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

基本信息

批准号：
10613583
负责人：
Jason Louis Stein
金额：
$ 57.2万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
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/10613583
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 Heterozygote Histone Acetylation Histone Fold Histones Human Imaging Techniques In Vitro Individual Knowledge Label Location Measures Mental disorders Methods Molecular Neuronal Differentiation Oligonucleotides Paint Pathogenesis Population Process Quantitative Trait Loci Regulator Genes Regulatory Element Reporter Risk Sorting 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 transcriptomic profiling transcriptomics

项目摘要

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.

神经精神疾病的绝大多数常见遗传变异是基本的遗传变异位于基因组的注释不足的非编码区域中，可能会影响调节基因表达。为了从与风险相关的基因组中的位置移动到监管机制，知识上有几个主要差距，包括：（a）因果关系相关基因座中的变体，（b）受到因果关系影响的调节元素变体（s），（c）因果变异的细胞类型（s）和发育时间周期（s）发挥其作用，（d）受到因果变异影响的基因。在这个建议中，我们将确定对染色质结构的两个特征的遗传影响（增强剂在人类皮质发育过程中，组蛋白标记及其30个相互作用）为了更多完全解释导致神经精神疾病风险的调节机制。大验尸后人类发育的皮质组织，以前经历过全基因组的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剂活动及其相互作用曲线（H3K27AC HICHIP）。然后，我们将确定对染色质的这两个特征的遗传影响和它们与以前的神经精神疾病全基因组关联的共同定位（GWAS）风险基因座。精神障碍风险变异可能会通过（1）发挥其调节性影响更改增强剂（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）。这种新颖的QTL类将增强我们对人类神经发育的基础分子过程以及该发展如何改变神经精神疾病。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的特异性。我们将执行特定的细胞类型大规模平行的记者测定（MPRA）以验证HAQTL的功能影响。在这个测定，包含增强剂相关等位基因的克隆的寡核体驱动条形码的表达可用于评估调节差异并确定因果变异的成绩单。我们还将应用单倍型特异性染色质成像技术来可视化调节变异会影响单个核中的染色质相互作用。这种技术绘制每个染色体的部分都带有等位基因特异性的寡核酸，以可视化和测量0NA分子的物理相互作用。完成本提案的目标将允许我们确定影响人脑发育和神经精神疾病的风险。