Novel combinatorial interactions of GATA-1, PU.1, and C/EBPepsilon isoforms regulate transcription of the gene encoding eosinophil granule major basic protein.
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).
Du
Jian
J
Department of Biochemistry and Molecular Biology, College of Medicine, University of Illinois, Chicago, Illinois 60612, USA.
Stankiewicz
Monika J
MJ
Liu
Yang
Y
Xi
Qing
Q
Schmitz
Jonathan E
JE
Lekstrom-Himes
Julie A
JA
Ackerman
Steven J
SJ
D020121:5' Untranslated Regions / Q000235:genetics; D001483:Base Sequence; D001782:Blood Donors; D001798:Blood Proteins / Q000235:genetics*; D022762:CCAAT-Enhancer-Binding Proteins / Q000097:blood*; D002467:Cell Nucleus / Q000502:physiology; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000097:blood*; D047091:Eosinophil Granule Proteins; D004804:Eosinophils / Q000502:physiology*; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D005786:Gene Expression Regulation; D006801:Humans; D016350:Leucine Zippers; D016296:Mutagenesis; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms / Q000097:blood; D011518:Proto-Oncogene Proteins / Q000097:blood*; D012260:Ribonucleases; D017384:Sequence Deletion; D015534:Trans-Activators / Q000097:blood*; D014157:Transcription Factors / Q000097:blood*; D000099232:Proto-Oncogene Protein Spi-1
D020121:5' Untranslated Regions; D001798:Blood Proteins; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D047091:Eosinophil Granule Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D020033:Protein Isoforms; D011518:Proto-Oncogene Proteins; D012260:Ribonucleases; D015534:Trans-Activators; D014157:Transcription Factors; D000099232:Proto-Oncogene Protein Spi-1; C494688:GATA1 protein, human; C070790:CEBPE protein, human