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

描述（由申请人提供）：慢性间歇性乙醇（CIE）暴露产生更持久的分子适应，包括NR 2B基因表达上调和持续性，这被视为乙醇依赖性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甲基化是可以改变基因表达的许多表观遗传修饰之一，因此，它代表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NR 2B基因5'区特定CpG位点的DNA去甲基化可能增加了转录因子对NR 2B基因5'区DNA的可及性。反复乙醇暴露可能促进细胞内信号传导机制，调节DNA甲基化并导致NR 2B基因表达的长期可塑性变化。这些都可能导致酒精依赖的发展。为了解决这一假设，我们计划在我们的实验室中使用现有的原代皮层神经元培养CIE模型，结合动物CIE模型，以a）通过定位NR 2B基因5'区域的单个CpG二核苷酸来检查CIE诱导的DNA甲基化状态的变化，所述CpG二核苷酸通过使用亚硫酸氢盐基因组测序响应于NR 2B基因转录的长期持续上调，并在小鼠CIE模型中检查这种变化与乙醇依赖性发展的相关性; B）通过检查CREB/AP-1的改变，确定CIE诱导的CREB和AP-1结合位点附近的特异性CpG位点的甲基化状态的变化如何影响转录因子CREB和AP-1结合复合物的形成。通过体外甲基化、转染、EMSA、定点突变和ChIP等方法检测DNA结合亲和力;和c）鉴定介导CIE至DNA甲基转移酶活性以及NR 2B基因转录的相关信号传导途径的作用，包括传统的信号通路cAMP/PKA和MAPK/通过使用特异性抑制剂，蛋白质印迹，ELISA和2-D凝胶分析。本研究结果将为乙醇通过表观遗传修饰调控NR 2B基因表达的分子机制提供新的有价值的见解，有望为酒精依赖和酒精戒断综合征的可能干预提供依据。

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.