title: C/EBPepsilon directly interacts with the DNA binding domain of c-myb and cooperatively activates transcription of myeloid promoters.
abstract: C/EBPepsilon is essential for granulocytic differentiation. We investigated the role of C/EBPepsilon in the transcriptional activation of various myeloid-specific genes. We found that two C/EBPepsilon isoforms, p32 and p30, possessing transcriptional activation domains were coexpressed in myeloid cells. Interestingly, isoform C/EBPepsilon p30 but not p32 was differentially upregulated in NB-4 promyelocytic leukemia cells treated with retinoids. Both isoforms bound specifically to C/EBP sites in myeloid promoters. The kd for C/EBPepsilon 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-specific 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 isoform lacked significant transcriptional activity, suggesting that other hematopoietic-specific factors were required for its function. Consistent with this prediction, transfections into the hematopoietic cell line Jurkat showed a 9.0- and 2.5-fold activation of the mim-1 promoter by the p32 and p30 isoforms, respectively. The additional 32 NH2-terminal residues made p32 a significantly more potent transcriptional activator than p30. T lymphoblasts (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/EBPepsilon in CV-1 cells cooperatively transactivated the mim-1 promoter by 20- and 16-fold, respectively, and the neutrophil elastase promoter by 10-and 7-fold, respectively. Pulldown assays showed that each C/EBPepsilon isoform interacted directly with the DNA binding domain of the c-myb protein. Further studies showed that Kcl22 myeloid cells only contained active C/EBPepsilon, but not C/EBPalpha, C/EBPbeta, or C/EBPdelta. A mutation of the C/EBP site in the neutrophil elastase promoter markedly decreased the transactivation of the promoter in Kcl22 myeloblasts. These results demonstrate a role for C/EBPepsilon in regulating myeloid promoters, such as neutrophil elastase, probably through a direct interaction with c-myb.
authors.affiliation: Division of Hematology/Oncology, Department of Medicine, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles CA, USA.
authors.forename: W
authors.initials: W
authors.lastname: Verbeek
authors.forename: A F
authors.initials: AF
authors.lastname: Gombart
authors.forename: A M
authors.initials: AM
authors.lastname: Chumakov
authors.forename: C
authors.initials: C
authors.lastname: Müller
authors.forename: A D
authors.initials: AD
authors.lastname: Friedman
authors.forename: H P
authors.initials: HP
authors.lastname: Koeffler
mesh_terms: D000818:Animals; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism; D017930:Genes, Reporter; D006410:Hematopoiesis / Q000235:genetics*; D006801:Humans; D019169:Jurkat Cells; D019272:Leukocyte Elastase / Q000235:genetics; D051379:Mice; D009857:Oncogenes; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism*; D020598:Proto-Oncogene Proteins c-myb; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D020298:U937 Cells
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; C489468:Cebpe protein, mouse; D004268:DNA-Binding Proteins; D020033:Protein Isoforms; D011518:Proto-Oncogene Proteins; D020598:Proto-Oncogene Proteins c-myb; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C070790:CEBPE protein, human; D019272:Leukocyte Elastase