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: 10360680
• 负责人: Francesca Telese
• 金额: $ 69.41万
• 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/10360680
• 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; 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. ! !

! 项目摘要 每年，每天有100多名美国人因过量服用阿片类药物而死亡。阿片类药物成瘾，包括 处方药如羟考酮，以及非法药物如海洛因和芬太尼，是一个国家危机， 影响公众健康和经济。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 鉴定与更高的易感性相关的细胞类型和细胞类型特异性基因表达模式， 强制性使用羟考酮的方法。我们将通过实施 单细胞测序分析，以测量基因表达和染色质的可及性，在数千个 单个细胞在一个实验中。我们将使用N/NIH异质性储备（HS）大鼠的大脑， 进行了羟考酮自我给药程序的扩展访问。我们专注于HS大鼠， 它们的基因是多样的，并表现出一种特殊的行为能力。这些老鼠的特征是 基于成瘾样的高级分析，对羟考酮强迫性摄入易受影响或耐药 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， 羟考酮使用障碍的预测和结构效度。这个项目利用了大脑组织 HS大鼠的储存库已经进行了基因分型，并被表征为易受强迫性行为的影响和抵抗。 使用羟考酮。羟考酮生物样本库（www.OxycodoneBioBank.org）将提供用于 这个项目我们将重点放在脑桥核，这是一个参与从中度到中度过渡的大脑区域。 吸毒过量我们对大脑皮层单细胞分析的初步研究提供了一个令人信服的 研究阿片成瘾生物学基础的战略。我们建议：1）使用单细胞RNA-seq来鉴定 羟考酮易患或耐药的HS大鼠脑中基因表达的变化 强迫使用（具体目标1）; 2）使用单细胞ATAC-seq来鉴定染色质可及性的变化 和转录因子的结合位点在同一群体的HS大鼠（具体目标2）; 3）使用H3 K27 Ac- PLAC-seq将远端调控元件连接到参与羟考酮成瘾相关行为的靶基因 （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 羟考酮生物库（U 01 DA 044451）和NIDA中心提供的远交大鼠GWAS数据 （P50DA037844）。我们认为，拟议的研究有可能导致开创性的 发现阿片类药物成瘾的机制基础。我们的测序数据集的综合分析 将为不同细胞类型对分子变化的贡献提供相当多的新见解 与成瘾相关表型相关。 ! !