The site specific demethylation in the 5'-regulatory area of NMDA receptor 2B subunit gene associated with CIE-induced up-regulation of transcription.
The site specific demethylation in the 5'-regulatory area of NMDA receptor 2B subunit gene associated with CIE-induced up-regulation of transcription.
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DOI：
10.1371/journal.pone.0008798
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发表时间：
2010-01-20
期刊：
影响因子：
3.7
通讯作者：
Henderson G
中科院分区：
文献类型：
--
作者：
Qiang M;Denny A;Chen J;Ticku MK;Yan B;Henderson G
NMDA 受体代表中枢神经系统中乙醇作用的一个特别重要的位点。我们最近报道，慢性间歇性乙醇 (CIE) 治疗后 NMDA 受体 2B (NR2B) 基因表达持续上调。越来越多的证据表明表观遗传机制参与多种神经适应过程中基因表达的动态和持久调节，促使我们研究 DNA 甲基化在介导 CIE 诱导的 NR2B 基因转录上调中的作用。为了剖析NR2B基因DNA甲基化的变化，我们在CIE处理后筛选了其5′调控区内的大量CpG位点。原代皮层培养神经元在 CIE 范例中接受乙醇处理。采用亚硫酸氢盐转化和焦磷酸测序对 NR2B 基因 5' 调控区内的 CpG 甲基化状态进行定量测量和分析；染色质免疫沉淀 (ChIP) 测定用于检查与甲基化和转录因子结合相关的 DNA 水平。进行电泳迁移率变动测定 (EMSA) 和体外 DNA 甲基化测定，以确定 DNA 甲基化对 DNA 和转录因子之间的相互作用以及启动子活性的直接影响。对 NR2B 5' 调控区内各个 CpG 甲基化位点的分析揭示了 CIE 治疗和停药后三个区域具有位点特异性 CpG 去甲基化簇。 ChIP 证实了这一点，显示相同区域中甲基化 DNA 的类似减少。 CIE 诱导的去甲基化的特征是位于某些转录因子结合序列 AP-1 和 CRE 附近，并且发生在治疗期间以及乙醇戒断后。此外，体外甲基化DNA的增加降低了转录因子结合活性和启动子活性。另一项 ChIP 测定表明，CIE 诱导的 DNA 去甲基化伴随着转录因子占据的增加。这些结果表明 DNA 去甲基化在介导 CIE 诱导的 NR2B 基因上调中发挥着重要作用，从而暗示了酒精作用的新分子位点。
The NMDA receptor represents a particularly important site of ethanol action in the CNS. We recently reported that NMDA receptor 2B (NR2B) gene expression was persistently up-regulated following chronic intermittent ethanol (CIE) treatment. Increasing evidence that epigenetic mechanisms are involved in dynamic and long-lasting regulation of gene expression in multiple neuroadaptive processes prompted us to investigate the role of DNA methylation in mediating CIE-induced up-regulation of NR2B gene transcription. To dissect the changes of DNA methylation in the NR2B gene, we have screened a large number of CpG sites within its 5′-regulatory area following CIE treatment. Primary cortical cultured neurons were subjected to ethanol treatment in a CIE paradigm. Bisulfite conversion followed by pyrosequencing was used for quantitative measurement and analysis of CpG methylation status within the 5′-regulatory area of the NR2B gene; chromatin immunoprecipitation (ChIP) assay was used to examine DNA levels associated with methylation and transcription factor binding. Electrophoretic mobility shift assay (EMSA) and in vitro DNA methylation assays were performed to determine the direct impact of DNA methylation on the interaction between DNA and transcription factor and promoter activity. Analysis of individual CpG methylation sites within the NR2B 5′regulatory area revealed three regions with clusters of site-specific CpG demethylation following CIE treatment and withdrawal. This was confirmed by ChIP showing similar decreases of methylated DNA in the same regions. The CIE-induced demethylation is characterized by being located near certain transcription factor binding sequences, AP-1 and CRE, and occurred during treatment as well as after ethanol withdrawal. Furthermore, the increase in vitro of methylated DNA decreased transcription factor binding activity and promoter activity. An additional ChIP assay indicated that the CIE-induced DNA demethylation is accompanied by increased occupation by transcription factors. These results suggest an important role of DNA demethylation in mediating CIE-induced NR2B gene up-regulation, thus implicating a novel molecular site of alcohol action.
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