Title: Novel Combinatorial Interactions of GATA-1, PU.1, and C/EBPε Isoforms Regulate Transcription of the Gene Encoding Eosinophil Granule Major Basic Protein
Abstract: GATA-1 and the ets factor PU.1 have been reported to functionally antagonize one another in the regulation of erythroidversus myeloid gene transcription and development. The CCAAT enhancer binding protein ε (C/EBPε) is expressed as multiple isoforms and has been shown to be essential to myeloid (granulocyte) terminal differentiation. We have defined a novel synergistic, as opposed to antagonistic, combinatorial interaction between GATA-1 and PU.1, and a unique repressor role for certain C/EBPε isoforms in the transcriptional regulation of a model eosinophil granulocyte gene, the major basic protein (MBP). The eosinophil-specific P2 promoter of the MBP gene contains GATA-1, C/EBP, and PU.1 consensus sites that bind these factors in nuclear extracts of the eosinophil myelocyte cell line, AML14.3D10. The promoter is transactivated by GATA-1 alone but is synergistically transactivated by low levels of PU.1 in the context of optimal levels of GATA-1. The C/EBPε27 isoform strongly represses GATA-1 activity and completely blocks GATA-1/PU.1 synergy. In vitro mutational analyses of the MBP-P2 promoter showed that both the GATA-1/PU.1 synergy, and repressor activity of C/EBPε27 are mediated via protein-protein interactions through the C/EBP and/or GATA-binding sites but not the PU.1 sites. Co-immunoprecipitations using lysates of AML14.3D10 eosinophils show that both C/EBPε32/30 and ε27 physically interact in vivo with PU.1 and GATA-1, demonstrating functional interactions among these factors in eosinophil progenitors. Our findings identify novel combinatorial protein-protein interactions for GATA-1, PU.1, and C/EBPε isoforms in eosinophil gene transcription that include GATA-1/PU.1 synergy and repressor activity for C/EBPε27. GATA-1 and the ets factor PU.1 have been reported to functionally antagonize one another in the regulation of erythroidversus myeloid gene transcription and development. The CCAAT enhancer binding protein ε (C/EBPε) is expressed as multiple isoforms and has been shown to be essential to myeloid (granulocyte) terminal differentiation. We have defined a novel synergistic, as opposed to antagonistic, combinatorial interaction between GATA-1 and PU.1, and a unique repressor role for certain C/EBPε isoforms in the transcriptional regulation of a model eosinophil granulocyte gene, the major basic protein (MBP). The eosinophil-specific P2 promoter of the MBP gene contains GATA-1, C/EBP, and PU.1 consensus sites that bind these factors in nuclear extracts of the eosinophil myelocyte cell line, AML14.3D10. The promoter is transactivated by GATA-1 alone but is synergistically transactivated by low levels of PU.1 in the context of optimal levels of GATA-1. The C/EBPε27 isoform strongly represses GATA-1 activity and completely blocks GATA-1/PU.1 synergy. In vitro mutational analyses of the MBP-P2 promoter showed that both the GATA-1/PU.1 synergy, and repressor activity of C/EBPε27 are mediated via protein-protein interactions through the C/EBP and/or GATA-binding sites but not the PU.1 sites. Co-immunoprecipitations using lysates of AML14.3D10 eosinophils show that both C/EBPε32/30 and ε27 physically interact in vivo with PU.1 and GATA-1, demonstrating functional interactions among these factors in eosinophil progenitors. Our findings identify novel combinatorial protein-protein interactions for GATA-1, PU.1, and C/EBPε isoforms in eosinophil gene transcription that include GATA-1/PU.1 synergy and repressor activity for C/EBPε27. CCAAT enhancer binding protein eosinophil granule major basic protein acute myeloid leukemia macrophage-colony stimulating factor receptor electrophoretic mobility shift assay immunoprecipitation GAPDH, glyceraldehyde-3-phosphate dehydrogenase interleukin granulocyte-macrophage colony stimulating factor granulocyte colony stimulating factor Hematopoietic development is regulated in part by the combinatorial actions of transcription factors that coordinate temporal and lineage-specific patterns of gene expression. The transcription factors GATA-1, PU.1, and members of the CCAAT enhancer binding protein (C/EBP)1 family (C/EBPα and C/EBPε in particular) are essential for the commitment and/or terminal differentiation of myeloid progenitors to the eosinophil lineage (1Nerlov C. Graf T. Genes Dev. 1998; 12: 2403-2412Crossref PubMed Google Scholar, 2McNagny K.M. Sieweke M.H. Doderlein G. Graf T. Nerlov C. EMBO J. 1998; 17: 3669-3680Crossref PubMed Scopus (103) Google Scholar, 3Nerlov C. 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Blood. 2000; 95: 2543-2551Crossref PubMed Google Scholar), and we have shown that GATA-1 is a key regulator of the MBP gene, we utilized the MBP-P2 promoter as a model to characterize further the combinatorial interactions and roles of GATA-1, PU.1, and C/EBPε in the eosinophil lineage. Consensus sites in the functional region of the MBP-P2 promoter were evaluated for their ability to bind GATA, PU.1, or C/EBP family members using nuclear extracts from the MBP-expressing eosinophil myelocyte cell line, AML14.3D10, and mature peripheral blood eosinophils. The functional relevance of these binding sites and transcription factor interactions were evaluated using mutagenesis, transactivation analyses in CV-1 cells, and transient transfection analyses in AML14.3D10 eosinophils. Co-immunoprecipitation (co-IP) experiments were performed to confirm the in vivo physical interactions of GATA-1, PU.1, and the various C/EBPε isoforms in eosinophil cell lines and mature peripheral blood eosinophils. Our results show that GATA-1, PU.1, and the various C/EBPε isoforms are differentially expressed, and physically and functionally interact during human eosinophil development to activate and then repress eosinophil gene transcription. In contrast to the functional antagonism reported for PU.1 and GATA-1 for activation of the M-CSFR promoter and various erythroid genes, we show that these factors cooperate synergistically in the activation of the eosinophil MBP-P2 promoter. Importantly, we demonstrate that PU.1 expression levels relative to GATA-1 determine whether the GATA-1/PU.1 interaction leads to synergy for GATA-1-regulated eosinophil target genes such as MBP. Moreover, we identify a novel role for the C/EBPε27 isoform as a repressor of GATA-1-mediated transactivation and GATA-1/PU.1 synergy. Finally, co-IP analyses demonstrate for the first time that the C/EBPε32/30 and C/EBPε27 isoforms physically interact with both PU.1 and GATA-1 in vivo, in eosinophils. Our findings demonstrate key combinatorial interactions of GATA-1, PU.1, and C/EBPε isoforms that may mediate either synergy or antagonism (repression) of granulocyte (eosinophil) gene transcription during myeloid development and terminal differentiation. Human blood eosinophils were isolated from a unit (475 ml) of blood obtained from normal, non-allergic, healthy donors. The use of normal human subjects as blood donors was in full compliance with all federal guidelines and was approved by the University of Illinois Institutional Review Board. Eosinophil isolation and purification were performed as described previously (71Du J. Alsayed Y.M. Xin F. Ackerman S.J. Platanias L.C. J. Biol. Chem. 2000; 275: 33167-33175Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) using a Miltenyi Biotec SuperMacs immunomagnetic sorting kit and apparatus according to manufacturer's specifications. Eosinophil preparations of >99% purity were routinely obtained and utilized for these studies. The human AML14.3D10 cell line is a fully differentiated eosinophil myelocyte line that contains eosinophil secondary (specific) granules and displays many characteristics of mature peripheral blood eosinophils. These include expression of the secondary granule cationic proteins major basic protein, eosinophil peroxidase, eosinophil-derived neurotoxin, and eosinophil cationic protein (72Paul C.C. Mahrer S. Tolbert M. Elbert B.L. Wong I.C. Ackerman S.J. Baumann M.A. Blood. 1995; 86: 3737-3744Crossref PubMed Google Scholar, 73Baumann M.A. Paul C.C. Stem Cells. 1998; 16: 16-24Crossref PubMed Google Scholar). AML14.3D10 cells can be induced to express chemokine receptors (e.g. for eotaxins) (74Zimmermann N. Daugherty B.L. Stark J.M. Rothenberg M.E. J. Immunol. 2000; 164: 1055-1064Crossref PubMed Scopus (41) Google Scholar) and express GM-CSF which drives their proliferation, differentiation, and survival in culture (75Pa