Replication stress impairs chromosome segregation and preimplantation development in human embryos.

Human cleavage-stage embryos frequently acquire chromosomal aneuploidies during mitosis due to unknown mechanisms. Here, we show that S phase at the 1-cell stage shows replication fork stalling, low fork speed, and DNA synthesis extending into G2 phase. DNA damage foci consistent with collapsed replication forks, DSBs, and incomplete replication form in G2 in an ATR- and MRE11-dependent manner, followed by spontaneous chromosome breakage and segmental aneuploidies. Entry into mitosis with incomplete replication results in chromosome breakage, whole and segmental chromosome errors, micronucleation, chromosome fragmentation, and poor embryo quality. Sites of spontaneous chromosome breakage are concordant with sites of DNA synthesis in G2 phase, locating to gene-poor regions with long neural genes, which are transcriptionally silent at this stage of development. Thus, DNA replication stress in mammalian preimplantation embryos predisposes gene-poor regions to fragility, and in particular in the human embryo, to the formation of aneuploidies, impairing developmental potential.

DNA Cross-Bridging Shapes a Single Nucleus from a Set of Mitotic Chromosomes.

Eukaryotic cells store their chromosomes in a single nucleus. This is important to maintain genomic integrity, as chromosomes packaged into separate nuclei (micronuclei) are prone to massive DNA damage. During mitosis, higher eukaryotes disassemble their nucleus and release individualized chromosomes for segregation. How numerous chromosomes subsequently reform a single nucleus has remained unclear. Using image-based screening of human cells, we identified barrier-to-autointegration factor (BAF) as a key factor guiding membranes to form a single nucleus. Unexpectedly, nuclear assembly does not require BAF's association with inner nuclear membrane proteins but instead relies on BAF's ability to bridge distant DNA sites. Live-cell imaging and in vitro reconstitution showed that BAF enriches around the mitotic chromosome ensemble to induce a densely cross-bridged chromatin layer that is mechanically stiff and limits membranes to the surface. Our study reveals that BAF-mediated changes in chromosome mechanics underlie nuclear assembly with broad implications for proper genome function.

Boveri and beyond: Chromothripsis and genomic instability from mitotic errors.

Mitotic cell division is tightly monitored by checkpoints that safeguard the genome from instability. Failures in accurate chromosome segregation during mitosis can cause numerical aneuploidy, which was hypothesized by Theodor Boveri over a century ago to promote tumorigenesis. Recent interrogation of pan-cancer genomes has identified unexpected classes of chromosomal abnormalities, including complex rearrangements arising through chromothripsis. This process is driven by mitotic errors that generate abnormal nuclear structures that provoke extensive yet localized shattering of mis-segregated chromosomes. Here, we discuss emerging mechanisms underlying chromothripsis from micronuclei and chromatin bridges, as well as highlight how this mutational cascade converges on the DNA damage response. A fundamental understanding of these catastrophic processes will provide insight into how initial errors in mitosis can precipitate rapid cancer genome evolution.

The proteomic landscape of genotoxic stress-induced micronuclei.

Micronuclei (MN) are induced by various genotoxic stressors and amass nuclear- and cytoplasmic-resident proteins, priming the cell for MN-driven signaling cascades. Here, we measured the proteome of micronuclear, cytoplasmic, and nuclear fractions from human cells exposed to a panel of six genotoxins, comprehensively profiling their MN protein landscape. We find that MN assemble a proteome distinct from both surrounding cytoplasm and parental nuclei, depleted of spliceosome and DNA damage repair components while enriched for a subset of the replisome. We show that the depletion of splicing machinery within transcriptionally active MN contributes to intra-MN DNA damage, a known precursor to chromothripsis. The presence of transcription machinery in MN is stress-dependent, causing a contextual induction of MN DNA damage through spliceosome deficiency. This dataset represents a unique resource detailing the global proteome of MN, guiding mechanistic studies of MN generation and MN-associated outcomes of genotoxic stress.

Micronuclei induced by radiation, replication stress, or chromosome segregation errors do not activate cGAS-STING.

The cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway plays a pivotal role in innate immune responses to viral infection and inhibition of autoimmunity. Recent studies have suggested that micronuclei formed by genotoxic stress can activate innate immune signaling via the cGAS-STING pathway. Here, we investigated cGAS localization, activation, and downstream signaling from micronuclei induced by ionizing radiation, replication stress, and chromosome segregation errors. Although cGAS localized to ruptured micronuclei via binding to self-DNA, we failed to observe cGAS activation; cGAMP production; downstream phosphorylation of STING, TBK1, or IRF3; nuclear accumulation of IRF3; or expression of interferon-stimulated genes. Failure to activate the cGAS-STING pathway was observed across primary and immortalized cell lines, which retained the ability to activate the cGAS-STING pathway in response to dsDNA or modified vaccinia virus infection. We provide evidence that micronuclei formed by genotoxic insults contain histone-bound self-DNA, which we show is inhibitory to cGAS activation in cells.

ATR promotes clearance of damaged DNA and damaged cells by rupturing micronuclei.

The human ataxia telangiectasia mutated and Rad3-related (ATR) kinase functions in the nucleus to protect genomic integrity. Micronuclei (MN) arise from genomic and chromosomal instability and cause aneuploidy and chromothripsis, but how MN are removed is poorly understood. Here, we show that ATR is active in MN and promotes their rupture in S phase by phosphorylating Lamin A/C at Ser395, which primes Ser392 for CDK1 phosphorylation and destabilizes the MN envelope. In cells harboring MN, ATR or CDK1 inhibition reduces MN rupture. Consequently, ATR inhibitor (ATRi) diminishes activation of the cytoplasmic DNA sensor cGAS and compromises cGAS-dependent autophagosome accumulation in MN and clearance of micronuclear DNA. Furthermore, ATRi reduces cGAS-mediated senescence and killing of MN-bearing cancer cells by natural killer cells. Thus, in addition to the canonical ATR signaling pathway, an ATR-CDK1-Lamin A/C axis promotes MN rupture to clear damaged DNA and cells, protecting the genome in cell populations through unexpected cell-autonomous and cell-non-autonomous mechanisms.

ER-directed TREX1 limits cGAS activation at micronuclei.

Micronuclei are aberrant nuclear compartments that can form as a result of chromosome mis-segregation. Frequent loss of micronuclear envelope integrity exposes DNA to the cytoplasm, leading to chromosome fragmentation and immune activation. Here, we use micronuclei purification to show that the endoplasmic reticulum (ER)-associated nuclease TREX1 inhibits cGAS activation at micronuclei by degrading micronuclear DNA upon micronuclear envelope rupture. We demonstrate that the ER accesses ruptured micronuclei and plays a critical role in enabling TREX1 nucleolytic attack. TREX1 mutations, previously implicated in immune disease, untether TREX1 from the ER, disrupt TREX1 localization to micronuclei, diminish micronuclear DNA damage, and enhance cGAS activation. These results establish ER-directed resection of micronuclear DNA by TREX1 as a critical regulator of cytosolic DNA sensing in chromosomally unstable cells and provide a mechanistic basis for the importance of TREX1 ER tethering in preventing autoimmunity.

T-Rex escaped from the cytosolic park: Re-thinking the impact of TREX1 exonuclease deficiencies on genomic stability.

The Three Prime Repair Exonuclease 1 (TREX1) has been implicated in several pathologies characterized by chronic and inborn inflammation. Aberrant innate immunity caused by DNA sensing through the cGAS-STING pathway has been proposed to play a major role in the etiology of these interferonopathies. However, the molecular source of this DNA sensing and the possible involvement of TREX1 in genome (in)stability remains poorly understood. Recent findings reignite the debate about the cellular functions performed by TREX1 nuclease, notably in chromosome biology and stability. Here I put into perspective recent findings that suggest that TREX1 is at the crossroads of DNA damage response and inflammation in different pathological contexts.

Nucleophagy contributes to genome stability through degradation of type II topoisomerases A and B and nucleolar components.

The nuclear architecture of mammalian cells can be altered as a consequence of anomalous accumulation of nuclear proteins or genomic alterations. Most of the knowledge about nuclear dynamics comes from studies on cancerous cells. How normal healthy cells maintain genome stability, avoiding accumulation of nuclear damaged material, is less understood. Here, we describe that primary mouse embryonic fibroblasts develop a basal level of nuclear buds and micronuclei, which increase after etoposide-induced DNA double-stranded breaks. Both basal and induced nuclear buds and micronuclei colocalize with the autophagic proteins BECN1 and LC3B (also known as MAP1LC3B) and with acidic vesicles, suggesting their clearance by nucleophagy. Some of the nuclear alterations also contain autophagic proteins and type II DNA topoisomerases (TOP2A and TOP2B), or the nucleolar protein fibrillarin, implying they are also targets of nucleophagy. We propose that basal nucleophagy contributes to genome and nuclear stability, as well as in response to DNA damage.

Molecular contribution to embryonic aneuploidy and karyotypic complexity in initial cleavage divisions of mammalian development.

Embryonic aneuploidy is highly complex, often leading to developmental arrest, implantation failure or spontaneous miscarriage in both natural and assisted reproduction. Despite our knowledge of mitotic mis-segregation in somatic cells, the molecular pathways regulating chromosome fidelity during the error-prone cleavage-stage of mammalian embryogenesis remain largely undefined. Using bovine embryos and live-cell fluorescent imaging, we observed frequent micro-/multi-nucleation of mis-segregated chromosomes in initial mitotic divisions that underwent unilateral inheritance, re-fused with the primary nucleus or formed a chromatin bridge with neighboring cells. A correlation between a lack of syngamy, multipolar divisions and asymmetric genome partitioning was also revealed, and single-cell DNA-seq showed propagation of primarily non-reciprocal mitotic errors. Depletion of the mitotic checkpoint protein BUB1B (also known as BUBR1) resulted in similarly abnormal nuclear structures and cell divisions, as well as chaotic aneuploidy and dysregulation of the kinase-substrate network that mediates mitotic progression, all before zygotic genome activation. This demonstrates that embryonic micronuclei sustain multiple fates, provides an explanation for blastomeres with uniparental origins, and substantiates defective checkpoints and likely other maternally derived factors as major contributors to the karyotypic complexity afflicting mammalian preimplantation development.

R-loops and impaired autophagy trigger cGAS-dependent inflammation via micronuclei formation in Senataxin-deficient cells.

Senataxin is an evolutionarily conserved DNA/RNA helicase, whose dysfunctions are linked to neurodegeneration and cancer. A main activity of this protein is the removal of R-loops, which are nucleic acid structures capable to promote DNA damage and replication stress. Here we found that Senataxin deficiency causes the release of damaged DNA into extranuclear bodies, called micronuclei, triggering the massive recruitment of cGAS, the apical sensor of the innate immunity pathway, and the downstream stimulation of interferon genes. Such cGAS-positive micronuclei are characterized by defective membrane envelope and are particularly abundant in cycling cells lacking Senataxin, but not after exposure to a DNA breaking agent or in absence of the tumor suppressor BRCA1 protein, a partner of Senataxin in R-loop removal. Micronuclei with a discontinuous membrane are normally cleared by autophagy, a process that we show is impaired in Senataxin-deficient cells. The formation of Senataxin-dependent inflamed micronuclei is promoted by the persistence of nuclear R-loops stimulated by the DSIF transcription elongation complex and the engagement of EXO1 nuclease activity on nuclear DNA. Coherently, high levels of EXO1 result in poor prognosis in a subset of tumors lacking Senataxin expression. Hence, R-loop homeostasis impairment, together with autophagy failure and unscheduled EXO1 activity, elicits innate immune response through micronuclei formation in cells lacking Senataxin.

How Cells Respond to DNA Breaks in Mitosis.

DNA double-strand breaks (DSBs) are highly toxic lesions that can lead to chromosomal instability if they are not repaired correctly. DSBs are especially dangerous in mitosis when cells go through the complex process of equal chromosome segregation into daughter cells. When cells encounter DSBs in interphase, they are able to arrest the cell cycle until the breaks are repaired before entering mitosis. However, when DSBs occur during mitosis, cells no longer arrest but prioritize completion of cell division over repair of DNA damage. This review focuses on recent progress in our understanding of the mechanisms that allow mitotic cells to postpone DSB repair without accumulating massive chromosomal instability. Additionally, we review possible physiological consequences of failed DSB responses in mitosis.

The second half of mitosis and its implications in cancer biology.

The nucleus undergoes dramatic structural and functional changes during cell division. With the entry into mitosis, in human cells the nuclear envelope breaks down, chromosomes rearrange into rod-like structures which are collected and segregated by the spindle apparatus. While these processes in the first half of mitosis have been intensively studied, much less is known about the second half of mitosis, when a functional nucleus reforms in each of the emerging cells. Here we review our current understanding of mitotic exit and nuclear reformation with spotlights on the links to cancer biology.

Breaking the vicious circle: Extrachromosomal circular DNA as an emerging player in tumour evolution.

Extrachromosomal circular DNA (ecDNA) or double minutes have gained renewed interest since its discovery more than five decades ago, emerging as potent drivers of tumour evolution. This has largely been motivated by recent discovery that the tumour-exclusive ecDNA are highly prevalent in almost all cancers unlike previously thought. EcDNAs contribute to elevated oncogene expression, intratumoural heterogeneity, tumour adaptation and therapy resistance independently of canonical chromosomal alterations. Importantly, ecDNAs play a critical role in patient survival as ecDNA-based oncogene amplification adversely affects clinical outcome to a significantly greater extent than intrachromosomal amplification. Chromothripsis, a major driver of ecDNA biogenesis and gene amplification, is a mutational process characterised by chromosomal shattering and localised complex genome rearrangement. Chemotherapeutic drugs can lead to chromothriptic rearrangements and therapy resistance. In this review, we examine how ecDNAs mediate oncogene overexpression, facilitate accelerated tumour malignancy and enhance rapid adaptation independently of linear chromosomes. We delve into discoveries pertaining to mechanisms of biogenesis, distinctive features of ecDNA, gene regulation and topological interactions with active chromatin. We also discuss the critical role of chromothripsis in engendering ecDNA amplification and evolution. One envisions that insights into ecDNA biology not only hold importance for the cancer genome and tumour evolutionary dynamics, but could also inform prognostication and clinical intervention, particularly for cancers characterised by high oncogene amplification.

Mechanistic origins of diverse genome rearrangements in cancer.

Cancer genomes frequently harbor structural chromosomal rearrangements that disrupt the linear DNA sequence order and copy number. To date, diverse classes of structural variants have been identified across multiple cancer types. These aberrations span a wide spectrum of complexity, ranging from simple translocations to intricate patterns of rearrangements involving multiple chromosomes. Although most somatic rearrangements are acquired gradually throughout tumorigenesis, recent interrogation of cancer genomes have uncovered novel categories of complex rearrangements that arises rapidly through a one-off catastrophic event, including chromothripsis and chromoplexy. Here we review the cellular and molecular mechanisms contributing to the formation of diverse structural rearrangement classes during cancer development. Genotoxic stress from a myriad of extrinsic and intrinsic sources can trigger DNA double-strand breaks that are subjected to DNA repair with potentially mutagenic outcomes. We also highlight how aberrant nuclear structures generated through mitotic cell division errors, such as rupture-prone micronuclei and chromosome bridges, can instigate massive DNA damage and the formation of complex rearrangements in cancer genomes.

Modest increase of KIF11 expression exposes fragilities in the mitotic spindle, causing chromosomal instability.

Chromosomal instability (CIN), the process of increased chromosomal alterations, compromises genomic integrity and has profound consequences on human health. Yet, our understanding of the molecular and mechanistic basis of CIN initiation remains limited. We developed a high-throughput, single-cell, image-based pipeline employing deep-learning and spot-counting models to detect CIN by automatically counting chromosomes and micronuclei. To identify CIN-initiating conditions, we used CRISPR activation in human diploid cells to upregulate, at physiologically relevant levels, 14 genes that are functionally important in cancer. We found that upregulation of CCND1, FOXA1 and NEK2 resulted in pronounced changes in chromosome counts, and KIF11 upregulation resulted in micronuclei formation. We identified KIF11-dependent fragilities within the mitotic spindle; increased levels of KIF11 caused centrosome fragmentation, higher microtubule stability, lagging chromosomes or mitotic catastrophe. Our findings demonstrate that even modest changes in the average expression of single genes in a karyotypically stable background are sufficient for initiating CIN by exposing fragilities of the mitotic spindle, which can lead to a genomically diverse cell population.

Polysaccharide from Helianthus tuberosus L. as a potential radioprotector.

INTRODUCTION: Radioprotectors help to protect the body or at least minimize the negative consequences of radiation exposure. The present study aimed to assess the radioprotective potential of Helianthus tuberosus L. polysaccharide (HTLP) in vitality and micronuclei tests. To assess the cytotoxic effects of HTLP, both vitality and MTT reductase assays were conducted. MATERIALS AND METHODS: RAW 264.7 cells viability was assessed 24 h after adding 200 µg/ml HTLP solution by staining cell cultures with propidium iodide and bis-benzimide to detect the nuclei of dead cells and the total number of cells in culture. To assess cell viability via cellular metabolic activity MTT test was used. In this work outbred 24-30 g 5-months old SHK mice have been used. Irradiation was provided with proton beams with an energy of 660 MeV at a dose rate of 80 Gy with doses 1.5 Gy for micronuclei test and 8.5 Gy for survival test. Whole body X-ray irradiation was conducted using the RUT-15 therapeutic X-ray unit with doses of 1.5 Gy for MN test and 6.5 Gy for survival. The HTLP sterile solution in dose 100 µg/animal was injected into the tail vein 15 min before X-ray or proton irradiation. RESULTS AND CONCLUSION: s: Vitality test showed no significant differences between the control group and cells treated with 200 µl of 200 µg/ml HTLP solution, though a greater variability was noted. In contrast, the MTT assay indicated enhanced cell viability in the HTLP-treated cells. HTLP does not exert any toxic effects in cell culture. Moreover, results of MTT reductase assay shows, that HTLP may enhance the cells' metabolic activity. Animals pre-treated with HTLP displayed a significant reduction in micronuclei formation, showing five times fewer micronuclei in bone marrow cells compared to the non-treated group. This comparison highlights HTLP's potential protective effect against radiation-induced chromosomal damage. HTLP treatment demonstrates a significant reduction in hazard compared to the control, indicating its protective effects against irradiation. Thus, it can be concluded that the use of HTLP increases the likelihood of animal survival under the ionizing effects of X-rays and protons. The survival analysis reveals that the HTLP-treated groups exhibit a higher survival rate compared to both the control and Cysteamine-treated groups, suggesting a significant protective effect of HTLP against irradiation, regardless of the type of irradiation (proton or X-ray) with p < 0.0001.

Maternal Smc3 protects the integrity of the zygotic genome through DNA replication and mitosis.

Aneuploidy is frequently observed in oocytes and early embryos, begging the question of how genome integrity is monitored and preserved during this crucial period. SMC3 is a subunit of the cohesin complex that supports genome integrity, but its role in maintaining the genome during this window of mammalian development is unknown. We discovered that, although depletion of Smc3 following meiotic S phase in mouse oocytes allowed accurate meiotic chromosome segregation, adult females were infertile. We provide evidence that DNA lesions accumulated following S phase in SMC3-deficient zygotes, followed by mitosis with lagging chromosomes, elongated spindles, micronuclei, and arrest at the two-cell stage. Remarkably, although centromeric cohesion was defective, the dosage of SMC3 was sufficient to enable embryogenesis in juvenile mutant females. Our findings suggest that, despite previous reports of aneuploidy in early embryos, chromosome missegregation in zygotes halts embryogenesis at the two-cell stage. Smc3 is a maternal gene with essential functions in the repair of spontaneous damage associated with DNA replication and subsequent chromosome segregation in zygotes, making cohesin a key protector of the zygotic genome.

Measurement of chromosomal instability and level of DNA damage in peripheral blood mononuclear cells of endometrial cancer patients.

Endometrial cancer is one of the most common invasive gynecologic malignancies in developed countries. The aim of this study was to evaluate chromosomal instability and level of DNA damage in peripheral blood mononuclear cells (PBMCs) of newly diagnosed endometrial cancer patients in relation to health status (diagnosis), age, histological grade of cancer, residence, smoking, number of pregnancies, miscarriages, and abortions. The analyzed sample consisted of 60 individuals, 30 endometrial cancer patients with an average age of 64.37 ±â€…7.08, and 30 healthy control women with an average age of 60.23 ±â€…11.55. Chromosomal instability was evaluated by the cytokinesis-block micronucleus (CBMN) assay, and the level of DNA damage by the single-cell gel electrophoresis (comet) assay in PBMCs. The average frequencies of micronuclei (MNi), nucleoplasmic bridges (NPBs) as well as nuclear buds (NBUDs) were significantly higher in cancer patients compared to controls (P < .0005). There was no difference in the nuclear division index (NDI) among the analyzed samples. The comet assay showed that the patients had a significantly increased genetic damage index (GDI) compared with controls (P < .0005). Using linear regression analysis, we found that health status (diagnosis) had the strongest influence on the MN frequency as well as GDI (P < .0005). Our results indicated that there is a high level of genetic damage in both the level of DNA and the level of chromosomes in the PBMCs of newly diagnosed patients with endometrial cancer, where the frequency and level of damage were significantly affected by health status, grade of cancer, residence, number of pregnancies, miscarriages, and abortions.

Twenty years of merotelic kinetochore attachments: a historical perspective.

Micronuclei, small DNA-containing structures separate from the main nucleus, were used for decades as an indicator of genotoxic damage. Micronuclei containing whole chromosomes were considered a biomarker of aneuploidy and were believed to form, upon mitotic exit, from chromosomes that lagged behind in anaphase as all other chromosomes segregated to the poles of the mitotic spindle. However, the mechanism responsible for inducing anaphase lagging chromosomes remained unknown until just over twenty years ago. Here, I summarize what preceded and what followed this discovery, highlighting some of the open questions and opportunities for future investigation.