Linking Micronuclei to Chromosome Fragmentation

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Linking Micronuclei to Chromosome Fragmentation Emily M. Hatch1 and Martin W. Hetzer1,* 1Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies 10010 North Torrey Pines Road, La Jolla, 92037 CA, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cell.2015.06.005

Human cancer cells bear complex chromosome rearrangements that can be potential drivers of cancer development. However, the molecular mechanisms underlying these rearrangements have been unclear. Zhang et al. use a new technique combining live-cell imaging and single-cell sequencing to demonstrate that chromosomes mis-segregated to micronuclei frequently undergo chromothripsis-like rearrangements in the subsequent cell cycle. Chromothripsis occurs when a chromosome fragments into many pieces all at once and is then stitched back together in a random order. It was originally identified by whole-genome sequencing of chronic lymphocytic leukemia cells (Stephens et al., 2011) and has been subsequently identified in many types of cancers and some congenital disorders (Kloosterman and Cuppen, 2013). Breakpoint analysis identified several characteristics that distinguish chromothripsis from progressive rearrangement processes and is the basis of the current model of chromothripsis. In a recent study, Zhang et al. (2015) identify a cellular mechanism that could cause chromothripsis in cancer cells. Previous work from the Pellman lab showed that chromosomes trapped in micronuclei can become fragmented and suggested that this could be the initial step of chromothripsis (Crasta et al., 2012). Their current paper solidifies and refines this model by demonstrating that damaged micronucleated chromatin frequently undergoes complex rearrangements within

the next cell cycle that meet the criteria for chromothripsis. Micronuclei occur in mammalian cells when chromosomes lag during anaphase or fail to align on the metaphase plate. If this chromosome, or chromosome fragment, is sufficiently far from the rest of the chromatin mass, it assembles a separate nuclear envelope (NE) at the end of mitosis. This results in an interphase cell with two nuclei: the primary nucleus, which contains the main chromatin mass, and the micronucleus, which contains the missegregated chromatin. Previous work had shown that micronucleated chromatin often undergoes extensive damage in interphase (Zhang et al., 2013), and the Pellman group linked delayed DNA replication in micronuclei to fragmentation of this chromatin in mitosis. Subsequently, it was shown that the NE in the majority of micronuclei collapses during interphase, and this instability was shown to trigger DNA damage and abrogate DNA replication (Hatch et al., 2013). Zhang et al. reinforce the connection between underreplication and the formation of double-

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stranded DNA breaks in micronucleated chromatin. They demonstrate that DNA damage following NE rupture only occurs after cells have left G1 phase, strengthening a previous observation (Hatch et al., 2013). In addition, they find that most damaged chromosomes have initiated DNA replication prior to NE rupture. To connect micronuclei to chromothripsis, the authors developed a live-cell imaging technique combined with single-cell sequencing to identify cells with ruptured micronuclei, track them through the next mitosis, and determine whether the micronucleated chromosome was rearranged. The authors take advantage of the fact that chromatin in ruptured micronuclei is significantly underreplicated to identify the previously micronucleated chromosome in the daughter cells. Underreplication means that a 2N cell with a micronucleus enters mitosis with an essentially 4N-1 genome, with one chromosome being unduplicated. When this cell divides, one daughter gets the full complement of chromosomes (2N) and one is missing a copy of the

Figure 1. Chromothripsis from Ruptured Micronuclei When a chromosome mis-segregates during mitosis, it can result in a daughter cell with two nuclei, the primary nucleus (PN) containing most of the genome and the micronucleus (MN), containing the missegregated chromatin (upper-left). After the cell enters S phase, DNA replication can occur on the micronucleated chromatin (upper-right). Rupture of the nuclear envelope during replication (middle-left) causes DNA damage, including double-stranded DNA breaks (middle-right). When the damaged chromatin is re-enclosed in a nuclear envelope after mitosis, DNA damage repair pathways can recognize the shattered chromatin and randomly reassemble the pieces to form a new chromosome (lower-right). Unassembled pieces are lost from the chromosome but may become circularized and persist in the genome. Because only one copy of the micronucleated chromatin is present at mitosis, due to incomplete DNA replication, only one daughter cell will be 2N. Identifying the chromosome missing from the other daughter identifies the chromosome that was previously in the micronucleus.

previously micronucleated chromosome (i.e., 2N-1). Analysis of the sequencing data from each individual daughter cell allowed the authors to identify which chromosome was present in only one copy and, thus, had been micronucleated in the previous cell cycle. Remarkably, after sequencing several daughter pairs derived from cells with ruptured micronuclei, almost all of the micronucleated chromosomes that they identified (eight out of nine) had undergone complex rearrangements. Some of these rearrangements exhibited the defining characteristics of chromothripsis, including clustering of breakpoints, alternating regions of sequence retention and loss, and rearrangements linked to a single haplotype (Korbel and Campbell, 2013; Stephens et al., 2011). These breakpoint characteristics indicate that the frag-

mented chromatin is reassembled through normal DNA repair processes, likely non-homologous end-joining. Similar to previous analyses (Stephens et al., 2011), the authors also identified microhomology at many of the breakpoints. Based on these data, the authors suggest that some rearrangements could occur by replication-based repair mechanisms, possibly in the replicating micronuclei prior to NE rupture. Another intriguing observation was that one daughter cell pair contained several circular chromosome pieces from the micronucleated chromosome in addition to the larger rearranged chromatid. This suggests that mis-segregation of chromosomes to micronuclei may also cause formation of double-minutes, highly amplified circular chromosome sequences found in cancer cells that

often contain an oncogene, which had been previously linked to chromothripsis (Rausch et al., 2012; Stephens et al., 2011; Zhang et al., 2013). In summary, this paper provides the first cellular mechanism for chromothripsis and outlines an experimental system for generating and analyzing micronucleated chromatin (Figure 1). This will facilitate further analysis of the molecular mechanisms of chromatin fragmentation and reassembly and the conditions required for it to occur. One immediate goal will be to determine what DNA repair pathways are used during chromosome reassembly and when they are acting on the chromatin. Another question is whether mutations in tumor suppressor genes, such as p53, that are associated with increased genome instability are also required for chromothripsis. Previous work from the Pellman group showed that the absence of p53 was important to bypass cell-cycle arrest after inducing micronucleation in their cell line. However, this loss may be necessary only for specific mechanisms of micronucleus formation (Uetake and Sluder, 2010) or in specific cell lines. Because extensive chromosome rearrangements are likely to be lethal, though, additional effects of reduced p53 activity, such as reduced apoptosis and increased aneuploidy frequency, may be required for continued cell growth after chromothripsis. Finally, the authors recognize that micronucleation may not be the only chromothripsis mechanism. An important question, then, is whether micronucleation is the main driver of chromothripsis in all cell types or whether other mechanisms can cause these rearrangements in cancer and sperm cells. A single chromothripsis event can simultaneously disrupt multiple cancer pathways, which bypasses the traditional model of a gradual accumulation of somatic mutations or translocations and could allow genomic landscapes to change rapidly. The identification of the micronuclei mechanism of chromothripsis enables experiments asking whether chromothripsis is a causal event in cancer initiation or development. Micronuclei are widespread in many cancer types and occur at an appreciable frequency in normal epithelium (Tolbert et al., 1992). In addition, ruptured micronuclei with

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extensive DNA damage are present in human tumor samples (Hatch et al., 2013). Thus, if the high frequency of genomic rearrangements after micronucleation reported in this study is representative of in vivo frequencies, it suggests that chromothripsis may be a common occurrence in human cells. The data presented by Zhang et al. demonstrate that underreplication of micronucleated chromatin can have outsized effects on chromosome structure and that continued research on micronucleated cells could yield important insights into the development of cancer and other diseases.

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Zhang, C.-Z., Spektor, A., Cornils, H., Francis, J.M., Jackson, E.K., Liu, S., Meyerson, M., and Pellman, D. (2015). Nature. Published online May 27, 2015. http://dx.doi.org/10.1038/nature14493.

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