鸡AChRγ亚基基因远上游增强子样作用依赖生肌因子

Activation Function of the Chick Muscle Acetylcholine Receptor γ-Subunit Distal Upstream Sequence Depends on the Expression of Myogenic Factors

先前曾报告鸡ＡＣｈＲγ亚基基因５＇连接区－５３８／－２８８片段（含一对Ｅ盒子）与生肌调节因子（ｍｙｏｇｅｎｉｎ）的相互作用，为进一步观察ＤＮＡ－ｍｙｏｇｅｎｉｎ相互作用对γ基因转录激活功能的影响，利用瞬时表达体系研究了鸡ｎＡＣｈＲγ亚基基因５＇端－５３８／－２８８片段调控机能。ＣＡＴ分析表明：γ基因－５３８／－２８８片段可显著激活ｔｋ启动子在分化的Ｃ２Ｃ１２肌细胞中的转录活性，但不能激活在未分化Ｃ２Ｃ１２肌细胞中的转录活性。这种组织特异性转录激活作用与距离无关，因而是一个典型的增强子；－５３８／－２８８片段的增强子样作用既依赖于两毗邻Ｅ盒子的完整性，又依赖于ｍｙｏｇｅｎｉｎ等生肌调节因子的存在。强化表达ｍｙｏｇｅｎｉｎ可激活ｐＢＬＣＡＴ２－γ（－５３８／－４３３）和ｐＢＬＣＡＴ２－γ（－４３２／－２８８）（各含一个Ｅ盒子）在分化肌细胞中的表达，究竟是克隆片段中的独立Ｅ盒子对这种表达起贡献还是出现在克隆位点附近的反向Ｅ盒子（ＧＴＣＧＡＣ）或ｔｋ启动子中的另一个Ｅ盆子起作用？尚不十分清楚。

The specific binding of distal upstream sequence of the chick nicotinic acetylcholine receptor (nAChR) γ-subunit gene with myogenic factor myogenin has been reported.To test the effect of the interaction between distal upstream sequence of γ-subunit gene and myogenin on the transcriptional activation of AChR γ-subunit gene, -538/-288 fragment was fused into upstream or downstream of the Herpes Simples Virus tk promoter in pBLCAT-2 to generate pBLCAT2-γ (-538/-288) expression vectors and transfection of these plasmids into C2C12 muscle cell lines was performed. Chloramphenicol acetyltransferase (CAT) assays indicated that -538/-288 fragment of γ gene could confer specific expression in differentiated C2C12 myotubes, but not in undifferentiated C2C12 myoblasts. The tissue-specific activation function of the -538/-288 fragment depended upon both E boxes located at -429/-424 and -463/-458 and myogenin expression in differentiated C2C12 cells. pBLCAT2-γ (-538/-433) and pBLCAT2-γ (-432/-288) which contain only single E box also expressed in C2C12 myotubes where forced expression of myogenin occurred. The possible explanation for this result is due to the existence of the reverse E box (GTCGAC) surrounding the cloning site or another E box (CAGCTG) in the tk promoter.