How phase-separation in the nucleus organizes 3D spatial assembly and gene regulation

项目摘要 基因调控是一个高度复杂的过程，涉及到许多调节因子的招募以及 DNA、RNA和蛋白质分子的动态三维空间重排， 定量控制基因调控的速率。然而，这些不同的组成部分如何同时相互作用， 它们对基因调控的定量贡献仍然没有得到解决，主要是因为缺乏 的方法，可以整合组合分子结合，空间信息，和定量 测量同一单个细胞内基因调控的各个方面。在这里我们将得到高度发展 创新的方法，使我们能够产生动态的分子电影，监测DNA的运动， 核糖核酸和蛋白质分子在高分辨率的方式，提供有关的空间信息， 分子沿着排列，同时具有关于转录速率和mRNA剪接的信息 在单个细胞内。为了实现这一目标，我们将开发开创性的新基因组方法来测量 RNA、DNA和蛋白质与单细胞能力的空间相互作用， 计算建模方法，以从从以下导出的快照生成高分辨率时间“电影”： 成千上万的同步单细胞。我们将使用这些方法来定量地了解 RNA-蛋白质复合物的动态组装、DNA定位和基因组DNA的结构动力学 以及这些整合的成分如何影响基因调控。具体来说，我们将剖析 三种RNA介导的过程的动态，这些过程将动态3D核结构和基因调控联系在一起， 生物学和疾病中的调控范例：（i）染色体范围的转录沉默，（ii）动力学偶联 的mRNA转录和剪接，和（iii）RNA诱导的聚集和神经退行性疾病的细胞毒性， 紊乱总之，这一建议的结果将产生高度创新的新方法， 测量各种调控因子的分子和空间动力学及其在基因调控中的作用。

Project Summary Gene regulation is a highly complex process that involves the recruitment of numerous regulatory factors as well as dynamic 3-dimensional spatial rearrangements of DNA, RNA, and protein molecules that are important for quantitatively controlling the rate of gene regulation. Yet, how these various components interact simultaneously and what their quantitative contributions are to gene regulation remains unresolved largely because of the lack of methods that can integrate combinatorial molecular binding, spatial information, and quantitative measurements of various aspects of gene regulation within the same individual cell. Here we will develop highly innovative methods that will allow us to generate dynamic molecular movies that monitor the movement of DNA, RNA. and protein molecules at high resolution in a manner that provides information about the spatial arrangement of molecules along with simultaneous information about transcription rates and mRNA splicing rates within single cells. To achieve this, we will develop pioneering new genomic methods for measuring the spatial interactions of RNA, DNA, and protein with single cell capabilities and build novel quantitative and computational modeling approaches to generate high resolution temporal “movies” from snapshots derived from tens of thousands of synchronized single cells. We will use these approaches to quantitatively understand the dynamic assembly of RNA-protein complexes, localization to DNA, and structural dynamics of genomic DNA and how these integrated components impact gene regulation across time. Specifically, we will dissect the dynamics of three RNA-mediated processes that link dynamic 3D nuclear structure and gene regulation in unique regulatory paradigms in biology and disease: (i) chromosome-wide transcriptional silencing, (ii) kinetic coupling of mRNA transcription and splicing, and (iii) RNA-induced aggregation and cellular toxicity in neurodegenerative disorders. Together, the results of this proposal will generate highly innovative new approaches for quantitatively measuring molecular and spatial dynamics of various regulators and their role in gene regulation.