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首页> 外文期刊>Molecular and Cellular Biology >Regulation of the human cardiac/slow-twitch troponin C gene by multiple, cooperative, cell-type-specific, and MyoD-responsive elements.
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Regulation of the human cardiac/slow-twitch troponin C gene by multiple, cooperative, cell-type-specific, and MyoD-responsive elements.

机译：人类心脏/慢肌钙蛋白C基因的调控是由多个，协同的，细胞类型特异性的和MyoD响应的元素组成的。

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The cardiac troponin C (cTnC) gene produces identical transcripts in slow-twitch skeletal muscle and in heart muscle (R. Gahlmann, R. Wade, P. Gunning, and L. Kedes, J. Mol. Biol. 201:379-391, 1988). A separate gene encodes the fast-twitch skeletal muscle troponin C and is not expressed in heart muscle. We have used transient transfection to characterize the regulatory elements responsible for skeletal and cardiac cell-type-specific expression of the human cTnC (HcTnC) gene. At least four separate elements cooperate to confer tissue-specific expression of this gene in differentiated myotubes; a basal promoter (between -61 and -13) augments transcription 9-fold, upstream major regulatory sequences (between -68 and -142 and between -1319 and -4500) augment transcription as much as 39-fold, and at least two enhancer-like elements in the first intron (between +58 and +1028 and between +1029 and +1523) independently augment transcription 4- to 5-fold. These enhancers in the first intron increase myotube-specific chloramphenicol acetyltransferase activity when linked to their own promoter elements or to the heterologous simian virus 40 promoter, and the effects are multiplicative rather than additive. Each of the major myotube regulatory regions is capable of responding directly or indirectly to the myogenic determination factor, MyoD.A MyoD expression vector in 10T1/2 cells induced constructs carrying either the upstream HcTnC promoter elements or the first intron of the gene 300- to 500-fold. Expression was inhibited by cotransfection with Id, a negative regulator of basic helix-loop-helix transcription factors. The basal promoter contains five tandem TGGGC repeats that interact with Sp1 or an Sp1-like factor in nuclear extracts. Mutational analysis of this element demonstrated that two of the five repeat sequences were sufficient to support basal level muscle cell-specific transcription. Whereas the basal promoter is also critical for expression in cardiac myocytes, the elements upstream of -67 appear to play little or no role. Major augmentation of expression in cardiomyocytes is also provided by sequences in the first intron, but these are upstream (between +58 and +1028). The downstream segment of the first intron has no enhancer activity in cardiomyocytes. A specific DNA-protein complex is formed by this C2 cell enhancer with extracts from C2 cells but not cardiomyocytes. These observations suggest that tissue-specific expression of the HcTnC gene is cooperatively regulated by the complex interactions of multiple regulatory elements and that different elements are used to regulate expression in myogenic and cardiac cells.
机译：心肌肌钙蛋白C（cTnC）基因在慢肌骨骼肌和心肌中产生相同的转录本（R.Gahlmann，R.Wade，P.Gunning和L.Kedes，J.Mol.Biol.201：379-391 （1988年）。单独的基因编码快肌骨骼肌肌钙蛋白C，但在心肌中不表达。我们已经使用瞬时转染来表征负责人cTnC（HcTnC）基因的骨骼和心脏细胞类型特异性表达的调控元件。至少四个单独的元件协同作用以赋予该基因在分化的肌管中的组织特异性表达。一个基础启动子（在-61和-13之间）将转录增加9倍，上游主要调控序列（在-68和-142之间以及在-1319和-4500之间）将转录增加39倍，并且至少两个增强子第一个内含子中的类似类元素（在+58和+1028之间以及在+1029和+1523之间）独立地将转录增加4至5倍。当第一个内含子中的这些增强子与它们自己的启动子元件或与异源猿猴病毒40启动子连接时，它们会增加肌管特异性氯霉素乙酰转移酶的活性，其作用是乘性的而不是累加的。每个主要的肌管调节区都能够直接或间接地对肌决定因子MyoD作出反应.10T1 / 2细胞诱导的构建物中的MyoD表达载体携带上游HcTnC启动子元件或基因300-的第一个内含子。 500倍与Id（碱性螺旋-环-螺旋转录因子的负调节剂）共转染可抑制表达。基础启动子包含五个串联的TGGGC重复序列，它们与核提取物中的Sp1或Sp1样因子相互作用。此元素的突变分析表明，五个重复序列中的两个足以支持基础水平的肌肉细胞特异性转录。尽管基础启动子对于在心肌细胞中表达也很关键，但-67上游的元件似乎起着很小的作用或没有作用。第一个内含子中的序列还提供了心肌细胞中表达的主要增强，但是这些序列是上游的（在+58和+1028之间）。第一内含子的下游区段在心肌细胞中没有增强子活性。这种C2细胞增强剂与C2细胞而非心肌细胞的提取物形成特异性的DNA-蛋白质复合物。这些观察结果表明，HcTnC基因的组织特异性表达受到多种调控元件复杂相互作用的共同调控，并且不同的元件被用于调控成肌细胞和心脏细胞的表达。

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