DNA damage can result in a transient cell-cycle arrest or lead to permanent cell-cycle withdrawal. Here we show that the decision to irreversibly withdraw from the cell cycle is made within a few ...hours following damage in G2 cells. This permanent arrest is dependent on induction of p53 and p21, resulting in the nuclear retention of Cyclin B1. This rapid response is followed by the activation of the APC/CCdh1 (the anaphase-promoting complex/cyclosome and its coactivator Cdh1) several hours later. Inhibition of APC/CCdh1 activity fails to prevent cell-cycle withdrawal, whereas preventing nuclear retention of Cyclin B1 does allow cells to remain in cycle. Importantly, transient induction of p53 in G2 cells is sufficient to induce senescence. Taken together, these results indicate that a rapid and transient pulse of p53 in G2 can drive nuclear retention of Cyclin B1 as the first irreversible step in the onset of senescence.
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•DNA damage can result in a transient cell-cycle arrest or permanent cell-cycle exit•DNA damage causes nuclear retention of Cyclin B1 followed by activation of APC/CCdh1•DNA-damage-induced nuclear Cyclin B1-Cdk1 is refractory to activation•Transient activation of p53 in G2 phase is sufficient to induce senescence
DNA damage triggers a checkpoint response that can result in a reversible or permanent cell-cycle arrest. Krenning et al. find that checkpoint reversibility in G2 is limited and that transient activation of p53 results in the nuclear retention of Cyclin B1, subsequent APC/CCdh1 activation, and the onset of senescence.
The spatial organization of chromosomes influences many nuclear processes including gene expression. The cohesin complex shapes the 3D genome by looping together CTCF sites along chromosomes. We show ...here that chromatin loop size can be increased and that the duration with which cohesin embraces DNA determines the degree to which loops are enlarged. Cohesin’s DNA release factor WAPL restricts this loop extension and also prevents looping between incorrectly oriented CTCF sites. We reveal that the SCC2/SCC4 complex promotes the extension of chromatin loops and the formation of topologically associated domains (TADs). Our data support the model that cohesin structures chromosomes through the processive enlargement of loops and that TADs reflect polyclonal collections of loops in the making. Finally, we find that whereas cohesin promotes chromosomal looping, it rather limits nuclear compartmentalization. We conclude that the balanced activity of SCC2/SCC4 and WAPL enables cohesin to correctly structure chromosomes.
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•Hi-C analysis demonstrates that chromatin loop size can be increased genome-wide•The duration with which cohesin embraces DNA determines the length of chromatin loops•Haploid genetics reveals that the SCC2/SCC4 complex promotes loop extension•Cohesin limits the compartmentalization of chromatin within the nucleus
Cohesin's dynamic association with DNA determines the length of chromatin loops and allows this complex to correctly structure chromosomes.
Cell cycle checkpoints activated by DNA double-strand breaks (DSBs) are essential for the maintenance of the genomic integrity of proliferating cells. Following DNA damage, cells must detect the ...break and either transiently block cell cycle progression, to allow time for repair, or exit the cell cycle. Reversal of a DNA-damage-induced checkpoint not only requires the repair of these lesions, but a cell must also prevent permanent exit from the cell cycle and actively terminate checkpoint signalling to allow cell cycle progression to resume. It is becoming increasingly clear that despite the shared mechanisms of DNA damage detection throughout the cell cycle, the checkpoint and its reversal are precisely tuned to each cell cycle phase. Furthermore, recent findings challenge the dogmatic view that complete repair is a precondition for cell cycle resumption. In this Commentary, we highlight cell-cycle-dependent differences in checkpoint signalling and recovery after a DNA DSB, and summarise the molecular mechanisms that underlie the reversal of DNA damage checkpoints, before discussing when and how cell fate decisions after a DSB are made.
DNA double-strand breaks (DSBs) pose a constant threat to genomic integrity. Such DSBs need to be repaired to preserve homeostasis at both the cellular and organismal levels. Hence, the DNA damage ...response (DDR) has evolved to repair these lesions and limit their toxicity. The initiation of DNA repair depends on the activation of the DDR, and we know that the strength of DDR signaling may differentially affect cellular viability. However, we do not fully understand what determines the cytotoxicity of a DSB. Recent work has identified genomic location, (in)correct DNA repair pathway usage, and cell-cycle position as contributors to DSB-induced cytotoxicity. In this review, we discuss how these determinants affect cytotoxicity, highlight recent discoveries, and identify open questions that could help to improve our understanding about cell fate decisions after a DNA DSB.
Krenning et al. give an overview of determinants that affect the cellular toxicity of DNA double-strand breaks and discuss how these determinants affect cytotoxicity. The authors highlight recent discoveries and identify open questions that could help to improve our understanding about cell fate decisions after a DNA double-strand break.
Various types of chromosomal aberrations, including numerical (aneuploidy) and structural (e.g., translocations, deletions), are commonly found in human tumors and are linked to tumorigenesis. ...Aneuploidy is a direct consequence of chromosome segregation errors in mitosis, whereas structural aberrations are caused by improperly repaired DNA breaks. Here, we demonstrate that chromosome segregation errors can also result in structural chromosome aberrations. Chromosomes that missegregate are frequently damaged during cytokinesis, triggering a DNA double-strand break response in the respective daughter cells involving ATM, Chk2, and p53. We show that these double-strand breaks can lead to unbalanced translocations in the daughter cells. Our data show that segregation errors can cause translocations and provide insights into the role of whole-chromosome instability in tumorigenesis.
Large numbers of inhibitors for polo-like kinases and aurora kinases are currently being evaluated as anticancer drugs. Interest in these drugs is fuelled by the idea that these kinases have unique ...functions in mitosis. Within the polo-like kinase family, the emphasis for targeted therapies has been on polo-like kinase 1 (PLK1), and in the aurora kinase family drugs have been developed to specifically target aurora kinase A (AURKA; also known as STK6) and/or aurora kinase B (AURKB; also known as STK12). Information on the selectivity of these compounds in vivo is limited, but it is likely that off-target effects within the same kinase families will affect efficacy and toxicity profiles. In addition, it is becoming clear that interplay between polo-like kinases and aurora kinases is much more extensive than initially anticipated, and that both kinase families are important factors in the response to classical chemotherapeutics that damage the genome or the mitotic spindle. In this Review we discuss the implications of these novel insights on the clinical applicability of polo-like kinase and aurora kinase inhibitors.
Accurate segregation of chromosomes during cell division is accomplished through the assembly of a bipolar microtubule-based structure called the mitotic spindle. Work over the past two decades has ...identified a core regulator of spindle bipolarity, the microtubule motor protein kinesin-5. However, an increasing body of evidence has emerged demonstrating that kinesin-5-independent mechanisms driving bipolar spindle assembly exist as well. Here, we discuss different pathways that promote initial centrosome separation and bipolar spindle assembly.
Wnt/β-catenin signaling is essential for stem cell regulation and tumorigenesis, but its molecular mechanisms are not fully understood. Here, we report that FoxM1 is a downstream component of Wnt ...signaling and is critical for β-catenin transcriptional function in tumor cells. Wnt3a increases the level and nuclear translocation of FoxM1, which binds directly to β-catenin and enhances β-catenin nuclear localization and transcriptional activity. Genetic deletion of FoxM1 in immortalized neural stem cells abolishes β-catenin nuclear localization. FoxM1 mutations that disrupt the FoxM1–β-catenin interaction or FoxM1 nuclear import prevent β-catenin nuclear accumulation in tumor cells. FoxM1–β-catenin interaction controls Wnt target gene expression, is required for glioma formation, and represents a mechanism for canonical Wnt signaling during tumorigenesis.
► FoxM1 binds β-catenin and promotes its nuclear localization ► FoxM1 is required for β-catenin activation and the expression of Wnt target genes ► FoxM1 is a downstream component of canonical Wnt signaling pathway ► FoxM1–β-catenin interaction controls Wnt target gene expression and tumorigenesis
Abstract
DNA damaging agents cause a variety of lesions, of which DNA double-strand breaks (DSBs) are the most genotoxic. Unbiased approaches aimed at investigating the relationship between the ...number of DSBs and outcome of the DNA damage response have been challenging due to the random nature in which damage is induced by classical DNA damaging agents. Here, we describe a CRISPR/Cas9-based system that permits us to efficiently introduce DSBs at defined sites in the genome. Using this system, we show that a guide RNA targeting only a single site in the human genome can trigger a checkpoint response that is potent enough to delay cell cycle progression. Abrogation of this checkpoint leads to DNA breaks in mitosis which gives rise to aneuploid progeny.
DNA double-strand break (DSB) repair is mediated by multiple pathways. It is thought that the local chromatin context affects the pathway choice, but the underlying principles are poorly understood. ...Using a multiplexed reporter assay in combination with Cas9 cutting, we systematically measure the relative activities of three DSB repair pathways as a function of chromatin context in >1,000 genomic locations. This reveals that non-homologous end-joining (NHEJ) is broadly biased toward euchromatin, while the contribution of microhomology-mediated end-joining (MMEJ) is higher in specific heterochromatin contexts. In H3K27me3-marked heterochromatin, inhibition of the H3K27 methyltransferase EZH2 reverts the balance toward NHEJ. Single-stranded template repair (SSTR), often used for precise CRISPR editing, competes with MMEJ and is moderately linked to chromatin context. These results provide insight into the impact of chromatin on DSB repair pathway balance and guidance for the design of Cas9-mediated genome editing experiments.
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•Sequencing-based reporter detects activities of multiple DSB repair pathways•Multiplexing approach to integrate reporter in >1,000 genomic locations•Overlay with epigenome data reveals effects of chromatin context on pathway balance•Pathway balance differs between heterochromatin and euchromatin
Schep et al. designed a reporter to probe the activities of three DNA double-strand break repair pathways. Integration of this reporter in >1,000 genomic locations in a human cell line provided a detailed view of the impact of local chromatin context on the balance between the repair pathways.
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