C/EBP epsilon directly interacts with the DNA binding domain of c-myb and cooperatively activates transcription of myeloid promoters

C/EBP epsilon is essential for granulocytic differentiation. We investigate d the role of C/EBP epsilon in the transcriptional activation of various my eloid-specific genes. We found that two C/EBP epsilon isoforms, p32 and p30 , possessing transcriptional activation domains were coexpressed in myeloid cells. Interestingly, isoform C/EBP epsilon p30 but not p32 was differenti ally upregulated in NB-4 promyelocytic leukemia cells treated with retinoid s. Both isoforms bound specifically to C/EBP sites in myeloid promoters. Th e kd for C/EBP epsilon binding to the C/EBP site of the neutrophil elastase promoter was 4.2 nmol/L. In transfection assays using the nonhematopoietic cell line, CV-1, the p32 isoform activated promoters from the myeloid-spec ific mim-1, neutrophil elastase, and granulocyte colony-stimulating factor (G-CSF) receptor genes by 2.5-, 1.8-, and 1.6-fold, respectively. The p30 i soform lacked significant transcriptional activity, suggesting that other h ematopoietic-specific factors were required for its function. Consistent wi th this prediction, transfections into the hematopoietic cell line Jurkat s howed a 9.0- and 2.5-fold activation of the mim-1 promoter by the p32 and p 30 isoforms, respectively. The additional 32 NH2-terminal residues made p32 a significantly more potent transcriptional activator than p30. T lymphobl asts (Jurkat cells) and immature myeloid cells (eg, Kcl22 cells) expressed high levels of the c-myb hematopoietic transcription factor. Cotransfection of c-myb with either the p32 or p30 isoform of C/EBP epsilon in CV-1 cells cooperatively transactivated the mim-1 promoter by 20- and 16-fold, respec tively, and the neutrophil elastase promoter by 10-and 7-fold, respectively . Pulldown assays showed that each C/EBP epsilon isoform interacted directl y with the DNA binding domain of the c-myb protein. Further studies showed that Kcl22 myeloid cells only contained active C/EBP epsilon, but not C/EBP alpha, C/EBP beta, or C/EBP delta. A mutation of the C/EBP site in the neu trophil elastase promoter markedly decreased the transactivation of the pro moter in Kcl22 myeloblasts. These results demonstrate a role for C/EBP epsi lon in regulating myeloid promoters, such as neutrophil elastase, probably through a direct interaction with c-myb. (C) 1999 by The American Society o f Hematology.