Laura A. Díaz-Martínez

Mutational inactivation of STAG2 causes aneuploidy in human cancer.

Tumors harbor mutations that disrupt chromatid separation during cell division, leading to chromosomal abnormalities. Most cancer cells are characterized by aneuploidy, an abnormal number of chromosomes. We have identified a clue to the mechanistic origins of aneuploidy through integrative genomic analyses of human tumors. A diverse range of tumor types were found to harbor deletions or inactivating mutations of STAG2, a gene encoding a subunit of the cohesin complex, which regulates the separation of sister chromatids during cell division. Because STAG2 is on the X chromosome, its inactivation requires only a single mutational event. Studying a near-diploid human cell line with a stable karyotype, we found that targeted inactivation of STAG2 led to chromatid cohesion defects and aneuploidy, whereas in two aneuploid human glioblastoma cell lines, targeted correction of the endogenous mutant alleles of STAG2 led to enhanced chromosomal stability. Thus, genetic disruption of cohesin is a cause of aneuploidy in human cancer.

PIASγ Is Required for Faithful Chromosome Segregation in Human Cells

Background The precision of the metaphase-anaphase transition ensures stable genetic inheritance. The spindle checkpoint blocks anaphase onset until the last chromosome biorients at metaphase plate, then the bonds between sister chromatids are removed and disjoined chromatids segregate to the spindle poles. But, how sister separation is triggered is not fully understood. Principal Findings We identify PIASγ as a human E3 sumo ligase required for timely and efficient sister chromatid separation. In cells lacking PIASγ, normal metaphase plates form, but the spindle checkpoint is activated, leading to a prolonged metaphase block. Sister chromatids remain cohered even if cohesin is removed by depletion of hSgo1, because DNA catenations persist at centromeres. PIASγ-depleted cells cannot properly localize Topoisomerase II at centromeres or in the cores of mitotic chromosomes, providing a functional link between PIASγ and Topoisomerase II. Conclusions PIASγ directs Topoisomerase II to specific chromosome regions that require efficient removal of DNA catenations prior to anaphase. The lack of this activity activates the spindle checkpoint, protecting cells from non-disjunction. Because DNA catenations persist without PIASγ in the absence of cohesin, removal of catenations and cohesin rings must be regulated in parallel.

Chromosome cohesion - Rings, knots, orcs and fellowship

Sister-chromatid cohesion is essential for accurate chromosome segregation. A key discovery towards our understanding of sister-chromatid cohesion was made 10 years ago with the identification of cohesins. Since then, cohesins have been shown to be involved in cohesion in numerous organisms, from yeast to mammals. Studies of the composition, regulation and structure of the cohesin complex led to a model in which cohesin loading during S-phase establishes cohesion, and cohesin cleavage at the onset of anaphase allows sister-chromatid separation. However, recent studies have revealed activities that provide cohesion in the absence of cohesin. Here we review these advances and propose an integrative model in which chromatid cohesion is a result of the combined activities of multiple cohesion mechanisms.

The Cdc20-binding Phe Box of the Spindle Checkpoint Protein BubR1 Maintains the Mitotic Checkpoint Complex during Mitosis

Background: The spindle checkpoint protein BubR1 inhibits the anaphase-promoting complex through binding to Cdc20. Results: We identify a new Cdc20-binding motif within BubR1 termed the Phe box. Conclusion: The Phe box maintains steady-state levels of BubR1-containing checkpoint complexes in human cells. Significance: Our study provides key insights into the homeostatic mechanisms of a key spindle checkpoint complex. The spindle checkpoint ensures accurate chromosome segregation by monitoring kinetochore-microtubule attachment. Unattached or tensionless kinetochores activate the checkpoint and enhance the production of the mitotic checkpoint complex (MCC) consisting of BubR1, Bub3, Mad2, and Cdc20. MCC is a critical checkpoint inhibitor of the anaphase-promoting complex/cyclosome, a ubiquitin ligase required for anaphase onset. The N-terminal region of BubR1 binds to both Cdc20 and Mad2, thus nucleating MCC formation. The middle region of human BubR1 (BubR1M) also interacts with Cdc20, but the nature and function of this interaction are not understood. Here we identify two critical motifs within BubR1M that contribute to Cdc20 binding and anaphase-promoting complex/cyclosome inhibition: a destruction box (D box) and a phenylalanine-containing motif termed the Phe box. A BubR1 mutant lacking these motifs is defective in MCC maintenance in mitotic human cells but is capable of supporting spindle-checkpoint function. Thus, the BubR1M-Cdc20 interaction indirectly contributes to MCC homeostasis. Its apparent dispensability in the spindle checkpoint might be due to functional duality or redundant, competing mechanisms.

Regulated separation of sister centromeres depends on the spindle assembly checkpoint but not on the anaphase promoting complex/cyclosome.

Key to faithful genetic inheritance is the cohesion between sister centromeres that physically links replicated sister chromatids and is then abruptly lost at the onset of anaphase. Misregulated cohesion causes aneuploidy, birth defects and perhaps initiates cancers. Loss of centromere cohesion is controlled by the spindle checkpoint and is thought to depend on a ubiquitin ligase, the Anaphase Promoting Complex/Cyclosome (APC). But here we present evidence that the APC pathway is dispensable for centromere separation at anaphase in mammals, and that anaphase proceeds in the presence of cyclin B and securin. Arm separation is perturbed in the absence of APC, compromising the fidelity of segregation, but full sister chromatid separation is achieved after a delayed anaphase. Thereafter, cells arrest terminally in telophase with high levels of cyclin B. Extending these findings we provide evidence that the spindle checkpoint regulates centromere cohesion through an APC-independent pathway. We propose that this Centromere Linkage Pathway (CLiP) is a second branch that stems from the spindle checkpoint to regulate cohesion preferentially at the centromeres and that Sgo1 is one of its components. Supplemental Figures

Yeast UBL-UBA proteins have partially redundant functions in cell cycle control

BackgroundProteins containing ubiquitin-like (UBL) and ubiquitin associated (UBA) domains have been suggested to shuttle ubiquitinated substrates to the proteasome for degradation. There are three UBL-UBA containing proteins in budding yeast: Ddi1, Dsk2 and Rad23, which have been demonstrated to play regulatory roles in targeting ubiquitinated substrates to the proteasome for degradation. An involvement of these proteins in cell cycle related events has also been reported. We tested whether these three proteins act redundantly in the cell cycle.ResultsHere we show that the UBL-UBA proteins are partially redundant for cell cycle related roles. RAD23 is redundant with DDI1 and DSK2, but DDI1 and DSK2 are not redundant with each other and the triple deletion shows a synthetic effect, suggesting the existence of at least two roles for RAD23 in cell cycle control. The rad23Δddi1Δdsk2Δ triple deletion strain delays both in G2/M-phase and in mid-anaphase at high temperatures with duplicated spindle pole bodies. Cell cycle progression in the triple deletion strain can only be partially rescued by a rad23 allele lacking the c-terminal UBA domain, suggesting that RAD23 requires its c-terminal UBA domain for full function. In addition to their ability to bind ubiquitin and the proteasome, the UBL-UBA proteins also share the ability to homodimerize. Rad23 and Dsk2 dimerization requires their UBL and/or UBA domains whereas Ddi1 dimerization does not. Here we show that Ddi1 homodimerization is necessary for its cell cycle related functions.ConclusionThe three yeast UBL-UBA proteins have partially redundant roles required for progression through mitosis.

Topoisomerase II Checkpoints: Universal Mechanisms that Regulate Mitosis

Checkpoint controls confer order to the cell cycle and help prevent genome instability. Here we discuss the Topoisomerase II (Decatenation) Checkpoint which functions to regulate mitotic progression so that chromosomes can be efficiently condensed in prophase and can be segregated with high fidelity in anaphase.

Cohesin is dispensable for centromere cohesion in human cells.

Background Proper regulation of the cohesion at the centromeres of human chromosomes is essential for accurate genome transmission. Exactly how cohesion is maintained and is then dissolved in anaphase is not understood. Principal Findings We have investigated the role of the cohesin complex at centromeres in human cells both by depleting cohesin subunits using RNA interference and also by expressing a non-cleavable version of the Rad21 cohesin protein. Rad21 depletion results in aberrant anaphase, during which the sister chromatids separate and segregate in an asynchronous fashion. However, centromere cohesion was maintained before anaphase in Rad21-depleted cells, and the primary constrictions at centromeres were indistinguishable from those in control cells. Expression of non-cleavable Rad21 (NC-Rad21), in which the sites normally cleaved by separase are mutated, resulted in delayed sister chromatid resolution in prophase and prometaphase, and a blockage of chromosome arm separation in anaphase, but did not impede centromere separation. Conclusions These data indicate that cohesin complexes are dispensable for sister cohesion in early mitosis, yet play an important part in the fidelity of sister separation and segregation during anaphase. Cleavage at the separase-sensitive sites of Rad21 is important for arm separation, but not for centromere separation.

Genome-wide siRNA screen reveals coupling between mitotic apoptosis and adaptation.

The antimitotic anti‐cancer drugs, including taxol, perturb spindle dynamics, and induce prolonged, spindle checkpoint‐dependent mitotic arrest in cancer cells. These cells then either undergo apoptosis triggered by the intrinsic mitochondrial pathway or exit mitosis without proper cell division in an adaptation pathway. Using a genome‐wide small interfering RNA (siRNA) screen in taxol‐treated HeLa cells, we systematically identify components of the mitotic apoptosis and adaptation pathways. We show that the Mad2 inhibitor p31comet actively promotes mitotic adaptation through cyclin B1 degradation and has a minor separate function in suppressing apoptosis. Conversely, the pro‐apoptotic Bcl2 family member, Noxa, is a critical initiator of mitotic cell death. Unexpectedly, the upstream components of the mitochondrial apoptosis pathway and the mitochondrial fission protein Drp1 contribute to mitotic adaption. Our results reveal crosstalk between the apoptosis and adaptation pathways during mitotic arrest.

Running on a treadmill: dynamic inhibition of APC/C by the spindle checkpoint

During mitosis, the genome duplicated during S-phase is synchronously and accurately segregated to the two daughter cells. The spindle checkpoint prevents premature sister-chromatid separation and mitotic exit. The anaphase-promoting complex/cyclosome (APC/C) is a key target of the spindle checkpoint. Upon checkpoint activation, the mitotic checkpoint complex (MCC) containing Mad2, Bub3, Mad3/BubR1 and Cdc20 inhibits APC/C. Two independent studies in budding yeast have now shed light on the mechanism by which MCC inhibits APC/C. These studies indicate that Mad3 binds to the mitotic activator of APC/C Cdc20 using peptide motifs commonly found in APC/C substrates and thus competes with APC/C substrates for APC/CCdc20 binding. In addition, Mad3 binding to APC/CCdc20 induces Cdc20 ubiquitination by APC/C, leading to the dissociation of MCC. Meanwhile, two other studies have shown that a deubiquitinating enzyme is required for the spindle checkpoint whereas APC/C-dependent ubiquitination is needed for checkpoint inactivation. Collectively, these studies suggest a dynamic model for APC/CCdc20 regulation by MCC in which APC/C- and Mad3-dependent ubiquitination of Cdc20 constitutes a self-regulated switch that rapidly inactivates the spindle checkpoint upon correct chromosome attachment.