Nucleome Positioning System for Spatiotemporal Genome Organization and Regulation

 产品说明：该提案旨在满足社区对全面的高分辨率基因组图谱平台的需求，该平台将能够调查人类基因组的结构，功能和时空组织。我们的最终目标是在3D基因组结构的背景下提供复杂的染色质相互作用网络图，从中可以监测和参考单个基因组元件的动态。在这里，我们建议开发一个核组定位系统（Nucleome Positioning System，简称NUS），包括1）一个强大的全基因组定位技术平台，2）先进的计算建模算法和3）最先进的核成像方法，这将允许全社区的用户揭示人类细胞中3D基因组组织的调控功能。NPS将基于已建立的ChIA-PET方法（1，2），并通过工艺优化进行增强-即，基于微流体的微型化和基于Tn 5转座酶的文库扩增，以促进在更广泛的人类细胞类型中由蛋白质因子介导的染色质相互作用的研究（目的1，另见绘图技术开发组件）。我们还将优化RICh-PET，用于非编码RNA介导的染色质相互作用的全面映射（Aim 1）。通过这些优化工作生成的高质量映射数据将由新的计算平台（三维核组建模引擎或3D-NOME）进行分析，该平台利用分层多尺度来建模3D基因组结构（目标2，数据分析和建模组件）。我们还将利用与遗传疾病（GWAS）相关的转录组，表观基因组和SNP数据补充3D建模，以提供结构单元的功能注释（Aim 2）。我们将继续制定策略，在人类细胞系和小鼠模型中，使用新的核成像技术在结构上验证3D-NOME预测的核组几何形状，并使用尖端的基因组和表观基因组编辑方法在功能上验证3D-NOME预测的核组几何形状（目标3，生物验证组件）。最后，我们将实施生物信息学，从广泛的人类细胞系和从全血中分选的原代免疫细胞中生成试点3D基因组图谱，以阐明人类基因组组织在主要发育和造血细胞谱系中的时空动态，以及参与免疫应答的分化淋巴细胞之间的时空动态（目标4，数据生成组件）。这些努力将为更大的研究社区提供一套强大的复杂，高质量的工具和映射数据，并将有助于建立未来3D/4D核组研究的标准。它们还将提供对组织结构和调节人类基因组功能的广泛机制的见解，以及在核水平上调节免疫反应的具体机制。

 DESCRIPTION: This proposal seeks to fulfill a community need for a comprehensive, high-resolution genome-mapping platform that will enable investigation of the structural, functional and spatiotemporal organization of the human genome. Our ultimate goal is to deliver complex chromatin interaction network maps in the context of 3D genome structures from which the dynamics of individual genomic elements can be monitored and referenced. Here, we propose to develop a Nucleome Positioning System (NPS)-comprised of 1) a robust genome- wide mapping technology platform, 2) advanced computational modeling algorithms and 3) state-of-the-art nuclear imaging methods-that will allow users community-wide to uncover the regulatory functions of 3D genome organization in human cells. NPS will be based upon the established ChIA-PET method (1,2), enhanced by process optimizations-i.e., microfluidic-based miniaturization and Tn5-transposase-based library preparation-to facilitate the study of chromatin interactions mediated by protein factors across a broader range of human cell types (Aim 1, see also Mapping Technology Development Component). We will also optimize RICh-PET for the comprehensive mapping of chromatin interactions mediated by non-coding RNAs (Aim 1). The high-quality mapping data generated through these optimization efforts will be analyzed by a new computational platform (Three-Dimensional Nucleome Modeling Engine, or 3D-NOME) that makes use of hierarchical multi-scaling to model 3D genome structures (Aim 2, Data Analysis and Modeling component). We will also complement the 3D modeling with transcriptome, epigenome and SNP data associated with genetic diseases (GWAS) to provide functional annotation to structural units (Aim 2). We will continue by developing strategies to validate the nucleome geometry predicted by 3D-NOME both structurally, using new nuclear imaging technologies, and functionally, using cutting-edge genome- and epigenome-editing approaches, in both human cell lines and mouse models (Aim 3, Biological Validation Component). Finally, we will implement NPS to generate pilot 3D genome maps from a wide range of human cell lines and primary immune cells sorted from whole blood, to elucidate the spatiotemporal dynamics of human genome organization over major developmental and hematopoietic cell lineages, as well as among differentiating lymphocytes involved in the immune response (Aim 4, Data Generation Component). Together, these efforts will yield a powerful set of sophisticated, high-quality tools and mapping data for the larger research community, and will help establish the standards for future 3D/4D nucleome studies. They will also provide insights into the broad mechanisms that organize the structure and regulate the function of the human genome, as well as the specific mechanisms by which immune responses are regulated at the nuclear level.