DNA Methylation/Demethylation Mechanisms in AUD

项目摘要 环境因素，包括酗酒和压力，会引起基因调控的长期变化。 通过表观遗传机制，如DNA甲基化在大脑中表达。与压力类似，酒精 刺激糖皮质激素的释放，糖皮质激素与特定受体结合，即，糖皮质激素受体（由 NR3C1）。到目前为止，对表观遗传DNA修饰在调节转录组中的作用知之甚少。 在人类前额叶皮层（PFC，BA 10）和大鼠PFC在慢性酒精暴露和戒断。的 研究部分#4的目标是询问新基因的DNA甲基化的全基因组变化 包括AUD患者的NR 3C 1基因网络。其他目标是研究是否以及如何 改变的DNA甲基化和/或羟甲基化标记是AUD病理生理学的基础。基因组- 宽DNA甲基化方法（InfiniumMethylationEPICBeadChip，Illumina）将用于前额皮质 从来自新南威尔士组织资源的30对对照和AUD受试者获得的样品 中心（澳大利亚悉尼大学）。在初步的研究中，我们发现了一种总DNA的差异模式， AUD中的5，254个基因的甲基化。然而，该技术不能区分5-甲基胞嘧啶（5 mC） 5-羟甲基胞嘧啶（5 hmC）。在这里，我们建议调查5 mC的全基因组分布， 和5 hmC，使用泰特亚硫酸氢盐转化法，然后进行人甲基化EPIC BeadChip测定。 因此，我们将研究5 hmC/5 mC的富集与新基因表达变化的关系。 通过RNA-seq测量。染色质可及性与先前鉴定的表观遗传标记有关， 通过转座酶可重复染色质测序测定法（ATAC-seq）进行评估。集成不同 全基因组方法，即全基因组DNA甲基化，RNA-seq和ATAC-seq将允许深入研究 AUD表观基因组状态的调查。此外，使用反向平移方法，我们 建议从机制上研究DNA甲基化对PFC中神经元功能的下游影响， 对慢性酒精治疗大鼠的焦虑样行为和酒精自我给药升级的影响， 24小时戒酒后。因为我们观察到DNA甲基化的增加与 下调泰特表达（催化5 mC转化为5 hmC的酶），我们提出了 使用dCas 9-Tet 1介导的蛋白质方法来校正NR 3C 1和其他水平的甲基化缺陷， 基因启动子在大鼠PFC，并确定其对基因表达，焦虑和饮酒行为的影响。 这项拟议中的研究将有助于在人类和大鼠大脑中识别新的表观遗传机制， 治疗AUD的新治疗靶点。

Project Summary Environmental factors, including alcohol abuse and stress, cause long-lasting changes in the regulation of gene expression in the brain via epigenetic mechanisms, such as DNA methylation. Similar to stress, alcohol stimulates glucocorticoid release that bind to specific receptors, i.e., the glucocorticoid receptor (encoded by NR3C1). As of today, little is known on the role of epigenetic DNA modifications in regulating the transcriptome in the human prefrontal cortex (PFC, BA10) and rat PFC during chronic alcohol exposure and withdrawal. The goal of research component #4 is to interrogate genome-wide changes in DNA methylation of novel gene networks, including the NR3C1 gene network in AUD patients. Additional goals are to study whether and how altered DNA methylation and/or hydroxymethylation marks underlie the pathophysiology of AUD. The genome- wide DNA methylation approach (Infinium MethylationEPICBeadChip, Illumina) will be used in prefrontal cortex samples obtained from 30 pairs of controls and AUD subjects from the New South Wales Tissue Resource Centre (University of Sydney, Australia). In preliminary studies we identified a differential pattern of total DNA methylation in AUD for 5,254 genes. However, this technique does not differentiate 5-methyl-cytosine (5mC) from 5-hydroxymethyl-cytosine (5hmC). Here, we propose to investigate the genome–wide distribution of 5mC and 5hmC using the TET bisulfite conversion method followed by the Human MethylationEPIC BeadChip assay. Hence, we will examine the enrichment of 5hmC/5mC in association with changes in novel gene expression measured by RNA-seq. Chromatin accessibility in association with previously identified epigenetic marks will be assessed by Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq). Integration of different whole-genome approaches, i.e. genome-wide DNA methylation, RNA-seq and ATAC-seq will allow an in-depth investigation of the status of the epigenome in AUD. Additionally, using a reverse-translational approach, we propose to mechanistically investigate downstream effects of DNA methylation on neuronal function in PFC and on anxiety-like behaviors and escalation of alcohol self-administration in rats treated chronically with alcohol or following a 24 h alcohol-withdrawal. Because we observed an increase of DNA methylation associated with a downregulation of TET expression (the enzyme that catalyzes the conversion of 5mC to 5hmC), we propose the use of a dCas9-Tet1-mediated protein approach to correct methylation deficits at the levels of NR3C1 and other gene promoters in the PFC of rats and determine their effect on gene expression, anxiety and drinking behaviors. The proposed study will help to identify in the human and rat brain novel epigenetic mechanisms that may provide new therapeutic targets for the treatment of AUD.