Shigehiro A. Kawashima

The conserved kinetochore protein shugoshin protects centromeric cohesion during meiosis.

Meiosis comprises a pair of specialized nuclear divisions that produce haploid germ cells. To accomplish this, sister chromatids must segregate together during the first meiotic division (meiosis I), which requires that sister chromatid cohesion persists at centromeres. The factors that protect centromeric cohesion during meiosis I have remained elusive. Here we identify Sgo1 (shugoshin), a protector of the centromeric cohesin Rec8 in fission yeast. We also identify a homologue of Sgo1 in budding yeast. We provide evidence that shugoshin is widely conserved among eukaryotes. Moreover, we identify Sgo2, a paralogue of shugoshin in fission yeast, which is required for faithful mitotic chromosome segregation. Localization of Sgo1 and Sgo2 at centromeres requires the kinase Bub1, identifying shugoshin as a crucial target for the kinetochore function of Bub1. These findings provide insights into the evolution of meiosis and kinetochore regulation during mitosis and meiosis.

Shugoshin collaborates with protein phosphatase 2A to protect cohesin

Sister chromatid cohesion, mediated by a complex called cohesin, is crucial—particularly at centromeres—for proper chromosome segregation in mitosis and meiosis. In animal mitotic cells, phosphorylation of cohesin promotes its dissociation from chromosomes, but centromeric cohesin is protected by shugoshin until kinetochores are properly captured by the spindle microtubules. However, the mechanism of shugoshin-dependent protection of cohesin is unknown. Here we find a specific subtype of serine/threonine protein phosphatase 2A (PP2A) associating with human shugoshin. PP2A colocalizes with shugoshin at centromeres and is required for centromeric protection. Purified shugoshin complex has an ability to reverse the phosphorylation of cohesin in vitro, suggesting that dephosphorylation of cohesin is the mechanism of protection at centromeres. Meiotic shugoshin of fission yeast also associates with PP2A, with both proteins collaboratively protecting Rec8-containing cohesin at centromeres. Thus, we have revealed a conserved mechanism of centromeric protection of eukaryotic chromosomes in mitosis and meiosis.

Phosphorylation of H2A by Bub1 Prevents Chromosomal Instability Through Localizing Shugoshin

Recruiting the Components for Cell Division A complete chromosome set must be apportioned to each daughter cell during cell division. A number of molecular mechanisms check that chromosome pairs or homologs are correctly aligned and attached to microtubules just before they separate to the two daughters; their spatial orientation ensures an even inheritance of the genome. One critical component of this system is the protein kinase Bub1. Kawashima et al. (p. 172, see the Perspective by Javerzat; published online 19 November) now show that the main substrate for Bub1 kinase activity in fission yeast is the chromatin protein histone H2A. Phosphorylation of H2A recruits the shugoshin proteins to chromatin, and especially to centromeres, where they also act to ensure correct chromosome segregation. This link between Bub1, histone H2A phosphorylation, and shugoshin is conserved in budding yeast and mammalian cells. Phosphorylation of the chromatin protein histone H2A plays a critical role in chromosome segregation during cell division. Bub1 is a multi-task protein kinase required for proper chromosome segregation in eukaryotes. Impairment of Bub1 in humans may lead to chromosomal instability (CIN) or tumorigenesis. Yet, the primary cellular substrate of Bub1 has remained elusive. Here, we show that Bub1 phosphorylates the conserved serine 121 of histone H2A in fission yeast Schizosaccharomyces pombe. The h2a-SA mutant, in which all cellular H2A-S121 is replaced by alanine, phenocopies the bub1 kinase-dead mutant (bub1-KD) in losing the centromeric localization of shugoshin proteins. Artificial tethering of shugoshin to centromeres largely restores the h2a-SA or bub1-KD–related CIN defects, a function that is evolutionally conserved. Thus, Bub1 kinase creates a mark for shugoshin localization and the correct partitioning of chromosomes.

Aurora controls sister kinetochore mono-orientation and homolog bi-orientation in meiosis-I

Aurora‐B kinases are important regulators of mitotic chromosome segregation, where they are required for the faithful bi‐orientation of sister chromatids. In contrast to mitosis, sister chromatids have to be oriented toward the same spindle pole in meiosis‐I, while homologous chromosomes are bi‐oriented. We find that the fission yeast Aurora kinase Ark1 is required for the faithful bi‐orientation of sister chromatids in mitosis and of homologous chromosomes in meiosis‐I. Unexpectedly, Ark1 is also necessary for the faithful mono‐orientation of sister chromatids in meiosis‐I, even though the canonical mono‐orientation pathway, which depends on Moa1 and Rec8, seems intact. Our data suggest that Ark1 prevents unified sister kinetochores during metaphase‐I from merotelic attachment to both spindle poles and thus from being torn apart during anaphase‐I, revealing a novel mechanism promoting monopolar attachment. Furthermore, our results provide an explanation for the previously enigmatic observation that fission yeast Shugoshin Sgo2, which assists in loading Aurora to centromeres, and its regulator Bub1 are required for the mono‐orientation of sister chromatids in meiosis‐I.

Examining post‐translational modification‐mediated protein–protein interactions using a chemical proteomics approach

Post‐translational modifications (PTM) of proteins can control complex and dynamic cellular processes via regulating interactions between key proteins. To understand these regulatory mechanisms, it is critical that we can profile the PTM‐dependent protein–protein interactions. However, identifying these interactions can be very difficult using available approaches, as PTMs can be dynamic and often mediate relatively weak protein–protein interactions. We have recently developed CLASPI (cross‐linking‐assisted and stable isotope labeling in cell culture‐based protein identification), a chemical proteomics approach to examine protein–protein interactions mediated by methylation in human cell lysates. Here, we report three extensions of the CLASPI approach. First, we show that CLASPI can be used to analyze methylation‐dependent protein–protein interactions in lysates of fission yeast, a genetically tractable model organism. For these studies, we examined trimethylated histone H3 lysine‐9 (H3K9Me3)‐dependent protein–protein interactions. Second, we demonstrate that CLASPI can be used to examine phosphorylation‐dependent protein–protein interactions. In particular, we profile proteins recognizing phosphorylated histone H3 threonine‐3 (H3T3‐Phos), a mitotic histone “mark” appearing exclusively during cell division. Our approach identified survivin, the only known H3T3‐Phos‐binding protein, as well as other proteins, such as MCAK and KIF2A, that are likely to be involved in weak but selective interactions with this histone phosphorylation “mark”. Finally, we demonstrate that the CLASPI approach can be used to study the interplay between histone H3T3‐Phos and trimethylation on the adjacent residue lysine 4 (H3K4Me3). Together, our findings indicate the CLASPI approach can be broadly applied to profile protein–protein interactions mediated by PTMs.

A chemical biology strategy to analyze rheostat-like protein kinase-dependent regulation.

Protein kinases may function more like variable rheostats rather than two-state switches. However, we lack approaches to properly analyze this aspect of kinase-dependent regulation. To address this, we develop a strategy in which a kinase inhibitor is identified using genetics-based screens, kinase mutations that confer resistance are characterized, and dose-dependent responses of isogenic drug-sensitive and resistant cells to inhibitor treatments are compared. This approach has the advantage that function of wild-type kinase, rather than mutants, is examined. To develop this approach, we focus on Ark1, the fission yeast member of the conserved Aurora kinase family. Applying this approach reveals that proper chromosome compaction in fission yeast needs high Ark1 activity, while other processes depend on significantly lower activity levels. Our strategy is general and can be used to examine the functions of other molecular rheostats.

Potent, Reversible, and Specific Chemical Inhibitors of Eukaryotic Ribosome Biogenesis

Summary All cellular proteins are synthesized by ribosomes, whose biogenesis in eukaryotes is a complex multi-step process completed within minutes. Several chemical inhibitors of ribosome function are available and used as tools or drugs. By contrast, we lack potent validated chemical probes to analyze the dynamics of eukaryotic ribosome assembly. Here, we combine chemical and genetic approaches to discover ribozinoindoles (or Rbins), potent and reversible triazinoindole-based inhibitors of eukaryotic ribosome biogenesis. Analyses of Rbin sensitivity and resistance conferring mutations in fission yeast, along with biochemical assays with recombinant proteins, provide evidence that Rbins’ physiological target is Midasin, an essential ∼540-kDa AAA+ (ATPases associated with diverse cellular activities) protein. Using Rbins to acutely inhibit or activate Midasin function, in parallel experiments with inhibitor-sensitive or inhibitor-resistant cells, we uncover Midasin’s role in assembling Nsa1 particles, nucleolar precursors of the 60S subunit. Together, our findings demonstrate that Rbins are powerful probes for eukaryotic ribosome assembly.

Supramolecular Ligands for Histone Tails by Employing a Multivalent Display of Trisulfonated Calix[4]arenes

Post‐translational modification of histone tails plays critical roles in gene regulation. Thus, molecules recognizing histone tails and controlling their epigenetic modification are desirable as biochemical tools to elucidate regulatory mechanisms. There are, however, only a few synthetic ligands that bind to histone tails with substantial affinity. We report CA2 and CA3, which exhibited sub‐micromolar affinity to histone tails (especially tails with a trimethylated lysine). Multivalent display of trisulfonated calix[4]arene was important for strong binding. CA2 was applicable not only to synthetic tail peptides but also to endogenous histone proteins, and was successfully used to pull‐down endogenous histones from nuclear extract. These findings indicate the utility of these supramolecular ligands as biochemical tools for studying chromatin regulator protein and as a targeting motif in ligand‐directed catalysis to control epigenetic modifications.

Dissecting the first and the second meiotic divisions using a marker-less drug-hypersensitive fission yeast.

Faithful chromosome segregation during meiosis is indispensable to prevent birth defects and infertility. Canonical genetic manipulations have not been very useful for studying meiosis II, since mutations of genes involved in cell cycle regulation or chromosome segregation may affect meiosis I, making interpretations of any defects observed in meiosis II complicated. Here we present a powerful strategy to dissect meiosis I and meiosis II, using chemical inhibitors in genetically tractable model organism fission yeast (Schizosaccharomyces pombe). As various chemical probes are not active in fission yeast, mainly due to an effective multidrug resistance (MDR) response, we have recently developed a drug-hypersensitive MDR-sup strain by suppression of the key genes responsible for MDR response. We further developed the MDR-supML (marker-less) strain by deleting 7 MDR genes without commonly used antibiotic markers. The new strain makes fluorescent tagging and gene deletion much simpler, which enables effective protein visualization in varied genetic backgrounds. Using the MDR-supML strain with chemical inhibitors and live cell fluorescence microscopy, we established cell cycle arrest at meiosis I and meiosis II and examined Aurora-dependent spindle assembly checkpoint (SAC) regulation during meiosis. We found that Aurora B/Ark1 kinase activity is required for recruitment of Bub1, an essential SAC kinase, to unattached kinetochore in prometaphase I and prometaphase II as in mitosis. Thus, Aurora’s role in SAC activation is likely conserved in mitosis, meiosis I, and meiosis II. Together, our MDR-supML strain will be useful to dissect complex molecular mechanisms in mitosis and 2 successive meiotic divisions.

Optimization of the analogue-sensitive Cdc2/Cdk1 mutant by in vivo selection eliminates physiological limitations to its use in cell cycle analysis

Analogue-sensitive (as) mutants of kinases are widely used to selectively inhibit a single kinase with few off-target effects. The analogue-sensitive mutant cdc2-as of fission yeast (Schizosaccharomyces pombe) is a powerful tool to study the cell cycle, but the strain displays meiotic defects, and is sensitive to high and low temperature even in the absence of ATP-analogue inhibitors. This has limited the use of the strain for use in these settings. Here, we used in vivo selection for intragenic suppressor mutations of cdc2-as that restore full function in the absence of ATP-analogues. The cdc2-asM17 underwent meiosis and produced viable spores to a similar degree to the wild-type strain. The suppressor mutation also rescued the sensitivity of the cdc2-as strain to high and low temperature, genotoxins and an anti-microtubule drug. We have used cdc2-asM17 to show that Cdc2 activity is required to maintain the activity of the spindle assembly checkpoint. Furthermore, we also demonstrate that maintenance of the Shugoshin Sgo1 at meiotic centromeres does not require Cdc2 activity, whereas localization of the kinase aurora does. The modified cdc2-asM17 allele can be thus used to analyse many aspects of cell-cycle-related events in fission yeast.