IDENTIFICATION OF A DNA ELEMENT DETERMINING SYNAPTIC EXPRESSION OF THE MOUSE ACETYLCHOLINE-RECEPTOR DELTA-SUBUNIT GENE
IDENTIFICATION OF A DNA ELEMENT DETERMINING SYNAPTIC EXPRESSION OF THE MOUSE ACETYLCHOLINE-RECEPTOR DELTA-SUBUNIT GENE
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DOI：
10.1073/pnas.92.23.10624
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发表时间：
1995-11-07
影响因子：
11.1
通讯作者：
CHANGEUX, JP
中科院分区：
文献类型：
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作者：
KOIKE, S;SCHAEFFER, L;CHANGEUX, JP
乙酰胆碱受体基因的mRNA高度集中在成人骨骼肌的终板区，这主要是由于转录限制在神经下核。为了鉴定所涉及的调节元件，我们使用了一种含有乙酰胆碱受体δ亚基基因启动子片段的质粒DNA注射，（位置-839至+45）连接到具有核定位信号的报告基因lacZ，将野生型构建体注射到小鼠腿部肌肉中产生了报告基因在突触区的优先表达，对各种突变启动子的分析导致鉴定出在神经下表达中起关键作用的DNA元件（位置-60至-49），称为N盒。在小鼠δ-亚基启动子的背景下，从位置-839至+45破坏该12-bp元件，使得报告基因在整个肌纤维中广泛表达，表明该元件是抑制接头外区域δ-亚基基因转录的沉默物。另一方面，该元件插入异源基础启动子的上游优先增强终板区域中的表达。因此，该元件既作为终板水平的增强子又作为接合外区域的沉默子调节δ亚基基因的受限表达。此外，小鼠肌肉提取物的凝胶迁移实验揭示了特异性结合该元件的6-bp序列TTCCGG的活性，表明转录因子通过该元件控制δ-亚基基因的表达。
mRNAs for acetylcholine receptor genes are highly concentrated in the endplate region of adult skeletal muscle largely as a result of a transcription restricted to the subneural nuclei, To identify the regulatory elements involved, we employed a DNA injection of a plasmid containing a fragment of the acetylcholine receptor delta-subunit gene promoter (positions -839 to +45) linked to the reporter gene lacZ with a nuclear localization signal, Injection of the wild-type construct into mouse leg muscles yielded preferential expression of the reporter gene in the synaptic region, Analysis of various mutant promoters resulted in the identification of a DNA element (positions -60 to -49), referred to as the N box, that plays a critical role in subneural expression. Disruption of this 12-bp element in the context of a mouse delta-subunit promoter from positions -839 to +45 gives widespread expression of the reporter gene throughout the entire muscle fiber, indicating that this element is a silencer that represses delta-subunit gene transcription in extrajunctional areas. On the other hand, this element inserted upstream of a heterologous basal promoter preferentially enhances expression in the endplate region. This element therefore regulates the restricted expression of the delta-subunit gene both as an enhancer at the endplate level and as a silencer in extrajunctional areas. Furthermore, gel-shift experiments with mouse muscle extracts reveal an activity that specifically binds the 6-bp sequence TTCCGG of this element, suggesting that a transcription factor(s) controls the expression of the delta-subunit gene via this element.