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

项目摘要 唐氏综合症(DS)是一种神经发育障碍，会导致包括学习障碍在内的认知障碍 以及记忆力和语言发展，每750名新生儿中就有一人受到影响。DS是由三倍以上的 21号染色体(T21)，导致基因剂量改变，脑细胞类型比例改变，以及 在神经元形态和成熟方面，提示神经发育病因学。然而，潜在的 导致观察到的神经病理和功能缺陷的分子机制在很大程度上仍不清楚。 由于大脑结构的整体复杂性，对细胞的不完全了解，进展受到了阻碍 DS的类型和分子通路在发育过程中调节失调，并限制与人类相关的实验 模特们。此外，批量转录组和表观基因组图谱表明，T21不仅改变了基因的剂量 在基因座内，也会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chr21基因剂量增加改变了全球基因表达。 在神经祖细胞中，改变神经细胞命运的指定和分化。在这里，我们利用新的基因组 包括联合单核转录组(SnRNAseq)、单核染色质可及性等技术 (SnATACseq)，以及单细胞联合染色质相互作用和甲基化分析(sc-M3C-seq) 作为人类初级神经前体细胞(PhNPC)，一种经过验证的人类皮质生成模型，来测试这一点 假设。我们将首先通过以下方式定义DS的细胞特异性分子和基因调控失调 一次将SnRNAseq、SnATACseq和sc-M3C-seq联合在控制和DS开发新皮质的集合中 神经发生的高峰期。这一全面的多组图谱将揭示细胞组成的变化 以及DS新皮质中细胞特异的基因表达特征，以及揭示细胞谱系的扰动 地图和规格。通过整合单细胞表达和表观遗传学图谱，我们将定义近端 以及远端基因调控元件，以及驱动DS疾病机制的转录因子。最后， 我们将利用一个独特的DS患者来源和对照phNPC细胞系的集合来在体外建立疾病模型 为了表征DS中神经前体细胞的增殖和规范，以及神经元的变化 形态发生和突触发生。我们在区分时间线上联合执行SnRNAseq和SnATACseq 它重述了胚胎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，并直接将该模型与体内DS机制进行比较。总而言之，我们 提出了一个全面的项目，提供了DS的深入细胞生物学和分子特征 利用活体组织和与人类相关的模型进行进展，并为未来的机制建立了这个模型 审问。这项研究的长期目标是提供基础的分子知识， 最终有助于DS治疗方法的发展。

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.