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障碍的潜在风险 驻留在基因组注释不佳的非编码区，并可能影响对 基因表达。为了从基因组中与风险相关的位置转移到 关于监管机制，在认识上存在几个主要差距，包括：(A)因果关系 相关基因座内的变异(S)，(B)受这些因果关系影响的调控因素 变异(S)，(C)细胞类型(S)和发育时间(S)，原因变异(S) 发挥它们的作用，以及(D)受这些因果变异影响的基因(S)。在这项提案中，我们 将确定对染色质结构的两个特征(增强子)的遗传影响 组蛋白标记及其30种相互作用)，以便在人类大脑皮质发育过程中 完整解释导致神经精神障碍风险的调节机制。在一个大的 以前经历过的人死后发育皮质组织的群体 全基因组基因分型和转录图谱，我们将利用一种技术， 使我们能够同时测量增强子的活性及其相互作用 (H3K27ac HiChIP)。然后我们将确定对染色质和染色质这两个特征的遗传影响 它们与既往和日益严重的神经精神障碍全基因组关联的共同定位 (Gwas)危险基因座。精神障碍风险变种可能通过(1)发挥其调节作用 改变增强子(H3K27ac QTL或组蛋白乙酰化QTL)和/或(2)染色质 互作(互作-QTL)。这类新颖的QTL将加深我们对 人类神经发育的分子过程以及这种发育是如何改变的 神经精神障碍。进一步，我们将采用两个正交法来验证影响 并评估它们的细胞类型特异性。我们将执行特定的细胞类型 大规模平行报告分析(MPRA)验证haQTL的功能影响。在这 检测，克隆的含有增强子相关等位基因的寡核苷酸驱动条形码的表达 可用于评估调控差异和识别因果变异的记录。我们 还将应用单倍型特定的染色质成像技术来可视化如何调节 变异会影响单个核染色质的相互作用。这项技术用来涂漆 每条染色体上带有等位基因特异性寡核苷酸的切片，以便可视化和 测量0NA分子的物理相互作用。完成这项提案的目标将 使我们能够确定影响人脑发育的基本完整的调控机制 神经精神障碍的风险。

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.