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: 10495000
• 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/10495000
• 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; Human; Human Genetics; Immune response; Individual; Interphase; Knowledge; Label; Lateral; Light; Microscopy; Mitosis; Mitotic; Molecular; Monitor; Movement; Mutagenesis; Nuclear Structure; Pathway interactions; Phase; Research; Shapes; Source; base; cancer genome; cancer genomics; chromothripsis; daughter cell; genome integrity; genome sequencing; human disease; insight; interdisciplinary approach; premature; programs; response; spatiotemporal; time use

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.

项目摘要/摘要 异常染色体是人类疾病和遗传疾病的标志性特征。癌症基因组 测序发现了一类复杂的局部基因组重排，称为Chromothripsis， 这是由单个染色体的灾难性碎片产生的。 Chromothripsis是由有丝分裂发起的 细胞分裂误差导致形成微核，异常的核结构 封装在细胞核之外的错误分离的染色体。微核作为热点 通过将DNA损伤限制为限制区域 基因组。然而，对铬骨的详细机械理解受到固有的限制 监测微核染色体的挑战以上一个以上的细胞周期。我们最近绕过了这个 通过开发一个平台来限制，该平台能够受控诱导染色体特异性的微核中 人类细胞。通过重建导致Chromothripsis的一系列事件，我们发现这损坏了 在过早的染色体凝结后，微核DNA易于破碎。 有丝分裂条目。这些片段在随后的细胞周期内经历容易出错的DSB修复 不同的染色体重排与癌症和基因组疾病中发现的染色体重排相同。 此外，我们确定夹在细胞质中的短DNA片段可以激活细胞自主 免疫反应。尽管有这些知识，我们目前对 染色体破碎的后果。例如，尚不清楚如何粉碎 微核在有丝分裂过程中重新组合成子细胞基因组，并被一个或多个重新组装 整个相间的DSB修复机制。另外，尚不清楚染色体碎片是否 可以引起非单元自动响应。在这里，我们概述了未来五年的研究计划 在了解细胞周期不同阶段的微核染色体及其的命运方面 诱变对基因组完整性的后果。使用延时灯表显微镜，我们将询问 染色体碎片，运动和重新组装的时空动力学在有丝分裂和重新组装 相间。这将通过设计基于CRISPR的标签策略来可视化微核的标签策略来实现这一目标 活细胞中染色体的染色体。接下来，我们将确定DNA损伤响应如何 独特的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，我们将研究染色体碎片如何居住 细胞质会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。这些研究旨在定义控制基本原则 微核的固有和外在命运在引发灾难性基因组改变时。拟议的研究 将填补我们对细胞周期误差如何快速驱动体细胞诱变的理解的关键空白。