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脂肪酸合酶启动子的胰岛素调节需要上游刺激因子与 -65 处的 E 盒结合。

Upstream stimulatory factor binding to the E-box at -65 is required for insulin regulation of the fatty acid synthase promoter.

作者信息

Wang D, Sul H S

机构信息

Department of Nutritional Sciences, University of California, Berkeley, California 94720-3104, USA.

出版信息

J Biol Chem. 1997 Oct 17;272(42):26367-74. doi: 10.1074/jbc.272.42.26367.

Abstract

Fatty acid synthase (FAS) plays a central role in de novo lipogenesis in mammals. We have shown that FAS transcription rate is induced dramatically when fasted animals are refed with a high carbohydrate diet or when streptozotocin-diabetic mice are given insulin. We also reported that FAS gene transcription was up-regulated by insulin through the proximal promoter region from -71 to -50 and that upstream stimulatory factors (USFs), including USF1 and USF2, interact with this region in vitro. In the present study, by using site-directed mutagenesis of the -71/-50 region and correlating functional assays of the mutated promoter with USF binding activities, we demonstrate that the -65/-60 E-box motif (5'-CATGTG-3') is functionally required for insulin regulation and that USFs are in vivo components of the insulin response complex. Mutation of the -65/-60 E-box sequence abolished insulin response in both transiently and stably transfected 3T3-L1 adipocytes in the -2. 1 kb promoter context, which contains all the necessary regulatory elements of the promoter based on our previous transgenic mice studies, and in the minimal -67 promoter context. Gel mobility shift assays demonstrated that USFs can no longer bind to the -71/-50 promoter region when the E-box is mutated. Cotransfection of USF1 and USF2 expression vectors with the FAS promoter-luciferase reporter constructs increased insulin-stimulated FAS promoter activity. Moreover, cotransfection of dominant negative USF1 and USF2 mutants lacking the DNA binding domain inhibited the insulin stimulation of the FAS promoter activity. On the other hand, site-directed mutagenesis of the -65/-60 E-box surrounding sequences within the overlapped tandem copies of sterol regulatory element-binding protein (SREBP) binding sites prevented SREBP from binding to -71/-50 promoter region in vitro but had no effect on insulin regulation of the FAS promoter in vivo. When rat liver nuclear extracts were used in gel mobility shift assays, only USF-containing protein-DNA complexes that can be supershifted by specific USF antibodies were observed. These results demonstrate that upstream stimulatory factor binding to the E-box at -65 is required for insulin regulation of the fatty acid synthase promoter.

摘要

脂肪酸合酶(FAS)在哺乳动物的从头脂肪生成中起核心作用。我们已经表明,当禁食动物重新喂食高碳水化合物饮食时,或给链脲佐菌素诱导的糖尿病小鼠注射胰岛素时,FAS转录速率会显著增加。我们还报道,胰岛素通过从 -71 到 -50 的近端启动子区域上调FAS基因转录,并且包括USF1和USF2在内的上游刺激因子(USFs)在体外与该区域相互作用。在本研究中,通过对 -71/-50 区域进行定点诱变,并将突变启动子的功能分析与USF结合活性相关联,我们证明 -65/-60 E-box 基序(5'-CATGTG-3')对于胰岛素调节在功能上是必需的,并且USFs是胰岛素反应复合物的体内组成部分。在 -2.1 kb 启动子背景下(根据我们之前的转基因小鼠研究,该启动子包含启动子的所有必要调控元件)以及在最小的 -67 启动子背景下,-65/-60 E-box 序列的突变消除了瞬时和稳定转染的 3T3-L1 脂肪细胞中的胰岛素反应。凝胶迁移率变动分析表明,当 E-box 突变时,USFs 不再能与 -71/-50 启动子区域结合。将 USF1 和 USF2 表达载体与 FAS 启动子 - 荧光素酶报告基因构建体共转染可增加胰岛素刺激的 FAS 启动子活性。此外,共转染缺乏 DNA 结合结构域的显性负性 USF1 和 USF2 突变体可抑制胰岛素对 FAS 启动子活性的刺激。另一方面,在固醇调节元件结合蛋白(SREBP)结合位点的重叠串联拷贝内对 -65/-60 E-box 周围序列进行定点诱变可阻止 SREBP 在体外与 -71/-50 启动子区域结合,但对体内 FAS 启动子的胰岛素调节没有影响。当在凝胶迁移率变动分析中使用大鼠肝核提取物时,仅观察到可被特异性 USF 抗体超迁移的含 USF 的蛋白质 - DNA 复合物。这些结果表明,上游刺激因子与 -65 处的 E-box 结合是胰岛素调节脂肪酸合酶启动子所必需的。

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