Cross-regulation between loop extrusion, chromatin fiber structure and chromatin-associated RNAs

环挤出、染色质纤维结构和染色质相关 RNA 之间的交叉调节

• 批准号: 10472889
• 负责人: Viviana I Risca
• 金额: $ 148.5万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10472889
• 关键词: 3-Dimensional; Acute; Affect; Architecture; Automobile Driving; Binding Sites; Cells; Chromatin; Chromatin Fiber; Chromatin Loop; Chromatin Structure; Chromosomes; Complex; DNA; DNA Packaging; DNA sequencing; DNA-Binding Proteins; Development; Epigenetic Process; Equilibrium; Gene Expression Profile; Genetic Transcription; Genome; Grain; Histones; Human; Human Genome; Knowledge; Lead; Light; Malignant Neoplasms; Measures; Methods; Modeling; Molecular; Mouse Cell Line; Mutate; Mutation; Nucleosomes; Pharmacology; Proteins; RNA; Regulation; Resolution; Shapes; Structure; Superhelical DNA; Testing; base; cohesin; developmental disease; epigenome; experimental study; frontier; mammalian genome; multi-scale modeling; new technology; simulation; single molecule; technology development; tool

The cohesin complex is a major factor driving the 3-D organization of mammalian genomes at the scale of tens to kilobases to megabases. Recent single-molecule experiments have shown that it can extrude loops of DNA, which are an organizing principle of genome architecture. Together with CTCF, a DNA-binding protein that stalls cohesin’s translocation and defines loop boundaries, and several regulators of cohesin that promote loading, such as NIPBL, or release from chromatin, such as WAPL, cohesin defines interaction domains in chromosomes that affect patterns of gene expression during development and can lead to developmental diseases or cancer when disrupted. Although loop extrusion on naked DNA has been studied, cohesin in cells must navigate nucleosome-packed chromatin fibers that restrict access to binding sites on DNA, self-organize into compartments of similar epigenetic state that are independent of and compete with loop domains, potentially regulate DNA supercoiling, and are decorated with chromatin-associated RNAs. How cohesin and CTCF interact with chromatin in cells is the next frontier in understanding 3-D genome organization. Progress in this arena will require a multi-scale approach, with methods that probe both nucleosome-scale and megabase-scale features. I propose experiments to probe (1) how loop extrusion by cohesin perturbs the local structure of the chromatin fiber; (2) how the local structure of the chromatin fiber, modulated by depletion of linker histones and destabilization of nucleosomes, regulates cohesin’s ability to load and extrude loops; (3) how changes in the balance of supercoiling due to excess cohesin looping affect local nucleosome-nucleosome interactions; and (4) the chromatin-associated RNA interactome of CTCF at RNA-dependent and RNA-independent loop boundaries. To dissect the specific effects of cohesin, its regulators and the chromatin fiber, we will use a combination of stable protein depletion, acute degradation, and pharmacological inhibition in human and mouse cell lines. We will read out changes in chromatin fiber structure and chromatin-associated RNAs using RICC-seq, a method I recently developed for measuring DNA-DNA contacts at sub-nucleosome resolution in intact cells, and using novel technology development to probe the chromatin-associated RNA interactome of specific proteins. These methods will be combined with more established epigenome and transcription profiling tools and with coarse- grained simulations to develop and test multi-scale models for the interaction of loop extrusion machinery with the chromatin fiber. I anticipate that the results of these experiments will shed new light on how loop extrusion and chromatin’s self-association interact in specific contexts, which models for cohesin’s engagement with DNA are relevant to loop extrusion, how supercoiling is disseminated across chromosomes, and how the local molecular context defines loop boundaries. This knowledge may reveal new strategies for compensating transcriptional dysregulation due to mutations in cohesin or its regulators using targetable factors that regulate the chromatin fiber, cohesin’s native substrate.

粘连蛋白复合物是驱动哺乳动物基因组在数十个尺度上进行 3D 组织的主要因素。 到千碱基到兆碱基。最近的单分子实验表明它可以挤出DNA环， 这是基因组架构的组织原则。与 CTCF 一起，一种 DNA 结合蛋白，可以阻止 粘连蛋白的易位并定义环边界，以及促进加载的粘连蛋白的几个调节剂， 例如 NIPBL，或从染色质释放，例如 WAPL，粘连蛋白定义染色体中的相互作用域 影响发育过程中基因表达模式并可能导致发育疾病或癌症 当受到干扰时。尽管已经研究了裸露 DNA 上的环挤出，但细胞中的粘连蛋白必须导航 核小体填充的染色质纤维限制进入 DNA 上的结合位点，自组织成 具有相似表观遗传状态的区室可能独立于环结构域并与环结构域竞争 调节 DNA 超螺旋，并用染色质相关 RNA 进行修饰。粘连蛋白和 CTCF 如何相互作用 细胞中染色质的研究是理解 3D 基因组组织的下一个前沿领域。这一领域的进展将 需要一种多尺度方法，采用同时探测核小体尺度和兆碱基尺度特征的方法。 我建议进行实验来探究（1）粘连蛋白的环挤出如何扰乱染色质的局部结构 纤维; (2) 染色质纤维的局部结构如何通过连接组蛋白的耗尽进行调节 核小体的不稳定，调节粘连蛋白加载和挤出环的能力； (3)如何变化 由于过度的粘连蛋白环而导致的超螺旋平衡影响局部核小体-核小体相互作用；和（4） CTCF 在 RNA 依赖性和 RNA 独立环边界处的染色质相关 RNA 相互作用组。 为了剖析粘连蛋白、其调节剂和染色质纤维的具体作用，我们将结合使用 人类和小鼠细胞系中的稳定蛋白质消耗、急性降解和药理抑制。我们 将使用 RICC-seq（方法 I）读出染色质纤维结构和染色质相关 RNA 的变化 最近开发用于测量完整细胞中亚核小体分辨率的 DNA-DNA 接触，并使用 开发新技术来探测特定蛋白质的染色质相关 RNA 相互作用组。这些 方法将与更成熟的表观基因组和转录分析工具以及粗略的相结合 粒度模拟，用于开发和测试环挤出机械与机械相互作用的多尺度模型 染色质纤维。我预计这些实验的结果将为环挤出如何产生新的启示 和染色质的自关联在特定环境中相互作用，这模拟了粘连蛋白与 DNA 的结合 与环挤出、超螺旋如何跨染色体传播以及局部如何传播有关。 分子上下文定义了循环边界。这些知识可能会揭示新的补偿策略 由于粘连蛋白或其调节因子的突变导致转录失调，使用可调节的靶向因子 染色质纤维，粘连蛋白的天然底物。