C/EBPβ and GATA-1 synergistically regulate activity of the eosinophil granule major basic protein promoter

Implication for C/EBPβ activity in eosinophil gene expression

Yuji Yamaguchi, Hitoshi Nishio, Kenji Kishi, Steven J. Ackerman, Toshio Suda

Research output: Contribution to journalArticle

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Abstract

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/EBPα mRNA expression decreased significantly concomitant with eosinophilic and neutrophilic differentiation, whereas C/EBPβ expression was markedly increased. Electrophoretic mobility shift assays (EMSAs) showed that recombinant C/EBPβ 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/EBPβ 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/EBPβ and GATA-1 can stimulate the MBP promoter in the C/EBPβ and GATA-1 negative Jurkat T-cell line. Cotransfection with C/EBPβ and GATA-1 expression vectors produced a 5- fold increase compared with cotransfection with the C/EBPβ or GATA-1 expression vectors individually. In addition, GST pull-down experiments demonstrated a physical interaction between human GATA-1 and C/EBPβ. Expression of FOG (Friend of GATA), 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/EBPβ 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.

Original languageEnglish
Pages (from-to)1429-1439
Number of pages11
JournalBlood
Volume94
Issue number4
Publication statusPublished - 1999 Aug 15

Fingerprint

Eosinophil Major Basic Protein
CCAAT-Enhancer-Binding Proteins
Eosinophils
Gene expression
Gene Expression
Proteins
Protein Binding
Binding Sites
Eosinophil Granule Proteins
Cell Line
Electrophoretic mobility
Jurkat Cells
Megakaryocytes
T-cells
Basophils

ASJC Scopus subject areas

  • Hematology

Cite this

C/EBPβ and GATA-1 synergistically regulate activity of the eosinophil granule major basic protein promoter : Implication for C/EBPβ activity in eosinophil gene expression. / Yamaguchi, Yuji; Nishio, Hitoshi; Kishi, Kenji; Ackerman, Steven J.; Suda, Toshio.

In: Blood, Vol. 94, No. 4, 15.08.1999, p. 1429-1439.

Research output: Contribution to journalArticle

Yamaguchi, Yuji ; Nishio, Hitoshi ; Kishi, Kenji ; Ackerman, Steven J. ; Suda, Toshio. / C/EBPβ and GATA-1 synergistically regulate activity of the eosinophil granule major basic protein promoter : Implication for C/EBPβ activity in eosinophil gene expression. In: Blood. 1999 ; Vol. 94, No. 4. pp. 1429-1439.
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abstract = "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/EBPα mRNA expression decreased significantly concomitant with eosinophilic and neutrophilic differentiation, whereas C/EBPβ expression was markedly increased. Electrophoretic mobility shift assays (EMSAs) showed that recombinant C/EBPβ 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/EBPβ 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/EBPβ and GATA-1 can stimulate the MBP promoter in the C/EBPβ and GATA-1 negative Jurkat T-cell line. Cotransfection with C/EBPβ and GATA-1 expression vectors produced a 5- fold increase compared with cotransfection with the C/EBPβ or GATA-1 expression vectors individually. In addition, GST pull-down experiments demonstrated a physical interaction between human GATA-1 and C/EBPβ. Expression of FOG (Friend of GATA), 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/EBPβ 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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AU - Kishi, Kenji

AU - Ackerman, Steven J.

AU - Suda, Toshio

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N2 - 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/EBPα mRNA expression decreased significantly concomitant with eosinophilic and neutrophilic differentiation, whereas C/EBPβ expression was markedly increased. Electrophoretic mobility shift assays (EMSAs) showed that recombinant C/EBPβ 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/EBPβ 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/EBPβ and GATA-1 can stimulate the MBP promoter in the C/EBPβ and GATA-1 negative Jurkat T-cell line. Cotransfection with C/EBPβ and GATA-1 expression vectors produced a 5- fold increase compared with cotransfection with the C/EBPβ or GATA-1 expression vectors individually. In addition, GST pull-down experiments demonstrated a physical interaction between human GATA-1 and C/EBPβ. Expression of FOG (Friend of GATA), 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/EBPβ 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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