Characterizing the role of tumor suppressor phase separation and chromatin organization in maintaining genomic integrity

表征肿瘤抑制相分离和染色质组织在维持基因组完整性中的作用

• 批准号: 10723739
• 负责人: Mikael Garabedian
• 金额: $ 12万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-05 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10723739
• 关键词: Address; Amino Acid Sequence; Aneuploidy; Architecture; Automobile Driving; BRCA1 gene; Binding Proteins; Biochemical; Biochemical Reaction; Biological Assay; Cell Cycle; Cell Nucleus; Cells; Cellular biology; Chromatin; Chromatin Loop; Chromosomes; Complex; DNA; DNA Damage; DNA Repair; DNA Repair Pathway; DNA lesion; Data; Defect; Development; Disease; Epigenetic Process; Equipment; Exons; Foundations; Functional disorder; Genomic Instability; Genotoxic Stress; Goals; Heterochromatin; Homeostasis; Human; Image; In Vitro; Knowledge; Lead; Link; Liquid substance; Maintenance; Malignant Neoplasms; Measures; Mediating; Modification; Mutation; Nonhomologous DNA End Joining; Nucleosomes; Organizational Change; Pathway interactions; Pennsylvania; Phase; Physical condensation; Physiological; Physiology; Play; Point Mutation; Poly(ADP-ribose) Polymerase Inhibitor; Positioning Attribute; Proteins; Public Health; Research; Resistance; Role; Stretching; System; TP53 gene; Testing; Tissues; Training; Tumor Suppressor Proteins; Universities; Visualization; Work; cancer therapy; cell transformation; chromatin modification; chromosome fusion; genome integrity; homologous recombination; insight; interdisciplinary approach; mutant; p53-binding protein 1; programs; reconstitution; recruit; repaired; response; self assembly; superresolution microscopy; therapy resistant; tool

ABSTRACT Cells must integrate competing DNA repair pathways and tightly control their chromatin landscapes to maintain genomic integrity. Disruption of these control systems or defects in any one pathway result in a mutational burden with profound physiological consequences to cells and tissues. DNA repair in human cells is primarily performed by two mutually exclusive pathways governed by two different, well- characterized tumor suppressors – non-homologous end joining (NHEJ) by 53BP1 and homologous recombination (HR) by BRCA1. 53BP1 and BRCA1 are examples of intrinsically disordered proteins (IDPs) containing large stretches of low complexity amino acid sequences. 53BP1 undergoes liquid-liquid phase separation to form biomolecular condensates in vitro and at DNA lesions. Further, recent studies hint that 53BP1 condensation also plays a major role in maintaining chromatin organization. Whether BRCA1 has similar phase separation activity has not been established. In preliminary data, I show the very first evidence that BRCA1 phase separates to form biomolecular in cells and in vitro. Chromatin landscapes also play a vital role in maintaining genomic integrity. DNA damage response requires dynamic rearrangements and specific chromatin modifications to elicit rapid recruitment of repair factors. Conversely, repair factors and their complexes can also modify chromatin to drive repair programs. Despite extensive study of 53BP1 and BRCA1 tumor suppressor activities and their repair mechanisms, it remains unknown by what mechanism 53BP1 or BRCA1 condense and what role condensations plays in DNA damage response. Further, the contributions of chromatin architecture to repair pathway selection and chromatin organization within repair condensates have not been elucidated. The goal of this work is to provide me with new training and expertise to address the proposed aims and establish an independent research program. In Aim 1 (K99 phase), I will identify the specific sequence determinants that promote 53BP1 and BRCA1 phase separation and determine the contribution of protein condensates to promoting DNA repair and fidelity in repair pathway selectivity (NHEJ vs. HR). I will test the hypothesis that the magnitude of tumor suppressor condensation contributes to pathway selection. In Aim 2 (K99/R00 phase), I will define the dynamic rearrangement of chromatin in response to DNA damage and how nucleosome clustering and DNA loop compaction contribute to chromatin dynamics in 53BP1- and BRCA1-mediated repair programs. Collectively, this work will address fundamental gaps in knowledge regarding the role of phase separations in genome integrity and uncover new paradigms that underlie tumor suppressor activities.

抽象的 细胞必须整合竞争的DNA修复途径，并将其染色质景观紧密地控制到 保持基因组完整性。这些控制系统的破坏或任何一个途径中的缺陷导致 突变烧伤，对细胞和组织产生深远的生理后果。人类的DNA修复 细胞主要由两种互斥途径进行，由两个不同的，良好 肿瘤补充剂的特征 - 非理论末端连接（NHEJ）由53BP1和同源 BRCA1重组（HR）。 53BP1和BRCA1是固有无序蛋白的示例 （IDP）包含大量低复杂性氨基酸序列。 53BP1经历液体液体 相位分离以在体外和DNA病变中形成生物分子冷凝物。此外，最近的研究 暗示53BP1凝结在维持染色质组织中也起着重要作用。无论 BRCA1具有相似的相分离活性。在初步数据中，我显示了 第一个证据表明，BRCA1相分离以在细胞和体外形成生物分子。 染色质景观在维持基因组完整性中也起着至关重要的作用。 DNA损伤响应 需要动态重排和特定的染色质修饰以快速募集修复 因解者。相反，维修因子及其复合物也可以修饰染色质以驱动修复 程序。尽管进行了53BP1和BRCA1肿瘤抑制活性的广泛研究及其修复 机制，哪种机制53BP1或BRCA1凝结以及什么作用仍然未知 凝结在DNA损伤反应中发挥作用。此外，染色质体系结构对 修复途径的选择和修复冷凝物中的染色质组织尚未阐明。 这项工作的目的是为我提供新的培训和专业知识，以解决拟议的目标和 建立一个独立的研究计划。在AIM 1（K99阶段）中，我将确定特定序列 确定促进53BP1和BRCA1相分离并确定蛋白质的贡献 凝结以促进DNA修复和维修途径选择性（NHEJ与HR）中的保真度。我会测试 肿瘤抑制的大小有助于途径选择的假设。 在AIM 2（K99/R00阶段）中，我将定义染色质的动态重排，以响应DNA 损伤以及核小体聚类和DNA环的压实如何有助于染色质动力学 53BP1和BRCA1介导的维修计划。总的来说，这项工作将解决 关于相位分离在基因组完整性中的作用并发现新范式的知识 肿瘤抑制活动的基础。