Berberich C, Dürr I, Koenen M, Witzemann V
Max-Planck-Institut für medizinische Forschung, Abteilung Zellphysiologie, Heidelberg, Germany.
Eur J Biochem. 1993 Sep 1;216(2):395-404. doi: 10.1111/j.1432-1033.1993.tb18157.x.
We have isolated and analysed the 5' flanking region of the rat acetylcholine receptor (AChR) beta subunit gene and determined regulatory elements that confer muscle specificity. Deletion mapping revealed a minimal TATA-box-less promoter region containing an initiator motif. An 85-bp fragment has been shown to promote high muscle-specific expression of a chloramphenicol acetyltransferase (CAT) reporter construct upon transfection in primary muscle cells. This sequence can be functionally dissected in a basal muscle-specific promoter element carrying a M-CAT box that is flanked at the 5' end by an enhancer element with two binding sites for myogenic factors. Point mutations in the M-CAT box cause the loss of transcriptional activity of the basal promoter fragment. The enhancer activity depends on the presence of both E boxes that cooperate in a synergistic fashion. We therefore conclude that the control of muscle-specific and developmental expression of the rat AChR beta subunit gene requires both regulatory elements, the M-CAT box and two adjacent E boxes, located in close proximity to each other. Cotransfection experiments in NIH3T3 cells demonstrate that the rat AChR beta subunit gene can be transactivated by myogenic factors displaying a preference for myogenin, as well as MRF4 and myf5 compared to a clearly weaker responsiveness to MyoD1.
我们分离并分析了大鼠乙酰胆碱受体（AChR）β亚基基因的5'侧翼区域，确定了赋予肌肉特异性的调控元件。缺失图谱分析揭示了一个最小的无TATA盒启动子区域，其中包含一个起始子基序。一个85bp的片段已被证明在转染原代肌肉细胞后能促进氯霉素乙酰转移酶（CAT）报告基因构建体的高肌肉特异性表达。该序列在一个携带M-CAT盒的基础肌肉特异性启动子元件中可进行功能剖析，该启动子元件在5'端侧翼有一个增强子元件，带有两个成肌因子结合位点。M-CAT盒中的点突变导致基础启动子片段转录活性丧失。增强子活性取决于两个以协同方式合作的E盒的存在。因此，我们得出结论，大鼠AChRβ亚基基因的肌肉特异性和发育表达的控制需要两个调控元件，即M-CAT盒和两个彼此紧邻的相邻E盒。在NIH3T3细胞中的共转染实验表明，与对MyoD1明显较弱的反应性相比，大鼠AChRβ亚基基因可被对肌细胞生成素以及MRF4和myf5表现出偏好的成肌因子反式激活。
