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: 10796728
• 负责人: Peter Ly
• 金额: $ 25万
• 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/10796728
• 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.

项目摘要/摘要 染色体异常是人类疾病和遗传性疾病的显著特征。癌症基因组 测序发现了一类复杂的局部性基因组重排，称为染色质疾病， 这是由个别染色体的灾难性碎裂引起的。染色体萎缩症是由有丝分裂引起的 细胞分裂错误导致微核的形成，即短暂的异常核结构 将错误分离的染色体包裹在核外。微核是人类免疫系统的热点 通过将DNA损伤限制在受限区域来积累广泛的DNA双链断裂(DSB) 基因组。然而，对嗜铬细胞症的详细机制理解受到固有因素的限制。 在多个细胞周期监测微核染色体方面的挑战。我们最近绕过了这个 通过开发一个平台来实现对染色体特异性微核的受控诱导 人类细胞。通过重建导致嗜铬细胞症的一连串事件，我们发现 微核DNA在染色体过早凝聚时容易发生片段化 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，以生成 不同的染色体重排，与癌症和基因组疾病中发现的相同。 此外，我们还发现，包裹在细胞质中的短dna片段可以激活细胞自主性。 免疫反应。尽管有这些知识，我们目前对这一问题的机械理解有限 染色体碎裂的后果。例如，目前还不清楚碎片是如何从 在有丝分裂过程中，微核重新整合到子细胞基因组中，并由一个或多个 整个间期的DSB修复机制。此外，目前还不清楚染色体是否会碎裂 可以引起非细胞的自主反应。在这里我们概述了我们未来五年的研究计划，目标是 在理解微核染色体在细胞周期的不同阶段的命运和它的 对基因组完整性的突变后果。使用延时光片显微镜，我们将询问 有丝分裂过程中染色体碎裂、移动和重组的时空动力学 间期。这将通过设计一种基于CRISPR的标记策略来实现，以可视化微核 活细胞中经历染色质分裂的染色体。接下来，我们将确定DNA损伤反应是如何 不同的DSB修复途径协调染色体片段的重组以塑造基因组 有丝分裂错误的重排图景。最后，我们将研究染色体片段是如何驻留在 细胞质可以引起与环境中相邻细胞的细胞间后果，包括 遗传物质的横向交换。总之，这些研究的目的是界定 微核在启动灾难性基因组改变中的内在和外在命运。拟议的研究 将填补我们在理解细胞周期错误如何快速驱动体细胞突变方面的一个关键空白。

项目成果

Induction of chromosome-specific micronuclei and chromothripsis by centromere inactivation.

通过着丝粒失活诱导染色体特异性微核和染色体碎裂。

• DOI: 10.1016/bs.mcb.2022.10.009
• 发表时间: 2024
• 期刊: Methods in cell biology
• 作者: Lin,Yu-Fen;Hu,Qing;Guyer,Alison;Fachinetti,Daniele;Ly,Peter
• 通讯作者: Ly,Peter

Non-homologous end joining shapes the genomic rearrangement landscape of chromothripsis from mitotic errors.

非同源末端连接塑造了有丝分裂错误导致的染色体碎裂的基因组重排景观。

• DOI: 10.1101/2023.08.10.552800
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Hu,Qing;Valle-Inclan,JoseEspejo;Dahiya,Rashmi;Guyer,Alison;Mazzagatti,Alice;Maurais,ElizabethG;Engel,JustinL;Cortés-Ciriano,Isidro;Ly,Peter
• 通讯作者: Ly,Peter