Abstract
GATA-1 and the ets factor PU.1 have been reported to functionally antagonize one another in the regulation of erythroid versus myeloid gene transcription and development. The CCAAT enhancer binding protein epsilon (C/EBPepsilon) 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/EBPepsilon 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/EBPepsilon(27) 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/EBPepsilon(27) 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/EBPepsilon(32/30) and epsilon(27) 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/EBPepsilon isoforms in eosinophil gene transcription that include GATA-1/PU.1 synergy and repressor activity for C/EBPepsilon(27).
Highlights
Novel Combinatorial Interactions of GATA-1, PU.1, and CCAAT/enhancer-binding proteins (C/EBPs)⑀ Isoforms Regulate Transcription of the Gene Encoding Eosinophil Granule Major Basic Protein*
PU.1, GATA-1, and C/EBP⑀ Isoforms Are Differentially Expressed in Eosinophilic Cell Lines and Mature Blood Eosinophils and Bind to the Their Consensus Sites in the Functional Region of the major basic protein (MBP)-P2 Promoter—We identified a number of additional GATA-1 and C/EBP consensus sites, as well as two PU.1 consensus sites not characterized previously in the MBP-P2 promoter, using subsequence homology searches of the bp Ϫ117 to ϩ1 functionally active region (Fig. 1)
Expression levels for GATA-1, PU.1, and the various C/EBP⑀ isoforms was compared for AML14 parental myeloblasts, AML14.3D10 eosinophil myelocytes, and peripheral blood eosinophils from normal donors, covering the range of differentiation stages from undifferentiated progenitors to terminally differentiated mature eosinophils
Summary
C/EBP, CCAAT enhancer binding proand C/EBP⑀ in particular) are essential for the commitment and/or terminal differentiation of myeloid progenitors to the eosinophil lineage [1,2,3,4,5,6,7,8,9]. Because PU. has been reported to antagonize GATA-1-mediated erythroid gene expression and development (10 –12), 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., and C/EBP⑀ in the eosinophil lineage. Our results show that GATA-1, PU., and the various C/EBP⑀ isoforms are differentially expressed, and physically and functionally interact during human eosinophil development to activate and repress eosinophil gene transcription. Our findings demonstrate key combinatorial interactions of GATA-1, PU., and C/EBP⑀ isoforms that may mediate either synergy or antagonism (repression) of granulocyte (eosinophil) gene transcription during myeloid development and terminal differentiation
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