Mechanisms of transcription in eosinophils: GATA-1, but not GATA-2, transactivates the promoter of the eosinophil granule major basic protein gene
Mechanisms of transcription in eosinophils: GATA-1, but not GATA-2, transactivates the promoter of the eosinophil granule major basic protein gene
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DOI：
10.1182/blood.v91.9.3447.3447_3447_3458
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发表时间：
1998-05-01
期刊：
影响因子：
20.3
通讯作者：
Suda, T
中科院分区：
文献类型：
--
作者：
Yamaguchi, Y;Ackerman, SJ;Suda, T
颗粒主要碱性蛋白（MBP）仅在嗜酸性粒细胞、嗜碱性粒细胞和胎盘滋养细胞中表达。为了鉴定参与调控MBP表达的顺式元件和转录因子，我们将第9外显子转录起始位点（P2启动子）上游的3.2 kb序列和一系列5'缺失亚克隆到pXP2荧光素酶报告载体中。当bp -117至-67之间的MBP序列被删除时，启动子活性降低80%。为了确定通过bp -117至-67区域结合和反激活的转录因子，我们首先比较了人类和小鼠MBP的上游基因组序列：在两个基因之间的50 bp区域保守了一个潜在的GATA结合共识位点。为了确定哪些GATA蛋白结合了这个共识位点，我们进行了电泳迁移量转移试验（EMSAs），结果表明GATA-1和GATA-2都可以结合这个共识位点。为了确定该位点的功能，我们测试了GATA-1和GATA-2单独或联合是否可以反激活Jurkat T细胞系中的MBP启动子。GATA-1表达载体的共转染使MBP启动子活性增加了20倍，而GATA-2没有活性。相比之下，GATA-1和GATA-2的联合共转染使GATA-1反激活MBP启动子的能力降低了约50%。我们的研究结果首次证明了GATA-1靶基因在嗜酸性粒细胞中存在，GATA-2在MBP表达中具有负调控作用，并可能在骨髓形成过程中普遍存在嗜酸性粒细胞基因转录。(C) 1998年由美国血液病学会出版。
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. (C) 1998 by The American Society of Hematology.