Sternberg E A, Spizz G, Perry W M, Vizard D, Weil T, Olson E N
Department of Biochemistry and Molecular Biology, University of Texas, M.D. Anderson Hospital and Tumor Institute, Houston 77030.
Mol Cell Biol. 1988 Jul;8(7):2896-909. doi: 10.1128/mcb.8.7.2896-2909.1988.
Terminal differentiation of skeletal myoblasts is accompanied by induction of a series of tissue-specific gene products, which includes the muscle isoenzyme of creatine kinase (MCK). To begin to define the sequences and signals involved in MCK regulation in developing muscle cells, the mouse MCK gene has been isolated. Sequence analysis of 4,147 bases of DNA surrounding the transcription initiation site revealed several interesting structural features, some of which are common to other muscle-specific genes and to cellular and viral enhancers. To test for sequences required for regulated expression, a region upstream of the MCK gene from -4800 to +1 base pairs, relative to the transcription initiation site, was linked to the coding sequences of the bacterial chloramphenicol acetyltransferase (CAT) gene. Introduction of this MCK-CAT fusion gene into C2 muscle cells resulted in high-level expression of CAT activity in differentiated myotubes and no detectable expression in proliferating undifferentiated myoblasts or in nonmyogenic cell lines. Deletion mutagenesis of sequences between -4800 and the transcription start site showed that the region between -1351 and -1050 was sufficient to confer cell type-specific and developmentally regulated expression on the MCK promoter. This upstream regulatory element functioned independently of position, orientation, or distance from the promoter and therefore exhibited the properties of a classical enhancer. This upstream enhancer also was able to confer muscle-specific regulation on the simian virus 40 promoter, although it exhibited a 3- to 5-fold preference for its own promoter. In contrast to the cell type- and differentiation-specific expression of the upstream enhancer, the MCK promoter was able to function in myoblasts and myotubes and in nonmyogenic cell lines when combined with the simian virus 40 enhancer. An additional positive regulatory element was identified within the first intron of the MCK gene. Like the upstream enhancer, this intragenic element functioned independently of position, orientation, and distance with respect to the MCK promoter and was active in differentiated myotubes but not in myoblasts. These results demonstrate that expression of the MCK gene in developing muscle cells is controlled by complex interactions among multiple upstream and intragenic regulatory elements that are functional only in the appropriate cellular context.
骨骼肌成肌细胞的终末分化伴随着一系列组织特异性基因产物的诱导，其中包括肌酸激酶的肌肉同工酶（MCK）。为了开始确定发育中的肌肉细胞中MCK调控所涉及的序列和信号，已分离出小鼠MCK基因。对转录起始位点周围4147个碱基的DNA进行序列分析，发现了几个有趣的结构特征，其中一些特征是其他肌肉特异性基因以及细胞和病毒增强子所共有的。为了测试调控表达所需的序列，将相对于转录起始位点从-4800到+1碱基对的MCK基因上游区域与细菌氯霉素乙酰转移酶（CAT）基因的编码序列相连。将此MCK-CAT融合基因导入C2肌肉细胞后，在分化的肌管中导致CAT活性的高水平表达，而在增殖的未分化成肌细胞或非肌源性细胞系中未检测到表达。对-4800和转录起始位点之间的序列进行缺失诱变表明，-1351至-1050之间的区域足以赋予MCK启动子细胞类型特异性和发育调控表达。这个上游调控元件的功能独立于其位置、方向或与启动子的距离，因此表现出经典增强子的特性。这个上游增强子也能够赋予猿猴病毒40启动子肌肉特异性调控，尽管它对自身启动子表现出3至5倍的偏好。与上游增强子的细胞类型和分化特异性表达相反，当与猿猴病毒40增强子结合时，MCK启动子能够在成肌细胞、肌管和非肌源性细胞系中发挥作用。在MCK基因的第一个内含子中鉴定出另一个正向调控元件。与上游增强子一样，这个基因内元件的功能独立于其相对于MCK启动子的位置、方向和距离，并且在分化的肌管中具有活性，但在成肌细胞中无活性。这些结果表明，发育中的肌肉细胞中MCK基因的表达受多个上游和基因内调控元件之间复杂相互作用的控制，这些元件仅在适当的细胞环境中起作用。
