Mitotic Segregation Research Articles

Condensin locates at transcriptional termination sites in mitosis, possibly releasing mitotic transcripts.

Condensin is an essential component of chromosome dynamics, including mitotic chromosome condensation and segregation, DNA repair, and development. Genome-wide localization of condensin is known to correlate with transcriptional activity. The functional relationship between condensin accumulation and transcription sites remains unclear, however. By constructing the auxin-inducible degron strain of condensin, herein we demonstrate that condensin does not affect transcription itself. Instead, RNA processing at transcriptional termination appears to define condensin accumulation sites during mitosis, in the fission yeast Schizosaccharomyces pombe. Combining the auxin-degron strain with the nda3 β-tubulin cold-sensitive (cs) mutant enabled us to inactivate condensin in mitotically arrested cells, without releasing the cells into anaphase. Transcriptional activation and termination were not affected by condensin's degron-mediated depletion, at heat-shock inducible genes or mitotically activated genes. On the other hand, condensin accumulation sites shifted approximately 500 bp downstream in the auxin-degron of 5′-3′ exoribonuclease Dhp1, in which transcripts became aberrantly elongated, suggesting that condensin accumulates at transcriptionally terminated DNA regions. Growth defects in mutant strains of 3′-processing ribonuclease and polyA cleavage factors were additive in condensin temperature-sensitive (ts) mutants. Considering condensin's in vitro activity to form double-stranded DNAs from unwound, single-stranded DNAs or DNA-RNA hybrids, condensin-mediated processing of mitotic transcripts at the 3′-end may be a prerequisite for faithful chromosome segregation.

Viral oncogenesis and genomic instability: the centr(osom)al connection

Currently, more than 10% of human cancers are associated with viral infection. Studies on oncoviruses led to the development of clinical intervention strategies and elucidated fundamental cellular events altered upon cell transformation. Cancer cells exhibit several hallmarks including genomic instability, defined as a high frequency of mutations including gain or loss of chromosomes. The centrosome is an organelle that governs mitotic chromosome segregation and that functions as a signaling platform downstream of the DNA damage response. Here, we review the current literature to highlight how oncoviruses induce genomic instability via the deregulation of the centrosome. Viral interference with the centrosome duplication cycle, leading to centrosome amplification, is illustrated, with a special emphasis on mechanisms shared by several viral families. In addition, we discuss how oncoviruses could alter the signaling functions of the centrosome, and we comment on the bibliographic gaps that could be addressed by future research.

TRAIP drives replisome disassembly and mitotic DNA repair synthesis at sites of incomplete DNA replication.

The faithful segregation of eukaryotic chromosomes in mitosis requires that the genome be duplicated completely prior to anaphase. However, cells with large genomes sometimes fail to complete replication during interphase and instead enter mitosis with regions of incompletely replicated DNA. These regions are processed in early mitosis via a process known as mitotic DNA repair synthesis (MiDAS), but little is known about how cells switch from conventional DNA replication to MiDAS. Using the early embryo of the nematode Caenorhabditis elegans as a model system, we show that the TRAIP ubiquitin ligase drives replisome disassembly in response to incomplete DNA replication, thereby providing access to replication forks for other factors. Moreover, TRAIP is essential for MiDAS in human cells, and is important in both systems to prevent mitotic segregation errors. Our data indicate that TRAIP is a master regulator of the processing of incomplete DNA replication during mitosis in metazoa.

Selective autophagy maintains centrosome integrity and accurate mitosis by turnover of centriolar satellites

The centrosome is the master orchestrator of mitotic spindle formation and chromosome segregation in animal cells. Centrosome abnormalities are frequently observed in cancer, but little is known of their origin and about pathways affecting centrosome homeostasis. Here we show that autophagy preserves centrosome organization and stability through selective turnover of centriolar satellite components, a process we termed doryphagy. Autophagy targets the satellite organizer PCM1 by interacting with GABARAPs via a C-terminal LIR motif. Accordingly, autophagy deficiency results in accumulation of large abnormal centriolar satellites and a resultant dysregulation of centrosome composition. These alterations have critical impact on centrosome stability and lead to mitotic centrosome fragmentation and unbalanced chromosome segregation. Our findings identify doryphagy as an important centrosome-regulating pathway and bring mechanistic insights to the link between autophagy dysfunction and chromosomal instability. In addition, we highlight the vital role of centriolar satellites in maintaining centrosome integrity.

Use of Drosophila S2 Cells for Live Imaging of Cell Division.

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a rapid and high-throughput manner. S2 cells have proven amenable to both fixed- and live-cell imaging applications. Notably, live-cell imaging can yield valuable information about how loss or knockdown of a gene can affect the kinetics and dynamics of key events during cell division, including mitotic spindle assembly, chromosome congression, and segregation, as well as overall cell cycle timing. Here we utilize S2 cells stably transfected with fluorescently tagged mCherry:α-tubulin to mark the mitotic spindle and GFP:CENP-A (referred to as 'CID' gene in Drosophila) to mark the centromere to analyze the effects of key mitotic genes on the timing of cell divisions, from prophase (specifically at Nuclear Envelope Breakdown; NEBD) to the onset of anaphase. This imaging protocol also allows for the visualization of the spindle microtubule and chromosome dynamics throughout mitosis. Herein, we aim to provide a simple yet comprehensive protocol that will allow readers to easily adapt S2 cells for live imaging experiments. Results obtained from such experiments should expand our understanding of genes involved in the cell division by defining their role in several simultaneous and dynamic events. Observations made in this cell culture system can be validated and further investigated in vivo using the impressive toolkit of genetic approaches in flies.

Quantification of the dynamic behaviour of ribosomal DNA genes and nucleolus during yeast Saccharomyces cerevisiae cell cycle

Spatial organisation of chromosomes is a determinant of genome stability and is required for proper mitotic segregation. However, visualization of individual chromatids in living cells and quantification of their geometry, remains technically challenging. Here, we used live cell imaging to quantitate the three-dimensional conformation of yeast Saccharomyces cerevisiae ribosomal DNA (rDNA). rDNA is confined within the nucleolus and is composed of about 200 copies representing about 10% of the yeast genome. To fluorescently label rDNA in living cells, we generated a set of nucleolar proteins fused to GFP or made use of a tagged rDNA, in which lacO repetitions were inserted in each repeat unit. We could show that nucleolus is not modified in appearance, shape or size during interphase while rDNA is highly reorganized. Computationally tracing 3D rDNA paths allowed us to quantitatively assess rDNA size, shape and geometry. During interphase, rDNA was progressively reorganized from a zig-zag segmented line of small size (5,5 µm) to a long, homogeneous, line-like structure of 8,7 µm in metaphase. Most importantly, whatever the cell-cycle stage considered, rDNA fibre could be decomposed in subdomains, as previously suggested for 3D chromatin organisation. Finally, we could determine that spatial reorganisation of these subdomains and establishment of rDNA mitotic organisation is under the control of the cohesin complex.

Chk2-dependent phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) regulates centrosome maturation

ABSTRACT Checkpoint kinase 2 (Chk2) is a pivotal effector kinase in the DNA damage response, with an emerging role in mitotic chromosome segregation. In this study, we show that Chk2 interacts with myosin phosphatase targeting subunit 1 (MYPT1), the targeting subunit of protein phosphatase 1cβ (PP1cβ). Previous studies have shown that MYPT1 is phosphorylated by CDK1 at S473 during mitosis, and subsequently docks to the polo-binding domain of PLK1 and dephosphorylates PLK1. Herein we present data that Chk2 phosphorylates MYPT1 at S507 in vitro and in vivo, which antagonizes pS473. Chk2 inhibition results in failure of γ-tubulin recruitment to the centrosomes, phenocopying Plk1 inhibition defects. These aberrancies were also observed in the MYPT1-S507A stable transfectants, suggesting that Chk2 exerts its effect on centrosomes via MYPT1. Collectively, we have identified a Chk2-MYPT1-PLK1 axis in regulating centrosome maturation. Abbreviations: Chk2: checkpoint kinase 2; MYPT1: myosin phosphatase targeting subunit 1; PP1cβ: protein phosphatase 1c β; Noc: nocodazole; IP: immunoprecipitation; IB: immunoblotting; LC-MS/MS: liquid chromatography-tandem mass spectrometry; Chk2: checkpoint kinase 2; KD: kinase domain; WT: wild type; Ub: ubiquitin; DAPI: 4′,6-diamidino-2-phenylindole; IF: Immunofluorescence; IR: ionizing radiation; siCHK2: siRNA targeting CHK2

Oncogenic MYC amplifies mitotic perturbations

The oncogenic transcription factor MYC modulates vast arrays of genes, thereby influencing numerous biological pathways including biogenesis, metabolism, proliferation, apoptosis and pluripotency. When deregulated, MYC drives genomic instability via several mechanisms including aberrant proliferation, replication stress and ROS production. Deregulated MYC also promotes chromosome instability, but less is known about how MYC influences mitosis. Here, we show that deregulating MYC modulates multiple aspects of mitotic chromosome segregation. Cells overexpressing MYC have altered spindle morphology, take longer to align their chromosomes at metaphase and enter anaphase sooner. When challenged with a variety of anti-mitotic drugs, cells overexpressing MYC display more anomalies, the net effect of which is increased micronuclei, a hallmark of chromosome instability. Proteomic analysis showed that MYC modulates multiple networks predicted to influence mitosis, with the mitotic kinase PLK1 identified as a central hub. In turn, we show that MYC modulates several PLK1-dependent processes, namely mitotic entry, spindle assembly and SAC satisfaction. These observations thus underpin the pervasive nature of oncogenic MYC and provide a mechanistic rationale for MYC's ability to drive chromosome instability.

Organelle Inheritance Control of Mitotic Entry and Progression: Implications for Tissue Homeostasis and Disease.

The Golgi complex (GC), in addition to its well-known role in membrane traffic, is also actively involved in the regulation of mitotic entry and progression. In particular, during the G2 phase of the cell cycle, the Golgi ribbon is unlinked into isolated stacks. Importantly, this ribbon cleavage is required for G2/M transition, indicating that a “Golgi mitotic checkpoint” controls the correct segregation of this organelle. Then, during mitosis, the isolated Golgi stacks are disassembled, and this process is required for spindle formation. Moreover, recent evidence indicates that also proper mitotic segregation of other organelles, such as mitochondria, endosomes, and peroxisomes, is required for correct mitotic progression and/or spindle formation. Collectively, these observations imply that in addition to the control of chromosomes segregation, which is required to preserve the genetic information, the cells actively monitor the disassembly and redistribution of subcellular organelles in mitosis. Here, we provide an overview of the major structural reorganization of the GC and other organelles during G2/M transition and of their regulatory mechanisms, focusing on novel findings that have shed light on the basic processes that link organelle inheritance to mitotic progression and spindle formation, and discussing their implications for tissue homeostasis and diseases.

Electroporated recombinant proteins as tools for in vivo functional complementation, imaging and chemical biology.

Delivery of native or chemically modified recombinant proteins into mammalian cells shows promise for functional investigations and various technological applications, but concerns that sub-cellular localization and functional integrity of delivered proteins may be affected remain high. Here, we surveyed batch electroporation as a delivery tool for single polypeptides and multi-subunit protein assemblies of the kinetochore, a spatially confined and well-studied subcellular structure. After electroporation into human cells, recombinant fluorescent Ndc80 and Mis12 multi-subunit complexes exhibited native localization, physically interacted with endogenous binding partners, and functionally complemented depleted endogenous counterparts to promote mitotic checkpoint signaling and chromosome segregation. Farnesylation is required for kinetochore localization of the Dynein adaptor Spindly. In cells with chronically inhibited farnesyl transferase activity, in vitro farnesylation and electroporation of recombinant Spindly faithfully resulted in robust kinetochore localization. Our data show that electroporation is well-suited to deliver synthetic and chemically modified versions of functional proteins, and, therefore, constitutes a promising tool for applications in chemical and synthetic biology.

Airborne particulate matter induces mitotic slippage and chromosomal missegregation through disruption of the spindle assembly checkpoint (SAC)

Particulate matter (PM) is a risk factor for lung cancer development and chromosomal missegregation and cell cycle disruptions are key cellular events that trigger tumorigenesis. We aimed to study the effect of PM10 (PM with an aerodynamic diameter ≤10 μm) on mitotic arrest and chromosomal segregation, evaluating the spindle assembly checkpoint (SAC) protein dynamics in the human lung A549 adenocarcinoma cell line. For this purpose, synchronized cells were exposed to PM10 for 24 h to obtain the frequency of micronucleated (MN) and trinucleated (TN) cells. Then, the efficiency of the mitotic arrest after PM10 exposure was analyzed. To elucidate the effect of PM10 in chromosomal segregation, the levels and subcellular localization of essential SAC proteins were evaluated. Results indicated that A549 cells exposed to PM10 exhibited an increase in MN and TN cells and a decrease in mitotic indexes and G2/M phase. A549 cells treated with PM10 showed reduced protein levels of MDC1 and NEK2 (38% and 35% respectively), which is required for MAD2 recruitment to kinetochores, MAD2 and BUBR1, effectors of the SAC (25% and 18% respectively), and CYCLIN B1, required during G2/M phase (35%). Besides, PM10 exposure increase the levels of AURORA B and SURVIVIN, required for SAC activation through chromosome-microtubule attachment errors (85% and 74% respectively). We suggest that PM10 causes mitotic slippage due to alterations in MAD2 localization. Thus, PM10 causes inadequate chromosomal segregation and deficient mitotic arrest by altering SAC protein levels, predisposing A549 cells to chromosomal instability, a common feature observed in cancer.

Abstract LB-323: Atrx deletion delays tumor formation and increases radiosensitivity of a primary mouse model of soft tissue sarcoma

Abstract Soft tissue sarcomas are tumors of the connective tissue that account for an estimated 12,000 new cancer cases annually and carry a poor prognosis with a five year survival rate of 50% despite treatment. An important research objective in improving therapy for soft tissue sarcoma patients is to understand how genetic mutations affect soft tissue sarcoma development and radiation response. Intriguingly, next-generation sequencing data from The Cancer Genome Atlas and other massive cancer sequencing efforts have identified Alpha Thalassemia and Mental Retardation X-linked, or ATRX, as the second most frequently mutated gene in soft tissue sarcoma. The vast majority of these ATRX mutations are frameshift mutations. To dissect the role of ATRX in soft tissue sarcoma development and therapeutic response, we generated a primary mouse model of soft tissue sarcoma. This model utilizes Cre-LoxP technology to conditionally activate oncogenic KrasG12D, delete p53, and delete both alleles of Atrx (KPA) in mouse hindlimb Pax7+ muscle satellite cells. When compared to mice with activation of oncogenic KrasG12D, deletion of p53 and at least one wild-type Atrx allele (KP), KPA mice showed a significant delay in time to tumor formation (35 vs. 58 days median), but no significant difference in tumor growth rate as measured by time to tumor quintupling. Next, we generated primary soft tissue sarcoma cell lines from our KP mouse model, and performed CRISPR-Cas9 mediated knockout of Atrx to generate isogenic cell lines. Testing of these isogenic cell lines revealed that Atrx deletion in soft tissue sarcoma cells increased sensitivity to double strand break inducing chemotherapeutics and ionizing radiation, but not to single strand break inducing chemotherapeutics in vitro. In clonogenic assays, Atrx deletion increased radiosensitivity of soft tissue sarcoma cell lines more than fourfold across all radiation doses tested. We next used our KP and KPA models to test the effect of Atrx deletion on radiosensitivity in vivo. Results showed that, relative to the KP model, Atrx deletion improved the response of soft tissue sarcomas to radiation therapy in vivo, increasing the time to tumor quintupling after radiation by 20%. Further analysis demonstrated that Atrx deletion significantly increases mitotic segregation defects after ionizing radiation, suggesting a potential mechanism for the radiosensitivity observed in our studies. Our in vivo study provides the first insight into how ATRX alters the development and therapeutic response of soft tissue sarcoma. Citation Format: Robert W. Floyd, Lixia Luo, David Kirsch. Atrx deletion delays tumor formation and increases radiosensitivity of a primary mouse model of soft tissue sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-323.

Overlapping Roles in Chromosome Segregation for Heterochromatin Protein 1 (Swi6) and DDK in Schizosaccharomyces pombe.

Fission yeast Swi6 is a human HP1 homolog that plays important roles in multiple cellular processes. In addition to its role in maintaining heterochromatin silencing, Swi6 is required for cohesin enrichment at the pericentromere. Loss of Swi6 leads to abnormal mitosis, including defects in the establishment of bioriented sister kinetochores and microtubule attachment. Swi6 interacts with Dfp1, a regulatory subunit of DBF4-dependent kinase (DDK), and failure to recruit Dfp1 to the pericentromere results in late DNA replication. Using the dfp1-3A mutant allele, which specifically disrupts Swi6-Dfp1 association, we investigated how interaction between Swi6 and Dfp1 affects chromosome dynamics. We find that disrupting the interaction between Swi6 and Dfp1 delays mitotic progression in a spindle assembly checkpoint-dependent manner. Artificially tethering Dfp1 back to the pericentromere is sufficient to restore normal spindle length and rescue segregation defects in swi6-deleted cells. However, Swi6 is necessary for centromeric localization of Rad21-GFP independent of DDK. Our data indicate that DDK contributes to mitotic chromosome segregation in pathways that partly overlap with, but can be separated from both, Swi6 and the other HP1 homolog, Chp2.

Centromere mechanical maturation during mammalian cell mitosis

During mitosis, tension develops across the centromere as a result of spindle-based forces. Metaphase tension may be critical in preventing mitotic chromosome segregation errors, however, the nature of force transmission at the centromere and the role of centromere mechanics in controlling metaphase tension remains unknown. We combined quantitative, biophysical microscopy with computational analysis to elucidate the mechanics of the centromere in unperturbed, mitotic human cells. We discovered that the mechanical stiffness of the human centromere matures during mitotic progression, which leads to amplified centromere tension specifically at metaphase. Centromere mechanical maturation is disrupted across multiple aneuploid cell lines, leading to a weak metaphase tension signal. Further, increasing deficiencies in centromere mechanical maturation are correlated with rising frequencies of lagging, merotelic chromosomes in anaphase, leading to segregation defects at telophase. Thus, we reveal a centromere maturation process that may be critical to the fidelity of chromosome segregation during mitosis.

The relationship between chromosomal mosaicism and maternal age in embryos TESTED WITH Next Generation Sequencing (NGS) platform

Introduction Chromosomal mosaicism refers to the presence of two or more cell lines with a differentchromosome content in an individual or tissue sample. Besides aneuploidy, the use of highresolution NGS for Pre-implantation Genetic Diagnosis (PGS) detects the presence of mosaicism introphectoderm biopsies. The transfer of mosaic embryos has been shown to result in lowerimplantation rates and higher miscarriage rates as compared to euploid transfer. This study aims to evaluate whether there is an association between mosaicism and maternal ageand the clinical pregnancy and implantation rate of mosaic embryos transfer. Materials and Method A retrospective observational study of 533 blastocyst-stage embryos analysed by NGS from IVFcycles from Sunfert International Fertility Centre over the course of 2015 to 2016 was performed. Ofthose analysed, 197 blastocysts were transferred. The clinical outcome obtained after the transfer ofwhole chromosomal mosaic embryos was compared with those resulting from a control group 190euploid blastocysts. Blastocyst-stage embryos underwent biopsy and trophectoderm cells were extracted for aneuploidyscreening using IIlumina NGS protocol. Mosaic information for each chromosome was obtained. Forthis study, only whole chromosomal mosaicism was considered; segmental mosaicism wascategorized as euploid embryos. These embryos were further stratified by maternal age group. Result The transfer of mosaic embryos resulted in higher clinical pregnancy rate (60% vs 57.5%) andimplantation rate (57% vs 52.9%) compared to euploid embryos. In our series, no miscarriage wasreported in this group of patients. Conclusion In concordance with most published studies, we found that mosaicism is not associated withadvanced maternal age. Unlike aneuploidy which arises from meiotic error, mosaicism on the otherhand is caused by impaired mitotic chromosome segregation which does not appear to increase withadvancing maternal age. Therefore, while there is a direct relationship between advanced maternalage and aneuploidy, age is not a contributor to mosaicism. Contrary to the experience of other groups, we have observed similar rate of implantation in mosaicembryo transfers as compared to euploid embryo transfers. This may be the consequence of thesmall sample size of mosaic embryos transfer (<10% of total blastocysts transferred) in our series. Similarly, no miscarriage was reported. PGS in its current form does have limitations. Implantation rates can potentially be further improvedby developing a clearer understanding of the impact of mosaic embryos. Chromosomal mosaicism refers to the presence of two or more cell lines with a differentchromosome content in an individual or tissue sample. Besides aneuploidy, the use of highresolution NGS for Pre-implantation Genetic Diagnosis (PGS) detects the presence of mosaicism introphectoderm biopsies. The transfer of mosaic embryos has been shown to result in lowerimplantation rates and higher miscarriage rates as compared to euploid transfer. This study aims to evaluate whether there is an association between mosaicism and maternal ageand the clinical pregnancy and implantation rate of mosaic embryos transfer. A retrospective observational study of 533 blastocyst-stage embryos analysed by NGS from IVFcycles from Sunfert International Fertility Centre over the course of 2015 to 2016 was performed. Ofthose analysed, 197 blastocysts were transferred. The clinical outcome obtained after the transfer ofwhole chromosomal mosaic embryos was compared with those resulting from a control group 190euploid blastocysts. Blastocyst-stage embryos underwent biopsy and trophectoderm cells were extracted for aneuploidyscreening using IIlumina NGS protocol. Mosaic information for each chromosome was obtained. Forthis study, only whole chromosomal mosaicism was considered; segmental mosaicism wascategorized as euploid embryos. These embryos were further stratified by maternal age group. The transfer of mosaic embryos resulted in higher clinical pregnancy rate (60% vs 57.5%) andimplantation rate (57% vs 52.9%) compared to euploid embryos. In our series, no miscarriage wasreported in this group of patients. In concordance with most published studies, we found that mosaicism is not associated withadvanced maternal age. Unlike aneuploidy which arises from meiotic error, mosaicism on the otherhand is caused by impaired mitotic chromosome segregation which does not appear to increase withadvancing maternal age. Therefore, while there is a direct relationship between advanced maternalage and aneuploidy, age is not a contributor to mosaicism.

Clinical outcome: the relationship between mosaicism and advanced maternal age with the use of Next Generation Sequencing (NGS)

Introduction Chromosomal mosaicism refers to the presence of two or more cell lines with a different chromosome content in an individual or tissue sample. The implementation of high resolution NGS for PGS has provided more information that it is not only used for embryonic aneuploidy screening, butdetects the presence of mosaicism in trophectoderm biopsies. Recent studies have shown that transfer of mosaic embryos resulted in lower implantation rates and higher miscarriage rates as compared to euploid transfer. This study aims to evaluate whether there is an association between chromosomal mosaicism and advanced maternal age. It also aims to compare the clinical pregnancy and implantation rate between euploid and whole chromosomal mosaic embryos transfer. Materials and Method Retrospective observational study of 533 blastocyst-stage embryos analysed by NGS from IVF cycles from Sunfert International Fertility Centre over the course of 2015 to 2016. Of those analysed, 197 blastocysts were transferred. The clinical outcome obtained after transfer of whole chromosomal mosaic embryos was compared with those resulting from a control group of 190 euploid blastocysts. Blastocyst-stage embryos underwent biopsy and trophectoderm cells are extracted for aneuploidy screening using IIlumina NGS protocol. Mosaic information for each chromosome was obtained. For this study, only whole chromosomal mosaicism is considered; segmental mosaicism were categorized under euploid embryos. These embryos were further stratified by maternal age group: 40 yo. Result Results are summarized in Figure 1. The result showed that mosaicism rates did not vary with advancing maternal age, staying at an average of 10.14%. Euploid and aneuploidy embryos, however, did show changes in regards to age factor as the former displayed a descending trend whereas the latter displayed an increasing trend. Download : Download high-res image (189KB) Download : Download full-size image Figure 1. Percentages of euploid, aneuploid and whole chromosomal mosaic embryos by age groups. Transfer of mosaic embryos resulted in higher clinical pregnancy rate (60% vs 57.5%) and implantation rate (57% vs 52.9%) than euploid embryos. No incidence of miscarriage was reported. Conclusion In accordance to most of the studies published, our findings showed that mosaicism is not associated with advanced maternal age. Unlike aneuploidy which arises from meiotic error, mosaicism, on the other hand is caused by impaired mitotic chromosome segregation which does not appear to increase with advancing maternal age. Therefore, while there is a direct relationship between advanced maternal age and aneuploidy, age is not a contributor to mosaicism. On the contrary, our findings showed conflicting result in the implantation rate of mosaic embryos. Similar rate of implantation as compared to euploid transfer embryos was found in our clinical study whereas in general, transfer of mosaic embryos have lower implantation rate. This might be the consequence of small sample size of mosaic embryos transfer (

The meiotic phosphatase GSP-2/PP1 promotes germline immortality and small RNA-mediated genome silencing.

Germ cell immortality, or transgenerational maintenance of the germ line, could be promoted by mechanisms that could occur in either mitotic or meiotic germ cells. Here we report for the first time that the GSP-2 PP1/Glc7 phosphatase promotes germ cell immortality. Small RNA-induced genome silencing is known to promote germ cell immortality, and we identified a separation-of-function allele of C. elegans gsp-2 that is compromised for germ cell immortality and is also defective for small RNA-induced genome silencing and meiotic but not mitotic chromosome segregation. Previous work has shown that GSP-2 is recruited to meiotic chromosomes by LAB-1, which also promoted germ cell immortality. At the generation of sterility, gsp-2 and lab-1 mutant adults displayed germline degeneration, univalents, histone methylation and histone phosphorylation defects in oocytes, phenotypes that mirror those observed in sterile small RNA-mediated genome silencing mutants. Our data suggest that a meiosis-specific function of GSP-2 ties small RNA-mediated silencing of the epigenome to germ cell immortality. We also show that transgenerational epigenomic silencing at hemizygous genetic elements requires the GSP-2 phosphatase, suggesting a functional link to small RNAs. Given that LAB-1 localizes to the interface between homologous chromosomes during pachytene, we hypothesize that small localized discontinuities at this interface could promote genomic silencing in a manner that depends on small RNAs and the GSP-2 phosphatase.

Structure-Specific Endonucleases and the Resolution of Chromosome Underreplication.

Complete genome duplication in every cell cycle is fundamental for genome stability and cell survival. However, chromosome replication is frequently challenged by obstacles that impede DNA replication fork (RF) progression, which subsequently causes replication stress (RS). Cells have evolved pathways of RF protection and restart that mitigate the consequences of RS and promote the completion of DNA synthesis prior to mitotic chromosome segregation. If there is entry into mitosis with underreplicated chromosomes, this results in sister-chromatid entanglements, chromosome breakage and rearrangements and aneuploidy in daughter cells. Here, we focus on the resolution of persistent replication intermediates by the structure-specific endonucleases (SSEs) MUS81, SLX1-SLX4 and GEN1. Their actions and a recently discovered pathway of mitotic DNA repair synthesis have emerged as important facilitators of replication completion and sister chromatid detachment in mitosis. As RS is induced by oncogene activation and is a common feature of cancer cells, any advances in our understanding of the molecular mechanisms related to chromosome underreplication have important biomedical implications.

Abstract P3-09-01: MYC dysregulates mitotic spindle function in triple-negative breast cancer creating a dependency on TPX2

Abstract Tumors that overexpress the MYC oncogene, including most receptor triple-negative breast cancers, frequently demonstrate aneuploidy, numerical chromosome alterations associated with highly aggressive cancers. Aneuploidy is also associated with rapid tumor evolution and poor patient outcome. We identify that MYC overexpression induces reversible defects in microtubule nucleation and mitotic spindle assembly, in TNBCs and other epithelial cells, promoting chromosome segregation defects, micronuclei and chromosomal instability (CIN). High TPX2 expression is permissive for mitotic spindle assembly and chromosome segregation in cells with MYC overexpression; whereas TPX2 depletion blocks mitotic progression, induces cell death and prevents tumor growth. Attenuating MYC expression reverses mitotic defects, even in established breast tumor cell lines, implicating an ongoing role for high MYC in the persistence of CIN in cancers. Our studies implicate the MYC oncogene as a regulator of spindle assembly and identify a new MYC-TPX2 synthetic-lethal interaction in TNBC that could represent a future therapeutic strategy in MYC-overexpressing cancers. Moreover, our studies suggest that blocking MYC activity can attenuate the emergence of CIN and tumor evolution. Citation Format: Goga A, Rohrberg J, Corella A, Taileb M, Kilinc S, Jokisch M-L, Camarda R, Zhou A, Balakrishnan S, Chang AN, Klein-Connolly H. MYC dysregulates mitotic spindle function in triple-negative breast cancer creating a dependency on TPX2 [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-09-01.

CDK1-mediated phosphorylation at H2B serine 6 is required for mitotic chromosome segregation.

Faithful mitotic chromosome segregation is required for the maintenance of genomic stability. We discovered the phosphorylation of histone H2B at serine 6 (H2B S6ph) as a new chromatin modification site and found that this modification occurs during the early mitotic phases at inner centromeres and pericentromeric heterochromatin. This modification is directly mediated by cyclin B1-associated CDK1, and indirectly by Aurora B, and is antagonized by PP1-mediated dephosphorylation. H2B S6ph impairs chromatin binding of the histone chaperone SET (I2PP2A), which is important for mitotic fidelity. Injection of phosphorylation-specific H2B S6 antibodies in mitotic cells caused anaphase defects with impaired chromosome segregation and incomplete cytokinesis. As H2B S6ph is important for faithful chromosome separation, this modification may contribute to the prevention chromosomal instability and aneuploidy which frequently occur in cancer cells.