Isolation of genes involved in the commitment of eosinophils and basophils

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3.2kb的MBP上游序列和一系列5'缺失亚克隆到pXP2荧光素酶报告载体中。当bp -117和-67之间的MBP序列被删除时，启动子活性降低了80%。为了鉴定结合并通过bp -117至-67区域反激活的转录因子，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50bp区域保守一个潜在的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（emsa），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，联合转染GATA-1和GATA-2会使GATA-1反激活MBP启动子的能力降低约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在骨髓形成过程中对MBP表达和嗜酸性粒细胞基因转录具有负调控作用。

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 3.2kb of MBP upstream sequence and serial 5' deletions into the pXP2 luciferase reporter vector. An 80% decrement in promoter activity was obtained when MBP sequences between bp -117 and -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 50bp region between the two genes. To determine which GATA proteins bind this consensus site, we performed electrophoretic mobility shift assays (EMSAs) which demonstrated 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 combinantion, can transactivate the MBP promoter in the Jurkat T cell line. Co-transfection with a GATA-1 expression vector produced 20-fold augmentation of MBP promoter activity, whereas GATA-2 had no activity. In contrast, combined co-transfection 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, and a negative regulatory role for GATA-2 in MBP expression, and possibly eosinophil gene transcription in general during myelopoiesis.