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.

粘附素复合体是驱动哺乳动物基因组三维组织的一个主要因素，其规模为TENS 从千基数到兆基数。最近的单分子实验表明，它可以挤出DNA环， 这是基因组结构的组织原则。与CTCF一起，一种DNA结合蛋白 粘附素的易位和定义环边界，以及促进负荷的粘附素的几个调节因子， 如NIPBL，或从染色质释放，如WAPL，粘附素定义了染色体中的相互作用区域 在发育过程中影响基因表达模式，并可能导致发育性疾病或癌症 当被打乱时。尽管裸露DNA上的环挤压已经被研究过，但细胞中的粘附素必须导航 核小体包裹的染色质纤维限制对DNA结合部位的访问，自组织成 类似表观遗传状态的隔间，它们独立于环域并与环域竞争，潜在地 调节DNA超级卷曲，并装饰着染色质相关的RNA。粘附素和CTCF如何相互作用 在细胞中发现染色质是理解三维基因组组织的下一个前沿。这一领域的进步将 需要多尺度的方法，方法既要探测核小体尺度的特征，又要探测兆基尺度的特征。 我提议用实验来探索(1)粘附素的环状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是如何受连接器组蛋白和 核小体的不稳定，调节粘附素加载和挤出环的能力；(3)如何改变 过度粘连素环导致的超螺旋平衡影响局部核小体-核小体相互作用；以及(4) CTCF染色质相关的RNA相互作用组在RNA依赖和RNA非依赖的环边界。 为了分析粘附素、它的调节器和染色质纤维的具体影响，我们将使用以下组合 人类和小鼠细胞系中稳定的蛋白质耗竭、急性降解和药理抑制。我们 将使用RICC-SEQ读取染色质纤维结构和染色质相关RNA的变化，这是一种方法I 最近开发的用于在完整细胞中以亚核小体分辨率测量DNA-DNA接触的方法，并使用 探索特定蛋白质染色质相关RNA相互作用组的新技术进展。这些 方法将与更成熟的表观基因组和转录图谱工具相结合，并与粗略- 开发和测试环流挤出机与多尺度模型相互作用的粒度模拟 染色质纤维。我预计，这些实验的结果将为环状挤出如何 染色质的自关联在特定的环境中相互作用，这为凝集素与DNA的结合提供了模型 与环挤出有关，超螺旋是如何在染色体上传播的，以及局部 分子背景定义了环的边界。这一知识可能会揭示补偿的新策略 使用靶向调节因子引起的粘附素或其调节器突变所致的转录失调 染色质纤维，粘附素的天然底物。