Mechanisms of transcription in eosinophils: GATA-1, but not GATA-2, transactivates the promoter of the eosinophil granule major basic protein gene.
Granule major basic protein (MBP) is expressed exclusively in eosinophils, basophils, and placental trophoblasts. To identify the cis-elements and transcription factors involved in regulating MBP expression, we subcloned 3.2 kb of sequence upstream of the exon 9 transcriptional start site (P2 promoter) and serial 5' deletions into the pXP2 luciferase reporter vector. An 80% decrement in promoter activity was obtained when MBP sequences between bp -117 to -67 were deleted. To identify transcription factors that bind to and transactivate through the bp -117 to -67 region, we first compared the upstream genomic sequences of human and murine MBP; a potential GATA binding consensus site was conserved in the 50-bp region between the two genes. To determine which GATA proteins bind this consensus site, we performed electrophoretic mobility shift assays (EMSAs), which showed that both GATA-1 and GATA-2 can bind to this consensus site. To determine the functionality of this site, we tested whether GATA-1 and GATA-2, either individually or in combination, can transactivate the MBP promoter in the Jurkat T cell line. Cotransfection with a GATA-1 expression vector produced 20-fold augmentation of MBP promoter activity, whereas GATA-2 had no activity. In contrast, combined cotransfection of GATA-1 and GATA-2 decreased the ability of GATA-1 to transactivate the MBP promoter by approximately 50%. Our results provide the first evidence for a GATA-1 target gene in eosinophils, a negative regulatory role for GATA-2 in MBP expression, and possibly eosinophil gene transcription in general during myelopoiesis.
Yamaguchi
Y
Y
Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, University School of Medicine, Kumamoto, Japan.
Ackerman
S J
SJ
Minegishi
N
N
Takiguchi
M
M
Yamamoto
M
M
Suda
T
T
D001665:Binding Sites; D001798:Blood Proteins / Q000235:genetics*; D001854:Bone Marrow Cells / Q000166:cytology; D002454:Cell Differentiation; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000502:physiology*; D047091:Eosinophil Granule Proteins; D004804:Eosinophils / Q000502:physiology*; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D050989:GATA2 Transcription Factor; D006410:Hematopoiesis / Q000187:drug effects; D006801:Humans; D015848:Interleukin-5 / Q000502:physiology; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D012260:Ribonucleases; D017384:Sequence Deletion; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D015854:Up-Regulation
D001798:Blood Proteins; D004268:DNA-Binding Proteins; D047091:Eosinophil Granule Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; D050989:GATA2 Transcription Factor; C494711:GATA2 protein, human; D015848:Interleukin-5; D012333:RNA, Messenger; D014157:Transcription Factors; D012260:Ribonucleases