Stability of the folded genome

项目摘要 由基因组结构缺陷引起的正常基因表达的扰动可导致 与衰老和各种疾病状态有关的细胞功能障碍。哺乳动物的基因组通常 组织成染色体、区室、拓扑关联结构域（TADs）和环。虽然 核反应堆和环的形成已被广泛研究，但对驱动核反应堆的过程知之甚少。 隔室形成已经提出，微相相分离驱动了 相似染色质状态的基因组结构域，导致A型（活性染色质）或B型的形成 （非活性染色质）区室。然而，由于缺乏工具， 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（HP 1）α和HP 1 β结合组成型异染色质并被 已知有助于核小体的桥接，这表明这些蛋白质在核小体的形成中起着关键作用。 异染色质区室化尽管最近的研究表明异染色质 压缩是独立的HP 1 α，我们的合作者的工作表明，这种蛋白质是必需的， 稳定异染色质基因座之间相互作用。有趣的是，HP 1蛋白和它们的一些相互作用 伴侣可以结合RNA。独立于HP 1功能，已知特异性RNA转录物在HP 1基因的转录中起重要作用。 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， 特别是在雌性细胞的核仁、斑点和不活跃的X染色体上。 我们最近开发了液体染色质Hi-C（LC-Hi-C），它可以定量染色质 全基因组相互作用稳定性测量。简言之，分离的细胞核受到原位限制， 消化.将基因组消化成特定的片段大小分布导致低聚物的损失。 密度/不稳定的相互作用，而更高的密度/稳定的相互作用被维持，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 染色质相互作用的程度在A和B区室以及区室亚结构之间变化很大。 Aim 1中“原位LC-HiC”的开发将允许对有丝分裂染色体进行稳定性测量， 简化现有的方案，并允许研究较小的细胞群。目标2将评估捐款 （HP 1）α和HP 1 β对异色性相互作用稳定性的影响。在目标3中，LC-Hi-C将允许识别 基因组区域因RNA耗竭而不稳定。然后将确定有助于稳定性的候选因素 使用原位染色质相关RNA测序（iMARGI），并通过扰动，然后通过LC-Hi- C.总之，这项研究旨在测量染色质相互作用的动态，并提供新的 关于基因组在整个细胞周期中是如何组织的机械论见解。

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.