Stability of the folded genome

折叠基因组的稳定性

• 批准号: 10471271
• 负责人: Denis Lafontaine
• 金额: $ 3.42万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-27 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10471271
• 关键词: Acute; Aging; Automobile Driving; Binding; Biological Assay; Cell Cycle; Cell Line; Cell Nucleus; Cells; Chromatin; Chromatin Loop; Chromosomes; Defect; Detergents; Development; Digestion; Disease; Female; Functional disorder; Gene Expression; Genome; Genomic Segment; Genomics; Goals; Heterochromatin; Hi-C; In Situ; Interphase; Kinetics; Lead; Link; Liquid substance; Maintenance; Malignant Neoplasms; Maps; Measurement; Measures; Mediating; Methods; Mitosis; Mitotic; Mitotic Chromosome; Nuclear; Nucleosomes; Pancreatic ribonuclease; Phase; Play; Population; Process; Protein Family; Proteins; Protocols documentation; RNA; RNA Binding; Role; Structure; Techniques; Testing; Transcript; Work; X Chromosome; X Inactivation; biophysical properties; chromatin isolation by RNA purification sequencing; chromosome conformation capture; cost effective; density; detection method; genome-wide; heterochromatin-specific nonhistone chromosomal protein HP-1; insight; malformation; mammalian genome; novel strategies; restriction enzyme; tool; transcriptome sequencing

PROJECT SUMMARY Perturbations in normal gene expression arising from defects in genome organization can lead to cellular dysfunctions linked to aging and various disease states. The mammalian genome is generally organized into chromosomes, compartments, topological associating domains (TADs) and loops. Although TAD and loop formation have been extensively studied, little is known about the processes that drive nuclear compartment formation. It has been proposed that microphase phase separation drives the association of genomic domains of similar chromatin state, resulting in the formation of either type A (active chromatin) or B (inactive chromatin) compartments. However, identifying factors involved has been limited by a lack of tools capable of quantifying the biophysical properties driving this phenomenon. Mammalian heterochromatin protein 1 (HP1) α and HP1β bind constitutive heterochromatin and are known to facilitate the bridging of nucleosomes, suggesting that these proteins play a key role in heterochromatin compartmentalization. Although a recent study has demonstrated that heterochromatin compaction is independent of HP1α, work from our collaborators suggest that this protein is required to stabilize interactions between heterochromatic loci. Interestingly, HP1 proteins and several of their interacting partners can bind RNAs. Independent of HP1 function, specific RNA transcripts are known to play important roles in the formation and maintenance of spatial genome organization and perhaps microphase separation, notably at nucleoli, speckles, and the inactive X chromosome of female cells. We recently developed liquid chromatin Hi-C (LC-Hi-C), which allows quantification of chromatin interaction stability measurements genome-wide. Briefly, isolated nuclei are subject to in situ restriction digestion. Digestion of the genome into a specific fragment size distribution results in the loss of low density/unstable interactions whereas higher density/stable interactions are maintained, which is quantifiable by genome-wide chromosome conformation capture (Hi-C). This technique reveals that the dissolution kinetics of chromatin interactions vary widely between A and B compartments as well as compartmental substructures. The development of “in situ LC-HiC” in Aim 1 will allow stability measurement on mitotic chromosomes, streamline the existing protocol and allow the study of smaller cell populations. Aim 2 will assess contributions of (HP1) α and HP1β to stability of heterochromatic interactions. In Aim 3, LC-Hi-C will allow identification of genomic regions destabilized by RNA depletion. Candidate factors contributing to stability will then be identified using in situ chromatin-associated RNA sequencing (iMARGI) and validated by perturbation followed by LC-Hi- C. Taken together, this study aims to measure the dynamics of chromatin interactions and to provide new mechanistic insight as to how the genome is organized throughout the cell cycle.

项目概要 基因组组织缺陷引起的正常基因表达的扰动可能导致 与衰老和各种疾病状态相关的细胞功能障碍通常是哺乳动物的基因组。 组织成染色体、区室、拓扑关联域 (TAD) 和环。 主要研究了 TAD 和环形成，但对驱动核的过程知之甚少。 有人提出，微相相分离驱动了缔合。 具有相似染色质状态的基因组结构域，导致形成 A 型（活性染色质）或 B 型 然而，由于缺乏工具，所涉及的因素的识别受到限制。 能够量化驱动这种现象的生物物理特性。 哺乳动物异染色质蛋白 1 (HP1) α 和 HP1β 结合组成型异染色质，并且是 已知促进核小体桥接，表明这些蛋白质在 尽管最近的一项研究表明异染色质区室化。 压缩与 HP1α 无关，我们合作者的工作表明，这种蛋白质需要 异染色质位点之间的相互作用。 伴侣可以结合 RNA，而与 HP1 功能无关，已知特定的 RNA 转录物发挥着重要作用。 在空间基因组组织的形成和维持以及可能的微相分离中的作用， 值得注意的是雌性细胞的核仁、斑点和不活跃的 X 染色体。 我们最近开发了液体染色质 Hi-C (LC-Hi-C)，它可以对染色质进行定量 简而言之，分离的细胞核受到原位限制。 将基因组消化成特定的片段大小分布会导致低的损失。 密度/不稳定的相互作用，而保持较高密度/稳定的相互作用，这是可量化的 通过全基因组染色体构象捕获（Hi-C）该技术揭示了溶解动力学。 A 区室和 B 区室以及区室亚结构之间的染色质相互作用差异很大。 目标 1 中“原位 LC-HiC”的开发将允许测量有丝分裂染色体的稳定性， 简化现有方案并允许研究较小的细胞群，目标 2 将评估贡献。 (HP1) α 和 HP1β 对异色相互作用稳定性的影响 在目标 3 中，LC-Hi-C 将允许识别 然后将鉴定因 RNA 消耗而不稳定的候选因素。 使用原位染色质相关 RNA 测序 (iMARGI) 并通过扰动和 LC-Hi- 进行验证 C. 综上所述，本研究旨在测量染色质相互作用的动态并提供新的 关于基因组如何在整个细胞周期中组织的机制见解。