Interplay Between Nuclear Organization and Function

项目总结/摘要 目的：着丝粒周围异染色质（PCH）是基因组稳定性/DNA修复，染色体 配对，核结构，转座子和基因沉默。以前的研究表明，组蛋白H3 赖氨酸9甲基化（H3 K9 me 2/3），异染色质蛋白1（HP 1）结合，HP 1相互作用蛋白 募集和染色质致密化足以解释PCH的形成和功能。2017年，我的实验室 Narlikar实验室发表的补充研究表明，3D PCH结构域通过液-液形成， 相分离（LLPS），产生无膜冷凝物，其具有不动的HP 1a核，所述核被 液体。我们提出了与高度网络化的相分离系统（例如， 流动性）对于理解PCH和其他染色质结构域如何形成和调节必需的核 功能协调发展的然而，我们缺乏对组织、动态和 生物物理/材料性质的PCH组分和缩合物在细胞和有机体的情况下。在 此外，我们需要确定生物物理特性是否以及如何调节基因组功能，如修复， 复制和转录，这是整个凝聚物生物学领域目前面临的主要挑战。 方法：该MIRA将询问LLPS和生物物理特性如何影响体内组织 以及异染色质和其他相关核小体的功能。我们将利用我们初步的 PCH生物学的结果和知识，结合先进的成像，生物化学，实验和 理论生物物理方法，以阐明1）负责PCH结构域的分子相互作用 形成; 2）结构域的结构、生物物理和化学性质;以及3）是否相 分离和流动性调节PCH功能并与其他核机构相互作用。 创新：尽管LLPS和生物冷凝物已成为研究的热门话题， 近年来的讨论中，我们对复合物的体内机制和功能的相关性知之甚少， 而是重要的细胞和有机体环境。这是一个新兴领域，具有独特的挑战， 要解决这些关键问题，就需要采取跨学科办法。因此，在这个MIRA提案中，我们将 联合收割机将我们在PCH生物学方面的数十年经验与合作者在实验和 理论生物物理学和高级生物成像。验证我们的假设将阐明重要信息 关于异染色质在细胞和动物中的组织和功能，可能提供一个范例- 改变了理解染色质结构域一般形式和功能的基础。 健康相关性：有缺陷的PCH会导致基因组不稳定和基因表达改变， 癌症、先天缺陷和衰老。了解PCH形成的生物物理特性， 在人类疾病中的功能改变可能会导致新的诊断和治疗方法。

PROJECT SUMMARY/ABSTRACT Goals: Peri-Centromeric Heterochromatin (PCH) is required for genome stability/DNA repair, chromosome pairing, nuclear architecture, and transposon and gene silencing. Previous studies suggested that histone H3 lysine 9 methylation (H3K9me2/3), Heterochromatin Protein 1 (HP1) binding, HP1-interacting protein recruitment and chromatin compaction are sufficient to explain PCH formation and function. In 2017, my lab and the Narlikar lab published complementary studies suggesting that 3D PCH domains form via liquid-liquid phase separation (LLPS), generating membrane-less condensates with an immobile HP1a core surrounded by a liquid. We proposed that novel properties associated with highly networked, phase separated systems (e.g. liquidity) are critical to understand how PCH, and other chromatin domains, form and regulate essential nuclear functions. However, we lack a mechanistic understanding of the organization, dynamics and biophysical/material properties of PCH components and condensates in a cellular and organismal context. In addition, we need to determine if and how biophysical properties regulate genome functions such as repair, replication and transcription, a current major challenge for the whole field of condensate biology. Approach: This MIRA will interrogate how LLPS and biophysical properties impact the in vivo organization and function of heterochromatin and other associated nuclear bodies. We will capitalize on our preliminary results and knowledge of PCH biology, combined with advanced imaging, biochemical, and experimental and theoretical biophysical approaches, to elucidate 1) the molecular interactions responsible for PCH domain formation; 2) the architectural, biophysical and chemical properties of the domain; and 3) whether or not phase separation and liquidity regulate PCH functions and interplay with other nuclear bodies. Innovation: Although LLPS and biological condensates have become a popular topic for study and discussion in recent years, we know little about in vivo mechanisms and relevance to function in the complex but important cellular and organismal contexts. This is an emerging field, with unique challenges, and an interdisciplinary approach is required to address these key questions. Thus in this MIRA proposal we will combine our decades of experience in PCH biology with the expertise of collaborators in experimental and theoretical biophysics, and advanced bioimaging. Testing our hypothesis will elucidate important information about the organization and function of heterochromatin in cells and animals, potentially providing a paradigm- shifting foundation for understanding how chromatin domains in general form and function. Health Relatedness: Defective PCH causes genome instability and altered gene expression, contributing to cancer, birth defects, and aging. Understanding how biophysical properties that underlie PCH formation and function are altered in human diseases will likely result in novel approaches to diagnosis and treatment.