RESUMEN
A hallmark of meiosis is chromosomal pairing, which requires telomere tethering and rotation on the nuclear envelope through microtubules, driving chromosome homology searches. Telomere pulling toward the centrosome forms the "zygotene chromosomal bouquet." Here, we identified the "zygotene cilium" in oocytes. This cilium provides a cable system for the bouquet machinery and extends throughout the germline cyst. Using zebrafish mutants and live manipulations, we demonstrate that the cilium anchors the centrosome to counterbalance telomere pulling. The cilium is essential for bouquet and synaptonemal complex formation, oogenesis, ovarian development, and fertility. Thus, a cilium represents a conserved player in zebrafish and mouse meiosis, which sheds light on reproductive aspects in ciliopathies and suggests that cilia can control chromosomal dynamics.
Asunto(s)
Emparejamiento Cromosómico , Cilios , Oocitos , Oogénesis , Ovario , Animales , Centrómero/genética , Centrómero/fisiología , Emparejamiento Cromosómico/genética , Emparejamiento Cromosómico/fisiología , Cilios/fisiología , Femenino , Fertilidad/fisiología , Ratones , Morfogénesis , Oocitos/crecimiento & desarrollo , Oogénesis/genética , Oogénesis/fisiología , Ovario/crecimiento & desarrollo , Telómero/genética , Telómero/fisiología , Pez Cebra/genética , Pez Cebra/fisiologíaRESUMEN
BACKGROUND: The centromere is the special region on a chromosome, which serves as the site for assembly of kinetochore complex and is essential for maintaining genomic integrity. Neocentromeres are new centromeres that form on the non-centromeric regions of the chromosome when the natural centromere is disrupted or inactivated. Although neocentromeres lack the typical features found in centromeres, cells with neocentromeres divide normally during mitosis and meiosis. Neocentromeres not only arise naturally but their formation can also be induced experimentally. Therefore, neocentromeres are a great tool for studying functions and formation of centromeres. OBJECTIVE: To study neocentromeres and use that knowledge to gain insights into the epigenetic regulation of canonical centromeres. DISCUSSION: Here, we review the characteristics of naturally occurring centromeres and neocentromeres and those of experimentally induced neocentromeres. We also discuss the mechanism of centromere formation and epigenetic regulation of centromere function, which we learned from studying the neocentromeres. Although neocentromeres lack main features of centromeres, such as presence of repetitive âº-satellite DNA and pericentric heterochromatin, they behave quite similar to the canonical centromere, indicating the epigenetic nature of the centromere. Still, further investigation will help to understand the formation and maintenance of the centromere, and the correlation to human diseases. CONCLUSION: Neocentromeres helped us to understand the formation of canonical centromeres. Also, since neocentromeres are associated with certain cancer types, knowledge about them could be helpful to treat cancer.
Asunto(s)
Centrómero , Epigénesis Genética , Centrómero/clasificación , Centrómero/genética , Centrómero/fisiología , Genómica , Humanos , MeiosisRESUMEN
Previous studies on grass species suggested that the total centromere size (sum of all centromere sizes in a cell) may be determined by the genome size, possibly because stable scaling is important for proper cell division. However, it is unclear whether this relationship is universal. Here we analyze the total centromere size using the CenH3-immunofluorescence area as a proxy in 130 taxa including plants, animals, fungi, and protists. We verified the reliability of our methodological approach by comparing our measurements with available ChIP-seq-based measurements of the size of CenH3-binding domains. Data based on these two independent methods showed the same positive relationship between the total centromere size and genome size. Our results demonstrate that the genome size is a strong predictor (R-squared = 0.964) of the total centromere size universally across Eukaryotes. We also show that this relationship is independent of phylogenetic relatedness and centromere type (monocentric, metapolycentric, and holocentric), implying a common mechanism maintaining stable total centromere size in Eukaryotes.
Asunto(s)
Centrómero/fisiología , Hongos/genética , Plantas/genética , Animales , Evolución Molecular , Tamaño del GenomaRESUMEN
Deletion of native centromeres in the human fungal pathogen Cryptococcus deuterogattii leads to neocentromere formation. Native centromeres span truncated transposable elements, while neocentromeres do not and instead span actively expressed genes. To explore the epigenetic organization of neocentromeres, we analyzed the distribution of the heterochromatic histone modification H3K9me2, 5mC DNA methylation and the euchromatin mark H3K4me2. Native centromeres are enriched for both H3K9me2 and 5mC DNA methylation marks and are devoid of H3K4me2, while neocentromeres do not exhibit any of these features. Neocentromeres in cen10Δ mutants are unstable and chromosome-chromosome fusions occur. After chromosome fusion, the neocentromere is inactivated and the native centromere of the chromosome fusion partner remains as the sole, active centromere. In the present study, the active centromere of a fused chromosome was deleted to investigate if epigenetic memory promoted the re-activation of the inactive neocentromere. Our results show that the inactive neocentromere is not re-activated and instead a novel neocentromere forms directly adjacent to the deleted centromere of the fused chromosome. To study the impact of transcription on centromere stability, the actively expressed URA5 gene was introduced into the CENP-A bound regions of a native centromere. The introduction of the URA5 gene led to a loss of CENP-A from the native centromere, and a neocentromere formed adjacent to the native centromere location. Remarkably, the inactive, native centromere remained enriched for heterochromatin, yet the integrated gene was expressed and devoid of H3K9me2. A cumulative analysis of multiple CENP-A distribution profiles revealed centromere drift in C. deuterogattii, a previously unreported phenomenon in fungi. The CENP-A-binding shifted within the ORF-free regions and showed a possible association with a truncated transposable element. Taken together, our findings reveal that neocentromeres in C. deuterogattii are highly unstable and are not marked with an epigenetic memory, distinguishing them from native centromeres.
Asunto(s)
Centrómero/genética , Centrómero/fisiología , Cryptococcus/genética , Autoantígenos/genética , Proteína A Centromérica/genética , Proteína A Centromérica/metabolismo , Cromatina , Proteínas Cromosómicas no Histona/genética , Cryptococcus/metabolismo , Metilación de ADN , Epigénesis Genética/genética , Epigénesis Genética/fisiología , Epigenómica , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismoRESUMEN
Chromosome instability (CIN) is a major hallmark of cancer cells and believed to drive tumor progression. Several cellular defects including weak centromeric cohesion are proposed to promote CIN, but the molecular mechanisms underlying these defects are poorly understood. In a screening for SET protein levels in various cancer cell lines, we found that most of the cancer cells exhibit higher SET protein levels than nontransformed cells, including RPE-1. Cancer cells with elevated SET often show weak centromeric cohesion, revealed by MG132-induced cohesion fatigue. Partial SET knockdown largely strengthens centromeric cohesion in cancer cells without increasing overall phosphatase 2A (PP2A) activity. Pharmacologically increased PP2A activity in these cancer cells barely ameliorates centromeric cohesion. These results suggest that compromised PP2A activity, a common phenomenon in cancer cells, may not be responsible for weak centromeric cohesion. Furthermore, centromeric cohesion in cancer cells can be strengthened by ectopic Sgo1 overexpression and weakened by SET WT, not by Sgo1-binding-deficient mutants. Altogether, these findings demonstrate that SET overexpression contributes to impaired centromeric cohesion in cancer cells and illustrate misregulated SET-Sgo1 pathway as an underlying mechanism.
Asunto(s)
Inestabilidad Cromosómica/fisiología , Segregación Cromosómica/genética , Proteínas de Unión al ADN/metabolismo , Chaperonas de Histonas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Línea Celular Tumoral , Centrómero/fisiología , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica/fisiología , Proteínas de Unión al ADN/fisiología , Chaperonas de Histonas/fisiología , Humanos , Mitosis , Proteínas Nucleares/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteína Fosfatasa 2/fisiología , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Histonas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiología , Línea Celular Tumoral , Centrómero/metabolismo , Centrómero/fisiología , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/fisiología , Segregación Cromosómica , Histonas/genética , Histonas/fisiología , Humanos , Cinetocoros/fisiología , Mitosis/fisiología , Unión Proteica , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Quinasa Tipo Polo 1RESUMEN
The aim of mitosis is to segregate duplicated chromosomes equally into daughter cells during cell division. Meiosis serves a similar purpose, but additionally separates homologous chromosomes to produce haploid gametes for sexual reproduction. Both mitosis and meiosis rely on centromeres for the segregation of chromosomes. Centromeres are the specialized regions of the chromosomes that are attached to microtubules during their segregation. In this review, we describe the adaptations and layers of regulation that are required for centromere function during meiosis, and their role in meiosis-specific processes such as homolog-pairing and recombination. Since female meiotic divisions are asymmetric, meiotic centromeres are hypothesized to evolve quickly in order to favor their own transmission to the offspring, resulting in the rapid evolution of many centromeric proteins. We discuss this observation using the example of the histone variant CENP-A, which marks the centromere and is essential for centromere function. Changes in both the size and the sequence of the CENP-A N-terminal tail have led to additional functions of the protein, which are likely related to its roles during meiosis. We highlight the importance of CENP-A in the inheritance of centromere identity, which is dependent on the stabilization, recycling, or re-establishment of CENP-A-containing chromatin during meiosis.
Asunto(s)
Proteína A Centromérica/fisiología , Centrómero/fisiología , Meiosis , Animales , Femenino , Humanos , Masculino , PlantasRESUMEN
The centromere and telomere are universal heterochromatic domains; however, the proper positioning of those domains in nuclear space during the mitotic interphase differs among eukaryotes. Consequently, the question arises how and why this difference occurs. Studies over the past 2 decades have identified several nuclear membrane proteins, nucleolar proteins, and the structural maintenance of a chromosome complex as factors involved in the positional control of centromeres and/or telomeres during the mitotic interphase in yeasts, animals, and plants. In this review, with a primary focus on plants, the roles of those factors are summarized, and the biological significance of proper centromere and telomere positionings during the mitotic interphase is discussed in an effort to provide guidance for this question.
Asunto(s)
Centrómero , Plantas , Telómero , Animales , Centrómero/genética , Centrómero/fisiología , Interfase , Plantas/genética , Telómero/genéticaRESUMEN
Nucleoli, the sites of ribosome biogenesis and the largest structures in human nuclei, form around nucleolar organizer regions (NORs) comprising ribosomal DNA (rDNA) arrays. NORs are located on the p-arms of the five human acrocentric chromosomes. Defining the rules of engagement between these p-arms and nucleoli takes on added significance as describing the three-dimensional organization of the human genome represents a major research goal. Here we used fluorescent in situ hybridization (FISH) and immuno-FISH on metaphase chromosomes from karyotypically normal primary and hTERT-immortalized human cell lines to catalog NORs in terms of their relative rDNA content and activity status. We demonstrate that a proportion of acrocentric p-arms in cell lines and from normal human donors have no detectable rDNA. Surprisingly, we found that all NORs with detectable rDNA are active, as defined by upstream binding factor loading. We determined the nucleolar association status of all NORs during interphase, and found that nucleolar association of acrocentric p-arms can occur independently of rDNA content, suggesting that sequences elsewhere on these chromosome arms drive nucleolar association. In established cancer lines, we characterize a variety of chromosomal rearrangements involving acrocentric p-arms and observe silent, rDNA-containing NORs that are dissociated from nucleoli. In conclusion, our findings indicate that within human nuclei, positioning of all 10 acrocentric chromosomes is dictated by nucleolar association. Furthermore, these nucleolar associations are buffered against interindividual variation in the distribution of rDNA.
Asunto(s)
ADN Ribosómico/genética , Región Organizadora del Nucléolo/metabolismo , Región Organizadora del Nucléolo/fisiología , Línea Celular , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Centrómero/fisiología , Cromosomas Humanos/metabolismo , ADN Ribosómico/metabolismo , Genoma Humano/genética , Genoma Humano/fisiología , Humanos , Hibridación Fluorescente in Situ/métodos , Región Organizadora del Nucléolo/genética , Ribosomas/metabolismoRESUMEN
Both the pericentromere and the nucleolus have unique characteristics that distinguish them amongst the rest of genome. Looping of pericentromeric DNA, due to structural maintenance of chromosome (SMC) proteins condensin and cohesin, drives its ability to maintain tension during metaphase. Similar loops are formed via condensin and cohesin in nucleolar ribosomal DNA (rDNA). Condensin and cohesin are also concentrated in transfer RNA (tRNA) genes, genes which may be located within the pericentromere as well as tethered to the nucleolus. Replication fork stalling, as well as downstream consequences such as genomic recombination, are characteristic of both the pericentromere and rDNA. Furthermore, emerging evidence suggests that the pericentromere may function as a liquid-liquid phase separated domain, similar to the nucleolus. We therefore propose that the pericentromere and nucleolus, in part due to their enrichment of SMC proteins and others, contain similar domains that drive important cellular activities such as segregation, stability, and repair.
Asunto(s)
Nucléolo Celular/genética , Nucléolo Celular/fisiología , Centrómero/fisiología , Adenosina Trifosfatasas , Proteínas de Ciclo Celular , Nucléolo Celular/metabolismo , Centrómero/metabolismo , Proteínas Cromosómicas no Histona , Segregación Cromosómica , Cromosomas/fisiología , Proteínas de Unión al ADN , Mitosis , Complejos Multiproteicos , Región Organizadora del Nucléolo/metabolismo , Región Organizadora del Nucléolo/fisiología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , CohesinasRESUMEN
Centromeres are rapidly evolving across eukaryotes, despite performing a conserved function to ensure high-fidelity chromosome segregation. CENP-A chromatin is a hallmark of a functional centromere in most organisms. Due to its critical role in kinetochore architecture, the loss of CENP-A is tolerated in only a few organisms, many of which possess holocentric chromosomes. Here, we characterize the consequence of the loss of CENP-A in the fungal kingdom. Mucor circinelloides, an opportunistic human pathogen, lacks CENP-A along with the evolutionarily conserved CENP-C but assembles a monocentric chromosome with a localized kinetochore complex throughout the cell cycle. Mis12 and Dsn1, two conserved kinetochore proteins, were found to co-localize to a short region, one in each of nine large scaffolds, composed of an â¼200-bp AT-rich sequence followed by a centromere-specific conserved motif that echoes the structure of budding yeast point centromeres. Resembling fungal regional centromeres, these core centromere regions are embedded in large genomic expanses devoid of genes yet marked by Grem-LINE1s, a novel retrotransposable element silenced by the Dicer-dependent RNAi pathway. Our results suggest that these hybrid features of point and regional centromeres arose from the absence of CENP-A, thus defining novel mosaic centromeres in this early-diverging fungus.
Asunto(s)
Centrómero/metabolismo , Cinetocoros/fisiología , Mucor/genética , Centrómero/fisiología , Proteína A Centromérica/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/fisiología , Segregación Cromosómica/fisiología , Histonas/metabolismo , Cinetocoros/metabolismo , Mucor/metabolismoRESUMEN
Chromosome segregation errors during female meiosis are a leading cause of pregnancy loss and human infertility. The segregation of chromosomes is driven by interactions between spindle microtubules and kinetochores. Kinetochores in mammalian oocytes are subjected to special challenges: they need to withstand microtubule pulling forces over multiple hours and are built on centromeric chromatin that in humans is decades old. In meiosis I, sister kinetochores are paired and oriented toward the same spindle pole. It is well established that they progressively separate from each other with advancing female age. However, whether aging also affects the internal architecture of centromeres and kinetochores is currently unclear. Here, we used super-resolution microscopy to study meiotic centromere and kinetochore organization in metaphase-II-arrested eggs from three mammalian species, including humans. We found that centromeric chromatin decompacts with advancing maternal age. Kinetochores built on decompacted centromeres frequently lost their integrity and fragmented into multiple lobes. Fragmentation extended across inner and outer kinetochore regions and affected over 30% of metaphase-II-arrested (MII) kinetochores in aged women and mice, making the lobular architecture a prominent feature of the female meiotic kinetochore. We demonstrate that a partial cohesin loss, as is known to occur in oocytes with advancing maternal age, is sufficient to trigger centromere decompaction and kinetochore fragmentation. Microtubule pulling forces further enhanced the fragmentation and shaped the arrangement of kinetochore lobes. Fragmented kinetochores were frequently abnormally attached to spindle microtubules, suggesting that kinetochore fragmentation could contribute to the maternal age effect in mammalian eggs.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Proteínas Cromosómicas no Histona/fisiología , Cinetocoros/metabolismo , Oocitos/metabolismo , Envejecimiento , Animales , Proteínas de Ciclo Celular/metabolismo , Centrómero/fisiología , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica/fisiología , Femenino , Células HEK293 , Humanos , Cinetocoros/fisiología , Meiosis/fisiología , Ratones , Microtúbulos/metabolismo , Células 3T3 NIH , Oocitos/fisiología , Óvulo/metabolismo , Óvulo/fisiología , Huso Acromático/fisiología , Porcinos , CohesinasRESUMEN
Centromeres and centrosomes are crucial mitotic players. Centromeres are unique chromosomal sites characterized by the presence of the histone H3-variant centromere protein A (CENP-A) [1]. CENP-A recruits the majority of centromere components, collectively named the constitutive centromere associated network (CCAN) [2]. The CCAN is necessary for kinetochore assembly, a multiprotein complex that attaches spindle microtubules (MTs) and is required for chromosome segregation [3]. In most animal cells, the dominant site for MT nucleation in mitosis are the centrosomes, which are composed of two centrioles, surrounded by a protein-rich matrix of electron-dense pericentriolar material (PCM) [4]. The PCM is the site of MT nucleation during mitosis [5]. Even if centromeres and centrosomes are connected via MTs in mitosis, it is not known whether defects in either one of the two structures have an impact on the function of the other. Here, using high-resolution microscopy combined with rapid removal of CENP-A in human cells, we found that perturbation of centromere function impacts mitotic spindle pole integrity. This includes release of MT minus-ends from the centrosome, leading to PCM dispersion and centriole mis-positioning at the spindle poles. Mechanistically, we show that these defects result from abnormal spindle MT dynamics due to defective kinetochore-MT attachments. Importantly, restoring mitotic spindle pole integrity following centromere inactivation lead to a decrease in the frequency of chromosome mis-segregation. Overall, our work identifies an unexpected relationship between centromeres and maintenance of the mitotic pole integrity necessary to ensure mitotic accuracy and thus to maintain genetic stability.
Asunto(s)
Proteína A Centromérica/metabolismo , Centrómero/metabolismo , Huso Acromático/metabolismo , Línea Celular , Centriolos/metabolismo , Centrómero/fisiología , Proteína A Centromérica/fisiología , Centrosoma/metabolismo , Centrosoma/fisiología , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica/fisiología , Histonas/metabolismo , Humanos , Cinetocoros/metabolismo , Cinetocoros/fisiología , Microtúbulos/metabolismo , Mitosis/fisiología , Huso Acromático/fisiología , Polos del Huso/metabolismoRESUMEN
Centromeres mediate the pairing of homologous chromosomes during meiosis; this pairing is particularly challenging for polyploid plants such as hexaploid bread wheat (Triticum aestivum), as their meiotic machinery must differentiate homologs from similar homoeologs. However, the sequence compositions (especially functional centromeric satellites) and evolutionary history of wheat centromeres are largely unknown. Here, we mapped T. aestivum centromeres by chromatin immunoprecipitation sequencing using antibodies to the centromeric-specific histone H3 variant (CENH3); this identified two types of functional centromeric satellites that are abundant in two of the three subgenomes. These centromeric satellites had unit sizes greater than 500 bp and contained specific sites with highly phased binding to CENH3 nucleosomes. Phylogenetic analysis revealed that the satellites have diverged in the three T. aestivum subgenomes, and the more homogeneous satellite arrays are associated with CENH3. Satellite signals decreased and the degree of satellites variation increased from diploid to hexaploid wheat. Moreover, several T. aestivum centromeres lack satellite repeats. Rearrangements, including local expansion and satellite variations, inversions, and changes in gene expression, occurred during the evolution from diploid to tetraploid and hexaploid wheat. These results reveal the asymmetry in centromere organization among the wheat subgenomes, which may play a role in proper homolog pairing during meiosis.
Asunto(s)
Centrómero/fisiología , Poliploidía , Triticum/genética , Triticum/fisiología , Centrómero/clasificación , Cromosomas de las Plantas , Evolución Molecular , Regulación de la Expresión Génica de las Plantas , Histonas/metabolismo , Meiosis , Nucleosomas/clasificación , Filogenia , Proteínas de Plantas , Especificidad de la Especie , Triticum/citologíaRESUMEN
The kinetochore is a large protein complex that ensures accurate chromosome segregation during mitosis by connecting the centromere and spindle microtubules. One of the kinetochore sub-complexes, the constitutive centromere-associated network (CCAN), associates with the centromere and recruits another sub-complex, the KMN (KNL1, Mis12, and Ndc80 complexes) network (KMN), which binds to spindle microtubules. The CCAN-KMN interaction is mediated by two parallel pathways (CENP-C- and CENP-T-pathways) in the kinetochore, which bridge the centromere and microtubules. Here, we discuss dynamic protein-interaction changes in the two pathways that couple the centromere with spindle microtubules during mitotic progression.
Asunto(s)
Polaridad Celular/fisiología , Centrómero/fisiología , Segregación Cromosómica/fisiología , Microtúbulos/metabolismo , Huso Acromático/fisiología , Animales , Humanos , Cinetocoros/fisiología , Mitosis/fisiologíaRESUMEN
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.
Asunto(s)
Centrómero/fisiología , Segregación Cromosómica/fisiología , Mitosis/fisiología , Aneuploidia , Línea Celular Tumoral , Células HeLa , Humanos , Metafase , Modelos Biológicos , Huso AcromáticoRESUMEN
CENP-A is the histone H3 variant necessary to specify the location of all eukaryotic centromeres via its CENP-A targeting domain and either one of its terminal regions. In humans, several post-translational modifications occur on CENP-A, but their role in centromere function remains controversial. One of these modifications of CENP-A, phosphorylation on serine 7, has been proposed to control centromere assembly and function. Here, using gene targeting at both endogenous CENP-A alleles and gene replacement in human cells, we demonstrate that a CENP-A variant that cannot be phosphorylated at serine 7 maintains correct CENP-C recruitment, faithful chromosome segregation and long-term cell viability. Thus, we conclude that phosphorylation of CENP-A on serine 7 is dispensable to maintain correct centromere dynamics and function.
Asunto(s)
Proteína A Centromérica/metabolismo , Centrómero/fisiología , Edición Génica , Células HeLa , Humanos , FosforilaciónRESUMEN
De novo kinetochore assembly, but not template-directed assembly, is dependent on COMA, the kinetochore complex engaged in cohesin recruitment. The slowing of replication fork progression by treatment with phleomycin (PHL), hydroxyurea, or deletion of the replication fork protection protein Csm3 can activate de novo kinetochore assembly in COMA mutants. Centromere DNA looping at the site of de novo kinetochore assembly can be detected shortly after exposure to PHL. Using simulations to explore the thermodynamics of DNA loops, we propose that loop formation is disfavored during bidirectional replication fork migration. One function of replication fork stalling upon encounters with DNA damage or other blockades may be to allow time for thermal fluctuations of the DNA chain to explore numerous configurations. Biasing thermodynamics provides a mechanism to facilitate macromolecular assembly, DNA repair, and other nucleic acid transactions at the replication fork. These loop configurations are essential for sister centromere separation and kinetochore assembly in the absence of the COMA complex.
Asunto(s)
Centrómero/fisiología , Replicación del ADN/fisiología , Cinetocoros/fisiología , Proteínas de Ciclo Celular , Centrómero/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona , ADN/metabolismo , Daño del ADN/fisiología , Reparación del ADN/fisiología , Cinetocoros/metabolismo , Fleomicinas , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Huso Acromático/metabolismo , Termodinámica , CohesinasRESUMEN
The approximately thirty core subunits of kinetochores assemble on centromeric chromatin containing the histone H3 variant CENP-A and connect chromosomes with spindle microtubules. The chromatin proximal 16-subunit CCAN (constitutive centromere associated network) creates a mechanically stable bridge between CENP-A and the kinetochore's microtubule-binding machinery, the 10-subunit KMN assembly. Here, we reconstituted a stoichiometric 11-subunit human CCAN core that forms when the CENP-OPQUR complex binds to a joint interface on the CENP-HIKM and CENP-LN complexes. The resulting CCAN particle is globular and connects KMN and CENP-A in a 26-subunit recombinant particle. The disordered, basic N-terminal tail of CENP-Q binds microtubules and promotes accurate chromosome alignment, cooperating with KMN in microtubule binding. The N-terminal basic tail of the NDC80 complex, the microtubule-binding subunit of KMN, can functionally replace the CENP-Q tail. Our work dissects the connectivity and architecture of CCAN and reveals unexpected functional similarities between CENP-OPQUR and the NDC80 complex.
Asunto(s)
Proteínas Cromosómicas no Histona/ultraestructura , Cinetocoros/fisiología , Cinetocoros/ultraestructura , Centrómero/fisiología , Proteína A Centromérica/metabolismo , Proteína A Centromérica/ultraestructura , Proteínas Cromosómicas no Histona/metabolismo , Proteínas del Citoesqueleto , Células HeLa , Humanos , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Microtúbulos/fisiología , Proteínas Nucleares/metabolismoRESUMEN
Histone H2A phosphorylation plays a role both in chromatin condensation during mitosis and in transcriptional activation during the G1/S transition. Bub1 and NHK1/VRK1 have been identified as histone H2A kinases. However, little is known about the importance of histone H2A phosphorylation in chromosome segregation. Here, we expressed recombinant hBUB1 and confirmed that it phosphorylates histone H2A T120 in the in vitro-assembled nucleosome. Knockdown (KD) of BUB1 decreases bulk H2A T120 phosphorylation in HeLa cells, whereas hBUB1 is upregulated during mitosis, which corresponds with H2A T120 phosphorylation. ChIP-qPCR of the DXZ1 centromeric and γ-ALR pericentromeric region showed that BUB1 localizes to this region and increases local H2A T120 phosphorylation during M phase. BUB1 KD did not induce apoptosis but increased the M phase cell population, as detected by flow cytometry. BUB1 KD also caused an abnormal metaphase and telophase, resulting in multinucleated cells and impaired cancer cell growth both in vitro and in vivo. Over-expression of the histone H2A T120D or T120E mutations, which mimic phosphorylated threonine, decreased the number of multinucleated cells caused by BUB1 KD. These results strengthen the apparent importance of BUB1-mediated H2A T120 phosphorylation in normal mitosis.