|
1000
|
Abstract/Summary
|
-
<jats:title>Abstract</jats:title><jats:p>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 (<jats:italic>SNCA</jats:italic>) in <jats:italic>cis</jats:italic> and <jats:italic>trans</jats:italic> 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 <jats:italic>SNCA</jats:italic> gene and regulates a-syn protein expression<jats:italic>.</jats:italic> Chromatin immunoprecipitation (ChIP) and electrophoretic mobility-shift assays (EMSA) were used to confirm binding of MeCP2 to regulatory regions of <jats:italic>SNCA</jats:italic>. 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 <jats:italic>SNCA</jats:italic> regulation was further investigated by overexpressing MeCP2 and mutated variants of MeCP2 in <jats:italic>MeCP2</jats:italic> knockout cells. We found that methylation-dependent binding of MeCP2 at a restricted region of intron 1 of <jats:italic>SNCA</jats:italic> 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 <jats:italic>SNCA</jats:italic> 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 <jats:italic>MeCP2</jats:italic> in SK-N-SH cells resulted in a significant increase in a-syn production, demonstrating that <jats:italic>SNCA</jats:italic> 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 <jats:italic>SNCA</jats:italic> gene plays an important role in the control of a-syn expression. In addition, the changes in <jats:italic>SNCA</jats:italic> 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 <jats:italic>MECP2</jats:italic>.</jats:p>
|
|
1000
|
Hinweis
|
-
DeepGreen-ID: 2b84afeec63b468cb23510079a9cc157 ; metadata provieded by: DeepGreen (https://www.oa-deepgreen.de/api/v1/), LIVIVO search scope life sciences (http://z3950.zbmed.de:6210/livivo), Crossref Unified Resource API (https://api.crossref.org/swagger-ui/index.html), to.science.api (https://frl.publisso.de/), ZDB JSON-API (beta) (https://zeitschriftendatenbank.de/api/), lobid - Dateninfrastruktur für Bibliotheken (https://lobid.org/resources/search)
|
