genes. However, binding to the promoter site in a gel shift assay was weak compared to the strong binding site in the neutrophil elastase promoter. To obtain further evidence for regulation of the PU.1 gene by C/EBPα, we have now investigated regulation of the 240 bp proximal segment of the PU.1 distal enhancer located at −14 kb. We focused on the proximal segment of the enhancer due to is strong transcriptional activating activity when linked to the PU.1 promoter, compared to the repressive effect of the distal enhancer segment. Five sites within the proximal enhancer (C1–C5) fit the C/EBP consensus site T(T/G)NNGNAA(T/G) or differed from this consensus by 1 bp. Each was evaluated for binding to C/EBPα expressed transiently in 293T cells. Site C1 bound C/EBPα weakly, and C2 and C5 each bound C/EBPα strongly. Binding of C/EBPα to the endogenous proximal enhancer segment was also demonstrated in the HF1 myeloid cell line using the chromosomal immunoprecipitation (ChIP) assay. HF1 cells were employed for this assay due to their high-level expression of C/EBPα. Sites C1, C2, and C5 were mutated individually within the enhancer, which was then linked to the PU.1 promoter and the luciferase cDNA. Mutation of C1 minimally reduced the activity of this reporter, mutation of C2 reduced activity 2-fold, and mutation of C5 reduced activity 5-fold, on average, upon transient transfection into the more readily tranfectable 32DPKCδ cell line. These data provide further support for the conclusion that C/EBPα directly activates the PU.1 gene and that induction of PU.1 by C/EBPα, potentially in cooperation with additional factors such as c-Jun, helps direct bipotent myeloid progenitors to commit to the monocytic lineage.