Defining molecular and gene-regulatory dysregulation in Down Syndrome tissues and models

项目摘要 唐氏综合征（DS）是一种神经发育障碍，导致认知缺陷，包括学习障碍 记忆力和语言发展，影响750个新生儿中的1个。DS是由三倍的 21号染色体（T21），导致基因剂量改变，并改变脑细胞类型的比例， 在神经元形态和成熟，提示神经发育病因。然而，底层 引起所观察到的神经病理学和功能缺陷的分子机制在很大程度上仍然是未知的。 由于大脑结构的整体复杂性，以及对细胞的不完全了解， 发育过程中DS的类型和分子途径失调，以及有限的人类相关实验 模型此外，大量转录组和表观基因组分析表明，T21不仅改变基因剂量， 在基因座内，但也导致基因表达的广泛变化，并可能导致基因调控的改变， 动力学基于这些数据，我们假设chr 21基因剂量的增加改变了整体基因表达。 在神经祖细胞中，改变神经细胞的命运规范和分化。在这里，我们利用新的基因组 包括联合单核转录组（snRNAseq）、单核染色质可及性 （snATACseq）分析，以及单细胞联合染色质相互作用和甲基化分析（sc-m3 C-seq）， 作为原代人神经祖细胞（phNPC），一种经验证的人皮质生成模型， 假说.我们将首先定义DS中的细胞特异性分子和基因调控失调， 联合snRNAseq、snATACseq和sc-m3 C-seq在对照和DS发育中的新皮层的集合中， 神经发生高峰期这种全面的多组学分析将揭示细胞组成的变化 和细胞特异性基因表达的签名在DS新皮层，以及揭示扰动细胞谱系 地图和规范。通过整合单细胞表达和表观遗传概况，我们将定义近端的 和远端基因调控元件，以及驱动DS疾病机制的转录因子。最后， 我们将利用DS患者来源的和对照phNPC细胞系的独特集合在体外模拟疾病， 为了表征DS中的神经祖细胞增殖和特化，以及神经元的变化， 形态发生和突触发生。我们在分化时间轴上执行联合snRNAseq和snATACseq 它概括了胚胎期到妊娠中期的皮质生成，以询问细胞、分子和基因 DS的调节失调，并直接比较该模型与体内DS机制。委员会共 提出一个全面的项目，提供深入的细胞生物学和分子表征的DS 使用体内组织和人类相关模型的进展，并建立该模型用于未来的机制 审讯这项研究的长期目标是提供基础分子知识， 最终有助于DS治疗的发展。

PROJECT ABSTRACT Down syndrome (DS) is a neurodevelopmental disorder causing cognitive deficits including impaired learning and memory, and language development, affecting 1 in 750 newborns. DS is caused by triplication of chromosome 21 (T21), leading to altered gene dosage, and to changes in the proportion of brain cell types, and in neuronal morphology and maturation, suggesting a neurodevelopmental etiology. However, the underlying molecular mechanisms causing the observed neuropathology and functional deficits are still largely unknown. Progress has been hindered by the overall complexity of brain architecture, an incomplete knowledge of the cell types and molecular pathways dysregulated in DS during development, and limited human-relevant experimental models. Moreover, bulk transcriptome and epigenome profiling indicates that T21 not only alters gene dosage within the locus, but also leads to broad changes in gene expression and may lead to altered gene regulatory dynamics. Based on these data, we hypothesize that increased chr21 gene dosage alters global gene expression in neural progenitors, changing neural cell fate specification and differentiation. Here, we leverage novel genomic technologies including joint single-nucleus transcriptome (snRNAseq), single-nucleus chromatin accessibility (snATACseq) profiling, and single-cell joint chromatin interaction and methylation profiling (sc-m3C-seq), as well as primary human neural progenitors (phNPCs), a validated model of human corticogenesis, to test this hypothesis. We will first define cell-specific molecular and gene-regulatory dysregulation in DS by performing joint snRNAseq, snATACseq and sc-m3C-seq in a collection of control and DS developing neocortex, at a time period of peak neurogenesis. This comprehensive multi-omic profiling will uncover changes in cell composition and cell-specific gene expression signatures in DS neocortex as well as reveal perturbations in cellular lineage maps and specification. By integrating single-cell expression and epigenetic profiles we will define the proximal and distal gene regulatory elements, as well as the transcription factors driving DS disease mechanisms. Finally, we will leverage a unique collection of DS patient-derived and control phNPC lines to model disease in vitro in order to characterize neural progenitor proliferation and specification in DS, as well as changes in neuronal morphogenesis and synaptogenesis. We perform joint snRNAseq and snATACseq over a differentiation timeline that recapitulates embryonic to mid-gestation corticogenesis in order to interrogate cellular, molecular and gene regulatory dysregulation in DS and directly compare this model with in vivo DS mechanisms. Altogether, we present a comprehensive project providing an in-depth cell biological and molecular characterization of DS progression using in vivo tissues and a human-relevant model, and establishes this model for future mechanistic interrogation. The long-term goal of this research is to provide the foundational molecular knowledge that will ultimately contribute to the development of treatments for DS.