MULTIPLE REGULATORY ELEMENTS CONTRIBUTE DIFFERENTIALLY TO MUSCLE CREATINE-KINASE ENHANCER ACTIVITY IN SKELETAL AND CARDIAC-MUSCLE
MULTIPLE REGULATORY ELEMENTS CONTRIBUTE DIFFERENTIALLY TO MUSCLE CREATINE-KINASE ENHANCER ACTIVITY IN SKELETAL AND CARDIAC-MUSCLE
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DOI：
10.1128/mcb.13.5.2753
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发表时间：
1993-05-01
影响因子：
5.3
通讯作者：
HAUSCHKA, SD
中科院分区：
文献类型：
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作者：
AMACHER, SL;BUSKIN, JN;HAUSCHKA, SD
我们使用瞬时转染MM14骨骼肌细胞、新生大鼠原代心室心肌细胞和非肌肉细胞来表征小鼠肌肌酸激酶（MCK）基因的调控元件。对mck5 '-侧翼序列的缺失分析显示，在-1256和-1020之间存在横纹肌特异性的正调控区。来自该区域的206bp片段作为骨骼肌增强剂，并在心肌细胞中赋予定向依赖性活性。110 bp的增强子亚片段在骨骼肌细胞中高水平表达，但在心肌细胞中不活跃，这表明骨骼肌和心肌MCK调节位点是可区分的。为了进一步描述肌肉调控序列，我们测试了MCK增强子中的六个位点，以确定它们的功能重要性。五个位点的突变降低了骨骼肌、心肌和非肌肉细胞的表达。其中两个位点（左E盒和MEF2）的突变导致所有三种细胞类型的相似减少。三个位点的突变对肌肉细胞的影响大于非肌肉细胞；富A/ t位点突变在两种横纹肌类型中都有明显的影响，MEF1（右E-box）位点的突变相对特异于骨骼肌的表达，而CArG位点的突变相对特异于心肌的表达。AP2位点的变化倾向于增加肌肉细胞的表达，而降低非肌肉细胞的表达。与表达肌生成决定因子MyoD的质粒共转染10T1/2细胞的报道相反，我们发现多聚化的MEF1位点的骨骼肌细胞活性比206 bp增强子的活性低30倍。因此，仅MyoD结合位点不足以在含有内源性MyoD和其他肌源性决定因子的骨骼肌细胞中实现高水平表达。
We have used transient transfections in MM14 skeletal muscle cells, newborn rat primary ventricular myocardiocytes, and nonmuscle cells to characterize regulatory elements of the mouse muscle creatine kinase (MCK) gene. Deletion analysis of MCK 5'-flanking sequence reveals a striated muscle-specific, positive regulatory region between -1256 and -1020. A 206-bp fragment from this region acts as a skeletal muscle enhancer and confers orientation-dependent activity in myocardiocytes. A 110-bp enhancer subfragment confers high-level expression in skeletal myocytes but is inactive in myocardiocytes, indicating that skeletal and cardiac muscle MCK regulatory sites are distinguishable. To further delineate muscle regulatory sequences, we tested six sites within the MCK enhancer for their functional importance. Mutations at five sites decrease expression in skeletal muscle, cardiac muscle, and nonmuscle cells. Mutations at two of these sites, Left E box and MEF2, cause similar decreases in all three cell types. Mutations at three sites have larger effects in muscle than nonmuscle cells; an A/T-rich site mutation has a pronounced effect in both striated muscle types, mutations at the MEF1 (Right E-box) site are relatively specific to expression in skeletal muscle, and mutations at the CArG site are relatively specific to expression in cardiac muscle. Changes at the AP2 site tend to increase expression in muscle cells but decrease it in nonmuscle cells. In contrast to reports involving cotransfection of 10T1/2 cells with plasmids expressing the myogenic determination factor MyoD, we show that the skeletal myocyte activity of multimerized MEF1 sites is 30-fold lower than that of the 206-bp enhancer. Thus, MyoD binding sites alone are not sufficient for high-level expression in skeletal myocytes containing endogenous levels of MyoD and other myogenic determination factors.