RESUMO
Understanding the basis for cellular growth, proliferation, and function requires determining the roles of essential genes in diverse cellular processes, including visualizing their contributions to cellular organization and morphology. Here, we combined pooled CRISPR-Cas9-based functional screening of 5,072 fitness-conferring genes in human HeLa cells with microscopy-based imaging of DNA, the DNA damage response, actin, and microtubules. Analysis of >31 million individual cells identified measurable phenotypes for >90% of gene knockouts, implicating gene targets in specific cellular processes. Clustering of phenotypic similarities based on hundreds of quantitative parameters further revealed co-functional genes across diverse cellular activities, providing predictions for gene functions and associations. By conducting pooled live-cell screening of â¼450,000 cell division events for 239 genes, we additionally identified diverse genes with functional contributions to chromosome segregation. Our work establishes a resource detailing the consequences of disrupting core cellular processes that represents the functional landscape of essential human genes.
Assuntos
Sistemas CRISPR-Cas , Genes Essenciais , Humanos , Células HeLa , Técnicas de Inativação de Genes , FenótipoRESUMO
To ensure the stable transmission of the genome during vertebrate cell division, the mitotic spindle must attach to a single locus on each chromosome, termed the centromere. The fundamental requirement for faithful centromere inheritance is the controlled deposition of the centromere-specifying histone, CENP-A. However, the regulatory mechanisms that ensure the precise control of CENP-A deposition have proven elusive. Here, we identify polo-like kinase 1 (Plk1) as a centromere-localized regulator required to initiate CENP-A deposition in human cells. We demonstrate that faithful CENP-A deposition requires integrated signals from Plk1 and cyclin-dependent kinase (CDK), with Plk1 promoting the localization of the key CENP-A deposition factor, the Mis18 complex, and CDK inhibiting Mis18 complex assembly. By bypassing these regulated steps, we uncoupled CENP-A deposition from cell-cycle progression, resulting in mitotic defects. Thus, CENP-A deposition is controlled by a two-step regulatory paradigm comprised of Plk1 and CDK that is crucial for genomic integrity.
Assuntos
Autoantígenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ciclo Celular , Linhagem Celular , Proteína Centromérica A , Quinases Ciclina-Dependentes/metabolismo , Instabilidade Genômica , Células HeLa , Humanos , Quinase 1 Polo-LikeRESUMO
Mitotic defects activate the spindle-assembly checkpoint, which inhibits the anaphase-promoting complex co-activator CDC20 to induce a prolonged cell cycle arrest1,2. Once errors are corrected, the spindle-assembly checkpoint is silenced, allowing anaphase onset to occur. However, in the presence of persistent unresolvable errors, cells can undergo 'mitotic slippage', exiting mitosis into a tetraploid G1 state and escaping the cell death that results from a prolonged arrest. The molecular logic that enables cells to balance these duelling mitotic arrest and slippage behaviours remains unclear. Here we demonstrate that human cells modulate the duration of their mitotic arrest through the presence of conserved, alternative CDC20 translational isoforms. Downstream translation initiation results in a truncated CDC20 isoform that is resistant to spindle-assembly-checkpoint-mediated inhibition and promotes mitotic exit even in the presence of mitotic perturbations. Our study supports a model in which the relative levels of CDC20 translational isoforms control the duration of mitotic arrest. During a prolonged mitotic arrest, new protein synthesis and differential CDC20 isoform turnover create a timer, with mitotic exit occurring once the truncated Met43 isoform achieves sufficient levels. Targeted molecular changes or naturally occurring cancer mutations that alter CDC20 isoform ratios or its translational control modulate mitotic arrest duration and anti-mitotic drug sensitivity, with potential implications for the diagnosis and treatment of human cancers.
Assuntos
Proteínas Cdc20 , Pontos de Checagem da Fase M do Ciclo Celular , Biossíntese de Proteínas , Humanos , Proteínas Cdc20/química , Proteínas Cdc20/genética , Proteínas Cdc20/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fuso Acromático/metabolismo , Iniciação Traducional da Cadeia PeptídicaRESUMO
The centromere is the region of the chromosome that directs its segregation in mitosis and meiosis. Although the functional importance of the centromere has been appreciated for more than 130 years, elucidating the molecular features and properties that enable centromeres to orchestrate chromosome segregation is an ongoing challenge. Most eukaryotic centromeres are defined epigenetically and require the presence of nucleosomes containing the histone H3 variant centromere protein A (CENP-A; also known as CENH3). Ongoing work is providing important molecular insights into the central requirements for centromere identity and propagation, and the mechanisms by which centromeres recruit kinetochores to connect to spindle microtubules.
Assuntos
Centrômero/metabolismo , Animais , DNA/química , DNA/metabolismo , Epigênese Genética , Humanos , Cinetocoros/metabolismo , Modelos BiológicosRESUMO
Mitotic spindle position defines the cell-cleavage site during cytokinesis. However, the mechanisms that control spindle positioning to generate equal-sized daughter cells remain poorly understood. Here, we demonstrate that two mechanisms act coordinately to center the spindle during anaphase in symmetrically dividing human cells. First, the spindle is positioned directly by the microtubule-based motor dynein, which we demonstrate is targeted to the cell cortex by two distinct pathways: a Gαi/LGN/NuMA-dependent pathway and a 4.1G/R and NuMA-dependent, anaphase-specific pathway. Second, we find that asymmetric plasma membrane elongation occurs in response to spindle mispositioning to alter the cellular boundaries relative to the spindle. Asymmetric membrane elongation is promoted by chromosome-derived Ran-GTP signals that locally reduce Anillin at the growing cell cortex. In asymmetrically elongating cells, dynein-dependent spindle anchoring at the stationary cell cortex ensures proper spindle positioning. Our results reveal the anaphase-specific spindle centering systems that achieve equal-sized cell division.
Assuntos
Anáfase , Membrana Celular/metabolismo , Dineínas/metabolismo , Fuso Acromático/metabolismo , Sequência de Aminoácidos , Animais , Antígenos Nucleares/metabolismo , Proteínas de Ciclo Celular , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Complexo Dinactina , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Dados de Sequência Molecular , Proteínas Associadas à Matriz Nuclear/metabolismo , Alinhamento de SequênciaRESUMO
As cells enter mitosis, the genome is restructured to facilitate chromosome segregation, accompanied by dramatic changes in gene expression. However, the mechanisms that underlie mitotic transcriptional regulation are unclear. In contrast to transcribed genes, centromere regions retain transcriptionally active RNA polymerase II (Pol II) in mitosis. Here, we demonstrate that chromatin-bound cohesin is necessary to retain elongating Pol II at centromeres. We find that WAPL-mediated removal of cohesin from chromosome arms during prophase is required for the dissociation of Pol II and nascent transcripts, and failure of this process dramatically alters mitotic gene expression. Removal of cohesin/Pol II from chromosome arms in prophase is important for accurate chromosome segregation and normal activation of gene expression in G1. We propose that prophase cohesin removal is a key step in reprogramming gene expression as cells transition from G2 through mitosis to G1.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas Cromossômicas não Histona/fisiologia , Regulação da Expressão Gênica , Mitose/genética , Transcrição Gênica , Anáfase/genética , Animais , Aurora Quinase B/análise , Ciclo Celular , Proteínas de Ciclo Celular/análise , Linhagem Celular , Centrômero/enzimologia , Segregação de Cromossomos , Fase G1/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Humanos , Metáfase/genética , Prófase , RNA Polimerase II/metabolismo , Xenopus laevis , CoesinasRESUMO
The multiprotein kinetochore complex must assemble at a specific site on each chromosome to achieve accurate chromosome segregation. Defining the nature of the DNA-protein interactions that specify the position of the kinetochore and provide a scaffold for kinetochore formation remain key goals. Here, we demonstrate that the centromeric histone-fold-containing CENP-T-W and CENP-S-X complexes coassemble to form a stable CENP-T-W-S-X heterotetramer. High-resolution structural analysis of the individual complexes and the heterotetramer reveals similarity to other histone fold-containing complexes including canonical histones within a nucleosome. The CENP-T-W-S-X heterotetramer binds to and supercoils DNA. Mutants designed to compromise heterotetramerization or the DNA-protein contacts around the heterotetramer strongly reduce the DNA binding and supercoiling activities in vitro and compromise kinetochore assembly in vivo. These data suggest that the CENP-T-W-S-X complex forms a unique nucleosome-like structure to generate contacts with DNA, extending the "histone code" beyond canonical nucleosome proteins.
Assuntos
Centrômero/química , Centrômero/metabolismo , Galinhas/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Sequência de Aminoácidos , Animais , Cromatina/química , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Histonas/metabolismo , Humanos , Cinetocoros/química , Cinetocoros/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , Difração de Raios XRESUMO
Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. Although prior work identified the centromeric histone H3-variant CENP-A as the important upstream factor necessary for centromere specification, in human cells CENP-A is not sufficient for kinetochore assembly. Here, we demonstrate that two constitutive DNA-binding kinetochore components, CENP-C and CENP-T, function to direct kinetochore formation. Replacing the DNA-binding regions of CENP-C and CENP-T with alternate chromosome-targeting domains recruits these proteins to ectopic loci, resulting in CENP-A-independent kinetochore assembly. These ectopic kinetochore-like foci are functional based on the stoichiometric assembly of multiple kinetochore components, including the microtubule-binding KMN network, the presence of microtubule attachments, the microtubule-sensitive recruitment of the spindle checkpoint protein Mad2, and the segregation behavior of foci-containing chromosomes. We additionally find that CENP-T phosphorylation regulates the mitotic assembly of both endogenous and ectopic kinetochores. Thus, CENP-C and CENP-T form a critical regulated platform for vertebrate kinetochore assembly.
Assuntos
Autoantígenos/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Cinetocoros/metabolismo , Nucleossomos/metabolismo , Vertebrados/metabolismo , Sequência de Aminoácidos , Animais , Proteína Centromérica A , Galinhas , Células HeLa , Humanos , Mitose , Dados de Sequência Molecular , FosforilaçãoRESUMO
The kinetochore plays an essential role in facilitating chromosome segregation during cell division. This massive protein complex assembles onto the centromere of chromosomes and enables their attachment to spindle microtubules during mitosis. The kinetochore also functions as a signaling hub to regulate cell cycle progression, and is crucial to ensuring the fidelity of chromosome segregation. Despite the fact that kinetochores are large and robust molecular assemblies, they are also highly dynamic structures that undergo structural and organizational changes throughout the cell cycle. This review will highlight our current understanding of kinetochore structure and function, focusing on the dynamic processes that underlie kinetochore assembly.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Fuso Acromático/metabolismo , HumanosRESUMO
Kinetochore specification and assembly requires the targeted deposition of specialized nucleosomes containing the histone H3 variant CENP-A at centromeres. However, CENP-A is not sufficient to drive full-kinetochore assembly, and it is not clear how centromeric chromatin is established. Here, we identify CENP-W as a component of the DNA-proximal constitutive centromere-associated network (CCAN) of proteins. We demonstrate that CENP-W forms a DNA-binding complex together with the CCAN component CENP-T. This complex directly associates with nucleosomal DNA and with canonical histone H3, but not with CENP-A, in centromeric regions. CENP-T/CENP-W functions upstream of other CCAN components with the exception of CENP-C, an additional putative DNA-binding protein. Our analysis indicates that CENP-T/CENP-W and CENP-C provide distinct pathways to connect the centromere with outer kinetochore assembly. In total, our results suggest that the CENP-T/CENP-W complex is directly involved in establishment of centromere chromatin structure coordinately with CENP-A.
Assuntos
Centrômero , Proteínas Cromossômicas não Histona/metabolismo , DNA/metabolismo , Cinetocoros/metabolismo , Sequência de Aminoácidos , Animais , Autoantígenos/genética , Autoantígenos/metabolismo , Proteína Centromérica A , Galinhas , Proteínas Cromossômicas não Histona/genética , Células HeLa , Histonas/metabolismo , Humanos , Mutação , Nucleossomos/metabolismoRESUMO
Kinetochores of mitotic chromosomes are coupled to spindle microtubules in ways that allow the energy from tubulin dynamics to drive chromosome motion. Most kinetochore-associated microtubule ends display curving "protofilaments," strands of tubulin dimers that bend away from the microtubule axis. Both a kinetochore "plate" and an encircling, ring-shaped protein complex have been proposed to link protofilament bending to poleward chromosome motion. Here we show by electron tomography that slender fibrils connect curved protofilaments directly to the inner kinetochore. Fibril-protofilament associations correlate with a local straightening of the flared protofilaments. Theoretical analysis reveals that protofilament-fibril connections would be efficient couplers for chromosome motion, and experimental work on two very different kinetochore components suggests that filamentous proteins can couple shortening microtubules to cargo movements. These analyses define a ring-independent mechanism for harnessing microtubule dynamics directly to chromosome movement.
Assuntos
Cromossomos/metabolismo , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Animais , Linhagem Celular , Cromossomos/ultraestrutura , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Cinetocoros/ultraestrutura , Microtúbulos/ultraestrutura , Potoroidae , Fuso Acromático/metabolismo , Fuso Acromático/ultraestrutura , Tubulina (Proteína)/metabolismoRESUMO
During mitosis, the macromolecular kinetochore complex assembles on the centromere to orchestrate chromosome segregation. The properties and architecture of the 16-subunit Constitutive Centromere-Associated Network (CCAN) that allow it to build a robust platform for kinetochore assembly are poorly understood. Here, we use inducible CRISPR knockouts and biochemical reconstitutions to define the interactions between the human CCAN proteins. We find that the CCAN does not assemble as a linear hierarchy, and instead, each sub-complex requires multiple non-redundant interactions for its localization to centromeres and the structural integrity of the overall assembly. We demonstrate that the CENP-L-N complex plays a crucial role at the core of this assembly through interactions with CENP-C and CENP-H-I-K-M. Finally, we show that the CCAN is remodeled over the cell cycle such that sub-complexes depend on their interactions differentially. Thus, an interdependent meshwork within the CCAN underlies the centromere specificity and stability of the kinetochore.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cinetocoros/metabolismo , Sistemas CRISPR-Cas , Ciclo Celular , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Células HeLa , Humanos , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismoRESUMO
Accurate chromosome segregation critically depends on the formation of attachments between microtubule polymers and each sister chromatid. The kinetochore is the macromolecular complex that assembles at the centromere of each chromosome during mitosis and serves as the link between the DNA and the microtubules. In this Cell Science at a Glance article and accompanying poster, we discuss the activities and molecular players that are involved in generating kinetochore-microtubule attachments, including the initial stages of lateral kinetochore-microtubule interactions and maturation to stabilized end-on attachments. We additionally explore the features that contribute to the ability of the kinetochore to track with dynamic microtubules. Finally, we examine the contributions of microtubule-associated proteins to the organization and stabilization of the mitotic spindle and the control of microtubule dynamics.
Assuntos
Cinetocoros/fisiologia , Microtúbulos/fisiologia , Animais , Centrômero/metabolismo , Centrômero/fisiologia , Segregação de Cromossomos/fisiologia , Humanos , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/metabolismo , Mitose/fisiologia , Fuso Acromático/metabolismoRESUMO
Segregation of the replicated genome during cell division in eukaryotes requires the kinetochore to link centromeric DNA to spindle microtubules. The kinetochore is composed of a number of conserved protein complexes that direct its specification and assembly, bind to spindle microtubules and regulate chromosome segregation. Recent studies have identified more than 80 kinetochore components, and are revealing how these proteins are organized into the higher order kinetochore structure, as well as how they function to achieve proper chromosome segregation.
Assuntos
Cinetocoros/fisiologia , Cinetocoros/ultraestrutura , Microtúbulos/fisiologia , Microtúbulos/ultraestrutura , Animais , Ciclo Celular/fisiologia , Centrômero/fisiologia , Centrômero/ultraestrutura , Cromossomos/fisiologia , HumanosRESUMO
Live-cell imaging and particle tracking provide rich information on mechanisms of intracellular transport. However, trajectory analysis procedures to infer complex transport dynamics involving stochastic switching between active transport and diffusive motion are lacking. We applied Bayesian model selection to hidden Markov modeling to infer transient transport states from trajectories of mRNA-protein complexes in live mouse hippocampal neurons and metaphase kinetochores in dividing human cells. The software is available at http://hmm-bayes.org/.
Assuntos
Actinas/metabolismo , Hipocampo/metabolismo , Modelos Biológicos , Imagem Molecular/métodos , Neurônios/citologia , Neurônios/metabolismo , Animais , Teorema de Bayes , Células Cultivadas , Simulação por Computador , Feminino , Células HeLa , Hipocampo/citologia , Humanos , Cadeias de Markov , Camundongos , MicroRNAs/metabolismo , Microscopia de Fluorescência/métodos , Modelos Estatísticos , Reconhecimento Automatizado de Padrão/métodos , Transporte Proteico/fisiologia , SoftwareRESUMO
Accurate chromosome segregation requires carefully regulated interactions between kinetochores and microtubules, but how plasticity is achieved to correct diverse attachment defects remains unclear. Here we demonstrate that Aurora B kinase phosphorylates three spatially distinct targets within the conserved outer kinetochore KNL1/Mis12 complex/Ndc80 complex (KMN) network, the key player in kinetochore-microtubule attachments. The combinatorial phosphorylation of the KMN network generates graded levels of microtubule-binding activity, with full phosphorylation severely compromising microtubule binding. Altering the phosphorylation state of each protein causes corresponding chromosome segregation defects. Importantly, the spatial distribution of these targets along the kinetochore axis leads to their differential phosphorylation in response to changes in tension and attachment state. In total, rather than generating exclusively binary changes in microtubule binding, our results suggest a mechanism for the tension-dependent fine-tuning of kinetochore-microtubule interactions.
Assuntos
Segregação de Cromossomos , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Aurora Quinase B , Aurora Quinases , Técnicas Biossensoriais , Proteínas de Caenorhabditis elegans/metabolismo , Galinhas , Segregação de Cromossomos/efeitos dos fármacos , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Cinetocoros/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/efeitos dos fármacos , Mutação , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Transdução Genética , Moduladores de Tubulina/farmacologiaRESUMO
The kinetochore forms a dynamic interface with microtubules from the mitotic spindle during mitosis. The Ndc80 complex acts as the key microtubule-binding complex at kinetochores. However, it is unclear how the Ndc80 complex associates with the inner kinetochore proteins that assemble upon centromeric chromatin. Here, based on a high-resolution structural analysis, we demonstrate that the N-terminal region of vertebrate CENP-T interacts with the 'RWD' domain in the Spc24/25 portion of the Ndc80 complex. Phosphorylation of CENP-T strengthens a cryptic hydrophobic interaction between CENP-T and Spc25 resulting in a phospho-regulated interaction that occurs without direct recognition of the phosphorylated residue. The Ndc80 complex interacts with both CENP-T and the Mis12 complex, but we find that these interactions are mutually exclusive, supporting a model in which two distinct pathways target the Ndc80 complex to kinetochores. Our results provide a model for how the multiple protein complexes at kinetochores associate in a phospho-regulated manner.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Animais , Calorimetria , Linhagem Celular Tumoral , Galinhas , Cromatografia em Gel , Proteínas Cromossômicas não Histona/química , Cristalização , Proteínas do Citoesqueleto , Humanos , Cinetocoros/química , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/química , Complexos Multiproteicos/química , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fosforilação , Especificidade da EspécieRESUMO
Successful execution of the meiotic program depends on the timely establishment and removal of sister chromatid cohesion. LAB-1 has been proposed to act in the latter by preventing the premature removal of the meiosis-specific cohesin REC-8 at metaphase I in C. elegans, yet the mechanism and scope of LAB-1 function remained unknown. Here we identify an unexpected earlier role for LAB-1 in promoting the establishment of sister chromatid cohesion in prophase I. LAB-1 and REC-8 are both required for the chromosomal association of the cohesin complex subunit SMC-3. Depletion of lab-1 results in partial loss of sister chromatid cohesion in rec-8 and coh-4 coh-3 mutants and further enhanced chromatid dissociation in worms where all three kleisins are mutated. Moreover, lab-1 depletion results in increased Aurora B kinase (AIR-2) signals in early prophase I nuclei, coupled with a parallel decrease in signals for the PP1 homolog, GSP-2. Finally, LAB-1 directly interacts with GSP-1 and GSP-2. We propose that LAB-1 targets the PP1 homologs to the chromatin at the onset of meiosis I, thereby antagonizing AIR-2 and cooperating with the cohesin complex to promote sister chromatid association and normal progression of the meiotic program.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Cromátides/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Prófase Meiótica I , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Aurora Quinase B , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromátides/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Troca Genética , Reparo do DNA , Ligação Proteica , Mapeamento de Interação de Proteínas/métodos , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Complexo Sinaptonêmico/genética , Complexo Sinaptonêmico/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Chromosomal rearrangements can radically alter gene products and their function, driving tumor formation or progression. However, the molecular origins and evolution of such rearrangements are varied and poorly understood, with cancer cells often containing multiple, complex rearrangements. One mechanism that can lead to genomic rearrangements is the formation of a "dicentric" chromosome containing two functional centromeres. Indeed, such dicentric chromosomes have been observed in cancer cells. Here, we tested the ability of a single dicentric chromosome to contribute to genomic instability and neoplastic conversion in vertebrate cells. We developed a system to transiently and reversibly induce dicentric chromosome formation on a single chromosome with high temporal control. We find that induced dicentric chromosomes are frequently damaged and mis-segregated during mitosis, and that this leads to extensive chromosomal rearrangements including translocations with other chromosomes. Populations of pre-neoplastic cells in which a single dicentric chromosome is induced acquire extensive genomic instability and display hallmarks of cellular transformation including anchorage-independent growth in soft agar. Our results suggest that a single dicentric chromosome could contribute to tumor initiation.
Assuntos
Carcinogênese/genética , Aberrações Cromossômicas , Rearranjo Gênico , Genômica/métodos , Animais , Linhagem Celular Tumoral , Centrômero/genética , Imunofluorescência , Instabilidade Genômica , Hibridização in Situ Fluorescente , Camundongos , Mitose/genética , Translocação GenéticaRESUMO
Alternative mRNA splicing can generate distinct protein isoforms to allow for the differential control of cell processes across cell types. However, alternative splice isoforms that differentially modulate distinct cell division programs have remained elusive. Here, we demonstrate that mammalian germ cells express an alternate mRNA splice isoform for the kinetochore component, DSN1, a subunit of the MIS12 complex that links the centromeres to spindle microtubules during chromosome segregation. This germline DSN1 isoform bypasses the requirement for Aurora kinase phosphorylation for its centromere localization due to the absence of a key regulatory region allowing DSN1 to display persistent centromere localization. Expression of the germline DSN1 isoform in somatic cells results in constitutive kinetochore localization, chromosome segregation errors, and growth defects, providing an explanation for its tight cell type-specific expression. Reciprocally, precisely eliminating expression of the germline DSN1 splice isoform in mouse models disrupts oocyte maturation and early embryonic divisions coupled with a reduction in fertility. Together, this work identifies a germline-specific splice isoform for a chromosome segregation component and implicates its role in mammalian fertility.