Displacement and re-accumulation of centromeric cohesin during transient pre-anaphase centromere splitting

The ring-shaped cohesin complex links sister chromatids until their timely segregation during mitosis. Cohesin is enriched at centromeres where it provides the cohesive counterforce to bipolar tension produced by the mitotic spindle. As a consequence of spindle tension, centromeric sequences transiently split in pre-anaphase cells, in some organisms up to several micrometers. This ‘centromere breathing’ presents a paradox, how sister sequences separate where cohesin is most enriched. We now show that in the budding yeast Saccharomyces cerevisiae, cohesin binding diminishes over centromeric sequences that split during breathing. We see no evidence for cohesin translocation to surrounding sequences, suggesting that cohesin is removed from centromeres during breathing. Two pools of cohesin can be distinguished. Cohesin loaded before DNA replication, which has established sister chromatid cohesion, disappears during breathing. In contrast, cohesin loaded after DNA replication is partly retained. As sister centromeres re-associate after transient separation, cohesin is reloaded in a manner independent of the canonical cohesin loader Scc2/Scc4. Efficient centromere re-association requires the cohesion establishment factor Eco1, suggesting that re-establishment of sister chromatid cohesion contributes to the dynamic behaviour of centromeres in mitosis. These findings provide new insights into cohesin behaviour at centromeres. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cohesin Associates with Spindle Poles in a Mitosis-specific Manner and Functions in Spindle Assembly in Vertebrate Cells

Cohesin is an essential protein complex required for sister chromatid cohesion. Cohesin associates with chromosomes and establishes sister chromatid cohesion during interphase. During metaphase, a small amount of cohesin remains at the chromosome-pairing domain, mainly at the centromeres, whereas the majority of cohesin resides in the cytoplasm, where its functions remain unclear. We describe the mitosis-specific recruitment of cohesin to the spindle poles through its association with centrosomes and interaction with nuclear mitotic apparatus protein (NuMA). Overexpression of NuMA enhances cohesin accumulation at spindle poles. Although transient cohesin depletion does not lead to visible impairment of normal spindle formation, recovery from nocodazole-induced spindle disruption was significantly impaired. Importantly, selective blocking of cohesin localization to centromeres, which disrupts centromeric sister chromatid cohesion, had no effect on this spindle reassembly process, clearly separating the roles of cohesin at kinetochores and spindle poles. In vitro, chromosome-independent spindle assembly using mitotic extracts was compromised by cohesin depletion, and it was rescued by addition of cohesin that was isolated from mitotic, but not S phase, cells. The combined results identify a novel spindle-associated role for human cohesin during mitosis, in addition to its function at the centromere/kinetochore regions.     

Roles and regulation of the Drosophila centromere cohesion protein MEI-S332 family

In meiosis, a physical attachment, or cohesion, between the centromeres of the sister chromatids is retained until their separation at anaphase II. This cohesion is essential for ensuring accurate segregation of the sister chromatids in meiosis II and avoiding aneuploidy, a condition that can lead to prenatal lethality or birth defects. The Drosophila MEI-S332 protein localizes to centromeres when sister chromatids are attached in mitosis and meiosis, and it is required to maintain cohesion at the centromeres after cohesion along the sister chromatid arms is lost at the metaphase I/anaphase I transition. MEI-S332 is the founding member of a family of proteins that protect centromeric cohesion but whose members also affect kinetochore behaviour and spindle microtubule dynamics. We compare the Drosophila MEI-S332 family members, evaluate the role of MEI-S332 in mitosis and meiosis I, and discuss the regulation of localization of MEI-S332 to the centromere and its dissociation at anaphase. We analyse the relationship between MEI-S332 and cohesin, a protein complex that is also necessary for sister-chromatid cohesion in mitosis and meiosis. In mitosis, centromere localization of 相似文献   

DNA loops generate intracentromere tension in mitosis

The centromere is the DNA locus that dictates kinetochore formation and is visibly apparent as heterochromatin that bridges sister kinetochores in metaphase. Sister centromeres are compacted and held together by cohesin, condensin, and topoisomerase-mediated entanglements until all sister chromosomes bi-orient along the spindle apparatus. The establishment of tension between sister chromatids is essential for quenching a checkpoint kinase signal generated from kinetochores lacking microtubule attachment or tension. How the centromere chromatin spring is organized and functions as a tensiometer is largely unexplored. We have discovered that centromere chromatin loops generate an extensional/poleward force sufficient to release nucleosomes proximal to the spindle axis. This study describes how the physical consequences of DNA looping directly underlie the biological mechanism for sister centromere separation and the spring-like properties of the centromere in mitosis.     

Cohesin, condensin, and the intramolecular centromere loop together generate the mitotic chromatin spring

Sister chromatid cohesion provides the mechanistic basis, together with spindle microtubules, for generating tension between bioriented chromosomes in metaphase. Pericentric chromatin forms an intramolecular loop that protrudes bidirectionally from the sister chromatid axis. The centromere lies on the surface of the chromosome at the apex of each loop. The cohesin and condensin structural maintenance of chromosomes (SMC) protein complexes are concentrated within the pericentric chromatin, but whether they contribute to tension-generating mechanisms is not known. To understand how pericentric chromatin is packaged and resists tension, we map the position of cohesin (SMC3), condensin (SMC4), and pericentric LacO arrays within the spindle. Condensin lies proximal to the spindle axis and is responsible for axial compaction of pericentric chromatin. Cohesin is radially displaced from the spindle axis and confines pericentric chromatin. Pericentric cohesin and condensin contribute to spindle length regulation and dynamics in metaphase. Together with the intramolecular centromere loop, these SMC complexes constitute a molecular spring that balances spindle microtubule force in metaphase.     

Coordinated requirements of human topo II and cohesin for metaphase centromere alignment under Mad2-dependent spindle checkpoint surveillance

Cohesin maintains sister chromatid cohesion until its Rad21/Scc1/Mcd1 is cleaved by separase during anaphase. DNA topoisomerase II (topo II) maintains the proper topology of chromatid DNAs and is essential for chromosome segregation. Here we report direct observations of mitotic progression in individual HeLa cells after functional disruptions of hRad21, NIPBL, a loading factor for hRad21, and topo II alpha,beta by RNAi and a topo II inhibitor, ICRF-193. Mitosis is delayed in a Mad2-dependent manner after disruption of either or both cohesin and topo II. In hRad21 depletion, interphase pericentric architecture becomes aberrant, and anaphase is virtually permanently delayed as preseparated chromosomes are misaligned on the metaphase spindle. Topo II disruption perturbs centromere organization leading to intense Bub1, but no Mad2, on kinetochores and sustains a Mad2-dependent delay in anaphase onset with persisting securin. Thus topo II impinges upon centromere/kinetochore function. Disruption of topo II by RNAi or ICRF-193 overrides the mitotic delay induced by cohesin depletion: sister centromeres are aligned and anaphase spindle movements occur. The ensuing accumulation of catenations in preseparated sister chromatids may overcome the reduced tension arising from cohesin depletion, causing the override. Cohesin and topo II have distinct, yet coordinated functions in metaphase alignment.     

Damage-induced reactivation of cohesin in postreplicative DNA repair

Cohesin establishes sister-chromatid cohesion during S phase to ensure proper chromosome segregation in mitosis. It also facilitates postreplicative homologous recombination repair of DNA double-strand breaks by promoting local pairing of damaged and intact sister chromatids. In G2 phase, cohesin that is not bound to chromatin is inactivated, but its reactivation can occur in response to DNA damage. Recent papers by Koshland's and Sj?gren's groups describe the critical role of the known cohesin cofactor Eco1 (Ctf7) and ATR checkpoint kinase in damage-induced reactivation of cohesin, revealing an intricate mechanism that regulates sister-chromatid pairing to maintain genome integrity.     

Cohesin ensures bipolar attachment of microtubules to sister centromeres and resists their precocious separation

The multisubunit protein complex cohesin is required to establish cohesion between sister chromatids during S phase and to maintain it during G2 and M phases. Cohesin is essential for mitosis, and even partial defects cause very high rates of chromosome loss. In budding yeast, cohesin associates with specific sites which are distributed along the entire length of a chromosome but are more dense in the vicinity of the centromere. Real-time imaging of individual centromeres tagged with green fluorescent protein suggests that cohesin bound to centromeres is important for bipolar attachment to microtubules. This cohesin is, however, incapable of resisting the consequent force, which leads to sister centromere splitting and chromosome stretching. Meanwhile, cohesin bound to sequences flanking the centromeres prevents sister chromatids from completely unzipping and is required to pull back together sister centromeres that have already split. Cohesin therefore has a central role in generating a dynamic tension between microtubules and sister chromatid cohesion at centromeres, which lasts until chromosome segregation is finally promoted by separin-dependent cleavage of the cohesin subunit Scc1p.     

Epigenetics regulate centromere formation and kinetochore function

The eukaryote centromere was initially defined cytologically as the primary constriction on vertebrate chromosomes and functionally as a chromosomal feature with a relatively low recombination frequency. Structurally, the centromere is the foundation for sister chromatid cohesion and kinetochore formation. Together these provide the basis for interaction between chromosomes and the mitotic spindle, allowing the efficient segregation of sister chromatids during cell division. Although centromeric (CEN) DNA is highly variable between species, in all cases the functional centromere forms in a chromatin domain defined by the substitution of histone H3 with the centromere specific H3 variant centromere protein A (CENP-A), also known as CENH3. Kinetochore formation and function are dependent on a variety of regional epigenetic modifications that appear to result in a loop chromatin conformation providing exterior CENH3 domains for kinetochore construction, and interior heterochromatin domains essential for sister chromatid cohesion. In addition pericentric heterochromatin provides a structural element required for spindle assembly checkpoint function. Advances in our understanding of CENH3 biology have resulted in a model where kinetochore location is specified by the epigenetic mark left after dilution of CENH3 to daughter DNA strands during S phase. This results in a self-renewing and self-reinforcing epigenetic state favorable to reliably mark centromere location, as well as to provide the optimal chromatin configuration for kinetochore formation and function.     

Control of Shugoshin function during fission-yeast meiosis

Meiosis consists of a single round of DNA replication followed by two consecutive nuclear divisions. During the first division (MI), sister kinetochores must orient toward the same pole to favor reductional segregation. Correct chromosome segregation during the second division (MII) requires the retention of centromeric cohesion until anaphase II. The spindle checkpoint protein Bub1 is essential for both processes in fission yeast . When bub1 is deleted, the Shugoshin protein Sgo1 is not recruited to centromeres, cohesin Rec8 does not persist at centromeres, and sister-chromatid cohesion is lost by the end of MI. Deletion of bub1 also affects kinetochore orientation because sister centromeres can move to opposite spindle poles in approximately 30% of MI divisions. We show here that these two functions are separable within the Bub1 protein. The N terminus of Bub1 is necessary and sufficient for Sgo1 targeting to centromeres and the protection of cohesion, whereas the C-terminal kinase domain acts together with Sgo2, the second fission-yeast Shugoshin protein, to promote sister-kinetochore co-orientation during MI. Additional analyses suggest that the protection of centromeric cohesion does not operate when sister kinetochores attach to opposite spindle poles during MI. Sgo1-mediated protection of centromere cohesion might therefore be regulated by the mode of kinetochore attachment.     

A matter of choice: the establishment of sister chromatid cohesion

Sister chromatid cohesion is the basis for the recognition of chromosomal DNA replication products for their bipolar segregation in mitosis. Fundamental to sister chromatid cohesion is the ring‐shaped cohesin complex, which is loaded onto chromosomes long before the initiation of DNA replication and is thought to hold replicated sister chromatids together by topological embrace. What happens to cohesin when the replication fork approaches, and how cohesin recognizes newly synthesized sister chromatids, is poorly understood. The characterization of a number of cohesion establishment factors has begun to provide hints as to the reactions involved. Cohesin is a member of the evolutionarily conserved family of Smc subunit‐based protein complexes that contribute to many aspects of chromosome biology by mediating long‐range DNA interactions. I propose that the establishment of cohesion equates to the selective stabilization of those cohesin‐mediated DNA interactions that link sister chromatids in the wake of replication forks.     

The cohesin complex: sequence homologies, interaction networks and shared motifs

Background  

Cohesin is a macromolecular complex that links sister chromatids together at the metaphase plate during mitosis. The links are formed during DNA replication and destroyed during the metaphase-to-anaphase transition. In budding yeast, the 14S cohesin complex comprises at least two classes of SMC (structural maintenance of chromosomes) proteins - Smc1 and Smc3 - and two SCC (sister-chromatid cohesion) proteins - Scc1 and Scc3. The exact function of these proteins is unknown.     

The spatial segregation of pericentric cohesin and condensin in the mitotic spindle

In mitosis, the pericentromere is organized into a spring composed of cohesin, condensin, and a rosette of intramolecular chromatin loops. Cohesin and condensin are enriched in the pericentromere, with spatially distinct patterns of localization. Using model convolution of computer simulations, we deduce the mechanistic consequences of their spatial segregation. Condensin lies proximal to the spindle axis, whereas cohesin is radially displaced from condensin and the interpolar microtubules. The histone deacetylase Sir2 is responsible for the axial position of condensin, while the radial displacement of chromatin loops dictates the position of cohesin. The heterogeneity in distribution of condensin is most accurately modeled by clusters along the spindle axis. In contrast, cohesin is evenly distributed (barrel of 500-nm width × 550-nm length). Models of cohesin gradients that decay from the centromere or sister cohesin axis, as previously suggested, do not match experimental images. The fine structures of cohesin and condensin deduced with subpixel localization accuracy reveal critical features of how these complexes mold pericentric chromatin into a functional spring.     

Holding chromatids together to ensure they go their separate ways

Association between sister chromatids is essential for their attachment and segregation to opposite poles of the spindle in mitosis and meiosis II. Sister-chromatid cohesion is also likely to be involved in linking homologous chromosomes together in meiosis I. Cytological observations provide evidence that attachment between sister chromatids is different in meiosis and mitosis and suggest that cohesion between the chromatid arms may differ mechanistically from that at the centromere. The physical nature of cohesion is addressed, and proteins that are candidates for holding sister chromatids together are discussed. Dissolution of sister-chromatid cohesion must be regulated precisely, and potential mechanisms to release cohesion are presented.     

Wapl controls the dynamic association of cohesin with chromatin

Cohesin establishes sister-chromatid cohesion from S phase until mitosis or meiosis. To allow chromosome segregation, cohesion has to be dissolved. In vertebrate cells, this process is mediated in part by the protease separase, which destroys a small amount of cohesin, but most cohesin is removed from chromosomes without proteolysis. How this is achieved is poorly understood. Here, we show that the interaction between cohesin and chromatin is controlled by Wapl, a protein implicated in heterochromatin formation and tumorigenesis. Wapl is associated with cohesin throughout the cell cycle, and its depletion blocks cohesin dissociation from chromosomes during the early stages of mitosis and prevents the resolution of sister chromatids until anaphase, which occurs after a delay. Wapl depletion also increases the residence time of cohesin on chromatin in interphase. Our data indicate that Wapl is required to unlock cohesin from a particular state in which it is stably bound to chromatin.     

The ‘kinetochore maintenance loop’—The mark of regulation?

Kinetochores can form and be maintained on DNA sequences that are normally non‐centromeric. The existence of these so‐called neo‐centromeres has posed the problem as to the nature of the epigenetic mechanisms that maintain the centromere. Here we highlight results that indicate that the amount of CENP‐A at human centromeres is tightly regulated. It is also known that kinetochore assembly requires sister chromatid cohesion at mitosis. We therefore suggest that separation or stretching between the sister chromatids at metaphase reciprocally determines the amount of centromere assembly in the subsequent interphase. This reciprocal relationship forms the basis of a negative feedback loop that could precisely control the amount of CENP‐A and faithfully maintain the presence of a kinetochore over many cell divisions. We describe how the feedback loop would work, propose how it could be tested experimentally and suggest possible components of its mechanism.     

Sister-chromatid cohesion via MEI-S332 and kinetochore assembly are separable functions of the Drosophila centromere

Attachment, or cohesion, between sister chromatids is essential for their proper segregation in mitosis and meiosis [1,2]. Sister chromatids are tightly apposed at their centromeric regions, but it is not known whether this is due to cohesion at the functional centromere or at flanking centric heterochromatin. The Drosophila MEI-S332 protein maintains sister-chromatid cohesion at the centromeric region [3]. By analyzing MEI-S332's localization requirements at the centromere on a set of minichromosome derivatives [4], we tested the role of heterochromatin and the relationship between cohesion and kinetochore formation in a complex centromere of a higher eukaryote. The frequency of MEI-S332 localization is decreased on minichromosomes with compromised inheritance, despite the consistent presence of two kinetochore proteins. Furthermore, MEI-S332 localization is not coincident with kinetochore outer-plate proteins, suggesting that it is located near the DNA. We conclude that MEI-S332 localization is driven by the functional centromeric chromatin, and binding of MEI-S332 is regulated independently of kinetochore formation. These results suggest that in higher eukaryotes cohesion is controlled by the functional centromere, and that, in contrast to yeast [5], the requirements for cohesion are separable from those for kinetochore assembly.     

Stable kinetochore-microtubule attachment constrains centromere positioning in metaphase

With a single microtubule attachment, budding-yeast kinetochores provide an excellent system for understanding the coordinated linkage to dynamic microtubule plus ends for chromosome oscillation and positioning. Fluorescent tagging of kinetochore proteins indicates that, on average, all centromeres are clustered, distinctly separated from their sisters, and positioned equidistant from their respective spindle poles during metaphase. However, individual fluorescent chromosome markers near the centromere transiently reassociate with their sisters and oscillate from one spindle half to the other. To reconcile the apparent disparity between the average centromere position and individual centromere proximal markers, we utilized fluorescence recovery after photobleaching to measure stability of the histone-H3 variant Cse4p/CENP-A. Newly synthesized Cse4p replaces old protein during DNA replication. Once assembled, Cse4-GFP is a physically stable component of centromeres during mitosis. This allowed us to follow centromere dynamics within each spindle half. Kinetochores remain stably attached to dynamic microtubules and exhibit a low incidence of switching orientation or position between the spindle halves. Switching of sister chromatid attachment may be contemporaneous with Cse4p exchange and early kinetochore assembly during S phase; this would promote mixing of chromosome attachment to each spindle pole. Once biorientation is attained, centromeres rarely make excursions beyond their proximal half spindle.     

A solid foundation: functional specialization of centromeric chromatin

Centromeres provide a distinctive mechanical function for the chromosomes as the site of kinetochore assembly and force generation in mitosis and meiosis. Recent studies show that a unique form of chromatin, based on the histone-H3-like protein CENP-A and homologues, provides a conserved foundation for this mechanical chromatin domain. CENP-A plays a role in templating kinetochore assembly and may be a central element in the epigenetic maintenance of centromere identity. Cohesion at the centromere, intimately linked to kinetochore assembly, is required for integrating spindle forces exerted across the centromere and for establishing the bipolar geometry of sister kinetochores.     

Function and Assembly of DNA Looping,Clustering, and Microtubule Attachment Complexes within a Eukaryotic Kinetochore

The kinetochore is a complex protein–DNA assembly that provides the mechanical linkage between microtubules and the centromere DNA of each chromosome. Centromere DNA in all eukaryotes is wrapped around a unique nucleosome that contains the histone H3 variant CENP-A (Cse4p in Saccharomyces cerevisiae). Here, we report that the inner kinetochore complex (CBF3) is required for pericentric DNA looping at the Cse4p-containing nucleosome. DNA within the pericentric loop occupies a spatially confined area that is radially displaced from the interpolar central spindle. Microtubule-binding kinetochore complexes are not involved in pericentric DNA looping but are required for the geometric organization of DNA loops around the spindle microtubules in metaphase. Thus, the mitotic segregation apparatus is a composite structure composed of kinetochore and interpolar microtubules, the kinetochore, and organized pericentric DNA loops. The linkage of microtubule-binding to centromere DNA-looping complexes positions the pericentric chromatin loops and stabilizes the dynamic properties of individual kinetochore complexes in mitosis.