Single-cell resolution analysis of chromatin accessibility and gene expression changes in a model of drug addiction

药物成瘾模型中染色质可及性和基因表达变化的单细胞分辨率分析

• 批准号: 9897370
• 负责人: Francesca Telese
• 金额: $ 71.02万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9897370
• 关键词: ATAC-seq; Address; Affect; American; Animals; Behavior; Behavioral; Behavioral Model; Behavioral Paradigm; Binding; Binding Sites; Biological; Biological Assay; Brain; Brain region; Cell Nucleus; Cells; Centers for Disease Control and Prevention (U.S.); Cerebral cortex; ChIP-seq; Chromatin; Complex; Computer Analysis; Country; DNA; DNA Sequence; Data; Data Set; Dependence; Development; Disease; Distal; Drug Addiction; Drug Modelings; Drug Prescriptions; Drug abuse; Drug usage; Epidemic; Epigenetic Process; Exhibits; Expression Profiling; Face; Fentanyl; Funding; Gene Expression; Gene Expression Profile; Genes; Genetic; Genetic Transcription; Genetic Variation; Genotype; Goals; Grant; Heroin; Human; Illicit Drugs; Individual; Individual Differences; Intake; Intravenous; Knowledge; Lead; Ligation; Link; Maps; Measures; Methods; Modeling; Molecular; Motivation; National Institute of Drug Abuse; Nucleus Accumbens; Opiate Addiction; Opioid; Overdose; Oxycodone; Pattern; Pharmaceutical Preparations; Phenotype; Population; Population Heterogeneity; Predisposition; Procedures; Public Health; Rattus; Recording of previous events; Regulatory Element; Resistance; Resolution; Resources; Sampling; Self Administration; Severities; Signal Transduction; Technology; Tissue Banks; Tissues; United States; United States National Institutes of Health; addiction; base; behavior measurement; biobank; brain tissue; cell type; cost; epigenomics; experience; experimental study; genetic association; genome wide association study; infancy; insight; molecular dynamics; multidisciplinary; neural circuit; neuroadaptation; novel therapeutics; opioid abuse; opioid overdose; prescription pain reliever; response; single cell analysis; single cell sequencing; single-cell RNA sequencing; trait; transcription factor; transcriptomics

! Project Summary Every year, more than 100 Americans a day die after overdosing on opiates. Addiction to opioids, including prescription drug such as oxycodone, and illicit drugs such a s heroin and fentanyl, is a national crisis that affects public health and the economy. Thus, there is an urgent need to develop better treatments for opiate addiction, which requires a better understanding of its biological basis. The primary goal of our proposal is to identify cell types and cell type-specific gene expression patterns associated with higher vulnerability to compulsive oxycodone use in an unbiased and quantitative way. We will accomplish this goal by implementing single-cell sequencing assays to measure gene expression and chromatin accessibility in thousands of individual cells in a single experiment. We will use brains of N/NIH heterogeneous stock (HS) rats that have undergone the extended access to oxycodone self-administration procedure. We focus on HS rats because they are genetically diverse and exhibit an exceptional behavioral repertoire. These rats are characterized as vulnerable or resistant to oxycodone compulsive intake based on advanced analysis of addiction-like behavioral traits, including tolerance, dependence, motivation, and compulsive drug intake. This behavioral paradigm recapitulates many of the key neuroadaptations observed in human addiction and has high face, predictive, and construct validity for oxycodone use disorders. This project takes advantage of a brain tissue repository of HS rats that have been genotyped and characterized as vulnerable and resistant to compulsive oxycodone use. The oxycodone biobank (www.OxycodoneBioBank.org) will provide the samples to be used in this project. We will focus on the nucleus accumbens, a brain region involved in the transition from moderate to excessive drug use. Our preliminary studies on single-cell analysis of the cerebral cortex provide a compelling strategy to study the biological basis of opiate addiction. We propose: 1) to use single-cell RNA-seq to identify gene expression changes in brains of HS rats that are characterized as prone or resistant to oxycodone compulsive use (Specific Aim 1); 2) to use single-cell ATAC-seq to identify changes in chromatin accessibility and transcription factors binding sites in the same population of HS rats (Specific Aim 2); 3) to use H3K27Ac- PLAC-seq to link distal regulatory elements to target genes involved in oxycodone addiction-related behaviors (Specific Aim 3). This project will benefit from multiple expertise and will leverage existing resources, including those provided by the oxycodone biobank (U01DA044451) and the NIDA center for GWAS in outbred rats (P50DA037844). We believe that the proposed studies have the potential to lead to groundbreaking discoveries in the mechanistic bases of opioids addiction. The integrative analysis of our sequencing datasets will provide considerable new insights concerning the contribution of distinct cell-types to molecular changes associated with addiction related phenotypes. ! !

！ 项目概要 每年，每天有超过 100 名美国人因服用过量阿片类药物而死亡。阿片类药物成瘾，包括 羟考酮等处方药以及海洛因和芬太尼等非法药物是一场国家危机 影响公共健康和经济。因此，迫切需要开发更好的阿片类药物治疗方法 成瘾，这需要更好地了解其生物学基础。我们提案的主要目标是 识别与较高易感性相关的细胞类型和细胞类型特异性基因表达模式 以公正和定量的方式强制使用羟考酮。我们将通过实施来实现这一目标 单细胞测序测定可测量数千个细胞的基因表达和染色质可及性 单个实验中的单个细胞。我们将使用 N/NIH 异种库存 (HS) 大鼠的大脑，这些大鼠具有 接受了羟考酮自我管理程序的扩展访问。我们关注 HS 大鼠是因为 他们基因多样，并表现出特殊的行为能力。这些大鼠的特征为 基于成瘾样高级分析，对羟考酮强迫性摄入易感或抵抗 行为特征，包括耐受性、依赖性、动机和强迫性药物摄入。这种行为 范式概括了在人类成瘾中观察到的许多关键神经适应，并且具有较高的面子， 羟考酮使用障碍的预测和构建有效性。该项目利用了脑组织 HS 大鼠的储存库，这些大鼠已被基因分型并具有易受攻击和对强迫具有抵抗力的特征 羟考酮的使用。羟考酮生物库 (www.OxycodoneBioBank.org) 将提供用于 这个项目。我们将重点关注伏隔核，这是一个参与从中度到中度过渡的大脑区域。 过度使用药物。我们对大脑皮层单细胞分析的初步研究提供了令人信服的证据 研究阿片成瘾生物学基础的策略。我们建议：1）使用单细胞RNA-seq来鉴定 HS 大鼠大脑中基因表达的变化，这些变化对羟考酮有倾向性或耐药性 强迫性使用（具体目标 1）； 2) 使用单细胞 ATAC-seq 识别染色质可及性的变化 同一 HS 大鼠群体中的转录因子结合位点（具体目标 2）； 3）使用H3K27Ac- PLAC-seq 将远端调控元件与参与羟考酮成瘾相关行为的靶基因连接起来 （具体目标 3）。该项目将受益于多种专业知识，并将利用现有资源，包括 由羟考酮生物库 (U01DA044451) 和 NIDA 远交大鼠 GWAS 中心提供 （P50DA037844）。我们相信所提出的研究有可能带来突破性的成果 阿片类药物成瘾机制基础的发现。我们的测序数据集的综合分析 将提供有关不同细胞类型对分子变化的贡献的大量新见解 与成瘾相关的表型有关。 ！ ！