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).