Single Cell and Single Molecule Technologies for Multiplex Chromatin Interaction Analysis

项目摘要 细胞核中的哺乳动物基因组DNA广泛折叠，形成复杂的三维（3D）结构。 染色质组织，包括涉及DNA的染色质相互作用的复杂和多价相互作用， RNA和蛋白质。3D基因组的排列是为了促进多种功能的相互作用，其中我们的 目前的理解是有限的，但最终用于调节细胞内的基因表达。一个 通过观察3D结构和功能， 染色质折叠的相互作用不是静态的，而是在给定细胞内随时间变化的动态的， 相同类型的细胞之间。了解这些复杂的功能相互作用及其变化， 这不仅是推进基础生物学知识的必要条件，也是提供新见解的必要条件。 人类疾病的研究，这可能会带来新的治疗模式。这个项目的科学前提是， 在任何给定的时间，通过复杂的3D基因组，多个染色质相互作用发生在多个位置 这些相互作用至少部分是由蛋白质和RNA因子介导的。 不幸的是，目前3D基因组技术的局限性使我们无法精确地揭示这一水平 在理想的单分子分辨率下的功能复杂性。在本建议中，我们寻求制定一套 单细胞和单分子技术，用于研究多个复杂的染色质相互作用， 3D基因组组织中的蛋白质和RNA调节因子。我们战略的基础在于 用于复合物的单细胞和单分子检测的基于液滴和条形码连接的微流体系统 染色质相互作用我们已经开发了一个用于分析单分子染色质相互作用的原型， 称为ChIA-Drop（通过液滴测序进行染色质相互作用分析），适用于相对较小的 果蝇基因组。为了开发和完善这种用于哺乳动物细胞的方法，并开始揭示 对于健康和疾病中的染色质拓扑结构和基因组功能至关重要的相互作用，我们建议 实现以下四个目标：目标1-基于果蝇的已发表结果的概念验证 我们将使用人类和小鼠细胞系对较大的哺乳动物基因组进行改进。目的 2 -我们将开发一种新的双索引策略（核特异性和染色质特异性）用于单细胞ChIA- 滴分析（scChIA-Drop），并将其应用于研究多重染色质与单细胞和 在人类和小鼠细胞中的单分子分辨率。在目标3 -我们将扩展ChlA-Drop检测 由蛋白质和RNA因子介导的多价染色质相互作用。最后，由于数据集产生 ChlA-Drop是新的数据类型，在目标4中，我们将建立强大的计算管道和工具， 解码染色质的相互作用，并使其公开。这些先进的发展 ChIA-Drop技术和工具将使染色质相互作用生物学的前所未有的探索， 推进我们对染色质拓扑结构和基因组调控功能的理解。

PROJECT SUMMARY Mammalian genome DNA in the cell nucleus is extensively folded to form a complex three-dimensional (3D) chromatin organization, comprising complex and multivalent interplays of chromatin interactions involving DNA, RNA and protein. The 3D genome is arranged so as to facilitate multiple functional interactions, of which our current understanding is limited, but that ultimately serve to regulate gene expression within a cell. An additional layer of complexity is introduced by the observations that the 3D structural and functional interactions of the chromatin folding are not static, but rather dynamic both over time within a given cell, and between cells of the same type. Understanding these complex functional interactions and their variations will be necessary not only for advancing fundamental biological knowledge, but also for providing novel insights into human disease that could lead to new treatment paradigms. The scientific premise of this project is that at any given time, multiple chromatin interactions are occurring at multiple locations through intricate 3D genome organization and that these interactions are mediated, at least in part, by protein and RNA factors. Unfortunately, the limitations of current 3D genome technologies prevent us from precisely revealing this level of functional complexity at the desirable single-molecule resolution. In this proposal we seek to develop a set of single cell and single molecule techniques for studying multiple, complex chromatin interactions involving protein and RNA regulatory factors within the 3D genome organization. The foundation of our strategy lies in a droplet-based and barcode-linked microfluidics system for single cell and single-molecule detection of complex chromatin interactions. We have developed a prototype for analysis of single-molecule chromatin interactions, called ChIA-Drop (Chromatin Interaction Analysis by Droplet sequencing) that works well for a relatively small Drosophila genome. To develop and refine this approach for mammalian cells, and to begin to uncover the interactions that are critical to chromatin topology and genome functions in health and disease, we propose to achieve the following four aims: Aim 1- Based on proof-of-concept of published results for the Drosophila genome we will use human and murine cell lines to make refinements for the larger mammalian genomes. Aim 2 - we will develop a novel dual-indexing strategy (nucleus-specific and chromatin-specific) for single-cell ChIA- Drop analysis (scChIA-Drop), and will apply it to study multiplex chromatin interactions with single-cell and single-molecule resolution in human and in mouse cells. In Aim 3 - we will extend ChlA-Drop to detect multivalent chromatin interactions mediated by protein and RNA factors. Finally, because datasets produced by ChlA-Drop are new data types, in Aim 4 - we will establish robust computational pipelines and tools for decoding chromatin interactions and make them publicly accessible. The development of these advanced ChIA-Drop technologies and tools will enable unprecedented exploration of chromatin interaction biology and advance our understanding of chromatin topology and genome regulatory functions.