Abstract：
Mounting evidence suggests a prominent role for alpha-synuclein (a-syn) in neuronal cell function. Alterations in the levels of cellular a-syn have been hypothesized to play a critical role in the development of Parkinson\'s disease (PD); however, mechanisms that control expression of the gene for a-syn (SNCA) in cis and trans as well as turnover of a-syn are not well understood. We analyzed whether methyl-CpG binding protein 2 (MeCP2), a protein that specifically binds methylated DNA, thus regulating transcription, binds at predicted binding sites in intron 1 of the SNCA gene and regulates a-syn protein expression. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility-shift assays (EMSA) were used to confirm binding of MeCP2 to regulatory regions of SNCA. Site-specific methylation and introduction of localized mutations by CRISPR/Cas9 were used to investigate the binding properties of MeCP2 in human SK-N-SH neuroblastoma cells. The significance of MeCP2 for SNCA regulation was further investigated by overexpressing MeCP2 and mutated variants of MeCP2 in MeCP2 knockout cells. We found that methylation-dependent binding of MeCP2 at a restricted region of intron 1 of SNCA had a significant impact on the production of a-syn. A single nucleotide substitution near to CpG1 strongly increased the binding of MeCP2 to intron 1 of SNCA and decreased a-syn protein expression by 60%. In contrast, deletion of a single nucleotide closed to CpG2 led to reduced binding of MeCP2 and significantly increased a-syn levels. In accordance, knockout of MeCP2 in SK-N-SH cells resulted in a significant increase in a-syn production, demonstrating that SNCA is a genomic target for MeCP2 regulation. In addition, the expression of two mutated MeCP2 variants found in Rett syndrome (RTT) showed a loss of their ability to reduce a-syn expression. This study demonstrates that methylation of CpGs and binding of MeCP2 to intron 1 of the SNCA gene plays an important role in the control of a-syn expression. In addition, the changes in SNCA regulation found by expression of MeCP2 variants carrying mutations found in RTT patients may be of importance for the elucidation of a new molecular pathway in RTT, a rare neurological disorder caused by mutations in MECP2.
摘要：
越来越多的证据表明，α-突触核蛋白（a-syn）在神经元细胞功能中起着重要作用。细胞a-syn水平的改变被认为在帕金森病（PD）的发展中起着关键作用；然而，控制a-syn（SNCA）基因顺式和反式表达以及a-syn周转的机制尚不清楚。我们分析了甲基-CpG结合蛋白2(MeCP2)一种特异性结合甲基化DNA的蛋白质，从而调节转录，在SNCA基因的内含子1中的预测结合位点处结合并调节a-syn蛋白表达。染色质免疫沉淀（ChIP）和电泳迁移率变化测定（EMSA）用于确认MeCP2与SNCA调节区的结合。通过CRISPR/Cas9的位点特异性甲基化和局部突变的引入用于研究MeCP2在人SK-N-SH神经母细胞瘤细胞中的结合特性。通过在MeCP2敲除细胞中过表达MeCP2和MeCP2的突变变体进一步研究了MeCP2对SNCA调节的意义。我们发现MeCP2在SNCA内含子1的限制性区域的甲基化依赖性结合对a-syn的产生具有显著影响。CpG1附近的单个核苷酸取代强烈增加了MeCP2与SNCA内含子1的结合，并将a-syn蛋白表达降低了60％。相比之下,对CpG2封闭的单个核苷酸的缺失导致MeCP2的结合减少并显着增加a-syn水平。InAccording,在SK-N-SH细胞中敲除MeCP2导致a-syn产生的显着增加，证明SNCA是MeCP2调节的基因组靶标。此外,在Rett综合征（RTT）中发现的两种突变的MeCP2变体的表达显示其降低a-syn表达的能力丧失。这项研究表明，CpG的甲基化和MeCP2与SNCA基因内含子1的结合在控制a-syn表达中起重要作用。此外,通过在RTT患者中发现的携带突变的MeCP2变体的表达发现的SNCA调节的变化可能对于阐明RTT中的新分子途径很重要，一种由MECP2突变引起的罕见神经系统疾病。
