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

表观遗传机制：CIE 诱导的酒精依赖中 NR2B 基因上调

• 批准号: 7504053
• 负责人: ALAN FRAZER
• 金额: $ 32.85万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7504053
• 关键词: 5' Flanking Region; 5' Untranslated Regions; Address; Affinity; Alcohol dependence; Alcohol withdrawal syndrome; Animal Model; Animals; Area; Binding; Binding Sites; Biological Assay; CREB1 gene; Cells; Chronic; Complex; CpG dinucleotide; Cyclic AMP; DNA; DNA Binding; DNA Methylation; DNA Methyltransferase; DNA Methyltransferase Inhibitor; DNA Modification Methylases; Development; EMSA; Enzyme-Linked Immunosorbent Assay; Epigenetic Process; Ethanol; Ethanol dependence; Gel; Gene Expression; Genes; Genetic Transcription; Genomics; In Vitro; Individual; Intervention; Laboratories; Lead; Link; Long-Term Effects; Luciferases; Maps; Mediating; Methylation; Modeling; Modification; Molecular; Mus; Mutation; NMDA receptor 2B; Neurobiology; Neurons; Pattern; Plastics; Relative (related person); Reporter; Reporter Genes; Role; SHPS-1 protein; Series; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Signaling Protein; Site; Testing; Transcription Factor AP-1; Transcriptional Activation; Transcriptional Regulation; Transfection; Up-Regulation; Western Blotting; alcohol exposure; base; bisulfite; deletion analysis; demethylation; genetic regulatory protein; in vitro Assay; in vivo; inhibitor/antagonist; insight; mRNA Expression; mouse model; novel; promoter; transcription factor

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.

描述（由申请人提供）：慢性间歇性乙醇（CIE）暴露会产生更持久的分子适应，包括 NR2B 基因表达的上调和持久性，这被视为乙醇依赖发展的重要神经生物学基础。然而，这种长期现象背后的确切机制仍不清楚。 DNA 甲基化是许多可以改变基因表达的表观遗传修饰之一，因此，它是 CIE 诱导的长期影响的理想候选机制。因此，我们假设 CIE 诱导的 NR2B 基因 5' 区域中特定 CpG 位点的 DNA 去甲基化可能会增加转录因子对 NR2B 基因 5' 区域中 DNA 的可及性。反复接触乙醇可能会促进细胞内信号传导机制，从而调节 DNA 甲基化并导致 NR2B 基因表达的长期持续塑性变化。这些可能会导致酒精依赖的发展。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 模型与动物 CIE 模型相结合，a) 通过绘制 NR2B 基因 5' 区域的单个 CpG 二核苷酸来检查 CIE 诱导的 DNA 甲基化状态变化，这些二核苷酸通过亚硫酸氢盐基因组测序对 NR2B 基因转录的长期持续上调作出反应，并检查 这种变化与小鼠 CIE 模型中乙醇依赖性发展的相关性； b) 通过使用体外甲基化、转染、EMSA、定点突变和 ChIP 检查 CREB/AP-1-DNA 结合亲和力的变化，确定 CIE 诱导的 CREB ​​和 AP-1 结合位点附近特定 CpG 位点甲基化状态的变化如何影响转录因子 CREB ​​和 AP-1 结合复合物的形成； c) 通过使用特异性抑制剂、蛋白质印迹、ELISA 和 2-D 凝胶分析，确定介导 CIE 的相关信号通路对 DNA 甲基转移酶活性以及 NR2B 基因转录的作用，包括传统信号通路 cAMP/PKA 和 MAPK/ERK 以及涉及 DNA 甲基化修饰和 NR2B 基因持续表达的新型信号蛋白。该提案的结果将为乙醇通过表观遗传修饰调节NR2B基因表达的分子机制提供新的、有价值的见解，有望作为可能干预酒精依赖和酒精戒断综合征的基础。