title: C/EBPbeta and GATA-1 synergistically regulate activity of the eosinophil granule major basic protein promoter: implication for C/EBPbeta activity in eosinophil gene expression.
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/EBPalpha mRNA expression decreased significantly concomitant with eosinophilic and neutrophilic differentiation, whereas C/EBPbeta expression was markedly increased. Electrophoretic mobility shift assays (EMSAs) showed that recombinant C/EBPbeta 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/EBPbeta 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/EBPbeta and GATA-1 can stimulate the MBP promoter in the C/EBPbeta and GATA-1 negative Jurkat T-cell line. Cotransfection with C/EBPbeta and GATA-1 expression vectors produced a 5-fold increase compared with cotransfection with the C/EBPbeta or GATA-1 expression vectors individually. In addition, GST pull-down experiments demonstrated a physical interaction between human GATA-1 and C/EBPbeta. 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/EBPbeta 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.
authors.affiliation: Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, Kumamoto, Japan. yujiya@gpo.kumamoto-u.ac.jp
authors.forename: Y
authors.initials: Y
authors.lastname: Yamaguchi
authors.forename: H
authors.initials: H
authors.lastname: Nishio
authors.forename: K
authors.initials: K
authors.lastname: Kishi
authors.forename: S J
authors.initials: SJ
authors.lastname: Ackerman
authors.forename: T
authors.initials: T
authors.lastname: Suda
mesh_terms: D001483:Base Sequence; D001798:Blood Proteins / Q000502:physiology*; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins / Q000502:physiology*; D047091:Eosinophil Granule Proteins; D004804:Eosinophils / Q000502:physiology*; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D005786:Gene Expression Regulation / Q000502:physiology*; D018922:HL-60 Cells; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000502:physiology*; D011401:Promoter Regions, Genetic; D012260:Ribonucleases; D015398:Signal Transduction; D014157:Transcription Factors / Q000502:physiology*
chemical_list: 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; C494688:GATA1 protein, human; D009687:Nuclear Proteins; D014157:Transcription Factors; D012260:Ribonucleases