Identification of a skeletal muscle-specific regulatory domain in the rat GLUT4/muscle-fat gene.
Identification of a skeletal muscle-specific regulatory domain in the rat GLUT4/muscle-fat gene.
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DOI：
10.1016/s0021-9258(19)36888-7
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发表时间：
1993-10
期刊：
影响因子：
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通讯作者：
Jeanne M. Richardson;J. Pessin
中科院分区：
文献类型：
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作者：
Jeanne M. Richardson;J. Pessin
为了确定负责大鼠GLUT4/肌肉脂肪基因肌肉特异性表达的序列，我们检测了该基因在分化小鼠C2C12骨骼肌细胞系中的转录调控。与未分化的成肌细胞相比，分化的肌纤维显示GLUT4 mRNA增加4-5倍，这与非肌肉β -肌动蛋白mRNA表达向肌肉特异性α -肌动蛋白mRNA表达的转化相似。瞬时转染GLUT4 5‘侧DNA的渐进5’和3'缺失，鉴定出位于相对于转录起始位点-517和-237之间的281个碱基对区域，该区域具有肌管特异性表达。在GLUT4最小启动子背景下，该区域以不依赖方向的方式增加了报告基因活性，此外，在异源胸苷激酶启动子上也增加了报告基因活性。GLUT4报告基因结构和内源性小鼠GLUT4 mRNA的肌管特异性表达也被观察到是甲状腺激素依赖的。此外，将含有281碱基对GLUT4分化特异性增强子的报告构建物与甲状腺激素受体共转染，可特异性地将肌管中的荧光素酶活性提高约12倍。因此，这些数据证明了近端骨骼肌特异性激活域的存在，这对于肌管特异性GLUT4表达和甲状腺激素反应都是必要的。
To identify sequences responsible for the muscle-specific expression of the rat GLUT4/muscle-fat gene, we examined the transcriptional regulation of this gene in the differentiating murine C2C12 skeletal muscle cell line. Differentiated myofibers displayed a 4-5-fold increase in GLUT4 mRNA compared with undifferentiated myoblasts which paralleled the conversion from non-muscle beta-actin mRNA to muscle-specific alpha-actin mRNA expression. Transient transfection of progressive 5' and 3' deletions of the GLUT4 5'-flanking DNA identified a 281-base pair region located between -517 and -237 relative to the transcription start site which conferred myotube-specific expression. This region increased reporter activity in the context of the GLUT4 minimal promoter in an orientation-independent manner and, in addition, onto the heterologous thymidine kinase promoter. Myotube-specific expression of both GLUT4 reporter constructs and the endogenous mouse GLUT4 mRNA was also observed to be thyroid hormone-dependent. Further, cotransfection of reporter constructs containing the 281-base pair GLUT4 differentiation-specific enhancer with the thyroid hormone receptor specifically increased luciferase activity in myotubes approximately 12-fold. Thus, these data demonstrate the presence of a proximal skeletal muscle-specific activation domain that is necessary for both myotube-specific GLUT4 expression and thyroid hormone responsiveness.