Epigenetics Core

项目摘要 表观遗传学核心的目标是提供一个强大的公共平台，用于识别和分析 大脑中受慢性酒精暴露戒断影响的基因。在本资助期内， 表观遗传学核心确定了酒精暴露或戒断引起的基因网络的诱导和抑制 杏仁核、海马体、腹侧被盖区和边缘皮质以及特定的靶基因 对RNA水平的变化以及H3K9/14ac水平的变化进行了功能验证。 在本意见书中，拟议的AIMS将允许开展临床前护理研究项目(项目1-3) 获得有关染色质可及性(atac-seq)和组蛋白全基因组变化的新信息 甲基化(H3K27me3芯片序列)。这些调查结果将与当前 资助期(RNA-SEQ和H3K9/14AC CHIP-SEQ)。我们还将执行atac-seq和rna-seq 酒精使用障碍(AUD)的前额叶皮质(PFC)/来自新南威尔士州的对照队列 组织资源中心(项目4)。此外，我们还将研究5个基因组水平上的 使用Illumina的EPIC甲基化阵列在同一组织中检测羟甲基胞嘧啶(5HmC)。来自这些的结果 研究将揭示大脑区域选择性基因的变化，这些基因可能导致酒精使用障碍。 上述研究构成了核心的目标1。目标2提出生物信息学管道用于分析 全基因组数据集和5hmC阵列，并集成临床前和临床后的大型数据集。 尸检研究。集成来自多个无偏见方法的数据集是创新的，并将提供 解释戒酒后基因调控格局变化的背景。在AIM 3，我们建议使用一种新的dCas9：P300乙酰转移酶融合蛋白来改变乙酰化水平 特定的基因组基因座。这将允许我们通过改变大脑中特定区域的关键基因的表达 选择性地在其相应的启动子上进行乙酰化。同样，我们将使用dCas9：Tet1融合蛋白来 改变糖皮质激素受体(NR3C1)的5mC和5hmC水平以调节mRNA的表达。 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，以提供 为每个护理组成部分提供额外资源，以解决其具体假设。酷睿将执行 在上述协议的许多关键步骤中采取质量控制措施。此外，在以下方面的主要发现 核心人员将与项目负责人协商，对全基因组措施进行验证。表观遗传学 CORE是一个重要的平台，不仅是个人关怀项目，也是整个大学范围内NIH资助的平台 表观遗传学研究人员。

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.