Muscat G E, Perry S, Prentice H, Kedes L
Department of Biochemistry, University of Queensland, St. Lucia, Australia.
Gene Expr. 1992;2(2):111-26.
The tissue-specific distal promoter of the human skeletal alpha-actin gene (-1282 to -708) induces transcription in myogenic cells approximately 10-fold and, with the most proximal promoter domain (-153 to -87), it synergistically increases transcription 100-fold (Muscat and Kedes 1987). We report here that it is a short fragment of the distal promoter, the distal regulatory element (DRE) from -1282 to -1177 that functions as a muscle-specific, composite enhancer. An internal deletion in the DRE (delta -1282/-1151) in the context of the full-length 2000 bp promoter, resulted in a 10-fold reduction in transcription. Three distinct nuclear proteins, DRF-1, DRF-2, and DRF-3, interact specifically with the DRE between positions -1260 and -1193. A site specific mutation that abolishes DRF-2 binding also results in a 10-fold reduction in transcriptional activity. The DRF-2 nuclear protein has characteristics similar to those of the muscle-specific regulatory factor, MEF-2 (Buskin and Hauschka 1989; Gossett et al., 1989). Like the MEF-2 binding site in the muscle creatine kinase enhancer, the critical DRF-2 binding site is also an A/T-rich sequence element. The DRF-2 nuclear protein binds equally well to the MCK MEF-2 binding site and to the A/T-rich regulatory element of the skeletal muscle fast-twitch troponin C gene (Gahlmann and Kedes 1990). Furthermore, this troponin C site competes in vivo for DRF-2 driven expression of the skeletal alpha-actin gene in C2 cells. The DRF-2 site alone, however, does not activate transcription in muscle cells when linked to the SV40 promoter. We conclude that the DRF-2 binding element is a MEF-2 binding site that is required but insufficient for regulation of muscle-specific skeletal alpha-actin gene expression by the DRE. Thus, muscle-specific regulation of the human skeletal alpha-actin gene appears to require interactions between the other elements of the composite DRE enhancer with the protein:DNA complex formed by DRF-2.
人类骨骼肌α-肌动蛋白基因的组织特异性远端启动子（-1282至-708）可诱导成肌细胞中的转录增加约10倍，并且与最近端的启动子结构域（-153至-87）协同作用，使转录增加100倍（马斯卡特和凯德斯，1987年）。我们在此报告，远端启动子的一个短片段，即从-1282至-1177的远端调控元件（DRE），作为一个肌肉特异性的复合增强子发挥作用。在全长2000 bp启动子的背景下，DRE中的内部缺失（δ-1282/-1151）导致转录减少10倍。三种不同的核蛋白，DRF-1、DRF-2和DRF-3，特异性地与-1260至-1193位之间的DRE相互作用。一个消除DRF-2结合的位点特异性突变也导致转录活性降低10倍。DRF-2核蛋白具有与肌肉特异性调控因子MEF-2相似的特征（巴斯金和豪施卡，1989年；戈塞特等人，1989年）。与肌肉肌酸激酶增强子中的MEF-2结合位点一样，关键的DRF-2结合位点也是一个富含A/T的序列元件。DRF-2核蛋白与MCK MEF-2结合位点以及骨骼肌快肌肌钙蛋白C基因的富含A/T的调控元件结合能力相当（加尔曼和凯德斯，1990年）。此外，这个肌钙蛋白C位点在体内竞争DRF-2驱动的C2细胞中骨骼肌α-肌动蛋白基因的表达。然而，单独的DRF-2位点与SV40启动子连接时，在肌肉细胞中不能激活转录。我们得出结论，DRF-2结合元件是一个MEF-2结合位点，它是DRE调节肌肉特异性骨骼肌α-肌动蛋白基因表达所必需的，但并不充分。因此，人类骨骼肌α-肌动蛋白基因的肌肉特异性调节似乎需要复合DRE增强子的其他元件与DRF-2形成的蛋白质:DNA复合物之间的相互作用。
