Christensen T H, Kedes L
Institute for Genetic Medicine, Department of Biochemistry and Molecular Biology, Keck School of Medicine of the University of Southern California, Los Angeles 90033, USA.
Gene Expr. 1999;8(4):247-61.
We have characterized the specific DNA regulatory elements responsible for the function of the human cardiac troponin C gene (cTnC) muscle-specific enhancer in myogenic cells. We used functional transient transfection assays with deletional and site-specific mutagenesis to evaluate the role of the conserved sequence elements. Gel electrophoresis mobility shift assays (EMSA) demonstrated the ability of the functional sites to interact with nuclear proteins. We demonstrate that three distinct transcription activator binding sites commonly found in muscle-specific enhancers (a MEF-2 site, a MEF-3 site, and at least four redundant E-box sites) all contribute to full enhancer activity but a CArG box does not. Mutation of either the MEF-2 or MEF-3 sites or deletion of the E-boxes reduces expression by 70% or more. Furthermore, the MEF-2 site and the E-boxes specifically bind, respectively, to MEF-2 and myogenic determination factors derived from nuclear extracts. EMSA assays using a MEF-3 containing oligonucleotide revealed indistinguishable separation patterns with extracts from myogenic cells and nonmyogenic cells. These data suggest that expression of the cTnC gene in slow-twitch skeletal muscle is sustained through complex interactions at the 3'Ile enhancer between muscle-specific and nontissue-specific transcription factors: either a myogenic bHLH complex or MEF-2 can activate transcription but only in the presence of a third transcriptional activator that appears not to be muscle specific. We conclude from these observations that the cTnC 3'Ile element is a composite enhancer that functions through the combined interactions of at least five regulatory elements and their cognate binding factors: three or four E-boxes, a MEF-2 site, and a MEF-3 site. The data support the notion that all of these sites contribute to enhancer function in cell systems in an additive way but that none are absolutely required for enhancer activity. The data imply that the levels of transcription of cTnC in myogenic tissues in which the activities of one of the transcriptional factors is lacking would be partially but not wholly suppressed. Our data support the critical role of E-box sites in conjunction with the adjacent elements. Hence, we assign CTnC gene regulation to the "ordinary" rather than to the "novel" category of transcriptional regulation during skeletal myogenesis.
我们已经鉴定了在成肌细胞中负责人类心肌肌钙蛋白C基因（cTnC）肌肉特异性增强子功能的特定DNA调控元件。我们使用了带有缺失和位点特异性诱变的功能性瞬时转染试验来评估保守序列元件的作用。凝胶电泳迁移率变动分析（EMSA）证明了功能位点与核蛋白相互作用的能力。我们证明，在肌肉特异性增强子中常见的三个不同的转录激活因子结合位点（一个MEF-2位点、一个MEF-3位点和至少四个冗余的E-box位点）都对增强子的完全活性有贡献，但一个CArG框没有。MEF-2或MEF-3位点的突变或E-boxes的缺失会使表达降低70%或更多。此外，MEF-2位点和E-boxes分别特异性地结合来自核提取物的MEF-2和生肌决定因子。使用含有MEF-3的寡核苷酸进行的EMSA分析显示，来自成肌细胞和非成肌细胞的提取物具有难以区分的分离模式。这些数据表明，慢肌骨骼肌中cTnC基因的表达是通过肌肉特异性和非组织特异性转录因子在3'Ile增强子处的复杂相互作用来维持的：生肌bHLH复合体或MEF-2都可以激活转录，但前提是存在一种似乎不是肌肉特异性的第三种转录激活因子。我们从这些观察结果中得出结论，cTnC 3'Ile元件是一个复合增强子，它通过至少五个调控元件及其同源结合因子的联合相互作用发挥功能：三个或四个E-boxes、一个MEF-2位点和一个MEF-3位点。数据支持这样一种观点，即所有这些位点都以累加的方式对细胞系统中的增强子功能有贡献，但没有一个位点是增强子活性绝对必需的。数据表明，在缺乏其中一种转录因子活性的成肌组织中，cTnC的转录水平将被部分但不是完全抑制。我们的数据支持E-box位点与相邻元件结合的关键作用。因此，我们将CTnC基因调控归为骨骼肌生成过程中“普通”而非“新颖”的转录调控类别。
