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，它还涉及到许多调控因子的招募。 作为DNA、RNA和蛋白质分子的动态三维空间重排，对 定量控制基因调控的速度。然而，这些不同的组件如何同时相互作用 它们对基因调控的数量贡献是什么仍未得到解决，这主要是因为缺乏 可以将组合分子结合、空间信息和定量 对同一个体细胞内基因调控的各个方面的测量。我们将在这里高度发展 创新的方法将使我们能够产生动态的分子电影来监控DNA的运动， 核糖核酸。和蛋白质分子以一种提供关于空间信息的方式以高分辨率 分子的排列以及有关转录速率和mRNA剪接的同时信息 单个单元格内的费率。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并建立新的定量和 计算建模方法，用于从源自于 数以万计的同步单细胞。我们将使用这些方法来定量地了解 RNA-蛋白质复合体的动态组装、DNA定位和基因组DNA的结构动力学 以及这些集成组件如何随时间影响基因调控。具体地说，我们将剖析 三种RNA介导的连接动态3D核结构和基因调控的过程的动力学 生物学和疾病中的调控范式：(I)全染色体转录沉默，(Ii)动力学耦合 MRNA转录和剪接，以及(Iii)RNA在神经退行性变中诱导的聚集和细胞毒性 精神错乱。总之，这项提议的结果将产生高度创新的新方法，用于 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.