Incomplete sister chromatid separation of long chromosome arms

Chromosome segregation ensures the equal partitioning of chromosomes at mitosis. However, long chromosome arms may pose a problem for complete sister chromatid separation. In this paper we report on the analysis of cell division in primary cells from field vole Microtus agrestis, a species with 52 chromosomes including two giant sex chromosomes. Dual chromosome painting with probes specific for the X and the Y chromosomes showed that these long chromosomes are prone to mis-segregate, producing DNA bridges between daughter nuclei and micronuclei. Analysis of mitotic cells with incomplete chromatid separation showed that reassembly of the nuclear membrane, deposition of INner CENtromere Protein (INCENP)/Aurora B to the spindle midzone and furrow formation occur while the two groups of daughter chromosomes are still connected by sex chromosome arms. Late cytokinetic processes are not efficiently inhibited by the incomplete segregation as in a significant number of cell divisions cytoplasmic abscission proceeds while Aurora B is at the midbody. Live-cell imaging during late mitotic stages also revealed abnormal cell division with persistent sister chromatid connections. We conclude that late mitotic regulatory events do not monitor incomplete sister chromatid separation of the large X and Y chromosomes of Microtus agrestis, leading to defective segregation of these chromosomes. These findings suggest a limit in chromosome arm length for efficient chromosome transmission through mitosis.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Regulation of sister chromatid cohesion during the mitotic cell cycle

Orderly execution of two critical events during the cell cycle––DNA replication and chromosome segregation––ensures the stable transmission of genetic materials. The cohesin complex physically connects sister chromatids during DNA replication in a process termed sister chromatid cohesion. Timely establishment and dissolution of sister chromatid cohesion is a prerequisite for accurate chromosome segregation, and is tight regulated by the cell cycle machinery and cohesin-associated proteins. In this review, we discuss recent progress in the molecular understanding of sister chromatid cohesion during the mitotic cell cycle.     

The mechanism of sister chromatid cohesion

Each of our cells inherit their genetic information in the form of chromosomes from a mother cell. In order that we obtain the full genetic complement, cells need to ensure that replicated chromosomes are accurately split and distributed during cell division. Mistakes in this process lead to aneuploidies, cells with supernumerous or missing chromosomes. Most aneuploid human embryos are not viable, and if they are, they develop severe birth defects. Aneuploidies later in human life are frequently found associated with the development of malignant cancer. DNA replication during S-phase is linked to segregation of the sister copies in mitosis by sister chromatid cohesion. A chromosomal protein complex, cohesin, holds replicated sister DNA strands together after their synthesis. This allows pairs of replication products to be recognised by the spindle apparatus in mitosis for segregation into opposite direction. At anaphase onset, cohesin is destroyed by a site-specific protease, separase. Here I review what we have learned about the molecular mechanism of sister chromatid cohesion. Cohesin forms a large proteinaceous ring that may hold sister chromatids by encircling and topological trapping. To understand how cohesin links newly synthesised replication products, biochemical assays to study the enzymology of cohesin will be required.     

Targeted sister chromatid cohesion by Sir2

The protein complex known as cohesin binds pericentric regions and other sites of eukaryotic genomes to mediate cohesion of sister chromatids. In budding yeast Saccharomyces cerevisiae, cohesin also binds silent chromatin, a repressive chromatin structure that functionally resembles heterochromatin of higher eukaryotes. We developed a protein-targeting assay to investigate the mechanistic basis for cohesion of silent chromatin domains. Individual silencing factors were tethered to sites where pairing of sister chromatids could be evaluated by fluorescence microscopy. We report that the evolutionarily conserved Sir2 histone deacetylase, an essential silent chromatin component, was both necessary and sufficient for cohesion. The cohesin genes were required, but the Sir2 deacetylase activity and other silencing factors were not. Binding of cohesin to silent chromatin was achieved with a small carboxyl terminal fragment of Sir2. Taken together, these data define a unique role for Sir2 in cohesion of silent chromatin that is distinct from the enzyme's role as a histone deacetylase.     

Minichromosome analysis of chromosome pairing, disjunction, and sister chromatid cohesion in maize

With the advent of engineered minichromosome technology in plants, an understanding of the properties of small chromosomes is desirable. Twenty-two minichromosomes of related origin but varying in size are described that provide a unique resource to study such behavior. Fourteen minichromosomes from this set could pair with each other in meiotic prophase at frequencies between 25 and 100%, but for the smaller chromosomes, the sister chromatids precociously separated in anaphase I. The other eight minichromosomes did not pair with themselves, and the sister chromatids divided equationally at meiosis I. In plants containing one minichromosome, the sister chromatids also separated at meiosis I. In anaphase II, the minichromosomes progressed to one pole or the other. The maize (Zea mays) Shugoshin protein, which has been hypothesized to protect centromere cohesion in meiosis I, is still present at anaphase I on minichromosomes that divide equationally. Also, there were no differences in the level of phosphorylation of Ser-10 of histone H3, a correlate of cohesion, in the minichromosomes in which sister chromatids separated during anaphase I compared with the normal chromosomes. These analyses suggest that meiotic centromeric cohesion is compromised in minichromosomes depending on their size and cannot be maintained by the mechanisms used by normal-sized chromosomes.     

Sister chromatid cohesion along arms and at centromeres

There is an obvious difference between the regulation of sister chromatid cohesion at centromeres and along chromosome arms during meiosis, because centromeric cohesion, but not arm cohesion, persists throughout anaphase of the first meiotic division. This regional difference of sister chromatid cohesion is also observed during mitosis; the cohesion is much more robust at the centromere at metaphase, where it antagonizes the pulling force of spindle microtubules that attach to the kinetochores from opposite poles. Recent studies have illuminated the underlying molecular mechanisms that strengthen and protect centromeric cohesion in mitosis and meiosis, and the central role of a conserved protein, shugoshin.     

Meiotic cohesin STAG3 is required for chromosome axis formation and sister chromatid cohesion

The cohesin complex is essential for mitosis and meiosis. The specific meiotic roles of individual cohesin proteins are incompletely understood. We report in vivo functions of the only meiosis‐specific STAG component of cohesin, STAG3. Newly generated STAG3‐deficient mice of both sexes are sterile with meiotic arrest. In these mice, meiotic chromosome architecture is severely disrupted as no bona fide axial elements (AE) form and homologous chromosomes do not synapse. Axial element protein SYCP3 forms dot‐like structures, many partially overlapping with centromeres. Asynapsis marker HORMAD1 is diffusely distributed throughout the chromatin, and SYCP1, which normally marks synapsed axes, is largely absent. Centromeric and telomeric sister chromatid cohesion are impaired. Centromere and telomere clustering occurs in the absence of STAG3, and telomere structure is not severely affected. Other cohesin proteins are present, localize throughout the STAG3‐devoid chromatin, and form complexes with cohesin SMC1β. No other deficiency in a single meiosis‐specific cohesin causes a phenotype as drastic as STAG3 deficiency. STAG3 emerges as the key STAG cohesin involved in major functions of meiotic cohesin.     

S-phase checkpoint genes safeguard high-fidelity sister chromatid cohesion

Cohesion establishment and maintenance are carried out by proteins that modify the activity of Cohesin, an essential complex that holds sister chromatids together. Constituents of the replication fork, such as the DNA polymerase alpha-binding protein Ctf4, contribute to cohesion in ways that are poorly understood. To identify additional cohesion components, we analyzed a ctf4Delta synthetic lethal screen performed on microarrays. We focused on a subset of ctf4Delta-interacting genes with genetic instability of their own. Our analyses revealed that 17 previously studied genes are also necessary for the maintenance of robust association of sisters in metaphase. Among these were subunits of the MRX complex, which forms a molecular structure similar to Cohesin. Further investigation indicated that the MRX complex did not contribute to metaphase cohesion independent of Cohesin, although an additional role may be contributed by XRS2. In general, results from the screen indicated a sister chromatid cohesion role for a specific subset of genes that function in DNA replication and repair. This subset is particularly enriched for genes that support the S-phase checkpoint. We suggest that these genes promote and protect a chromatin environment conducive to robust cohesion.     

Sororin mediates sister chromatid cohesion by antagonizing Wapl

Sister chromatid cohesion is essential for chromosome segregation and is mediated by cohesin bound to DNA. Cohesin-DNA interactions can be reversed by the cohesion-associated protein Wapl, whereas a stably DNA-bound form of cohesin is thought to mediate cohesion. In vertebrates, Sororin is essential for cohesion and stable cohesin-DNA interactions, but how Sororin performs these functions is unknown. We show that DNA replication and cohesin acetylation promote binding of Sororin to cohesin, and that Sororin displaces Wapl from its binding partner Pds5. In the absence of Wapl, Sororin becomes dispensable for cohesion. We propose that Sororin maintains cohesion by inhibiting Wapl's ability to dissociate cohesin from DNA. Sororin has only been identified in vertebrates, but we show that many invertebrate species contain Sororin-related proteins, and that one of these, Dalmatian, is essential for cohesion in Drosophila. The mechanism we describe here may therefore be widely conserved among different species.     

Calpain 2 is required for sister chromatid cohesion

Calpains form a family of Ca²⁺-dependent cysteine proteases involved in diverse cellular processes. However, the specific functions of each calpain isoform remain unknown. Recent reports have shown that calpain 2 (Capn2) is essential for cell viability. We have recently shown that Capn2 is a nuclear protease associated with chromosomes during mitosis in mammalian embryonic cells. We now report that Capn2 depletion impairs mitosis and induces apoptosis in murine cells. Low Capn2 levels induce chromosome alignment defects, the loss of histone H3 threonine 3 phosphorylation at centromeres, and premature sister chromatid separation. Thus, Capn2 may play a role in fundamental mitotic functions, such as the maintenance of sister chromatid cohesion.     

A physical assay for sister chromatid cohesion in vitro

Cohesion between sister chromatids depends on a multiprotein complex called cohesin that has been proposed to hold sister DNAs together by trapping them inside a large tripartite ring. Sister chromatid cohesion has hitherto only been detected by using cytological methods in living cells. We show here that cohesion between the sister DNAs of circular minichromosomes established in vivo can be detected in vitro by velocity gradient sedimentation and agarose-gel electrophoresis. This ex vivo cohesion does not depend on intercatenation of sister DNAs but is destroyed by cleavage of cohesin's Scc1 subunit or minichromosome linearization. These data represent the best evidence so far that the cohesin ring physically holds sister DNAs together and are consistent with the notion that it does so by using a topological principle involving the trapping DNAs inside its ring.     

A multi-step pathway for the establishment of sister chromatid cohesion

The cohesion of sister chromatids is mediated by cohesin, a protein complex containing members of the structural maintenance of chromosome (Smc) family. How cohesins tether sister chromatids is not yet understood. Here, we mutate SMC1, the gene encoding a cohesin subunit of budding yeast, by random insertion dominant negative mutagenesis to generate alleles that are highly informative for cohesin assembly and function. Cohesins mutated in the Hinge or Loop1 regions of Smc1 bind chromatin by a mechanism similar to wild-type cohesin, but fail to enrich at cohesin-associated regions (CARs) and pericentric regions. Hence, the Hinge and Loop1 regions of Smc1 are essential for the specific chromatin binding of cohesin. This specific binding and a subsequent Ctf7/Eco1-dependent step are both required for the establishment of cohesion. We propose that a cohesin or cohesin oligomer tethers the sister chromatids through two chromatin-binding events that are regulated spatially by CAR binding and temporally by Ctf7 activation, to ensure cohesins crosslink only sister chromatids.     

Identification of protein complexes required for efficient sister chromatid cohesion

Ctf8p is a component of Ctf18-RFC, an alternative replication factor C-like complex required for efficient sister chromatid cohesion in Saccharomyces cerevisiae. We performed synthetic genetic array (SGA) analysis with a ctf8 deletion strain as a primary screen to identify other nonessential genes required for efficient sister chromatid cohesion. We then assessed proficiency of cohesion at three chromosomal loci in strains containing deletions of the genes identified in the ctf8 SGA screen. Deletion of seven genes (CHL1, CSM3, BIM1, KAR3, TOF1, CTF4, and VIK1) resulted in defective sister chromatid cohesion. Mass spectrometric analysis of immunoprecipitated complexes identified a physical association between Kar3p and Vik1p and an interaction between Csm3p and Tof1p that we confirmed by coimmunoprecipitation from cell extracts. These data indicate that synthetic genetic array analysis coupled with specific secondary screens can effectively identify protein complexes functionally related to a reference gene. Furthermore, we find that genes involved in mitotic spindle integrity and positioning have a previously unrecognized role in sister chromatid cohesion.     

PCNA controls establishment of sister chromatid cohesion during S phase

Accurate segregation of the genetic material during cell division requires that sister chromatids are kept together by cohesion proteins until anaphase, when the chromatids become separated and distributed to the two daughter cells. Studies in yeast revealed that chromatid cohesion is essential for viability and is triggered by the conserved protein Eco1 (Ctf7). Cohesion must be established already in S phase in order to tie up sister chromatids instantly after replication, but how this crucial timing is achieved remains enigmatic. Here, we report that in yeast and humans Eco1 is directly physically coupled to the replication protein PCNA, a ring-shaped cofactor of DNA polymerases. Binding to PCNA is crucial, as yeast Eco1 mutants deficient in Eco1-PCNA interaction are defective in cohesion and inviable. Our study thus indicates that PCNA, a central matchmaker for replication-linked functions, is also crucially involved in the establishment of cohesion in S phase.     

Colchicine promotes a change in chromosome structure without loss of sister chromatid cohesion in prometaphase I-arrested bivalents

In somatic cells colchicine promotes the arrest of cell division at prometaphase, and chromosomes show a sequential loss of sister chromatid arm and centromere cohesion. In this study we used colchicine to analyse possible changes in chromosome structure and sister chromatid cohesion in prometaphase I-arrested bivalents of the katydid Pycnogaster cucullata. After silver staining we observed that in colchicine-arrested prometaphase I bivalents, and in contrast to what was found in control bivalents, sister kinetochores appeared individualised and sister chromatid axes were completely separated all along their length. However, this change in chromosome structure occurred without loss of sister chromatid arm cohesion. We also employed the MPM-2 monoclonal antibody against mitotic phosphoproteins on control and colchicine-treated spermatocytes. In control metaphase I bivalents this antibody labelled the tightly associated sister kinetochores and the interchromatid domain. By contrast, in colchicine-treated prometaphase I bivalents individualised sister kinetochores appeared labelled, but the interchromatid domain did not show labelling. These results support the notion that MPM-2 phosphoproteins, probably DNA topoisomerase IIalpha, located in the interchromatid domain act as "chromosomal staples" associating sister chromatid axes in metaphase I bivalents. The disappearance of these chromosomal staples would induce a change in chromosome structure, as reflected by the separation of sister kinetochores and sister axes, but without a concomitant loss of sister chromatid cohesion.     

Reduced sister chromatid cohesion acts as a tumor penetrance modifier

Sister chromatid cohesion (SCC) is an important process in chromosome segregation. ESCO2 is essential for establishment of SCC and is often deleted/altered in human cancers. We demonstrate that esco2 haploinsufficiency results in reduced SCC and accelerates the timing of tumor onset in both zebrafish and mouse p53 heterozygous null models, but not in p53 homozygous mutant or wild-type animals. These data indicate that esco2 haploinsufficiency accelerates tumor onset in a loss of heterozygosity (LOH) sensitive background. Analysis of The Cancer Genome Atlas (TCGA) confirmed ESCO2 deficient tumors have elevated number of LOH events throughout the genome. Further, we demonstrated heterozygous loss of sgo1, important in maintaining SCC, also results in reduced SCC and accelerated tumor formation in a p53 heterozygous background. Surprisingly, while we did observe elevated levels of chromosome missegregation and micronuclei formation in esco2 heterozygous mutant animals, this chromosomal instability did not contribute to the accelerated tumor onset in a p53 heterozygous background. Interestingly, SCC also plays a role in homologous recombination, and we did observe elevated levels of mitotic recombination derived p53 LOH in tumors from esco2 haploinsufficient animals; as well as elevated levels of mitotic recombination throughout the genome of human ESCO2 deficient tumors. Together these data suggest that reduced SCC contributes to accelerated tumor penetrance through elevated mitotic recombination.     

Pds5 cooperates with cohesin in maintaining sister chromatid cohesion

BACKGROUND: Sister chromatid cohesion depends on a complex called cohesin, which contains at least four subunits: Smc1, Smc3, Scc1 and Scc3. Cohesion is established during DNA replication, is partially dismantled in many, but not all, organisms during prophase, and is finally destroyed at the metaphase-to-anaphase transition. A quite separate protein called Spo76 is required for sister chromatid cohesion during meiosis in the ascomycete Sordaria. Spo76-like proteins are highly conserved amongst eukaryotes and a homologue in Aspergillus nidulans, called BimD, is required for the completion of mitosis. The isolation of the cohesin subunit Smc3 as a suppressor of BimD mutations suggests that Spo76/BimD might function in the same process as cohesin. RESULTS: We show here that the yeast homologue of Spo76, called Pds5, is essential for establishing sister chromatid cohesion and maintaining it during metaphase. We also show that Pds5 co-localizes with cohesin on chromosomes, that the chromosomal association of Pds5 and cohesin is interdependent, that Scc1 recruits Pds5 to chromosomes in G1 and that its cleavage causes dissociation of Pds5 from chromosomes at the metaphase-to-anaphase transition. CONCLUSIONS: Our data show that Pds5 functions as part of the same process as cohesin. Sequence similarities and secondary structure predictions indicate that Pds5 consists of tandemly repeated HEAT repeats, and might therefore function as a protein-protein interaction scaffold, possibly in the cohesin-DNA complex assembly.