Epigenetic mechanisms: CIE-induced NR2B gene up-regulation in alcohol dependence

描述(申请人提供)：慢性间歇性乙醇(CIE)暴露可产生更持久的分子适应，包括上调NR2B基因的表达和持久性，这被视为酒精依赖发展的重要神经生物学基础。然而，这种长期存在的现象背后的确切机制仍不清楚。DNA甲基化是许多可改变基因表达的表观遗传修饰之一，因此，它是CIE诱导的长期效应的理想候选机制。因此，我们推测CIE诱导的NR2B基因5‘区特异性CpG位点的DNA去甲基化可能增加转录因子对NR2B基因5’区DNA的可及性。反复的酒精暴露可能促进细胞内的信号机制，这些机制调节DNA甲基化，并导致NR2B基因表达的长期可塑性变化。这些都可能导致酒精依赖的形成。为了解决这一假设，我们计划使用我们实验室现有的原代皮质神经元培养CIE模型结合动物CIE模型来检测CIE诱导的DNA甲基化状态的变化，通过使用亚硫酸氢盐基因组测序来定位NR2B基因5‘区的单个CpG二核苷酸，以响应NR2B基因转录的长期上调，并研究这种变化与小鼠CIE模型中酒精依赖的发展的相关性；B)通过体外甲基化、转染法、EMSA、定点突变和芯片检测CIE诱导的CREB和AP-1结合位点附近特定CpG位点甲基化状态的改变如何影响转录因子CREB和AP-1结合复合体的形成；C)通过特异性抑制物、免疫印迹、酶联免疫吸附试验和双向凝胶分析，确定介导CIE对DNA甲基转移酶活性和NR2B基因转录的相关信号通路的作用，包括传统的信号通路cAMP/PKA和MAPK/ERK以及参与DNA甲基化修饰和NR2B基因持续表达的新信号蛋白。这一研究结果将为乙醇通过表观遗传修饰调控NR2B基因表达的分子机制提供新的有价值的见解，有望成为可能干预酒精依赖和酒精戒断综合征的基础。

DESCRIPTION (provided by applicant): Chronic intermittent ethanol (CIE) exposure produces longer-lasting molecular adaptations including up-regulation of NR2B gene expression and persistence, which is viewed as an important neurobiological basis for the development of ethanol dependence. However, the exact mechanisms underlying this long lasting phenomenon are still unclear. DNA methylation is one of many epigenetic modifications that can alter gene expression, thus, it represents as an ideal candidate mechanism for CIE-induced long-term effects. Therefore, we hypothesize that CIE-induced DNA demethylation of specific CpG sites in the NR2B gene 5' region may increase accessibility of transcription factors to DNA in 5' region of NR2B gene. Repeated ethanol exposures may promote intracellular signaling mechanisms, which regulate DNA methylation and lead to the long lasting plastic changes in NR2B gene expression. These may contribute to the development of alcohol dependence. To address this hypothesis, we plan to use an existing primary cortical neuronal culture CIE model in our laboratory combining with an animal CIE model to a) examine CIE-induced changes in DNA methylation state by mapping individual CpG dinucleotide of the NR2B gene 5' region, which are responsive to long lasting up-regulation of NR2B gene transcription by using bisulfite genomic sequencing, and also examine the correlation of this changes to the development of ethanol-dependence in mice CIE model; b) determine how CIE-induced changes in methylation state of specific CpG sites near the CREB and AP-1 binding sites may impact transcription factors CREB and AP-1 binding complex formation via examining the alterations in CREB/AP-1-DNA binding affinity by using in vitro methylation, transfection, EMSA, site-direct mutation and ChIP; and c) identify the role of relative signaling pathways mediating CIE to DNA methyltransferase activity as well as NR2B gene transcription, including traditional signaling pathways cAMP/PKA and MAPK/ERK and novel signaling proteins involving in modifications of DNA methylation and persistent expression of the NR2B gene by using specific inhibitors, western blot, ELISA and 2-D gel analysis. The results from this proposal will provide new and valuable insight of molecular mechanisms of ethanol regulating NR2B gene expression through epigenetic modifications, which are expected to serve as a basis for possible intervention of alcohol dependence and alcohol withdrawal syndrome.