Genomic Instability from Fragmented Chromosomes in Micronuclei

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性特征。癌症基因组 测序已经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引起的。有丝分裂起始于 细胞分裂错误导致形成微核，异常的核结构， 将错误分离的染色体包裹在细胞核外。古核是 通过将DNA损伤限制在一个有限的区域， 基因组然而，对chromothripsis的详细机制理解受到固有的限制。 监测一个以上细胞周期的微核染色体的挑战。我们最近绕过了这个 通过开发一个平台，使染色体特异性微核的可控诱导， 人体细胞通过重建导致染色体断裂的一连串事件，我们发现， 微核DNA在由以下因素引发的过早染色体凝聚时容易断裂： 有丝分裂进入这些片段在随后的细胞周期中经历易错的DSB修复，以产生 与癌症和基因组疾病中发现的那些相同的多种染色体重排。 此外，我们发现，细胞质中捕获的短DNA片段可以激活细胞自主 免疫反应尽管有这些知识，我们目前对机械的理解仍然有限 染色体断裂的后果。例如，目前尚不清楚， 微核在有丝分裂期间重新并入子细胞基因组，并通过一个或多个 整个间期的DSB修复机制。此外，尚不清楚染色体断裂是否 可以引发非细胞自主反应。在这里，我们概述了我们在未来五年的研究计划，旨在 在理解微核染色体在细胞周期不同阶段的命运及其 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 有丝分裂过程中染色体断裂、运动和重组的时空动力学， 间期。这将通过设计基于CRISPR的标记策略来实现，以可视化微核细胞。 在活细胞中经历染色体断裂的染色体。接下来，我们将确定DNA损伤如何响应 不同的DSB修复途径协调染色体片段的重组，以形成基因组 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 细胞质可以引发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。总而言之，这些研究旨在确定管理 微核在引发灾难性基因组改变中的内在和外在命运。拟议研究 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中的关键空白。

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.