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.

粘蛋白复合物是驱动哺乳动物基因组3-D组织的主要因素 到千座到巨型BABASE。最近的单分子实验表明，它可以挤出DNA的环， 这是基因组建筑的组织原理。与CTCF一起，一种停滞的DNA结合蛋白 粘着蛋白的易位并定义循环边界，以及促进负载的几个调节剂， 例如NIPBL或从染色质中释放（例如WAPL），粘蛋白定义了染色体中的相互作用域 这会影响发育过程中基因表达的模式，并可能导致发育疾病或癌症 尽管裸体DNA上的循环延伸已经研究了，但细胞中的粘着蛋白必须导航 核糖体包装的染色质纤维限制了对DNA上结合位点的访问，自我组织 与环域无关并竞争类似表观遗传状态的隔室，可能 调节DNA超螺旋，并用染色质相关的RNA装饰。粘着蛋白和CTCF如何相互作用 细胞中的染色质是理解3-D基因组组织的下一个领域。在这个领域的进步将 需要采用多尺度方法，并采用探测核小体规模和巨型尺度特征的方法。 我提出了探测探测的实验（1）如何通过粘着蛋白延伸循环延伸 纤维; （2）染色质纤维的局部结构是如何通过链接器Hisons和 核体的不稳定，调节粘蛋白负载和挤压环的能力； （3）如何变化 由于过量的粘着蛋白循环而导致的超串联影响局部核小体核体相互作用； （4） CTCF在RNA依赖性和RNA独立的环边界上的染色质相关RNA相互作用。 为了剖析粘着蛋白，调节剂和染色质纤维的特定作用，我们将结合使用 人和小鼠细胞系中稳定的蛋白质消耗，急性降解和药物抑制。我们 将使用RICC-Seq读取染色质纤维结构和染色质相关的RNA的变化，方法I 最近开发用于测量完整细胞中亚核小体分辨率下的DNA-DNA接触，并使用 新型技术开发以探测特定蛋白质的染色质相关RNA相互作用组。这些 方法将与更具成熟的表观基因组和转录分析工具相结合，并与粗糙 用于开发和测试多尺度模型的粒度模拟，以使环路延伸机械与与 染色质纤维。我预计这些实验的结果将为循环延伸方式提供新的启示 染色质的自我关联在特定环境中相互作用，该环境模型以粘合剂与DNA的互动模型 与循环扩展，如何在染色体之间传播超涂层以及局部 分子环境定义了环边界。这些知识可能会揭示补偿的新策略 由于粘蛋白或其调节剂的突变引起的转录失调，使用调节的可定位因素 染色质纤维，粘着蛋白的本地底物。