REGULATION OF THE HUMAN CARDIAC SLOW-TWITCH TROPONIN-C GENE BY MULTIPLE, COOPERATIVE, CELL-TYPE-SPECIFIC, AND MYOD-RESPONSIVE ELEMENTS
REGULATION OF THE HUMAN CARDIAC SLOW-TWITCH TROPONIN-C GENE BY MULTIPLE, COOPERATIVE, CELL-TYPE-SPECIFIC, AND MYOD-RESPONSIVE ELEMENTS
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DOI：
10.1128/mcb.13.11.6752
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发表时间：
1993-11-01
影响因子：
5.3
通讯作者：
KEDES, L
中科院分区：
文献类型：
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作者：
CHRISTENSEN, TH;PRENTICE, H;KEDES, L
心肌肌钙蛋白C（cTnC）基因在慢收缩骨骼肌和心肌中产生相同的转录本（R。加尔曼河Wade，P. Gunning，and L. Kedes，J. Mol. 201：379-391，1988）。一个单独的基因编码快速收缩骨骼肌肌钙蛋白C，在心肌中不表达。我们已经使用瞬时转染来表征负责骨骼和心脏细胞类型特异性表达的人cTnC（HcTnC）基因的调控元件。至少有四个独立的元件协同作用，以赋予该基因在分化的肌管中的组织特异性表达;基础启动子（-61和-13之间）增加转录9倍，上游主要调控序列（在-68和-142之间以及在-1319和-4500之间）使转录增加多达39倍，并且第一内含子中的至少两个增强子样元件（+58和+1028之间以及+1029和+1523之间）独立地使转录增加4至5倍。这些增强子在第一内含子增加肌管特异性氯霉素乙酰转移酶的活性时，连接到自己的启动子元件或异源猿猴病毒40启动子，效果是乘法，而不是加和。每个主要的肌管调节区都能够直接或间接地对生肌决定因子MyoD作出反应。10 T1/2细胞中的MyoD表达载体诱导了携带上游HcTnC启动子元件或基因第一内含子300至500倍的构建体。通过与Id共转染抑制表达，Id是碱性螺旋-环-螺旋转录因子的负调节因子。基础启动子包含5个串联TGGGC重复序列，与核提取物中的Spl或Spl-like因子相互作用。该元件的突变分析表明，五个重复序列中的两个足以支持基础水平的肌细胞特异性转录。尽管基础启动子对于心肌细胞中的表达也是关键的，但-67上游的元件似乎起很少或没有作用。心肌细胞中表达的主要增强也由第一内含子中的序列提供，但这些序列位于上游（+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细胞增强剂与来自C2细胞而不是心肌细胞的提取物形成特异性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节元件复杂相互作用的协同调节，并且不同的元件用于调节肌源性和心脏细胞的表达。
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 Spl or an Spl-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.