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Blood, Vol. 94 No. 4 (August 15), 1999:
pp. 1429-1439
C/EBP and GATA-1 Synergistically Regulate Activity of the
Eosinophil Granule Major Basic Protein Promoter: Implication for
C/EBP Activity in Eosinophil Gene Expression
Yuji Yamaguchi,
Hitoshi Nishio,
Kenji Kishi,
Steven J. Ackerman, and
Toshio Suda
From the Department of Cell Differentiation, Institute of Molecular
Embryology and Genetics, Kumamoto University School of Medicine,
Kumamoto, Japan; the Department of Hematology and Rheumatology, Tokai
University School of Medicine, Isehara, Japan; and the Department of
Biochemistry and Molecular Biology, University of Illinois at Chicago,
Chicago, IL.
Eosinophil granule major basic protein (MBP) is expressed
exclusively in eosinophils and basophils in hematopoietic cells. In our
previous study, we demonstrated a major positive regulatory role for
GATA-1 and a negative regulatory role for GATA-2 in MBP gene
transcription. Further analysis of the MBP promoter region identified a
C/EBP (CCAAT/enhancer-binding protein) consensus binding site 6 bp
upstream of the functional GATA-binding site in the MBP gene. In the
cell line HT93A, which is capable of differentiating towards both the
eosinophil and neutrophil lineages in response to retinoic acid (RA),
C/EBP mRNA expression decreased significantly concomitant with
eosinophilic and neutrophilic differentiation, whereas C/EBP
expression was markedly increased. Electrophoretic mobility shift
assays (EMSAs) showed that recombinant C/EBP protein could bind to
the potential C/EBP-binding site (bp  90 to 82) in the MBP
promoter. Furthermore, we have demonstrated that both C/EBP and
GATA-1 can bind simultaneously to the C/EBP- and GATA-binding sites in
the MBP promoter. To determine the functionality of both the C/EBP- and
GATA-binding sites, we analyzed whether C/EBP and GATA-1 can
stimulate the MBP promoter in the C/EBP and GATA-1 negative Jurkat
T-cell line. Cotransfection with C/EBP and GATA-1 expression vectors
produced a 5-fold increase compared with cotransfection with the
C/EBP or GATA-1 expression vectors individually. In addition, GST
pull-down experiments demonstrated a physical interaction between human
GATA-1 and C/EBP. Expression of FOG
( riend
  ATA), which binds to GATA-1 and acts
as a cofactor for GATA-binding proteins, decreased transactivation
activity of GATA-1 for the MBP promoter in a dose-dependent manner. Our
results provide the first evidence that both GATA-1 and C/EBP
synergistically transactivate the promoter of an eosinophil-specific
granule protein gene and that FOG may act as a negative cofactor for
the eosinophil lineage, unlike its positively regulatory function for
the erythroid and megakaryocyte lineages.
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