Zhang X, Xing G, Fraizer G C, Saunders G F
Department of Biochemistry and Molecular Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
J Biol Chem. 1997 Nov 14;272(46):29272-80. doi: 10.1074/jbc.272.46.29272.
The Wilms' tumor 1 gene (WT1) encodes a zinc-finger transcription factor which is expressed in a tissue-specific manner. Our studies indicate that in addition to the promoter, other regulatory elements are required for tissue-specific expression of this gene. A 258-base pair hematopoietic specific enhancer in intron 3 of the WT1 gene increased the transcriptional activity of the WT1 promoter by 8-10-fold in K562 and HL60 cells. Sequence analysis revealed both a GATA and a c-Myb motif in the enhancer fragment. Mutation of the GATA motif decreased the enhancer activity by 60% in K562 cells. Electrophoretic mobility shift assays showed that the GATA-1 protein in K562 nuclear extracts binds to this motif. Cotransfection of the enhancer containing reporter construct with a GATA-1 expression vector showed that GATA-1 transactivated this enhancer, increasing the CAT reporter activity 10-15-fold. Similar analysis of the c-Myb motif by cotransfection with the enhancer CAT reporter construct and a c-Myb expression vector showed that c-Myb transactivated the enhancer by 5-fold. A DNase I-hypersensitive site has also been mapped in the 258-base pair enhancer region. These data suggest that GATA-1 and c-Myb are responsible for the activity of this enhancer in hematopoietic cells and may bind to the enhancer in vivo.
威尔姆斯瘤1基因（WT1）编码一种以组织特异性方式表达的锌指转录因子。我们的研究表明，除启动子外，该基因的组织特异性表达还需要其他调控元件。WT1基因第3内含子中的一个258个碱基对的造血特异性增强子可使WT1启动子在K562和HL60细胞中的转录活性提高8至10倍。序列分析显示该增强子片段中存在一个GATA基序和一个c-Myb基序。GATA基序的突变使K562细胞中的增强子活性降低了60%。电泳迁移率变动分析表明，K562细胞核提取物中的GATA-1蛋白与该基序结合。将含增强子的报告基因构建体与GATA-1表达载体共转染表明，GATA-1可反式激活该增强子，使CAT报告基因活性提高10至15倍。通过将增强子CAT报告基因构建体与c-Myb表达载体共转染对c-Myb基序进行类似分析表明，c-Myb可使增强子的活性提高5倍。在258个碱基对的增强子区域也绘制了一个DNase I超敏位点。这些数据表明，GATA-1和c-Myb负责该增强子在造血细胞中的活性，并且可能在体内与该增强子结合。
