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Regulated start-codon selection has the potential to reshape the proteome through the differential production of uORFs, canonical proteins, and alternative translational isoforms. However, conditions under which start-codon selection is altered remain poorly defined. Here, using transcriptome-wide translation initiation site profiling, we reveal a global increase in the stringency of start-codon selection during mammalian mitosis. Low-efficiency initiation sites are preferentially repressed in mitosis, resulting in pervasive changes in the translation of thousands of start sites and their corresponding protein products. This increased stringency of start-codon selection during mitosis results from increased interactions between the key regulator of start-codon selection, eIF1, and the 40S ribosome. We find that increased eIF1-40S ribosome interactions during mitosis are mediated by the release of a nuclear pool of eIF1 upon nuclear envelope breakdown. Selectively depleting the nuclear pool of eIF1 eliminates the changes to translational stringency during mitosis, resulting in altered mitotic proteome composition. In addition, preventing mitotic translational rewiring results in substantially increased cell death and decreased mitotic slippage following treatment with anti-mitotic chemotherapeutics. Thus, cells globally control translation initiation stringency with critical roles during the mammalian cell cycle to preserve mitotic cell physiology.
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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.
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Microtubules play essential roles in diverse cellular processes and are important pharmacological targets for treating human disease. Here, we sought to identify cellular factors that modulate the sensitivity of cells to anti-microtubule drugs. We conducted a genome-wide CRISPR/Cas9-based functional genetics screen in human cells treated with the microtubule-destabilizing drug nocodazole or the microtubule-stabilizing drug taxol. We further conducted a focused secondary screen to test drug sensitivity for ~1400 gene targets across two distinct human cell lines and to additionally test sensitivity to the Kif11-inhibitor, STLC. These screens defined gene targets whose loss enhances or suppresses sensitivity to anti-microtubule drugs. In addition to gene targets whose loss sensitized cells to multiple compounds, we observed cases of differential sensitivity to specific compounds and differing requirements between cell lines. Our downstream molecular analysis further revealed additional roles for established microtubule-associated proteins and identified new players in microtubule function.
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Formation of macromolecular cellular structures relies on recruitment of multiple proteins, requiring the precisely controlled pairwise binding interactions. At human kinetochores, our recent work found that the high molecular density environment enables strong bonding between the Ndc80 complex and its two binding sites at the CENP-T receptor. However, the mechanistic basis for this unusual density-dependent facilitation remains unknown. Here, using quantitative single-molecule approaches, we reveal two distinct mechanisms that drive preferential recruitment of the Ndc80 complex to higher-order structures of CENP-T, as opposed to CENP-T monomers. First, the Ndc80 binding sites within the disordered tail of the CENP-T mature over time, leading to a stronger grip on the Spc24/25 heads of the Ndc80 complexes. Second, the maturation of Ndc80 binding sites is accelerated when CENP-T molecules are clustered in close proximity. The rates of the clustering-induced maturation are remarkably different for two binding sites within CENP-T, correlating with different interfaces formed by the corresponding CENP-T sequences as they wrap around the Spc24/25 heads. The differential clustering-dependent regulation of these sites is preserved in dividing human cells, suggesting a distinct regulatory entry point to control kinetochore-microtubule interactions. The tunable acceleration of slowly maturing binding sites by a high molecular-density environment may represent a fundamental physicochemical mechanism to assist the assembly of mitotic kinetochores and other macromolecular structures.
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To faithfully segregate chromosomes during vertebrate mitosis, kinetochore-microtubule interactions must be restricted to a single site on each chromosome. Prior work on pair-wise kinetochore protein interactions has been unable to identify the mechanisms that prevent outer kinetochore formation in regions with a low density of CENP-A nucleosomes. To investigate the impact of higher-order assembly on kinetochore formation, we generated oligomers of the inner kinetochore protein CENP-T using two distinct, genetically engineered systems in human cells. Although individual CENP-T molecules interact poorly with outer kinetochore proteins, oligomers that mimic centromeric CENP-T density trigger the robust formation of functional, cytoplasmic kinetochore-like particles. Both in cells and in vitro, each molecule of oligomerized CENP-T recruits substantially higher levels of outer kinetochore components than monomeric CENP-T molecules. Our work suggests that the density dependence of CENP-T restricts outer kinetochore recruitment to centromeres, where densely packed CENP-A recruits a high local concentration of inner kinetochore proteins.
Assuntos
Proteínas Cromossômicas não Histona , Cinetocoros , Humanos , Proteína Centromérica A/genética , Cinetocoros/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Centrômero/genética , Centrômero/metabolismo , Nucleossomos , MitoseRESUMO
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.
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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
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.
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Sistemas CRISPR-Cas , Genes Essenciais , Humanos , Células HeLa , Técnicas de Inativação de Genes , FenótipoRESUMO
Prior work has identified signal sequences and motifs that are necessary and sufficient to target proteins to specific subcellular regions and organelles such as the plasma membrane, nucleus, endoplasmic reticulum, and mitochondria. In contrast, minimal sequence motifs that are sufficient for Golgi localization remain largely elusive. In this work, we identified a 37-amino acid alternative open reading frame (altORF) within the mRNA of the centromere protein CENP-R. This altORF peptide localizes specifically to the cytoplasmic surface of the Golgi apparatus. Through mutational analysis, we identify a minimal 10-amino acid sequence and a critical cysteine residue that are necessary and sufficient for Golgi localization. Pharmacological perturbations suggest that this peptide undergoes lipid modification to promote its localization. Together, our work defines a minimal sequence that is sufficient for Golgi targeting and provide a valuable Golgi marker for live cell imaging.
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Cisteína , Complexo de Golgi , Cisteína/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Lipídeos , Sinais Direcionadores de Proteínas , RNA Mensageiro/metabolismoRESUMO
The kinetochore is a macromolecular structure that is needed to ensure proper chromosome segregation during each cellular division. The kinetochore is assembled upon a platform of the 16-subunit constitutive centromere-associated network (CCAN), which is present at centromeres throughout the cell cycle. The nature and regulation of CCAN assembly, interactions, and dynamics needed to facilitate changing centromere properties and requirements remain to be fully elucidated. The CENP-LN complex is a CCAN component that displays unique cell cycle-dependent localization behavior, peaking in the S phase. Here, we demonstrate that phosphorylation of CENP-L and CENP-N controls CENP-LN complex formation and localization in a cell cycle-dependent manner. Mimicking constitutive phosphorylation of either CENP-L or CENP-N or simultaneously preventing phosphorylation of both proteins prevents CENP-LN localization and disrupts chromosome segregation. Our work suggests that cycles of phosphorylation and dephosphorylation are critical for CENP-LN complex recruitment and dynamics at kinetochores to enable cell cycle-dependent CCAN reorganization.
Assuntos
Centrômero , Proteínas Cromossômicas não Histona , Ciclo Celular , Centrômero/metabolismo , Proteína Centromérica A/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cinetocoros/metabolismo , FosforilaçãoRESUMO
The organismal body axes that are formed during embryogenesis are intimately linked to intrinsic asymmetries established at the cellular scale in oocytes.1 However, the mechanisms that generate cellular asymmetries within the oocyte and then transduce that polarity to organismal scale body axes are poorly understood outside of select model organisms. Here, we report an axis-defining event in meiotic oocytes of the sea star Patiria miniata. Dishevelled (Dvl) is a cytoplasmic Wnt pathway effector required for axis development in diverse species,2-4 but the mechanisms governing its function and distribution remain poorly defined. Using time-lapse imaging, we find that Dvl localizes uniformly to puncta throughout the cell cortex in Prophase I-arrested oocytes but becomes enriched at the vegetal pole following meiotic resumption through a dissolution-reassembly mechanism. This process is driven by an initial disassembly phase of Dvl puncta, followed by selective reformation of Dvl assemblies at the vegetal pole. Rather than being driven by Wnt signaling, this localization behavior is coupled to meiotic cell cycle progression and influenced by Lamp1+ endosome association and Frizzled receptors pre-localized within the oocyte cortex. Our results reveal a cell cycle-linked mechanism by which maternal cellular polarity is transduced to the embryo through spatially regulated Dvl dynamics.
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Padronização Corporal , Estrelas-do-Mar , Animais , Desenvolvimento Embrionário , Oócitos/metabolismo , SolubilidadeRESUMO
Meiosis is a specialized cell cycle that requires sequential changes to the cell division machinery to facilitate changing functions. To define the mechanisms that enable the oocyte-to-embryo transition, we performed time-course proteomics in synchronized sea star oocytes from prophase I through the first embryonic cleavage. Although we found that protein levels were broadly stable, our analysis reveals that dynamic waves of phosphorylation underlie each meiotic stage. We found that the phosphatase PP2A-B55 is reactivated at the meiosis I/meiosis II (MI/MII) transition, resulting in the preferential dephosphorylation of threonine residues. Selective dephosphorylation is critical for directing the MI/MII transition as altering PP2A-B55 substrate preferences disrupts key cell cycle events after MI. In addition, threonine to serine substitution of a conserved phosphorylation site in the substrate INCENP prevents its relocalization at anaphase I. Thus, through its inherent phospho-threonine preference, PP2A-B55 imposes specific phosphoregulated behaviors that distinguish the two meiotic divisions.
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Meiose , Oócitos/fisiologia , Monoéster Fosfórico Hidrolases/metabolismo , Estrelas-do-Mar/genética , Animais , Fosforilação , Estrelas-do-Mar/enzimologiaRESUMO
To generate haploid gametes, germ cells undergo two consecutive meiotic divisions requiring key changes to the cell division machinery. Here, we demonstrate that the protease separase rewires key cell division processes at the meiosis I/II transition by cleaving the meiosis-specific protein Meikin. Separase proteolysis does not inactivate Meikin but instead alters its function to create a distinct activity state. Full-length Meikin and the C-terminal Meikin separase cleavage product both localize to kinetochores, bind to Plk1 kinase, and promote Rec8 cleavage, but our results reveal distinct roles for these proteins in controlling meiosis. Mutations that prevent Meikin cleavage or that conditionally inactivate Meikin at anaphase I result in defective meiosis II chromosome alignment in mouse oocytes. Finally, as oocytes exit meiosis, C-Meikin is eliminated by APC/C-mediated degradation prior to the first mitotic division. Thus, multiple regulatory events irreversibly modulate Meikin activity during successive meiotic divisions to rewire the cell division machinery at two distinct transitions.
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Proteínas Cromossômicas não Histona/metabolismo , Meiose/fisiologia , Separase/metabolismo , Animais , Animais não Endogâmicos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Divisão do Núcleo Celular , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/fisiologia , Segregação de Cromossomos , Feminino , Células HeLa , Humanos , Cinetocoros/metabolismo , Camundongos , Oócitos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Separase/fisiologia , Quinase 1 Polo-LikeRESUMO
Mitotic kinetochores assemble via the hierarchical recruitment of numerous cytosolic components to the centromere region of each chromosome. However, how these orderly and localized interactions are achieved without spurious macromolecular assemblies forming from soluble kinetochore components in the cell cytosol remains poorly understood. We developed assembly assays to monitor the recruitment of green fluorescent protein-tagged recombinant proteins and native proteins from human cell extracts to inner kinetochore components immobilized on microbeads. In contrast to prior work in yeast and Xenopus egg extracts, we find that human mitotic cell extracts fail to support de novo assembly of microtubule-binding subcomplexes. A subset of interactions, such as those between CENP-A-containing nucleosomes and CENP-C, are permissive under these conditions. However, the subsequent phospho-dependent binding of the Mis12 complex is less efficient, whereas recruitment of the Ndc80 complex is blocked, leading to weak microtubule-binding activity of assembled particles. Using molecular variants of the Ndc80 complex, we show that auto-inhibition of native Ndc80 complex restricts its ability to bind to the CENP-T/W complex, whereas inhibition of the Ndc80 microtubule binding is driven by a different mechanism. Together, our work reveals regulatory mechanisms that guard against the spurious formation of cytosolic microtubule-binding kinetochore particles.
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Centrômero/metabolismo , Cinetocoros/metabolismo , Mitose/fisiologia , Extratos Celulares , Proteína Centromérica A/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/fisiologia , Proteínas do Citoesqueleto/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Cinetocoros/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismoRESUMO
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.
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Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Fuso Acromático/metabolismo , HumanosRESUMO
Similar to other core biological processes, the vast majority of cell division components are essential for viability across human cell lines. However, recent genome-wide screens have identified a number of proteins that exhibit cell line-specific essentiality. Defining the behaviors of these proteins is critical to our understanding of complex biological processes. Here, we harness differential essentiality to reveal the contributions of the four-subunit centromere-localized CENP-O complex, whose precise function has been difficult to define. Our results support a model in which the CENP-O complex and BUB1 act in parallel pathways to recruit a threshold level of PLK1 to mitotic kinetochores, ensuring accurate chromosome segregation. We demonstrate that targeted changes to either pathway sensitizes cells to the loss of the other component, resulting in cell-state dependent requirements. This approach also highlights the advantage of comparing phenotypes across diverse cell lines to define critical functional contributions and behaviors that could be exploited for the targeted treatment of disease.
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Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Histonas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Centrômero/metabolismo , Centrômero/fisiologia , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/fisiologia , Segregação de Cromossomos , Histonas/genética , Histonas/fisiologia , Humanos , Cinetocoros/fisiologia , Mitose/fisiologia , Ligação Proteica , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Quinase 1 Polo-LikeRESUMO
Quiescence is a state of reversible proliferative arrest in which cells are not actively dividing and yet retain the capacity to reenter the cell cycle upon receiving an appropriate stimulus. Quiescent cells are remarkably diverse-they reside in different locations throughout the body, serve distinct roles, and are activated by a variety of signals. Despite this diversity, all quiescent cells must be able to persist in a nondividing state without compromising their proliferative potential, which requires changes to core cellular programs. How drastically different cell types are able to implement extensive changes to their gene-expression programs, metabolism, and cellular structures to induce a common cellular state is a fascinating question in cell and developmental biology. In this review, we explore the diversity of quiescent cells and highlight the unifying characteristics that define the quiescent state.
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Ciclo Celular , Células/metabolismo , Animais , Ciclo Celular/genética , Proliferação de Células , Humanos , Modelos Biológicos , Transdução de Sinais , Transcrição GênicaRESUMO
Although originally thought to be silent chromosomal regions, centromeres are instead actively transcribed. However, the behavior and contributions of centromere-derived RNAs have remained unclear. Here, we used single-molecule fluorescence in-situ hybridization (smFISH) to detect alpha-satellite RNA transcripts in intact human cells. We find that alpha-satellite RNA-smFISH foci levels vary across cell lines and over the cell cycle, but do not remain associated with centromeres, displaying localization consistent with other long non-coding RNAs. Alpha-satellite expression occurs through RNA polymerase II-dependent transcription, but does not require established centromere or cell division components. Instead, our work implicates centromere-nucleolar interactions as repressing alpha-satellite expression. The fraction of nucleolar-localized centromeres inversely correlates with alpha-satellite transcripts levels across cell lines and transcript levels increase substantially when the nucleolus is disrupted. The control of alpha-satellite transcripts by centromere-nucleolar contacts provides a mechanism to modulate centromere transcription and chromatin dynamics across diverse cell states and conditions.
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Nucléolo Celular/genética , Centrômero/metabolismo , RNA Satélite/genética , Transcrição Gênica , Linhagem Celular , Nucléolo Celular/metabolismo , Centrômero/genética , Cromatina/genética , Cromatina/metabolismo , Humanos , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Satélite/metabolismoRESUMO
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.
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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
Despite a conserved requirement in mediating chromosome segregation, kinetochores display remarkable plasticity in their structure and composition. New work in holocentric insect species highlights the molecular rewiring that occurs when key structural components of the kinetochore are lost and centromere structure is changed.
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Segregação de Cromossomos , Lepidópteros , Animais , Centrômero , Cinetocoros , Microtúbulos , PlásticosRESUMO
Centromeres provide a robust model for epigenetic inheritance as they are specified by sequence-independent mechanisms involving the histone H3-variant centromere protein A (CENP-A). Prevailing models indicate that the high intrinsic stability of CENP-A nucleosomes maintains centromere identity indefinitely. Here, we demonstrate that CENP-A is not stable at centromeres but is instead gradually and continuously incorporated in quiescent cells including G0-arrested tissue culture cells and prophase I-arrested oocytes. Quiescent CENP-A incorporation involves the canonical CENP-A deposition machinery but displays distinct requirements from cell cycle-dependent deposition. We demonstrate that Plk1 is required specifically for G1 CENP-A deposition, whereas transcription promotes CENP-A incorporation in quiescent oocytes. Preventing CENP-A deposition during quiescence results in significantly reduced CENP-A levels and perturbs chromosome segregation following the resumption of cell division. In contrast to quiescent cells, terminally differentiated cells fail to maintain CENP-A levels. Our work reveals that quiescent cells actively maintain centromere identity providing an indicator of proliferative potential.