Epigenetics Core

项目摘要 表观遗传学核心的目标是提供一个强大的通用平台，用于识别和分析 大脑中受慢性乙醇暴露影响的基因。在本资助期内， 表观遗传学核心鉴定了在成年人中由酒精暴露或戒断诱导和抑制的基因网络。 杏仁核，海马，腹侧被盖区和边缘皮层和特定的靶基因， RNA水平以及H3 K9/14 ac水平的变化在功能上得到验证。 在当前提交文件中，拟定的目标将允许临床前CARE研究项目（项目1-3） 为了获得关于染色质可及性（ATAC-seq）和组蛋白的全基因组变化的新信息， 甲基化（H3 K27 me 3 ChIP-seq）。这些调查结果将与本报告期间获得的数据相结合。 （RNA-seq和H3K9/14ac ChIP-seq）。我们还将在实验室中进行ATAC-seq和RNA-seq。 来自新南威尔士州的酒精使用障碍（AUD）/对照队列的前额叶皮层（PFC） 组织资源中心（项目4）。此外，我们还将研究5- 使用Illumina的EPIC甲基化阵列在相同组织中检测羟甲基胞嘧啶（5 hmC）。从这些 研究将揭示可能导致酒精使用障碍的基因的大脑区域选择性变化。 上述研究构成了《核心》的目标1。目标2提出了生物信息学管道，用于分析 全基因组数据集和5 hmC阵列，并整合临床前和临床后的大型数据集， 尸检从多个无偏见的方法的数据集的整合是创新的，将提供 背景下解释的变化，基因调控景观，以响应从酒精戒断。在Aim中 3，我们提出采用新的dCas 9：P300乙酰转移酶融合蛋白来改变乙酰化水平， 特定的基因位点这将使我们能够通过改变大脑中特定区域的关键基因来激活它们的表达。 在其相应的启动子处选择性地乙酰化。类似地，我们将使用dCas 9：Tet 1融合蛋白来 改变糖皮质激素受体（Nr 3c 1）的5 mC和5 hmC水平以调节mRNA表达。 表观遗传学核心正在使用最先进的技术来获得全基因组信息， 为每个援外社组成部分提供额外资源，以解决其具体假设。核心将执行 在上述方案的许多关键步骤中进行质量控制措施。此外， 全基因组测量将由核心人员与项目负责人协商后进行验证。表观遗传学 核心是一个重要的平台，不仅对个人的关怀项目，而且对大学范围内的NIH资助 表观遗传学研究者

Project Summary The goal of the Epigenetics Core is to provide a robust common platform for the identification and analysis of genes affected by withdrawal from chronic ethanol exposure in the brain. During the current funding period, the Epigenetics Core identified gene networks both induced and repressed by alcohol exposure or withdrawal in the amygdala, hippocampus, ventral tegmental area and limbic cortex and specific target genes that showed changes in RNA levels, as well as, in H3K9/14ac levels were functionally validated. In the current submission, the proposed Aims will allow preclinical CARE Research Projects (Projects 1-3) to obtain novel information regarding genome-wide changes in chromatin accessibility (ATAC-seq) and histone methylation (H3K27me3 ChIP-seq). These findings will be integrated with data obtained during the current funding period (RNA-seq and H3K9/14ac ChIP-seq). We will also perform ATAC-seq and RNA-seq in the prefrontal cortex (PFC) of the alcohol use disorder (AUD)/control cohort obtained from the New South Wales Tissue Resource Center (Project 4). In addition, we will investigate the across genome levels of 5- hydroxymethylcytosine (5hmC) in the same tissue using Illumina’s EPIC Methylation Array. Results from these studies will reveal brain region-selective changes in genes that are likely to contribute to alcohol use disorders. The above studies constitute Aim 1 of the Core. Aim 2 proposes bioinformatics pipelines for the analyses of the genome-wide datasets and the 5hmC array and integrating the large datasets for both preclinical and post- mortem studies. The integration of datasets from multiple non-biased approaches is innovative and will provide context for interpreting changes in the gene regulatory landscape in response to withdrawal from alcohol. In Aim 3, we propose to employ a novel dCas9:P300 acetyl transferase fusion protein to alter acetylation levels at specific genomic loci. This will allow us to activate expression of key genes in specific brain regions by altering acetylation selectively at their corresponding promoters. Similarly, we will use a dCas9:Tet1 fusion protein to alter 5mC and 5hmC levels at glucocorticoid receptor (Nr3c1) to modulate mRNA expression. The Epigenetics Core is using state-of-the-art technology to obtain genome-wide information to provide additional resources for each CARE component to address their specific hypotheses. The Core will perform quality control measures at many of the key steps of the protocols described above. In addition, key findings in the genome-wide measures will be validated by Core personnel in consult with Project leaders. The Epigenetics Core is an important platform, not only for individual CARE Projects, but also for University-wide NIH-funded epigenetics investigators.