Discovery and validation of genetic variation impacting the gene regulatory landscape during human cortical development

发现并验证影响人类皮质发育过程中基因调控景观的遗传变异

• 批准号: 9948273
• 负责人: Jason Louis Stein
• 金额: $ 61.55万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9948273
• 关键词: 3-Dimensional; Adult; Alleles; Architecture; Autopsy; Bar Codes; Biological Assay; Brain; Cell Nucleus; Chromatin; Chromosomes; Complex; DNA; Development; Disease; Enhancers; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Variation; Genome; Genotype; Haplotypes; Histone Acetylation; Histone Fold; Histones; Human; Imaging Techniques; In Vitro; Individual; Knowledge; Label; Lead; Location; Measures; Mental disorders; Methods; Molecular; Neuronal Differentiation; Oligonucleotides; Paint; Pathogenesis; Population; Process; Quantitative Trait Loci; Regulator Genes; Regulatory Element; Reporter; Risk; Specificity; Stimulus; Techniques; Technology; Time; Tissue Donors; Tissues; Transcript; Untranslated RNA; Validation; Variant; Visualization; Work; brain tissue; causal variant; cell type; fetal; genetic variant; genome wide association study; genome-wide; improved; nerve stem cell; neurodevelopment; neuropsychiatric disorder; novel; risk variant; transcriptomics

The vast majority of common genetic variation underlying risk for neuropsychiatric disorders resides in poorly annotated non-coding regions of the genome and likely impacts the regulation of gene expression. In order to move from a location in the genome associated with risk to a regulatory mechanism, there are several major gaps in knowledge including: (a) the causal variant(s) within the associated locus, (b) the regulatory elements impacted by those causal variant(s), (c) the cell-type(s) and developmental time period(s) at which the causal variants(s) exert their effects, and (d) the gene(s) impacted by those causal variant(s). In this proposal, we will identify genetic influences on two features of chromatin architecture (enhancer histone marks and their 30 interactions) during human cortical development in order to more completely explain regulatory mechanisms leading to risk for neuropsychiatric disorders. In a large population of post-mortem human developing cortical tissue that has previously undergone genome-wide genotyping and transcriptomic profiling, we will utilize a technique that allows us to simultaneously measure enhancer activity and its interaction profile (H3K27ac HiChIP). We will then identify genetic influences on these two features of chromatin and their co-localization with previous and growing neuropsychiatric disorder genome-wide association (GWAS) risk loci. Psychiatric disorder risk variants may exert their regulatory impact by (1) changing enhancers (H3K27ac QTLs or histone acetylation (ha)QTLs) and/or (2) chromatin interaction (interaction-QTLs). This novel class of QTLs will enhance our understanding of the molecular processes underlying human neurodevelopment and how that development is altered in neuropsychiatric disorders. Further, we will conduct two orthogonal methods to validate the impact of the genetic variants and assess their cell-type specificity. We will perform cell-type specific massively parallel reporter assays (MPRA) to validate the functional impact of haQTLs. In this assay, cloned oligos containing the enhancer associated alleles drive expression of barcoded transcripts that can be used to assess regulatory differences and identify causal variants. We will also apply a haplotype-specific chromatin imaging technique to visualize how regulatory variation impacts chromatin interactions in individual nuclei. This technique paints sections of each chromosome with allele-specific oligos in order to visualize and measure the physical interactions of the 0NA molecule. Completing the aims of this proposal will allow us to identify largely complete regulatory mechanisms impacting human brain development and risk for neuropsychiatric disorders.

绝大多数常见的遗传变异存在神经精神疾病的风险 存在于基因组注释不良的非编码区域，并可能影响 基因表达。为了从基因组中与风险相关的位置移动到 监管机制方面，存在几个主要的知识空白，包括：(a) 因果关系 相关位点内的变异，(b) 受这些因果影响的调控元件 变异，(c) 引起因果变异的细胞类型和发育时间段 发挥其影响，以及 (d) 受这些因果变异影响的基因。在这个提案中，我们 将识别遗传对染色质结构两个特征的影响（增强子 组蛋白标记及其 30 种相互作用）在人类皮质发育过程中，以便更多 完全解释导致神经精神疾病风险的调节机制。在一个大 死后人类发育中的皮质组织群体，这些组织之前曾经历过 全基因组基因分型和转录组分析，我们将利用一种技术 允许我们同时测量增强子活性及其相互作用概况 （H3K27ac HiChIP）。然后我们将确定遗传对染色质和染色质这两个特征的影响 它们与先前和不断发展的神经精神疾病全基因组关联的共定位 （GWAS）风险位点。精神疾病风险变异可能通过以下方式发挥监管影响：(1) 改变增强子（H3K27ac QTL 或组蛋白乙酰化 (ha)QTL）和/或 (2) 染色质 相互作用（相互作用-QTL）。这类新颖的 QTL 将增强我们对 人类神经发育的分子过程以及这种发育是如何改变的 神经精神疾病。此外，我们将进行两种正交方法来验证影响 遗传变异并评估其细胞类型特异性。我们将进行细胞类型特异性 大规模并行报告分析 (MPRA) 来验证 haQTL 的功能影响。在这个 测定中，含有增强子相关等位基因的克隆寡核苷酸驱动条形码表达 可用于评估监管差异并识别因果变异的转录本。我们 还将应用单倍型特异性染色质成像技术来可视化监管如何 变异影响单个细胞核中染色质的相互作用。这种技术画 每个染色体的部分带有等位基因特异性寡核苷酸，以便可视化和 测量 0NA 分子的物理相互作用。完成本提案的目标将 使我们能够确定影响人类大脑发育的基本完整的调节机制 神经精神疾病的风险。