Regulatory Mechanisms Linking Spatial Gene Control and Genome Organization

项目总结 细胞动态平衡是通过信号事件快速启动下游转录因子来维持的。 激活或抑制数百个基因。下一代测序技术和超级计算机的应用 过去十年的分辨率成像揭示了非编码调控元件的核心作用，称为 哺乳动物基因表达时空调控中的增强剂。最近的证据表明，急性 信号事件将增强子结合的转录复合体组装成已知的无膜室 作为冷凝物。一个重要的问题是这些细胞器如何与较大的核体相互作用以形成 三维基因组组织和空间基因控制。我们已经确定了三个关键挑战 这限制了我们对空间基因控制的理解。我们的研究计划致力于解决这些挑战 填补信号诱导基因调控中的关键知识空白。挑战1)了解增强器如何 沟通是有效的。除了控制主要的靶基因外，几种途径特异性增强子 参与远距离互动。我们的数据表明，增强子中心促进了全基因组的协调 信令程序。这一机制在信号激活基因中的流行及其调控特征 项目才刚刚开始探索。我们将使用典型的I型和II型核激素受体 研究增强子中心在转录协调中的作用。挑战2)阐明非 基因控制中的编码基因组和无序蛋白质组。我们已经报道了配体诱导的 增强子缩合物由带有固有无序区(IDR)的蛋白质和RNA组成。虽然 许多转录复合体的一个共同特征是组装的分子和酶调节， 转录缩合物的溶解和物质性质在很大程度上是未知的。我们将使用单细胞 CRISPR筛查策略、突变扫描、生物物理和基因组学分析以鉴定酶 通过修改IDR结构控制信号激活的增强子。挑战3)拆解细胞 空间基因组组织的生物学基础。真核生物基因组的划分是基于 染色质的转录状态。我们的数据表明，几个核建筑结构和 转录缩合物作为固态锚促进长距离增强子相互作用和 组织染色质架构。获得对基因组中常见核体的作用的机械性见解 ，我们将使用遗传策略来暂时降解这些细胞器并评估其影响 用成像和基因组学工具研究染色质的三维结构和增强子功能。通过 在非编码基因组、核凝聚体和基因的交叉点上解决这些问题 调控，我们将揭开空间基因调控的细胞生物学基础。这项研究还将有助于 开发创新的研究工具以填补非编码基因组调控作用方面的知识空白 同时促进多学科科学领域下一代研究人员的培训和指导。

PROJECT SUMMARY Cellular homeostasis is maintained by signaling events that turn on downstream transcription factors to rapidly activate or repress hundreds of genes. The application of next-generation sequencing technologies and super- resolution imaging in the past decade has revealed the central role of non-coding regulatory elements called enhancers in the spatio-temporal control of mammalian gene expression. Recent evidence indicates that acute signaling events assemble the enhancer-bound transcriptional complex as membraneless compartments known as condensates. An important question is how these organelles interact with larger nuclear bodies to shape three-dimensional genome organization and spatial gene control. We have identified three critical challenges that limit our understanding of spatial gene control. Our research program is focused on solving these challenges to fill the critical knowledge gaps in signal-induced gene regulation. Challenge 1) Understand how enhancer communication works. In addition to controlling primary target genes, several pathway-specific enhancers engage in long-distance interactions. Our data indicate that enhancer hubs facilitate genome-wide coordination of signaling programs. The prevalence and regulatory features of this mechanism in signal-activated gene programs are just beginning to be explored. We will use prototypic type I and type II nuclear hormone receptors to study the role of enhancer hubs in transcription coordination. Challenge 2) Elucidate the interaction of non- coding genome and disordered proteome in gene control. We have reported that the ligand-induced enhancer condensates are composed of proteins with intrinsically disordered regions (IDR) and RNAs. Although a common feature of many transcriptional complexes, the molecular and enzymatic regulation of the assembly, dissolution, and material properties of transcriptional condensates are largely unknown. We will use single-cell CRISPR screening strategies, mutational scans, biophysical and genomics assays to identify enzymes controlling signaling-activated enhancers by modifying the IDR structure. Challenge 3) Unravel the cell biological basis of the spatial genome organization. The eukaryotic genome is compartmentalized based on the transcriptional states of the chromatin. Our data indicate that several nuclear architectural structures and transcriptional condensates act as solid-state anchors to facilitate long-distance enhancer interaction and organize chromatin architecture. To gain mechanistic insights into the role of common nuclear bodies in genome organization, we will employ genetic strategies to transiently degrade these organelles and assess the impact on three-dimensional chromatin structure and enhancer function using imaging and genomics tools. By addressing these questions at the intersection of non-coding genome, nuclear condensates, and gene regulation, we will unravel a cell biological basis of spatial gene control. This research will also contribute to developing innovative research tools to fill the knowledge gaps in the regulatory roles of the non-coding genome while facilitating the training and mentoring of the next generation of researchers in multidisciplinary science.