DNA damage in Mitosis: bringing together 2 (un)-related fields/ Les dommages de l’ADN dans la mitose: rapprocher 2 domaines (non) liés

Seminar organized by Renata Basto and Marie-Hélène Verlhac from 17 to 22 July, 2023

Participants

Adel AL JORD (Collège de France – Paris, France), Renata BASTO (Institut Curie – Paris, France), Uri BEN-DAVID (Tel Aviv University – Tel-Aviv, Israël), Arnaud ECHARD (Institut Pasteur – CNRS, Paris – France), Greg FITZHARRIS (CRCHUM/Université de Montréal – Montreal, Canada), Floris FOIJER (University Medical Center Groningen – Groningen, Pays-Bas), Simon GEMBLE (Institut Curie – Paris, France), Sarah LAMBERT (Institut Curie, Paris, France), Gaëlle LEGUBE (CBI MCD Université de Toulouse – Toulouse, France), Valeria NAIM (CNRS Paris-Saclay Gustave Roussy – Villejuif, France), David PELLMAN (Dana-Faber Cancer Institute – Boston, États-Unis), Jonathon PINES (Institute of Cancer Research – London, Royaume-Uni), Marie-Hélène VERLHAC (CIRB, Collège de France – Paris, France), Sara WICKSTRÖM (Max Planck Institute for Molecular Biomedicine – Muenster, Finlande)

Résumé

Cassure chromosomique (cf. le petit bout de chromosome tout en bas) d’un ovocyte de souris issu du travail de Manil-Segalen et al. J Cell Biol 2018.

Le 17 juillet, nous nous sommes réunis à la Fondation des Treilles pour discuter des processus pouvant conduire à des dommages de l’ADN durant la mitose. Ces dommages sont délétères pour les cellules filles et souvent à l’origine d’erreurs de ségrégation des chromosomes et de processus de cancérisation. Ce séminaire a rassemblé un groupe de 14 chercheurs, dont deux jeunes chercheurs. Le but de cette réunion était de rassembler des scientifiques qui ne se rencontrent normalement pas dans les réunions conventionnelles, car les dommages à l’ADN et la réparation des dommages à l’ADN sont principalement étudiés pendant l’interphase, qui précède la division cellulaire en mitose.

Le temps était magnifique, même si les températures étaient un peu trop chaudes. Cependant, nous avons passé un excellent moment tous ensemble. Les séminaires étaient sous la forme de présentations relativement formelles, et ont donné lieu à énormément d’échanges sur les travaux menés dans nos laboratoires. Ces discussions se sont prolongées de manière informelle bien en deçà des moments de séminaires, notamment en nous asseyant ensemble en fin de journée ou durant les excellents repas. Les prévisions des organisateurs ont été atteintes et même dépassées, les participants ayant toutes et tous été très satisfaits de l’ensemble de cette rencontre.

Summary

On the 17^th of July, we met at the Fondation des Treilles to discuss the processes that can lead to DNA damage in mitosis. This meeting assembled a group of 14 researchers, including two young researchers. The purpose of this meeting was to bring scientists together that normally do not meet in conventional meetings as DNA damage and DNA damage repair are mainly studied during interphase, which precede cell division in mitosis. The weather was beautiful even if slightly on the “too hot” temperatures. However, together we had a wonderful time. While seminars set the stage for more formal presentations about the work carried out in our labs, whilst seating together at the end of the day, or during the spectacular meals, we had the opportunity to discuss about our work in a very informal manner. The predictions of the organizers were encountered and exceeded as the two seemingly unrelated fields managed to interact and discuss beyond any expectation.

Report

The meeting started with peculiar cell divisions, the meiotic ones of the female germline in oocytes. Marie Hélène Verlhac (CIRB, Collège de France, Paris in France) showed that unlike somatic cells that enter M-phase with a thinner cell cortex, mouse oocytes, display a thicker actin cortex in an Arp2/3 complex dependent manner. In the absence of this complex, oocytes show prominent cortical contractions that last for many hours, only subsiding before anaphase I. Although cortical contractions are common in oocytes and embryos, contractions of this magnitude have never been observed in this model before. These contractions actively stir the oocyte cytoplasm, resulting in the clustering of lipid particles, misplacement of mitochondria and cortical granules. These defects ultimately impact oocyte quality and its capacity to be fertilized by sperm. Interestingly, similar contractions were observed in human oocytes collected as byproducts of in vitro fertilization considered of low quality, suggesting that these findings are relevant to human fertility research. Adel Al Jord, (also from the CIRB), presented that growing mouse oocytes intensify actomyosin-based stirring forces in their cytoplasm to remodel nuclear droplet-like organelles known as biomolecular condensates. These cytoplasmic stirring forces function simultaneously as nuclear condensate colliders, boosting mesoscale reorganization in the nucleus, and as condensate core particle accelerators, enhancing enzymatic catalysis associated with condensates such as mRNA-processing. Altogether, these findings implies that cells evolved a mechanism, based on cytoplasmic force tuning, to functionally regulate a broad range of nuclear condensates across scales. Greg Fitzharris from Montreal University, Canada, presented recent lab work on the mechanisms of cytokinesis in the mammalian embryo. Unexpectedly, in embryos the final step of cytokinesis, called abscission, is dramatically delayed such that cells exist as pairs connected by cytoplasmic bridges. It was discussed that this unusual feature of embryos may relate to the unusual Aurora Kinase signalling environment in the early embryo. Experiments exploring the relationship between cytoplasmic bridges and cellular differentiation were presented. Possible physiological benefits of sister pairs existing as mini-syncytia in the embryo were discussed. Sara Wickström (Max Plank Institute, Muenster, Germany) addressed the consequences of nuclear deformations after mechanical stress. Cells are constantly subjected to a spectrum of mechanical cues, such as shear stress, compression, differential tissue rigidity, and strain, to which they respond to by engaging mechanisms of mechanotransduction. These forces function as important morphogenetic ques that are transmitted to the nucleus to alter genetic programs. On the other hand, excessive mechanical stresses have the potential to damage cells and tissues. In her presentation, Sara Wickström discussed how dynamic changes in chromatin condensation in response to nuclear deformation alter the mechanical properties of the nucleus and chromatin to prevent damage. She also showed how changes in nuclear shape, volume and envelope tension regulate transcription through a combination of global effects on transcription and activation of specific enhancers in a manner depending on the biochemical context. These mechanisms allow cells to adapt to differential force environments by changing their structure and state. The importance of an intact and well-shaped nucleus was also addressed by Simon Gemble (Institut Curie, Paris) in cells after whole genome duplication. He presented that premature mitotic exit generates tetraploid cells with abnormal nuclear shape. This is due to a decreased nuclear stiffness that allows microtubules to generate nuclear invaginations. To support this model, he showed that restoring nuclear stiffness or depolymerizing microtubules are sufficient to generate tetraploid cells without nuclear deformations. Since about 40% of human tumors are tetraploid, this work will contribute to a better understanding of how tetraploidy participates to tumorigenesis. Considering DNA damage and DNA damage repair, Gaëlle Legube (CBI, Toulouse, France) presented her recent work on the dynamics of chromosome during DNA Double Strand break (DSB) repair. Using the DIvA system, a human transformed cell line in which sequence-specific DSBs can be induced at annotated positions throughout the genome, her lab performed HiC experiments to analyse chromosome changes induced by DSBs. They could demonstrate that cohesin-mediated loop extrusion takes place at DSBs and that this process allows to establish g-H2AX foci. They further identify a novel DSB-induced chromatin compartment (called the “D” Compartment) that is essential for optimal activation of the DNA Damage Response. Finally, they also found that, as for stalled replication fork in S. pombe, or persistent/refractory DSB in S. cerevisiae and Drosophila, DSB induced in transcribed loci also reposition to the nuclear envelope in human cells. Sarah Lambert (Institut Curie, Orsay, France) presented her current research work on the mobility of stalled DNA replication forks within the fission yeast nucleus. She introduced the topic of replication stress and how the homologous recombination pathway contributes to replication competence and integrity of stalled fork to avoid chromosome breakage during mitosis. She further presented results to demonstrate that stalled fork shift away from the nucleoplasm compartment to relocate and anchor to the nuclear periphery. Unpublished results were shared to further discuss the benefit of such mobility phenomenon. She presented that two activities, enriched at the nuclear periphery, are engaged at stalled forks to promote the dynamic restart of stalled fork by homologous recombination. First, the SUMO SENP protease Ulp1, which is known to be anchored at the nuclear periphery, is required to sustain the efficiency of fork restart. Second, the proteasome, which activity is enriched at the nuclear periphery, is necessary to ensure the dynamic speed of the restarted forks. Overall, Sarah’s presentation opened up scientific discussions on the fact that DNA repair pathways are spatially segregated in the nucleus architecture and that key questions about the functions and the mechanisms of such spatial regulation remain unanswered and may involve key components of the nuclear envelop. David Pellman (Dana-Farber Cancer Institute, Boston, USA) first addressed the question of whether mitosis imposes unique constraints on DNA replication that requires mitotic reorganization of the replisome. He reviewed published work showing that at sites of mitotic replication stalling, the CMG helicase is stripped off the replisome by a mitosis-specific cascade involving the E3 ubiquitin ligase, TRAIP and the p97 ATPase. Loss of CMG exposes single-stranded DNA that is cleaved by a yet unidentified nuclease(s). The resulting fork collapse is followed by polymerase theta-mediated end joining.  David discussed a model that such “controlled” fork breakage could be beneficial at under-replicated DNA at common fragile sites and generate the signatures of rearrangements known to occur at fragile sites in cancer genomes. He presented new data suggesting that the distinct functions of TRAIP in interphase and mitosis are mediated by a specific series of events that reorient TRAIP activity on the replisome. In interphase, TRAIP ubiquitylates substrates in front of the replisome (e.g., DNA- protein crosslinks or the CMG from a converging fork). In mitosis, in a CDK1 phosphorylation-dependent manner, TRAIP binds an ATPase called TTF2 in a way that positions TRAIP for ubiquitylation of the replisome that it travels with.

TTF2 only associates with the replisome during mitosis. The talk by Valeria Naim (Gustave Roussy Institute, Villejuif, France) addressed the link between DNA replication stress and chromosomal instability in the context of Fanconi anemia disease, a bone marrow failure and cancer predisposition disorder. Valeria presented an overview of her work showing that members of the Fanconi anemia DNA repair pathway maintain the stability of difficult-toreplicate genomic loci, which are called chromosome fragile sites from S-phase through mitosis. This role prevents defects in chromosome segregation and the appearance of chromosome bridges; She has then highlighted a signaling pathway that connects mitochondria with the nucleus and the role of the Fanconi anemia pathway in this crosstalk. Finally, she has shown that factors counteracting endogenous replication stress may be harnessed to induce mitotic catastrophe and selectively impair cancer cell proliferation. Renata Basto (Institut Curie Paris) presented the role of GEN1 nuclease in preventing the proliferation of cells after whole genome duplication. GEN1 is a nuclease with established roles in holiday junction processing. In diploid cells, this protein seems to act redundantly with other nucleases. But in cells with double the genome, it becomes absolutely essentially. In the absence of GEN1, cells accumulate higher levels of DNA damage and display multiple mitotic defects consistent with unrepaired DNA damage. Jonathon Pines (ICR, London, UK) addressed the question of how mitosis is regulated by the Anaphase Promoting Complex/Cyclosome (APC/C) that ubiquitylates the key mitotic regulator, Cyclin B1. He presented evidence from fluorescence cross correlation microscopy to support the idea that the APC/C is primarily active at the chromosomes, and that Cyclin B1 is targeted to chromosomes through binding the acidic patch on nucleosomes. He showed that this is necessary for Cyclin B1 to be degraded on time; if it is not, this leads to aneuploidy. Still related with cell division, Uri Ben-David (Tel Aviv University, Israel) discussed the interactions between aneuploidy (when cells gain or lose at least one chromosome) and DNA damage in the context of cancer. He first discussed how DNA damage can lead to aneuploidy, and then highlighted the main ways by which aneuploidy can lead to DNA damage. Finally, he discussed how this reciprocal interaction might be used in cancer therapy. Uri elaborated on three unpublished ongoing studies from his lab: (1) The discovery that highly aneuploid cells depend on the RAF/MEK/ERK pathway for overcoming ongoing DNA damage. (2) A systematic approach to identify mechanisms of aneuploidy tolerance, which identified genes involved in the DNA damage response as one of the major categories required for aneuploidy tolerance. (3) A study that characterizes chromothrispsis, resulting from fragmentation and abnormal stitching of chromosome  in human cancer cell lines, in an attempt to identify chromothripsis-induced cellular vulnerabilities. Together, these studies highlight the intricate relationship between DNA damage and aneuploidy, suggesting this relationship has important implications for cancer treatments. Floris Foijer (Groningen University, Netherlands) described how chromosomal instability (CIN) is a hallmark of cancer cells, and how this can be exploited in cancer therapy. To get to such therapies, Floris first described  the need to understand what is the impact for cancer cells. He described tools develop in his lab to quantify CIN, using single cell genomics and presented some of these tools and their usefulness to quantify CIN and DNA damage in primary cancers. Furthermore, Floris showed that cancers with CIN  circumvent immune recognition by alleviating STAT1 signaling, suggesting  that reinstating inflammatory signaling at the level of STAT1 could be a powerful strategy to awake the immune system towards killing cancer cells with CIN. Arnaud Echard (Pasteur Institute, Paris) presented new results regarding the mechanisms involved in cytokinesis, the last step of cell division leading to the physical separation of the daughter cells. Cytokinetic failure often leads to the formation of tetraploid, genetically unstable cells that can be the starting point of tumors in vivo. He showed how new candidates for cytokinetic abscission can be identified by quantitative proteomics of the “midbody remnant”, an organelle that recruits the scission machinery. He reported how the localization of the ESCRT-III polymers responsible for the membrane scission is controlled by branched F-actin to promote cytokinetic abscission and thereby to prevent the formation of tetraploid cells.

 At the end of this meeting we all felt that we had learned enormously from the interactions among all the scientists. A special word goes to Olivia Gay, a photographer that we met at the foundation des Treilles. We invited Olivia to give a presentation on the last day of the meeting. It was very well received and we were impressed by Olivia’s work and portfolio. We shared very good moments by contrasting the approaches taken by scientists and photographers. The credits of the picture below go to Olivia. Finally, we would like to thank la Fondation des Treilles for this unique opportunity, this was an unforgettable meeting.