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.

摘要 细胞必须整合竞争性DNA修复途径，并严格控制其染色质景观， 保持基因组的完整性。这些控制系统的中断或任何一个途径的缺陷都会导致 突变负担对细胞和组织具有深远的生理后果。人类DNA修复 细胞主要是由两个相互排斥的途径，由两个不同的，良好的， 表征的肿瘤抑制子-通过53 BP 1的非同源末端连接（NHEJ）和通过53 BP 1的同源末端连接（NHEJ） BRCA 1基因重组（HR）。53 BP 1和BRCA 1是内在无序蛋白质的例子 在一些实施方案中，多肽是含有大量低复杂性氨基酸序列的内源性多肽（IDP）。53 BP 1经历液-液 相分离以在体外和DNA损伤处形成生物分子凝聚物。此外，最近的研究 提示53 BP 1缩合在维持染色质组织中也起主要作用。是否 BRCA 1具有类似的相分离活性尚未确定。在初步数据中，我显示了 这是BRCA 1在细胞和体外分离形成生物分子的第一个证据。 染色质景观在维持基因组完整性方面也起着至关重要的作用。DNA损伤反应 需要动态重排和特异性染色质修饰以引起修复的快速募集 因素相反，修复因子及其复合物也可以修饰染色质以驱动修复 程序.尽管对53 BP 1和BRCA 1肿瘤抑制活性及其修复进行了广泛的研究， 然而，53 BP 1或BRCA 1通过何种机制缩合以及在其中的作用仍然未知。 缩合在DNA损伤反应中起作用。此外，染色质结构对 修复途径选择和修复浓缩物中的染色质组织尚未阐明。 这项工作的目标是为我提供新的培训和专业知识，以解决拟议的目标， 建立独立的研究计划。在目标1（K99阶段），我将确定特定的序列 促进53 BP 1和BRCA 1相分离并决定蛋白质贡献的决定因素 在修复途径选择性（NHEJ vs. HR）中，DNA聚合物缩合物促进DNA修复和保真度。我将测试 肿瘤抑制因子浓缩的程度有助于途径选择的假设。 在目标2（K99/R 00阶段）中，我将定义染色质响应DNA的动态重排 损伤以及核小体聚集和DNA环压缩如何有助于染色质动力学， 53 BP 1和BRCA 1介导的修复程序。总的来说，这项工作将解决以下方面的根本差距： 有关相分离在基因组完整性中的作用的知识，并发现新的范式， 是肿瘤抑制活性的基础。