Formation Of Chromatin Bridges Research Articles

Tryptophanyl-tRNA synthetase-1 (WARS-1) depletion and high tryptophan concentration lead to genomic instability in Caenorhabditis elegans

The fidelity of translation is ensured by a family of proteins named aminoacyl-tRNA synthetases (ARSs), making them crucial for development and survival. More recently, mutations in the tryptophanyl-tRNA synthetase 1 (WARS1) have been linked to various human diseases, from intellectual disability to various types of cancer. To understand the function of WARS1, we investigated the effect of WARS-1 depletion during the mitotic and meiotic cell cycle in the developing germline of Caenorhabditis elegans (C. elegans) and demonstrated the role of WARS-1 in genome integrity. wars-1 knockdown results in cell cycle arrest of the mitotically active germ cells. Such mitotic arrest is also associated with canonical DNA damage-induced checkpoint signaling in mitotic and meiotic germ cells. Significantly, such DNA checkpoint activation is associated with the morphological anomalies in chromatin structures that are the hallmarks of genome instability, such as the formation of chromatin bridges, micronuclei, and chromatin buds. We demonstrated that knocking down wars-1 results in an elevation of the intracellular concentration of tryptophan and its catabolites, a surprising finding emphasizing the impact of cellular amino acid availability and organismal/individual dietary uptake on genome integrity. Our result demonstrates that exposing C. elegans to a high tryptophan dosage leads to DNA damage checkpoint activation and a significant increase in the tryptophan metabolites. Targeting tryptophan catabolism, the least utilized amino acid in nature, can be important in developing new cancer therapeutic approaches. All in all, we have strong evidence that knocking down wars-1 results in defects in genomic integrity.

Disruption of CDK7 signaling leads to catastrophic chromosomal instability coupled with a loss of condensin-mediated chromatin compaction

Chromatin organization is highly dynamic and modulates DNA replication, transcription, and chromosome segregation. Condensin is essential for chromosome assembly during mitosis and meiosis, as well as maintenance of chromosome structure during interphase. While it is well established that sustained condensin expression is necessary to ensure chromosome stability, the mechanisms that control its expression are not yet known. Herein, we report that disruption of cyclin-dependent kinase 7 (CDK7), the core catalytic subunit of CDK-activating kinase, leads to reduced transcription of several condensin subunits, including structural maintenance of chromosomes 2 (SMC2). Live and static microscopy revealed that inhibiting CDK7 signaling prolongs mitosis and induces chromatin bridge formation, DNA double-strand breaks, and abnormal nuclear features, all of which are indicative of mitotic catastrophe and chromosome instability. Affirming the importance of condensin regulation by CDK7, genetic suppression of the expression of SMC2, a core subunit of this complex, phenocopies CDK7 inhibition. Moreover, analysis of genome-wide chromatin conformation using Hi-C revealed that sustained activity of CDK7 is necessary to maintain chromatin sublooping, a function that is ascribed to condensin. Notably, the regulation of condensin subunit gene expression is independent of superenhancers. Together, these studies reveal a new role for CDK7 in sustaining chromatin configuration by ensuring the expression of condensin genes, including SMC2.

Telomerase inhibition is an effective therapeutic strategy in TERT promoter-mutant glioblastoma models with low tumor volume.

Glioblastoma is one of the most lethal forms of cancer, with 5-year survival rates of only 6%. Glioblastoma-targeted therapeutics have been challenging to develop due to significant inter- and intra-tumoral heterogeneity. Telomerase reverse transcriptase gene (TERT) promoter mutations are the most common known clonal oncogenic mutations in glioblastoma. Telomerase is therefore considered to be a promising therapeutic target against this tumor. However, an important limitation of this strategy is that cell death does not occur immediately after telomerase ablation, but rather after several cell divisions required to reach critically short telomeres. We, therefore, hypothesize that telomerase inhibition would only be effective in glioblastomas with low tumor burden. We used CRISPR interference to knock down TERT expression in TERT promoter-mutant glioblastoma cell lines and patient-derived models. We then measured viability using serial proliferation assays. We also assessed for features of telomere crisis by measuring telomere length and chromatin bridge formation. Finally, we used a doxycycline-inducible CRISPR interference system to knock down TERT expression in vivo early and late in tumor development. Upon TERT inactivation, glioblastoma cells lose their proliferative ability over time and exhibit telomere shortening and chromatin bridge formation. In vivo, survival is only prolonged when TERT knockdown is induced shortly after tumor implantation, but not when the tumor burden is high. Our results support the idea that telomerase inhibition would be most effective at treating glioblastomas with low tumor burden, for example in the adjuvant setting after surgical debulking and chemoradiation.

EXTH-11. TELOMERASE INHIBITION IS AN EFFECTIVE THERAPEUTIC STRATEGY IN TERT PROMOTER-MUTANT GLIOBLASTOMAS MODELS WITH LOW TUMOR BURDEN

Abstract Glioblastoma targeted therapeutics have been challenging to develop due to significant inter- and intra-tumoral heterogeneity. While many activated oncogenes in glioblastoma are subclonal, TERT promoter mutations commonly occur as clonal events and are found in up to 80% of IDH-wildtype glioblastomas. Given the high prevalence and clonal nature of TERT promoter mutations in glioblastoma, telomerase is considered a promising therapeutic target for this deadly cancer. Prior studies have validated this hypothesis, demonstrating that knockout of the transcription factor GABPA, which selectively binds to the mutant TERT promoter, as well as base editing-mediated correction of TERT promoter mutations, are selectively toxic to TERTpromoter mutant glioblastomas. However, an important limitation of this strategy is that cancer cell death does not occur immediately after telomerase ablation, but rather after several cell divisions required to reach critically short telomeres. We therefore hypothesize that telomerase inhibition would only be effective in low tumor burden glioblastomas. In this study, we used CRISPR interference to knock down TERT expression in TERT promoter-mutant glioblastoma cell lines and patient derived models. We then measured cell viability and assessed for features of telomere crisis by measuring telomere length and chromatin bridge formation. Lastly, we used a doxycycline inducible CRISPR interference system to knock down TERT expression in vivo early and late in the tumor formation process. We demonstrated that TERT promoter-mutant glioblastoma cells are sensitive to telomerase inhibition and undergo telomere crisis. In vivo, tumor formation is only inhibited when TERT knockdown is induced shortly after tumor implantation, but not when tumor burden is high. This work supports the idea that telomerase inhibition would be a suitable therapeutic strategy for glioblastoma patients with low tumor burden, for example in the adjuvant setting after surgical debulking and chemoradiation.

Centromeric chromatin integrity is compromised by loss of Cdk5rap2, a transcriptional activator of CENP-A

Centromeres are chromosomal loci where kinetochores assemble to ensure faithful chromosome segregation during mitosis. CENP-A defines the loci by serving as an epigenetic marker that recruits other centromere components for a functional structure. However, the mechanism that controls CENP-A regulation of centromeric chromatin integrity remains to be explored. Separate studies have shown that loss of CENP-A or the Cdk5 regulatory subunit associated protein 2 (Cdk5rap2), a key player in mitotic progression, triggers the occurrence of lagging chromosomes. This prompted us to investigate a potential link between CENP-A and Cdk5rap2 in the maintenance of centromeric chromatin integrity. Here, we demonstrate that loss of Cdk5rap2 causes reduced CENP-A expression while exogenous Cdk5rap2 expression in cells depleted of endogenous Cdk5rap2 restores CENP-A expression. Indeed, we show that Cdk5rap2 is a nuclear protein that acts as a positive transcriptional regulator of CENP-A. Cdk5rap2 interacts with the CENP-A promoter and upregulates CENP-A transcription. Accordingly, loss of Cdk5rap2 causes reduced level of centromeric CENP-A. Exogenous CENP-A expression partially inhibits the occurrence of lagging chromosomes in Cdk5rap2 knockdown cells, indicating that lagging chromosomes induced by loss of Cdk5rap2 is due, in part, to loss of CENP-A. Aside from manifesting lagging chromosomes, cells depleted of Cdk5rap2, and thus CENP-A, show increased micronuclei and chromatin bridge formation. Altogether, our findings indicate that Cdk5rap2 serves to maintain centromeric chromatin integrity partly through CENP-A.

CBMT-21. TERT PROMOTER-MUTANT GLIOBLASTOMAS EXHIBIT DEPENDENCY ON TELOMERASE

Abstract TERT promoter mutations are among the most common somatic alterations in cancer and they occur in about 80% of IDH-wildtype glioblastomas. TERT promoter mutations were found to reactivate telomerase by providing a novel binding site for the GABP transcription factor. While the effects of telomerase ablation are well understood in mice and somatic human cells, these effects in cancer are yet to be fully elucidated. In this study, we used a genetic approach with CRISPR-interference to knock down telomerase in TERT promoter-mutant glioblastoma cell lines. We show that this leads to a gradual and significant reduction in proliferation. This phenotype ultimately culminates in telomere crisis, with telomere shortening, activation of the DNA damage response pathway and formation of chromatin bridges. These data suggest that anti-telomerase therapy is a potential effective approach for glioblastoma tumors.

Premature mitotic entry induced by ATR inhibition potentiates olaparib inhibition-mediated genomic instability, inflammatory signaling, and cytotoxicity in BRCA2-deficient cancer cells.

Poly(ADP‐ribose) polymerase (PARP) inhibitors are selectively cytotoxic in cancer cells with defects in homologous recombination (HR) (e.g., due to BRCA1/2 mutations). However, not all HR‐deficient tumors efficiently respond to PARP inhibition and often acquire resistance. It is therefore important to uncover how PARP inhibitors induce cytotoxicity and develop combination strategies to potentiate PARP inhibitor efficacy in HR‐deficient tumors. In this study, we found that forced mitotic entry upon ATR inhibition potentiates cytotoxic effects of PARP inhibition using olaparib in BRCA2‐depleted and Brca2 knockout cancer cell line models. Single DNA fiber analysis showed that ATR inhibition does not exacerbate replication fork degradation. Instead, we find ATR inhibitors accelerate mitotic entry, resulting in the formation of chromatin bridges and lagging chromosomes. Furthermore, using genome‐wide single‐cell sequencing, we show that ATR inhibition enhances genomic instability of olaparib‐treated BRCA2‐depleted cells. Inhibition of CDK1 to delay mitotic entry mitigated mitotic aberrancies and genomic instability upon ATR inhibition, underscoring the role of ATR in coordinating proper cell cycle timing in situations of DNA damage. Additionally, we show that olaparib treatment leads to increased numbers of micronuclei, which is accompanied by a cGAS/STING‐associated inflammatory response in BRCA2‐deficient cells. ATR inhibition further increased the numbers of cGAS‐positive micronuclei and the extent of cytokine production in olaparib‐treated BRCA2‐deficient cancer cells. Altogether, we show that ATR inhibition induces premature mitotic entry and mediates synergistic cytotoxicity with PARP inhibition in HR‐deficient cancer cells, which involves enhanced genomic instability and inflammatory signaling.

Loss of PICH promotes chromosome instability and cell death in triple-negative breast cancer

Triple-negative breast cancer (TNBC), defined by the lack of expression of estrogen, progesterone, and ERBB2 receptors, has the worst prognosis of all breast cancers. It is difficult to treat owing to a lack of effective molecular targets. Here, we report that the growth of TNBC cells is exceptionally dependent on PICH, a DNA-dependent ATPase. Clinical samples analysis showed that PICH is highly expressed in TNBC compared to other breast cancer subtypes. Importantly, its high expression correlates with higher risk of distal metastasis and worse clinical outcomes. Further analysis revealed that PICH depletion selectively impairs the proliferation of TNBC cells, but not that of luminal breast cancer cells, in vitro and in vivo. In addition, knockdown of PICH in TNBC cells induces the formation of chromatin bridges and lagging chromosomes in anaphase, frequently resulting in micronucleation or binucleation, finally leading to mitotic catastrophe and apoptosis. Collectively, our findings show the dependency of TNBC cells on PICH for faithful chromosome segregation and the clinical potential of PICH inhibition to improve treatment of patients with high-risk TNBC.

Mutations in NCAPG2 Cause a Severe Neurodevelopmental Syndrome that Expands the Phenotypic Spectrum of Condensinopathies

The use of whole-exome and whole-genome sequencing has been a catalyst for a genotype-first approach to diagnostics. Under this paradigm, we have implemented systematic sequencing of neonates and young children with a suspected genetic disorder. Here, we report on two families with recessive mutations in NCAPG2 and overlapping clinical phenotypes that include severe neurodevelopmental defects, failure to thrive, ocular abnormalities, and defects in urogenital and limb morphogenesis. NCAPG2 encodes a member of the condensin II complex, necessary for the condensation of chromosomes prior to cell division. Consistent with a causal role for NCAPG2, we found abnormal chromosome condensation, augmented anaphase chromatin-bridge formation, and micronuclei in daughter cells of proband skin fibroblasts. To test the functional relevance of the discovered variants, we generated an ncapg2 zebrafish model. Morphants displayed clinically relevant phenotypes, such as renal anomalies, microcephaly, and concomitant increases in apoptosis and altered mitotic progression. These could be rescued by wild-type but not mutant human NCAPG2 mRNA and were recapitulated in CRISPR-Cas9 F0 mutants. Finally, we noted that the individual with a complex urogenital defect also harbored a heterozygous NPHP1 deletion, a common contributor to nephronophthisis. To test whether sensitization at the NPHP1 locus might contribute to a more severe renal phenotype, we co-suppressed nphp1 and ncapg2, which resulted in significantly more dysplastic renal tubules in zebrafish larvae. Together, our data suggest that impaired function of NCAPG2 results in a severe condensinopathy, and they highlight the potential utility of examining candidate pathogenic lesions beyond the primary disease locus.

Mechanism of cytokinesis failure in ovarian cystadenomas with defective BRCA1 and P53 pathways.

We previously described an in vitro model in which serous ovarian cystadenomas were transfected with SV40 large T antigen, resulting in loss of RB and P53 functions and thus mimicking genetic defects present in early high-grade serous extra-uterine Müllerian (traditionally called high-grade serous ovarian) carcinomas including those associated with the BRCA1 mutation carrier state. We showed that replicative aging in this cell culture model leads to a mitotic arrest at the spindle assembly checkpoint. Here we show that this arrest is due to a reduction in microtubule anchoring that coincides with decreased expression of the BUB1 kinase and of the phosphorylated form of its substrate, BUB3. The ensuing prolonged mitotic arrest leads to cohesion fatigue resulting in cell death or, in cells that recover from this arrest, in cytokinesis failure and polyploidy. Down-regulation of BRCA1 to levels similar to those present in BRCA1 mutation carriers leads to increased and uncontrolled microtubule anchoring to the kinetochore resulting in overcoming the spindle assembly checkpoint. Progression to anaphase under those conditions is associated with formation of chromatin bridges between chromosomal plates due to abnormal attachments to the kinetochore, significantly increasing the risk of cytokinesis failure. The dependence of this scenario on accelerated replicative aging can, at least in part, account for the site specificity of the cancers associated with the BRCA1 mutation carrier state, as epithelia of the mammary gland and of the reproductive tract are targets of cell-nonautonomous consequences of this carrier state on cellular proliferation associated with menstrual cycle progressions.

The Pseudo-Pelger huët Cell as a Retrospective Dosimeter: Analysis of a Radium Dial Painter Cohort.

Recently, the pseudo-Pelger Huët anomaly in peripheral blood neutrophils has been described as a new radiation-induced, stable biomarker. In this study, pseudo-Pelger Huët anomaly was examined in peripheral blood slides from a cohort of 166 former radium dial painters and ancillary personnel in the radium dial industry, 35 of whom had a marrow dose of zero above background. Members of the radium dial painter cohort ingested Ra and Ra at an early age (average age 20.6 ± 5.4 y; range 13-40 y) during the years 1914-1955. Exposure duration ranged from 1-1,820 wk with marrow dose 1.5-6,750 mGy. Pseudo-Pelger Huët anomaly expressed as a percentage of total neutrophils in this cohort rises in a sigmoidal fashion over five decades of red marrow dose. Six subjects in this cohort eventually developed malignancies: five osteosarcomas and one mastoid cell neoplasm. The pseudo-Pelger Huët anomaly percentage in these cases of neoplasm increases with marrow dose and is best fit with a sigmoid function, suggestive of a threshold effect. No sarcomas are seen for a marrow dose under 2 Gy. These results indicate that pseudo-Pelger Huët anomaly in peripheral blood is a reasonable surrogate for the estimation of alpha dose to bone marrow in historic radiation cases. Hypotheses are discussed to explain late (months to years), early (hours to days), and intermediate (weeks to months) effects of ionizing radiation, respectively, on the expression of genes encoding inner nuclear membrane proteins and their receptors, on the structure and function of nuclear membrane proteins and lipids, and on cytokinesis through chromatin bridge formation.

Cytological analysis of cytoplasmic incompatibility induced by Cardinium suggests convergent evolution with its distant cousin Wolbachia.

Cytoplasmic incompatibility (CI) is a conditional sterility in numerous arthropods that is caused by inherited, intracellular bacteria such as Wolbachia Matings between males carrying CI-inducing Wolbachia and uninfected females, or between males and females infected with different Wolbachia strains, result in progeny that die during very early embryogenesis. Multiple studies in diploid (Drosophila) and haplodiploid (Nasonia) insects have shown that CI-Wolbachia cause a failure of the paternally derived chromatin from resolving into distinct chromosomes. This leads to the formation of chromatin bridges and other mitotic defects as early as the first mitotic division, and to early mitotic arrest. It is currently unknown if CI-inducing symbionts other than Wolbachia affect similar cellular processes. Here, we investigated CI caused by an unrelated bacterium, Cardinium, which naturally infects a parasitic wasp, Encarsia suzannae CI crosses in this host-symbiont system resulted in early mitotic defects including asynchrony of paternal and maternal chromosome sets as they enter mitosis, chromatin bridges and improper chromosome segregation that spanned across multiple mitotic divisions, triggering embryonic death through accumulated aneuploidy. We highlight small differences with CI-Wolbachia, which could be due to the underlying CI mechanism or host-specific effects. Our results suggest a convergence of CI-related cellular phenotypes between these two unrelated symbionts.

DNA double-strand breaks and Aurora B mislocalization induced by exposure of early mitotic cells to H2O2 appear to increase chromatin bridges and resultant cytokinesis failure

Aneuploidy, an abnormal number of chromosomes that is a hallmark of cancer cells, can arise from tetraploid/binucleated cells through a failure of cytokinesis. Reactive oxygen species (ROS) have been implicated in various diseases, including cancer. However, the nature and role of ROS in cytokinesis progression and related mechanisms has not been clearly elucidated. Here, using time-lapse analysis of asynchronously growing cells and immunocytochemical analyses of synchronized cells, we found that hydrogen peroxide (H2O2) treatment at early mitosis (primarily prometaphase) significantly induced cytokinesis failure. Cytokinesis failure and the resultant formation of binucleated cells containing nucleoplasmic bridges (NPBs) seemed to be caused by increases in DNA double-strand breaks (DSBs) and subsequent unresolved chromatin bridges. We further found that H2O2 induced mislocalization of Aurora B during mitosis. All of these effects were attenuated by pretreatment with N-acetyl-L-cysteine (NAC) or overexpression of Catalase. Surprisingly, the PARP inhibitor PJ34 also reduced H2O2-induced Aurora B mislocalization and binucleated cell formation. Results of parallel experiments with etoposide, a topoisomerase IIα inhibitor that triggers DNA DSBs, suggested that both DNA DSBs and Aurora B mislocalization contribute to chromatin bridge formation. Aurora B mislocalization also appeared to weaken the “abscission checkpoint”. Finally, we showed that KRAS–induced binucleated cell formation appeared to be also H2O2-dependent. In conclusion, we propose that a ROS, mainly H2O2 increases binucleation through unresolved chromatin bridges caused by DNA damage and mislocalization of Aurora B, the latter of which appears to augment the effect of DNA damage on chromatin bridge formation.

Mutations in genes encoding condensin complex proteins cause microcephaly through decatenation failure at mitosis.

Compaction of chromosomes is essential for accurate segregation of the genome during mitosis. In vertebrates, two condensin complexes ensure timely chromosome condensation, sister chromatid disentanglement, and maintenance of mitotic chromosome structure. Here, we report that biallelic mutations in NCAPD2, NCAPH, or NCAPD3, encoding subunits of these complexes, cause microcephaly. In addition, hypomorphic Ncaph2 mice have significantly reduced brain size, with frequent anaphase chromatin bridge formation observed in apical neural progenitors during neurogenesis. Such DNA bridges also arise in condensin-deficient patient cells, where they are the consequence of failed sister chromatid disentanglement during chromosome compaction. This results in chromosome segregation errors, leading to micronucleus formation and increased aneuploidy in daughter cells. These findings establish "condensinopathies" as microcephalic disorders, with decatenation failure as an additional disease mechanism for microcephaly, implicating mitotic chromosome condensation as a key process ensuring mammalian cerebral cortex size.

Loss ofCaenorhabditis elegansBRCA1 Promotes Genome Stability During Replication insmc-5Mutants

DNA damage by ultraviolet (UV) light poses a risk for mutagenesis and a potential hindrance for cell cycle progression. Cells cope with UV-induced DNA damage through two general strategies to repair the damaged nucleotides and to promote cell cycle progression in the presence of UV-damaged DNA. Defining the genetic pathways and understanding how they function together to enable effective tolerance to UV remains an important area of research. The structural maintenance of chromosomes (SMC) proteins form distinct complexes that maintain genome stability during chromosome segregation, homologous recombination, and DNA replication. Using a forward genetic screen, we identified two alleles of smc-5 that exacerbate UV sensitivity in Caenorhabditis elegans. Germ cells of smc-5-defective animals show reduced proliferation, sensitivity to perturbed replication, chromatin bridge formation, and accumulation of RAD-51 foci that indicate the activation of homologous recombination at DNA double-strand breaks. Mutations in the translesion synthesis polymerase polh-1 act synergistically with smc-5 mutations in provoking genome instability after UV-induced DNA damage. In contrast, the DNA damage accumulation and sensitivity of smc-5 mutant strains to replication impediments are suppressed by mutations in the C. elegans BRCA1/BARD1 homologs, brc-1 and brd-1. We propose that SMC-5/6 promotes replication fork stability and facilitates recombination-dependent repair when the BRC-1/BRD-1 complex initiates homologous recombination at stalled replication forks. Our data suggest that BRC-1/BRD-1 can both promote and antagonize genome stability depending on whether homologous recombination is initiated during DNA double-strand break repair or during replication stalling.

Chk1 protects against chromatin bridges by constitutively phosphorylating BLM serine 502 to inhibit BLM degradation.

Chromatin bridges represent incompletely segregated chromosomal DNA connecting the anaphase poles and can result in chromosome breakage. The Bloom's syndrome protein helicase (BLM, also known as BLMH) suppresses formation of chromatin bridges. Here, we show that cells deficient in checkpoint kinase 1 (Chk1, also known as CHEK1) exhibit higher frequency of chromatin bridges and reduced BLM protein levels compared to controls. Chk1 inhibition leads to BLM ubiquitylation and proteasomal degradation during interphase. Furthermore, Chk1 constitutively phosphorylates human BLM at serine 502 (S502) and phosphorylated BLM localises to chromatin bridges. Mutation of S502 to a non-phosphorylatable alanine residue (BLM-S502A) reduces the stability of BLM, whereas expression of a phospho-mimicking BLM-S502D, in which S502 is mutated to aspartic acid, stabilises BLM and prevents chromatin bridges in Chk1-deficient cells. In addition, wild-type but not BLM-S502D associates with cullin 3, and cullin 3 depletion rescues BLM accumulation and localisation to chromatin bridges after Chk1 inhibition. We propose that Chk1 phosphorylates BLM-S502 to inhibit cullin-3-mediated BLM degradation during interphase. These results suggest that Chk1 prevents deleterious anaphase bridges by stabilising BLM.

Protective role of RAD50 on chromatin bridges during abnormal cytokinesis

Faithful chromosome segregation is required for preserving genomic integrity. Failure of this process may entail chromatin bridges preventing normal cytokinesis. To test whether RAD50, a protein normally involved in DNA double-strand break repair, is involved in abnormal cytokinesis and formation of chromatin bridges, we used immunocytochemical and protein interaction assays. RAD50 localizes to chromatin bridges during aberrant cytokinesis and subsequent stages of the cell cycle, either decorating the entire bridge or focally accumulating at the midbody zone. Ionizing radiation led to an ∼4-fold increase in the rate of chromatin bridges in an ataxia telangiectatica mutated (ATM)-dependent manner in human RAD50-proficient fibroblasts but not in RAD50-deficient cells. Cells with a RAD50-positive chromatin bridge were able to continue cell cycling and to progress through S phase (44%), whereas RAD50 knockdown caused a deficiency in chromatin bridges as well as an ∼4-fold prolonged duration of mitosis. RAD50 colocalized and directly interacted with Aurora B kinase and phospho-histone H3, and Aurora B kinase inhibition led to a deficiency in RAD50-positive bridges. Based on these observations, we propose that RAD50 is a crucial factor for the stabilization and shielding of chromatin bridges. Our study provides evidence for a hitherto unknown role of RAD50 in abnormal cytokinesis.

On the regulation, function, and localization of the DNA-dependent ATPase PICH

The putative chromatin remodeling enzyme Plk1-interacting checkpoint helicase (PICH) was discovered as an interaction partner and substrate of the mitotic kinase Plk1. During mitosis PICH associates with centromeres and kinetochores and, most interestingly, constitutes a robust marker for ultrafine DNA bridges (UFBs) that connect separating chromatids in anaphase cells. The precise roles of PICH remain to be clarified. Here, we have used antibody microinjection and siRNA-rescue experiments to study PICH function and localization during M phase progression, with particular emphasis on the role of the predicted ATPase domain and the regulation of PICH localization by Plk1. We show that interference with PICH function results in chromatin bridge formation and micronucleation and that ATPase activity is critical for PICH function. Interestingly, an intact ATPase domain of PICH is required for prevention of chromatin bridge formation but not for UFB resolution, and quantitative analyses of UFB and chromatin bridge frequencies suggest that these structures are of different etiologies. We also show that the ATPase activity of PICH is required for temporal and spatial control of PICH localization to chromatin and that Plk1 likely controls PICH localization through phosphorylation of proteins distinct from PICH itself. This work strengthens the view that PICH is an important, Plk1-regulated enzyme, whose ATPase activity is essential for maintenance of genome integrity. Although not required for the spindle assembly checkpoint, PICH is clearly important for faithful chromosome segregation.Electronic supplementary materialThe online version of this article (doi:10.1007/s00412-012-0370-0) contains supplementary material, which is available to authorized users.

The PP2A Regulatory Subunit Tap46, a Component of the TOR Signaling Pathway, Modulates Growth and Metabolism in Plants

Tap42/α4, a regulatory subunit of protein phosphatase 2A, is a downstream effector of the target of rapamycin (TOR) protein kinase, which regulates cell growth in coordination with nutrient and environmental conditions in yeast and mammals. In this study, we characterized the functions and phosphatase regulation of plant Tap46. Depletion of Tap46 resulted in growth arrest and acute plant death with morphological markers of programmed cell death. Tap46 interacted with PP2A and PP2A-like phosphatases PP4 and PP6. Tap46 silencing modulated cellular PP2A activities in a time-dependent fashion similar to TOR silencing. Immunoprecipitated full-length and deletion forms of Arabidopsis thaliana TOR phosphorylated recombinant Tap46 protein in vitro, supporting a functional link between Tap46 and TOR. Tap46 depletion reproduced the signature phenotypes of TOR inactivation, such as dramatic repression of global translation and activation of autophagy and nitrogen mobilization, indicating that Tap46 may act as a positive effector of TOR signaling in controlling those processes. Additionally, Tap46 silencing in tobacco (Nicotiana tabacum) BY-2 cells caused chromatin bridge formation at anaphase, indicating its role in sister chromatid segregation. These findings suggest that Tap46, in conjunction with associated phosphatases, plays an essential role in plant growth and development as a component of the TOR signaling pathway.

Relationship of mRNA expressions of RanBP2 and topoisomerase II isoforms to cytotoxicity of amrubicin in human lung cancer cell lines

RanBP2 is a small ubiquitin-like modifier ligase for DNA topoisomerase II (TopoII) and plays a role in maintaining chromosome stability by recruiting TopoII to centromeres during mitosis. Engineered-mice with low amounts of RanBP2 have been reported to form lung adenocarcinomas. Furthermore, in the murine embryonic fibroblasts, formation of chromatin bridges in anaphase, a distinctive feature of cells with impaired DNA decatenation by chemical inhibition of TopoII, has been reported. In this study, we tested whether the association between mRNA expression of the RanBP2 gene and chemosensitivity of a TopoII inhibitor, amrubicin could be seen. Using a panel of 20 lung cancer cell lines, the mRNA expression levels of the RanBP2, TopoII-alpha and TopoII-beta genes were examined by quantitative real-time reverse transcription PCR. The in vitro cytotoxicity of amrubicin was assessed using a tetrazolium-based colorimetric assay (MTT assay). Although RanBP2 mRNA expression was infrequently downregulated in human lung cancer cell lines, significantly higher RanBP2 transcripts were observed in small cell lung cancer than non-small cell lung cancer. There were no correlations between chemosensitivity of amrubicin and mRNA expression levels of the RanBP2, TopoII-alpha and TopoII-beta genes. Our in vitro results suggest that mRNA expressions of RanBP2 and TopoII isoforms are unlikely to be a predictive biomarker for the sensitivity to amrubicin.