Structural Integrity of Centromeric Chromatin and Faithful Chromosome Segregation Requires Pat1

The kinetochore (centromeric DNA and associated protein complex) is essential for faithful chromosome segregation and maintenance of genome stability. Here we report that an evolutionarily conserved protein Pat1 is a structural component of Saccharomyces cerevisiae kinetochore and associates with centromeres in a NDC10-dependent manner. Consistent with a role for Pat1 in kinetochore structure and function, a deletion of PAT1 results in delay in sister chromatid separation, errors in chromosome segregation, and defects in structural integrity of centromeric chromatin. Pat1 is involved in topological regulation of minichromosomes as altered patterns of DNA supercoiling were observed in pat1Δ cells. Studies with pat1 alleles uncovered an evolutionarily conserved region within the central domain of Pat1 that is required for its association with centromeres, sister chromatid separation, and faithful chromosome segregation. Taken together, our data have uncovered a novel role for Pat1 in maintaining the structural integrity of centromeric chromatin to facilitate faithful chromosome segregation and proper kinetochore function.     

Cdp1, a Novel Saccharomyces Cerevisiae Gene Required for Proper Nuclear Division and Chromosome Segregation

To identify new gene products involved in chromosome segregation, we isolated Saccharomyces cerevisiae mutants that require centromere binding factor I (Cbf1p) for viability. One Cbf1p-dependent mutant (denoted cdp1-1) was selected for further analysis. The CDP1 gene encodes a novel 125-kD protein that is notably similar to previously identified mouse, human and Caenorhabditis elegans proteins. CDP1Δ and cdp1-1 mutant cells were temperature sensitive for growth. At the permissive temperature, cdp1-1 and cdp1Δ cells lost chromosomes at a frequencies ~20-fold and ~110-fold higher than wild-type cells, respectively. These mutants also displayed unusually long and numerous bundles of cytoplasmic microtubules as revealed by immunofluorescent staining. In addition, we occasionally observed improperly oriented mitotic spindles, residing entirely within one of the cells. Presumably as a result of undergoing nuclear division with improperly oriented spindles, a large percentage of cdp1 cells had accumulated multiple nuclei. While cdp1 mutant cells were hypersensitive to the microtubule-disrupting compound thiabendazole, they showed increased resistance to the closely related compound benomyl relative to wild-type cells. Taken together, these results suggest that Cdp1p plays a role in governing tubulin dynamics within the cell and may interact directly with microtubules or tubulin.     

Human Condensin Function Is Essential for Centromeric Chromatin Assembly and Proper Sister Kinetochore Orientation

Condensins I and II in vertebrates are essential ATP-dependent complexes necessary for chromosome condensation in mitosis. Condensins depletion is known to perturb structure and function of centromeres, however the mechanism of this functional link remains elusive. Depletion of condensin activity is now shown to result in a significant loss of loading of CENP-A, the histone H3 variant found at active centromeres and the proposed epigenetic mark of centromere identity. Absence of condensins and/or CENP-A insufficiency produced a specific kinetochore defect, such that a functional mitotic checkpoint cannot prevent chromosome missegregation resulting from improper attachment of sister kinetochores to spindle microtubules. Spindle microtubule-dependent deformation of both inner kinetochores and the HEC1/Ndc80 microtubule-capturing module, then results in kinetochore separation from the Aurora B pool and ensuing reduced kinase activity at centromeres. Moreover, recovery from mitosis-inhibition by monastrol revealed a high incidence of merotelic attachment that was nearly identical with condensin depletion, Aurora B inactivation, or both, indicating that the Aurora B dysfunction is the key defect leading to chromosome missegregation in condensin-depleted cells. Thus, beyond a requirement for global chromosome condensation, condensins play a pivotal role in centromere assembly, proper spatial positioning of microtubule-capturing modules and positioning complexes of the inner centromere versus kinetochore plates.     

XMAP215–EB1 Interaction Is Required for Proper Spindle Assembly and Chromosome Segregation in Xenopus Egg Extract

In metaphase Xenopus egg extracts, global microtubule growth is mainly promoted by two unrelated microtubule stabilizers, end-binding protein 1 (EB1) and XMAP215. Here, we explore their role and potential redundancy in the regulation of spindle assembly and function. We find that at physiological expression levels, both proteins are required for proper spindle architecture: Spindles assembled in the absence of EB1 or at decreased XMAP215 levels are short and frequently multipolar. Moreover, the reduced density of microtubules at the equator of ΔEB1 or ΔXMAP215 spindles leads to faulty kinetochore–microtubule attachments. These spindles also display diminished poleward flux rates and, upon anaphase induction, they neither segregate chromosomes nor reorganize into interphasic microtubule arrays. However, EB1 and XMAP215 nonredundantly regulate spindle assembly because an excess of XMAP215 can compensate for the absence of EB1, whereas the overexpression of EB1 cannot substitute for reduced XMAP215 levels. Our data indicate that EB1 could positively regulate XMAP215 by promoting its binding to the microtubules. Finally, we show that disruption of the mitosis-specific XMAP215–EB1 interaction produces a phenotype similar to that of either EB1 or XMAP215 depletion. Therefore, the XMAP215–EB1 interaction is required for proper spindle organization and chromosome segregation in Xenopus egg extracts.     

The Post-anaphase SUMO Pathway Ensures the Maintenance of Centromeric Cohesion through Meiosis I-II Transition in Mammalian Oocytes

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Centromeric Regions Control Autonomous Segregation Tendencies in Single-Division Meiosis of Saccharomyces Cerevisiae

We have previously shown that yeast cdc5 or cdc14 homozygotes can be led through a single-division meiosis in which some of the chromosomes segregate reductionally whereas others, within the same cell, segregate equationally. Chromosomes XI tend to segregate reductionally, whereas chromosomes IV tend to segregate equationally. In this report we present experiments with cdc5 homozygous strains, in which the centromeres of one or both chromosomes XI was replaced by the centromeric region from chromosome IV. Analysis of the products of single-division meioses in these strains demonstrates that the choice between reductional or equational segregation is directed by sequences in the vicinity of the centromeres. Although the choice is made separately for each individual chromosome, the analysis also reveals the existence of a system responsible for coordinated segregation of the two chromosomes of a given pair.     

染色体着丝点结构变化与习惯性流产的关系

为探讨染色体Cd结构变化与习惯性流产关系,采用Cd-NOR同步银染技术,对38例习惯性流产患者和42例正常人Cd结构变异、Cd结构消失、Cd结构最大横径和Cd-NOR融合进行测量和比较分析。发现习惯性流产患者的Cd结构变异和Cd结构消失的频率明显高于正常人,Cd结构最大横径明显小于正常人。Cd结构消失和Cd结构变异频率的增高以及Cd结构最大横径变小可能是影响习惯性流产的相关因素。 Abstract:To study the correlation between chromosome centromeric dots and habitual abortions,Cd variation,Cd loss,maximum diameter of Cd and Cd-NOR of 38 habitual abortion patients and 42 healthy persons were measured,compared and analysed with Cd-banding technique.It was found that the frequencies of Cd variation and Cd loss were obviously higher and maximum diameter of Cd was smaller in habitual abortion patients than those in healthy persons.The increase of frequencies of Cd variation and Cd loss and the decrease of maximum diameter of Cd might be the causes affecting habitual abortions.     

A Role for the Chaperone Complex BAG3-HSPB8 in Actin Dynamics,Spindle Orientation and Proper Chromosome Segregation during Mitosis

The co-chaperone BAG3, in complex with the heat shock protein HSPB8, plays a role in protein quality control during mechanical strain. It is part of a multichaperone complex that senses damaged cytoskeletal proteins and orchestrates their seclusion and/or degradation by selective autophagy. Here we describe a novel role for the BAG3-HSPB8 complex in mitosis, a process involving profound changes in cell tension homeostasis. BAG3 is hyperphosphorylated at mitotic entry and localizes to centrosomal regions. BAG3 regulates, in an HSPB8-dependent manner, the timely congression of chromosomes to the metaphase plate by influencing the three-dimensional positioning of the mitotic spindle. Depletion of BAG3 caused defects in cell rounding at metaphase and dramatic blebbing of the cortex associated with abnormal spindle rotations. Similar defects were observed upon silencing of the autophagic receptor p62/SQSTM1 that contributes to BAG3-mediated selective autophagy pathway. Mitotic cells depleted of BAG3, HSPB8 or p62/SQSTM1 exhibited disorganized actin-rich retraction fibres, which are proposed to guide spindle orientation. Proper spindle positioning was rescued in BAG3-depleted cells upon addition of the lectin concanavalin A, which restores cortex rigidity. Together, our findings suggest the existence of a so-far unrecognized quality control mechanism involving BAG3, HSPB8 and p62/SQSTM1 for accurate remodelling of actin-based mitotic structures that guide spindle orientation.     

TIN2 binds TRF1 and TRF2 simultaneously and stabilizes the TRF2 complex on telomeres

Human telomeres contain two related telomeric DNA-binding proteins, TRF1 and TRF2. The TRF1 complex contains the TRF1 interacting partner, TIN2, as well as PIP1 and POT1 and regulates telomere-length homeostasis. The TRF2 complex is primarily involved in telomere protection and contains the TRF2 interacting partner human (h)Rap1 as well as several factors involved in the DNA damage response. A prior report showed that conditional deletion of murine TRF1 reduced the presence of TRF2 on telomeres. Here we showed that TRF2 is also lost from human telomeres upon TRF1 depletion with small interfering RNA prompting a search for the connection between the TRF1 and TRF2 complexes. Using mass spectrometry and co-immunoprecipitation, we found that TRF1, TIN2, PIP1, and POT1 are associated with the TRF2-hRap1 complex. Gel filtration identified a TRF2 complex containing TIN2 and POT1 but not TRF1 indicating that TRF1 is not required for this interaction. Co-immunoprecipitation, Far-Western assays, and two-hybrid assays showed that TIN2, but not POT1 or PIP1, interacts directly with TRF2. Furthermore, TIN2 was found to bind TRF1 and TRF2 simultaneously, showing that TIN2 can link these telomeric proteins. This connection appeared to stabilize TRF2 on the telomeres as the treatment of cells with TIN2 small interfering RNA resulted in a decreased presence of TRF2 and hRap1 at chromosome ends. The TIN2-mediated cooperative binding of TRF1 and TRF2 to telomeres has important implications for the mechanism of telomere length regulation and protection.     

Control of human telomere length by TRF1 and TRF2

Telomere length in human cells is controlled by a homeostasis mechanism that involves telomerase and the negative regulator of telomere length, TRF1 (TTAGGG repeat binding factor 1). Here we report that TRF2, a TRF1-related protein previously implicated in protection of chromosome ends, is a second negative regulator of telomere length. Overexpression of TRF2 results in the progressive shortening of telomere length, similar to the phenotype observed with TRF1. However, while induction of TRF1 could be maintained over more than 300 population doublings and resulted in stable, short telomeres, the expression of exogenous TRF2 was extinguished and the telomeres eventually regained their original length. Consistent with their role in measuring telomere length, indirect immunofluorescence indicated that both TRF1 and TRF2 bind to duplex telomeric DNA in vivo and are more abundant on telomeres with long TTAGGG repeat tracts. Neither TRF1 nor TRF2 affected the expression level of telomerase. Furthermore, the presence of TRF1 or TRF2 on a short linear telomerase substrate did not inhibit the enzymatic activity of telomerase in vitro. These findings are consistent with the recently proposed t loop model of telomere length homeostasis in which telomerase-dependent telomere elongation is blocked by sequestration of the 3' telomere terminus in TRF1- and TRF2-induced telomeric loops.     

Dis1: A Yeast Gene Required for Proper Meiotic Chromosome Disjunction

Mutants at a newly identified locus, DIS1 (disjunction), were detected by screening for mutants that generate aneuploid spores (chromosome VIII disomes) at an increased frequency. Strains carrying the partially dominant alleles, DIS1-1 or DIS1-2, generate disomes at rates up to 100 times the background level. Mitotic nondisjunction is also increased 10- to 50-fold over background. Half-tetrad analysis of disomes for a marked interval on chromosome VIII yields wild-type map distances, indicating that a general recombination deficiency is not the cause of nondisjunction. Meiotic nondisjunction in DIS1 mutants is not chromosome specific; 5% of the spores disomic for chromosome VIII are also disomic for chromosome III. Although only one disomic spore is found per exceptional ascus most of the disomes appear to be generated in the first meiotic division because recovered chromosome VIII disomes contain mostly nonsister chromosomes. We propose that disome generation in the DIS1 mutants results from precocious separation of sister centromeres.