Genomic Instability from Fragmented Chromosomes in Micronuclei

微核中染色体碎片导致的基因组不稳定性

• 批准号: 10673104
• 负责人: Peter Ly
• 金额: $ 41万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10673104
• 关键词: Automobile Driving; Bypass; Cell Cycle; Cell Nucleus; Cell division; Cells; Chromosomal Rearrangement; Chromosome Condensation; Chromosome abnormality; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cytoplasm; DNA; DNA Damage; DNA Double Strand Break; DNA Sequence Alteration; DNA Sequence Rearrangement; Disease; Double Strand Break Repair; Encapsulated; Engineering; Environment; Event; Genetic Diseases; Genetic Materials; Genome; Genomic Instability; Genomics; Human; Human Genetics; Immune response; Individual; Interphase; Knowledge; Label; Lateral; Light; Malignant Neoplasms; Microscopy; Mitosis; Mitotic; Molecular; Monitor; Movement; Mutagenesis; Nuclear; Nuclear Structure; Pathway interactions; Phase; Predisposition; Research; Shapes; Source; Visualization; cancer genome; chromosome missegregation; chromothripsis; daughter cell; genome integrity; genome sequencing; human disease; insight; interdisciplinary approach; micronucleus; premature; programs; response; spatiotemporal

Project Summary/Abstract Abnormal chromosomes are hallmark features of human diseases and genetic disorders. Cancer genome sequencing has uncovered a complex class of localized genomic rearrangements, known as chromothripsis, that arises from the catastrophic fragmentation of individual chromosomes. Chromothripsis is initiated by mitotic cell division errors resulting in the formation of micronuclei, aberrant nuclear structures that transiently encapsulate mis-segregated chromosomes outside of the nucleus. Micronuclei serve as hotspots for the accumulation of extensive DNA double-strand breaks (DSBs) by restricting DNA damage to a confined region of the genome. A detailed mechanistic understanding of chromothripsis, however, has been limited by inherent challenges in monitoring micronucleated chromosomes for more than one cell cycle. We recently bypassed this limitation by developing a platform that enables the controlled induction of chromosome-specific micronuclei in human cells. By reconstructing the cascade of events resulting in chromothripsis, we found that damaged micronuclear DNAs are susceptible to fragmentation upon premature chromosome condensation triggered by mitotic entry. These fragments undergo error-prone DSB repair during the subsequent cell cycle to generate diverse chromosomal rearrangements that are identical to those found in cancers and genomic disorders. Moreover, we identified that short DNA fragments entrapped in the cytoplasm can activate a cell-autonomous immune response. Despite this knowledge, we currently have a limited mechanistic understanding of the consequences of chromosome fragmentation. For example, it remains unclear how pulverized fragments from micronuclei re-incorporate into daughter cell genomes during mitosis and become reassembled by one or more DSB repair mechanisms throughout interphase. Additionally, it is unknown whether chromosome fragmentation can elicit a non-cell autonomous response. Here we outline our research program over the next five years aimed at understanding the fate of micronucleated chromosomes across different phases of the cell cycle and its mutagenic consequences on genome integrity. Using time-lapse light-sheet microscopy, we will interrogate the spatiotemporal dynamics of chromosome fragmentation, movement, and reassembly during mitosis and interphase. This will be achieved by engineering a CRISPR-based labeling strategy to visualize micronucleated chromosomes undergoing chromothripsis in living cells. Next, we will identify how the DNA damage response and distinct DSB repair pathways orchestrate the reassembly of chromosome fragments to shape the genomic rearrangement landscape of mitotic errors. Lastly, we will investigate how chromosome fragments residing in the cytoplasm can elicit inter-cellular consequences with neighboring cells in the environment, including the lateral exchange of genetic material. Altogether, these studies aim to define fundamental principles governing the intrinsic and extrinsic fate of micronuclei in initiating catastrophic genomic alterations. The proposed research will fill a critical gap in our understanding of how cell cycle errors can rapidly drive somatic mutagenesis.

项目概要/摘要 染色体异常是人类疾病和遗传性疾病的标志特征。癌症基因组 测序发现了一类复杂的局部基因组重排，称为染色体碎裂， 这是由单个染色体的灾难性碎片引起的。染色体碎裂是由有丝分裂引发的 细胞分裂错误导致微核、异常核结构的形成， 将错误分离的染色体封装在细胞核外。微核作为热点 通过将 DNA 损伤限制在有限的区域，从而积累广泛的 DNA 双链断裂 (DSB) 基因组。然而，对染色体碎裂的详细机制理解受到固有的限制。 监测多个细胞周期的微核染色体面临的挑战。我们最近绕过了这个 通过开发一个能够控制诱导染色体特异性微核的平台来克服限制 人体细胞。通过重建导致染色体碎裂的级联事件，我们发现受损的 微核 DNA 很容易因染色体过早凝结而产生碎片 有丝分裂进入。这些片段在随后的细胞周期中经历容易出错的 DSB 修复，生成 与癌症和基因组疾病中发现的相同的多种染色体重排。 此外，我们还发现，截留在细胞质中的短 DNA 片段可以激活细胞自主 免疫反应。尽管有了这些知识，我们目前对这一机制的了解还很有限。 染色体断裂的后果。例如，目前尚不清楚粉碎的碎片是如何从 微核在有丝分裂过程中重新整合到子细胞基因组中，并由一个或多个细胞重新组装 DSB 修复机制贯穿整个间期。此外，尚不清楚染色体是否断裂。 可以引发非细胞自主反应。在这里，我们概述了未来五年的研究计划，旨在 了解细胞周期不同阶段微核染色体的命运及其 对基因组完整性的诱变后果。使用延时光片显微镜，我们将询问 有丝分裂和分裂过程中染色体断裂、运动和重组的时空动态 间期。这将通过设计基于 CRISPR 的标记策略来实现微核可视化 活细胞中的染色体经历染色体碎裂。接下来，我们将确定 DNA 损伤如何反应 不同的 DSB 修复途径协调染色体片段的重新组装以塑造基因组 有丝分裂错误的重排景观。最后，我们将研究染色体片段如何驻留在 细胞质可以引起与环境中邻近细胞的细胞间后果，包括 遗传物质的横向交换。总而言之，这些研究旨在定义管理的基本原则 微核在引发灾难性基因组改变中的内在和外在命运。拟议的研究 将填补我们对细胞周期错误如何快速驱动体细胞突变的理解的关键空白。