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

绝大多数常见的遗传变异是神经精神疾病的潜在风险 存在于基因组的注释不佳的非编码区，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，有几个主要的知识空白，包括：（一）因果关系 相关基因座内的变异，（B）受那些致病基因影响的调节元件 （c）致病性变体发生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。在本提案中，我们 将确定遗传对染色质结构（增强子）的两个特征的影响 组蛋白标记及其相互作用）在人类皮层发育过程中的作用， 完全解释导致神经精神疾病风险的调节机制。在一个大 一群死后人类发育中的皮质组织， 全基因组基因分型和转录组分析，我们将利用一种技术， 使我们能够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这两个特征的影响， 它们与先前和正在发展的神经精神障碍的共定位 （GWAS）风险位点。精神疾病风险变体可能通过以下方式发挥其调节作用：（1） 改变增强子（H3 K27 ac QTL或组蛋白乙酰化（ha）QTL）和/或（2）染色质 互作（interaction-QTL）。这类新的QTL将增强我们对 人类神经发育的分子过程，以及这种发展是如何在 神经精神障碍此外，我们将进行两个正交的方法来验证的影响 的遗传变异，并评估其细胞类型的特异性。我们将进行细胞类型特异性 大规模平行报告基因测定（MPRA）来验证haQTL的功能影响。在这 在该测定中，含有增强子相关等位基因的克隆寡核苷酸驱动条形码化的寡核苷酸的表达。 转录本，可用于评估监管差异和确定因果变异。我们 还将应用单倍型特异性染色质成像技术，以可视化如何调节 变异影响单个细胞核中的染色质相互作用。这项技术描绘了 每个染色体的切片与等位基因特异性寡核苷酸，以便可视化， 测量0 NA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。

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.