Identification of a novel cis-element required for the constitutive activity and osmotic response of the rat aldose reductase promoter
Identification of a novel cis-element required for the constitutive activity and osmotic response of the rat aldose reductase promoter
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DOI：
10.1074/jbc.272.51.32500
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发表时间：
1997-12-19
影响因子：
4.8
通讯作者：
Carper, D
中科院分区：
文献类型：
--
作者：
Iwata, T;Minucci, S;Carper, D
在大鼠肝细胞系中发现了一种新的必需的顺式元件AEE(醛糖还原酶增强元件)，它对大鼠肝细胞系中大鼠醛糖还原酶转录的构成活性和渗透应激反应是必需的。在瞬时转基因试验中，使用荧光素酶报告基因构建的荧光素酶报告基因结构，在渗透胁迫(600mom/kg H2O)下，启动子的活性增加了3.8倍，其中的醛糖还原酶启动子序列从-1,094个碱基对(BP)到+23个碱基对(+23)。在-1,071到-895bp之间的缺失降低了构成活性，并取消了启动子的渗透反应。核酸外切酶III介导的体内DNA足迹和体内硫酸二甲酯足迹分别显示了该区域的一个32bP区域和该区域内的两个鸟苷(G)的DNA保护作用。使用全肝细胞提取物的凝胶迁移率变化分析表明，在正常和应激条件下，蛋白质都是结合的。受体内硫酸二甲酯DNA足迹(GAAGAGTG)保护的两个鸟苷之间的序列在荧光素酶构建体(-1,094bp到+23bp)中的缺失取消了构成启动子的活性。一个拷贝的AEE融合到胸苷激酶启动子上，其最大构成活性为7.7倍，最大渗透反应活性为6.7倍。
A new and essential cis-element AEE (aldose reductase enhancer element), necessary for the constitutive activity and the osmotic stress response of rat aldose reductase transcription in a rat liver cell line, has been identified. In transient transfection assays, an increase in promoter activity, up to 3.8-fold, was observed with osmotic stress (600 mosm/kg H2O) using a luciferase reporter gene construct containing aldose reductase promoter sequence from -1,094 base pair (bp) to +23 bp. A deletion between -1,071 and -895 bp reduced the constitutive activity and abolished the osmotic response of the promoter. Exonuclease III mediated in vivo DNA footprinting and dimethyl sulfate in vivo footprinting revealed DNA protection of a 32-bp region and two guanosines (G) within this region protected from methylation, respectively. Electrophoretic gel mobility shift assays using whole liver cell extracts showed protein binding, under both normal and stressed conditions. Deletion of the sequence between the two guanosines protected by in vivo dimethyl sulfate DNA footprinting (GAAGAGTG) in a luciferase construct (-1,094 bp to +23 bp) abolished the constitutive promoter activity. One copy of AEE fused to the thymidine kinase promoter gave a maximum constitutive activity of 7.7-fold and a maximum osmotic response activity of 6.7-fold.