Interplay Between Nuclear Organization and Function

项目摘要/摘要 目的：外着丝粒异染色质(PCH)是基因组稳定/DNA修复、染色体所必需的 配对、核结构、转座子和基因沉默。先前的研究表明，组蛋白H3 赖氨酸9甲基化(H3K9me2/3)，异染色质蛋白1(HP1)结合，HP1相互作用蛋白 重新募集和染色质压缩足以解释PCH的形成和功能。2017年，我的实验室 纳利卡实验室发表的补充研究表明，3D PCH结构域通过液体-液体形成 相分离(LLP)，产生无膜冷凝液，其周围有固定的HP1a核 一种液体。我们提出了与高度网络化的、相分离的系统相关的新性质(例如， 流动性)是理解PCH和其他染色质结构域如何形成和调节基本核的关键 功能。然而，我们缺乏对组织、动态和 细胞和生物体环境中PCH组分和冷凝物的生物物理/材料特性。在……里面 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，目前凝析油生物学整个领域面临的主要挑战。 方法：此Mira将询问LLP和生物物理特性如何影响体内组织 异染色质和其他相关核体的功能。我们将利用我们的初步经验 PCH生物学的结果和知识，结合先进的成像、生化和实验和 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)是否为相 分离和流动性调节PCH的功能，并与其他核机构相互作用。 创新：尽管有限合伙人和生物冷凝物已经成为一个热门的研究和 近年来的讨论中，我们对体内机制以及与复合体功能的相关性知之甚少 而是重要的细胞和生物体环境。这是一个新兴领域，具有独特的挑战， 解决这些关键问题需要跨学科的方法。因此，在这个Mira提案中，我们将 将我们在五氯环己烷生物学领域数十年的经验与合作者在实验和研究方面的专业知识结合起来 理论生物物理学和高级生物成像。检验我们的假设将阐明重要的信息 关于异染色质在细胞和动物中的组织和功能，潜在地提供了一个范例- 为了解染色质结构域的一般形式和功能奠定了基础。 与健康相关：PCH缺陷会导致基因组不稳定和基因表达改变，这是 癌症、先天缺陷和衰老。了解PCH形成和形成的生物物理性质 人类疾病中的功能改变可能会导致诊断和治疗的新方法。

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.