Beyond Gene Dosage: Understanding Down Syndrome via 4D Genome Organization

摘要 染色体非整倍体是多种人类疾病的基础。其中最突出的范例 唐氏综合症(DS)是由人类第21号染色体(HSA21)的额外拷贝引起的。作为 最常见的人类认知障碍遗传病，DS大约每750名活产婴儿中就有1名受到影响 在美国，它预先使患者患上肌肉张力低下、畸形特征、先天性心脏病和 早发性阿尔茨海默氏症。尽管取得了许多进展，但我们对病理的概念性理解 到目前为止，这种染色体异常的基础在很大程度上局限于“基因剂量假说”，即 然而，不能解释在特定细胞类型的整个基因组中发生的广泛的基因去调控。 3D基因组错误折叠是否可能在DS和其他疾病中发挥尚未实现的作用尚不清楚 非整倍体。在这里，我们组建了一个强大的团队来测试一个总体假设，即21三体的存在 将3D基因组作为一个整体解除管制，并改变DS细胞的基因表达，特别是通过形成 染色体间异常相互作用(ICIS)。我们有两个具体目标。在AIM-1中，在多对 同基因的IPSC细胞及其衍生的神经元/神经胶质细胞含有二体和三体HSA21，我们将 进行分析以系统地描述它们的3D基因组(原位Hi-C和Plac-Seq)、转录组 一维表观基因组(Pro-Seq、ATAC-Seq和组蛋白修饰芯片-Seq)。综合分析将 解剖异常的染色质相互作用，特别是在三体中改变的染色体间相互作用。 细胞核，并与特定发育阶段或细胞类型(神经元、 星形胶质细胞或小胶质细胞)。我们将使用基于长阅读测序的前沿新技术来进一步 描述异常的染色体间相互作用，并将使用DNA和/或RNA FISH来验证它们。在……里面 Aim-2，我们重点从功能上剖析了染色体间异常相互作用在基因中的作用 放松管制。这将首先通过化学和表观遗传扰动在两种培养的原代细胞中进行研究 神经前体细胞和大脑皮质器官中。然后我们将使用新的光遗传工具来模拟 与疾病相关的ICIS的形成，以推断它们在基因放松调控中的潜在因果作用。预期中的 这一提议的结果不仅对我们理解4D基因组具有重要意义，而且对人类也具有重要意义 大脑发育障碍。这里产生的知识将揭示许多形式的非整倍体， 提供一个超越“基因剂量效应”的新概念框架，以理解基因放松管制，以及 启发通过恢复3D基因组结构来改善这些疾病的策略。

ABSTRACT Chromosomal aneuploidy underlies a variety of human diseases. The most prominent paradigm among these is Down syndrome (DS) that is caused by an extra copy of homo sapiens chromosome 21 (HSA21). As the most common genetic disease of human cognitive impairment, DS affects about 1 in 750 live-born infants in the US, and it pre-disposes patients to muscle hypotonia, dysmorphic features, congenital heart defects and early onset Alzheimer's disease. Despite many progress, our conceptual understanding of the pathological basis of such chromosomal abnormality is so far largely limited to the “gene dosage hypothesis”, which however cannot explain broad gene deregulation that takes place throughout the genome in specific cell types. It has been unexplored that whether 3D genome mal-folding may play yet unrealized roles in DS and other aneuploidies. Here, we assembled a strong team to test an overall hypothesis that the presence of trisomy 21 deregulates 3D genome as an entirety and changes gene expression in DS cells, particularly via forming aberrant inter-chromosomal interactions (ICIs). We have two specific aims. In Aim-1, in multiple pairs of isogenic iPSC cells and their derived neuron/glia cells that contain disomic versus trisomy HSA21, we will conduct assays to systematically characterize their 3D genome (in situ Hi-C and PLAC-Seq), transcriptome and 1D epigenome (PRO-Seq, ATAC-Seq, and histone modification ChIP-Seq). Integrative analyses will dissect the aberrant chromatin interactomes, particularly these interchromosomal interactions altered in trisomy nucleus, and correlate those with gene deregulation in specific developmental stages or cell types (neurons, astrocytes or microglia). We will use leading-edge new techniques based on long reads sequencing to further characterize aberrant inter-chromosomal interactions, and will validate them using DNA and/or RNA FISH. In Aim-2, we focus on functionally dissecting the roles of aberrant inter-chromosomal interactions in gene deregulation. This will be first investigated by chemical and epigenetic perturbation in both cultured primary neural progenitor cells and in brain cortical organoids. We will then use novel optogenetic tools to model disease-relevant formation of ICIs to deduce their potential causal roles in gene deregulation. The expected results from this proposal are significant not only to our understanding of the 4D genome, but also to human brain developmental disorders. The knowledge generated here will shed light on many forms of aneuploidy, providing a new conceptual framework beyond “gene dosage effects” to understand gene deregulation, and inspire strategies to ameliorate these diseases via restoring 3D genome architecture.