Mieda M, Haga T, Saffen D W
Department of Biochemistry, Institute for Brain Research, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.
J Biol Chem. 1997 Feb 28;272(9):5854-60. doi: 10.1074/jbc.272.9.5854.
Neuronal cell-specific expression of the rat m4 muscarinic acetylcholine receptor (mAChR) is regulated by a silencer element. A likely mediator of this silencing is the neuron-restrictive silencer element/repressor element 1 (NRSE/RE1), which is present 837 base pairs (bp) upstream from the transcription initiation site of the m4 mAChR gene (Wood, I. C., Roopra, A., Harrington, C., and Buckley, N. J. (1995) J. Biol. Chem. 270, 30933-30940; Mieda, M., Haga, T., and Saffen, D. W. (1996) J. Biol. Chem. 271, 5177-5182). In the present study, we examined whether this putative NRSE/RE1 functions as a silencer. Transient expression assays using m4 mAChR promoter/luciferase expression vectors showed that the m4 NRSE/RE1 is necessary and sufficient to repress m4 promoter activity in non-neuronal L6 cells. m4 promoter activity was only partially repressed, however, in neuronal NG108-15 cells exogenously expressing the neuronal-restrictive silencer factor/RE1-silencing transcription factor (NRSF/REST). By contrast, the promoter activity of the type II sodium channel (NaII) gene was nearly completely repressed in NRSF/REST-expressing NG108-15 cells. Experiments with expression vectors containing chimeric promoters revealed that the NRSE/RE1 elements derived from both the m4 and NaII genes are independently sufficient to silence NaII gene promoter activity, but only partially repress m4 mAChR gene promoter activity in NRSF/REST-expressing NG108-15 cells. Thus, the repression activity of NRSF/REST depends upon the species of promoter to which it is linked. Gel-shift assays showed that the NRSF/REST is the only protein that binds to a 92-bp segment from the m4 mAChR promoter containing NRSE/RE1. This and the fact that m4 promoter activity was completely repressed in L6 cells suggest that the proteins that bind to the m4 constitutive promoter may be different from those in NG108-15 cells. Deletion analysis of the m4 constitutive promoter revealed that a 90-bp segment immediately upstream from the transcription initiation site contains significant promoter activity. Gel-shift assays revealed that several proteins in nuclear extracts prepared from L6 and NG108-15 cells bind to this 90-bp segment and that some of these proteins are L6 or NG108-15 cell-specific. These data support the idea that the repression activity of NRSF/REST depends upon the species of promoter to which it is linked and upon the proteins that bind to those promoters.
大鼠M4毒蕈碱型乙酰胆碱受体（mAChR）的神经元细胞特异性表达受一个沉默子元件调控。这种沉默作用的一个可能介导因子是神经元限制性沉默子元件/阻遏元件1（NRSE/RE1），它位于M4 mAChR基因转录起始位点上游837个碱基对（bp）处（伍德，I.C.，鲁普拉，A.，哈林顿，C.，和巴克利，N.J.（1995年）《生物化学杂志》270，30933 - 30940；三枝，M.，羽贺，T.，和萨芬，D.W.（1996年）《生物化学杂志》271，5177 - 5182）。在本研究中，我们检测了这个假定的NRSE/RE1是否作为一个沉默子发挥作用。使用M4 mAChR启动子/荧光素酶表达载体的瞬时表达分析表明，M4 NRSE/RE1对于抑制非神经元L6细胞中的M4启动子活性是必要且充分的。然而，在体外表达神经元限制性沉默因子/RE1沉默转录因子（NRSF/REST）的神经元NG108 - 15细胞中，M4启动子活性仅被部分抑制。相比之下，在表达NRSF/REST的NG108 - 15细胞中，II型钠通道（NaII）基因的启动子活性几乎被完全抑制。对含有嵌合启动子的表达载体进行的实验表明，源自M4和NaII基因的NRSE/RE1元件各自足以使NaII基因启动子活性沉默，但在表达NRSF/REST的NG108 - 15细胞中仅部分抑制M4 mAChR基因启动子活性。因此，NRSF/REST的抑制活性取决于与之相连的启动子种类。凝胶迁移分析表明，NRSF/REST是唯一能与M4 mAChR启动子中包含NRSE/RE1的92 - bp片段结合的蛋白质。这一点以及M4启动子活性在L6细胞中被完全抑制这一事实表明，与M4组成型启动子结合的蛋白质可能与NG108 - 15细胞中的不同。对M4组成型启动子的缺失分析表明，转录起始位点上游紧邻的一个90 - bp片段具有显著的启动子活性。凝胶迁移分析表明，从L6和NG108 - 15细胞制备的核提取物中的几种蛋白质与这个90 - bp片段结合，其中一些蛋白质是L6或NG108 - 15细胞特异性的。这些数据支持这样一种观点，即NRSF/REST的抑制活性取决于与之相连的启动子种类以及与这些启动子结合的蛋白质。
