van de Klundert F A, Jansen H J, Bloemendal H
University of Nijmegen, Faculty of Science, Department of Biochemistry, The Netherlands.
J Biol Chem. 1994 Jan 7;269(1):220-5.
The muscle-specific intermediate filament protein desmin is up-regulated during skeletal muscle differentiation. When myoblasts leave the cell cycle and fusion into multinucleated myotubes starts, genes associated with myogenesis become activated. The activation is believed to be mediated by the muscle-specific determination factors. We present evidence that both MyoD and myogenin are able to activate the transcription of the hamster desmin gene. A proximal promoter fragment of 89 base pairs is sufficient for this transactivation process. The single E-box in this region is essential for desmin promoter activity in mouse C2 skeletal muscle cells and upon co-transfection of a myogenin expression vector also in human primary fibroblasts. Mutation of this MyoD binding site abrogates desmin transcription, and transactivation of the promoter no longer occurs. By using gel electrophoretic mobility shift assays, we were able to demonstrate that nuclear proteins from C2 muscle cells and myogenin/E12 glutathione S-transferase fusion proteins are able to bind to the functional E-box consensus sequence. A second E-box, situated in a more upstream regulatory region, which also binds to purified Helix-Loop-Helix proteins in vitro is only moderately affected by site-directed in vitro mutagenesis.
肌肉特异性中间丝蛋白结蛋白在骨骼肌分化过程中上调。当成肌细胞退出细胞周期并开始融合形成多核肌管时，与肌生成相关的基因被激活。据信这种激活是由肌肉特异性决定因子介导的。我们提供的证据表明，MyoD和肌细胞生成素都能够激活仓鼠结蛋白基因的转录。一个89个碱基对的近端启动子片段足以进行这种反式激活过程。该区域中的单个E盒对于小鼠C2骨骼肌细胞中的结蛋白启动子活性至关重要，并且在共转染肌细胞生成素表达载体时对人原代成纤维细胞中的启动子活性也至关重要。这个MyoD结合位点的突变消除了结蛋白转录，并且启动子的反式激活不再发生。通过使用凝胶电泳迁移率变动分析，我们能够证明来自C2肌肉细胞的核蛋白和肌细胞生成素/E12谷胱甘肽S-转移酶融合蛋白能够结合到功能性E盒共有序列上。位于更上游调节区域中的第二个E盒，其在体外也与纯化的螺旋-环-螺旋蛋白结合，仅受到定点体外诱变的中度影响。
