Genomic Instability from Fragmented Chromosomes in Micronuclei

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.