CHARACTERIZATION OF THE FUNCTIONAL-ROLE OF E-BOX ELEMENTS FOR THE TRANSCRIPTIONAL ACTIVITY OF RAT ACETYLCHOLINE-RECEPTOR EPSILON-SUBUNIT AND GAMMA-SUBUNIT GENE PROMOTERS IN PRIMARY MUSCLE-CELL CULTURES
CHARACTERIZATION OF THE FUNCTIONAL-ROLE OF E-BOX ELEMENTS FOR THE TRANSCRIPTIONAL ACTIVITY OF RAT ACETYLCHOLINE-RECEPTOR EPSILON-SUBUNIT AND GAMMA-SUBUNIT GENE PROMOTERS IN PRIMARY MUSCLE-CELL CULTURES
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DOI：
10.1111/j.1432-1033.1994.00353.x
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发表时间：
1994-09-01
期刊：
影响因子：
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通讯作者：
WITZEMANN, V
中科院分区：
文献类型：
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作者：
DURR, I;NUMBERGER, M;WITZEMANN, V
哺乳动物骨骼肌乙酰胆碱受体γ亚基和epsilon亚基的表达在肌源性分化和神经支配过程中受到独立调控。含有ε -亚基和γ -亚基基因5′侧序列的基因组DNA片段的特征是与氯霉素-乙酰转移酶基因融合的一系列5′缺失，并通过转染大鼠肌肉细胞和非肌肉细胞的原代培养短暂表达。一个6.3 kb的epsilon亚基片段可以减少为270 bp的片段，其在肌肉细胞中的表达水平比在非肌肉细胞中的表达水平高5-10倍。由核苷酸-185至-128组成的区域适度增加转录活性，而在核苷酸-88至-83处含有单个E盒的14 bp回文可能与启动子相互作用，但在肌肉细胞中没有增强子特性。γ -亚基基因的1.1 kb基因组片段中，167 bp足以用于肌肉特异性表达。两个启动子-近端E-box元件增强肌肉中的启动子活性并介导肌生成因子的转激活。Myogenin和myf5比MRF4和MyoD1更有效，而MRF4和MyoD1仅发挥少量的转激活作用。共转染实验表明，原代肌细胞中Id表达的增加抑制了γ -亚单位基因启动子介导的氯霉素乙酰转移酶的表达，支持了成肌因子在γ -亚单位基因转录调控中发挥重要作用的观点。
The expression of gamma and epsilon subunits of the acetylcholine receptor from mammalian skeletal muscle is regulated independently during myogenic differentiation and innervation. Genomic DNA fragments containing 5'-flanking sequences of the epsilon-subunit and gamma-subunit genes were characterised by a series of 5' deletions fused to the chloramphenicol-acetyltransferase gene and transiently expressed by transfection of primary cultures of rat muscle cells and non-muscle cells. A 6.3-kb epsilon-subunit fragment can be reduced to yield a 270-bp fragment that confers 5-10-times higher expression levels in muscle cells compared to in non-muscle cells. The region composed of nucleotides -185 to -128 increases the transcriptional activity moderately while the 14-bp palindrome containing a single E box at nucleotides -88 to -83 may interact with the promoter but has no enhancer properties in muscle cells. From a 1.1-kb genomic fragment of the gamma-subunit gene, 167 bp were sufficient for muscle-specific expression. Two promoter-proximal E-box elements enhance promoter activity in muscle and mediate transactivation by myogenic factors. Myogenin and myf5 were much more efficient than MRF4 or MyoD1 which exerted only little transactivation. Cotransfection experiments show that increased expression of Id in primary muscle cells inhibits chloramphenicol-acetyltransferase expression mediated by the gamma-subunit gene promoter and support the view that myogenic factors play an important role in the transcriptional regulation of the gamma-subunit gene.