Yuya Yamagishi

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.

Two Histone Marks Establish the Inner Centromere and Chromosome Bi-Orientation

Location, Location, Location Cell division is orchestrated by a complex signaling pathway that ensures the correct segregation of newly replicated chromosomes to the two daughter cells. The pathway is controlled in part by restricting the activity of critical regulators to specific subcellular locations. For example, the chromosomal passenger complex (CPC) is recruited to chromosomes during mitosis where it oversees kinetochore activity and cytokinesis (see Perspective by Musacchio). Wang et al. (p. 231, published online 12 August), Kelly et al. (p. 235, published online 12 August), and Yamagishi et al. (p. 239) now show that the phosphorylation of the chromatin protein, histone H3, acts to bring the CPC to chromosomes, thereby activating its aurora B kinase subunit. The Survivin subunit of CPC binds specifically to phosphorylated H3, with the phosphorylation at centromeres being carried out by the mitosis-specific kinase, haspin. Furthermore, Bub1 phosphorylation of histone H2A recruits shugoshin, a centromeric CPC adapter. Thus, these two histone marks in combination define the inner centromere. Phosphorylation of histones recruits proteins critical for the segregation of chromosomes during cell division. For proper partitioning of chromosomes in mitosis, the chromosomal passenger complex (CPC) including Aurora B and survivin must be localized at the center of paired kinetochores, at the site called the inner centromere. It is largely unknown what defines the inner centromere and how the CPC is targeted to this site. Here, we show that the phosphorylation of histone H3–threonine 3 (H3-pT3) mediated by Haspin cooperates with Bub1-mediated histone 2A–serine 121 (H2A-S121) phosphorylation in targeting the CPC to the inner centromere in fission yeast and human cells. H3-pT3 promotes nucleosome binding of survivin, whereas phosphorylated H2A-S121 facilitates the binding of shugoshin, the centromeric CPC adaptor. Haspin colocalizes with cohesin by associating with Pds5, whereas Bub1 localizes at kinetochores. Thus, the inner centromere is defined by intersection of two histone kinases.

A Neuronal Identity Code for the Odorant Receptor-Specific and Activity-Dependent Axon Sorting

In the mouse, olfactory sensory neurons (OSNs) expressing the same odorant receptor (OR) converge their axons to a specific set of glomeruli in the olfactory bulb. To study how OR-instructed axonal fasciculation is controlled, we searched for genes whose expression profiles are correlated with the expressed ORs. Using the transgenic mouse in which the majority of OSNs express a particular OR, we identified such genes coding for the homophilic adhesive molecules Kirrel2/Kirrel3 and repulsive molecules ephrin-A5/EphA5. In the CNGA2 knockout mouse, where the odor-evoked cation influx is disrupted, Kirrel2 and EphA5 were downregulated, while Kirrel3 and ephrin-A5 were upregulated, indicating that these genes are transcribed in an activity-dependent manner. Mosaic analysis demonstrated that gain of function of these genes generates duplicated glomeruli. We propose that a specific set of adhesive/repulsive molecules, whose expression levels are determined by OR molecules, regulate the axonal fasciculation of OSNs during the process of glomerular map formation.

MPS1/Mph1 phosphorylates the kinetochore protein KNL1/Spc7 to recruit SAC components

The genomic stability of all organisms depends on the precise partition of chromosomes to daughter cells. The spindle assembly checkpoint (SAC) senses unattached kinetochores and prevents premature entry to anaphase, thus ensuring that all chromosomes attach to opposite spindle poles (bi-orientation) during mitosis. MPS1 is an evolutionarily conserved protein kinase required for the SAC and chromosome bi-orientation. Yet, its primary cellular substrate has remained elusive. We show that fission yeast Mph1 (MPS1 homologue) phosphorylates the kinetochore protein Spc7 (KNL1/Blinkin homologue) at the MELT repeat sequences. This phosphorylation promotes the in vitro binding to the Bub1–Bub3 complex, which is required for kinetochore-based SAC activation (Mad1–Mad2–Mad3 localization) and chromosome alignment. Accordingly, a non-phosphorylatable spc7-12A mutation abolishes kinetochore targeting of Bub1–Bub3, whereas a phospho-mimetic spc7-12E mutation forces them to localize at kinetochores throughout the entire cell cycle, even in the absence of Mph1. Thus, MPS1/Mph1 kinase locating at the unattached kinetochores initially creates a mark, which is crucial for SAC activation and chromosome bi-orientation. This mechanism seems to be conserved in human cells.

Heterochromatin links to centromeric protection by recruiting shugoshin

The centromere of a chromosome is composed mainly of two domains, a kinetochore assembling core centromere and peri-centromeric heterochromatin regions. The crucial role of centromeric heterochromatin is still unknown, because even in simpler unicellular organisms such as the fission yeast Schizosaccharomyces pombe, the heterochromatin protein Swi6 (HP1 homologue) has several functions at centromeres, including silencing gene expression and recombination, enriching cohesin, promoting kinetochore assembly, and, ultimately, preventing erroneous microtubule attachment to the kinetochores. Here we show that the requirement of heterochromatin for mitotic chromosome segregation is largely replaced by forcibly enriching cohesin at centromeres in fission yeast. However, this enrichment of cohesin is not sufficient to replace the meiotic requirement for heterochromatin. We find that the heterochromatin protein Swi6 associates directly with meiosis-specific shugoshin Sgo1, a protector of cohesin at centromeres. A point mutation of Sgo1 (V242E), which abolishes the interaction with Swi6, impairs the centromeric localization and function of Sgo1. The forced centromeric localization of Sgo1 restores proper meiotic chromosome segregation in swi6Δ cells. We also show that the direct link between HP1 and shugoshin is conserved in human cells. Taken together, our findings suggest that the recruitment of shugoshin is the important primary role for centromeric heterochromatin in ensuring eukaryotic chromosome segregation.

Kinetochore composition and its function: lessons from yeasts

Proper chromosome segregation during cell division is essential for proliferation, and this is facilitated by kinetochores, large protein complexes assembled on the centromeric region of the chromosomes. Although the sequences of centromeric DNA differ totally among organisms, many components of the kinetochores assembled on centromeres are very well conserved among eukaryotes. To define the identity of centromeres, centromere protein A (CENP-A), which is homologous to canonical histone H3, acts as a landmark for kinetochore assembly. Kinetochores mediate spindle–microtubule attachment and control the movement of chromosomes during mitosis and meiosis. To conduct faithful chromosome segregation, kinetochore assembly and microtubule attachment are elaborately regulated. Here we review the current understanding of the composition, assembly, functions and regulation of kinetochores revealed mainly through studies on fission and budding yeasts. Moreover, because recent cumulative evidence suggests the importance of the regulation of the orientation of kinetochore–microtubule attachment, which differs distinctly between mitosis and meiosis, we focus especially on the molecular mechanisms underlying this regulation.

Acetylation regulates monopolar attachment at multiple levels during meiosis I in fission yeast

In fission yeast, meiotic mono‐orientation of sister kinetochores is established by cohesion at the core centromere, which is established by a meiotic cohesin complex and the kinetochore protein Moa1. The cohesin subunit Psm3 is acetylated by Eso1 and deacetylated by Clr6. We show that in meiosis, Eso1 is required for establishing core centromere cohesion during S phase, whereas Moa1 is required for maintaining this cohesion after S phase. The clr6‐1 mutation suppresses the mono‐orientation defect of moa1Δ cells, although the Clr6 target for this suppression is not Psm3. Thus, several acetylations are crucial for establishing and maintaining core centromere cohesion.

Pds5 Regulates Sister-Chromatid Cohesion and Chromosome Bi-orientation through a Conserved Protein Interaction Module

Sister-chromatid cohesion is established by the cohesin complex in S phase and persists until metaphase, when sister chromatids are captured by microtubules emanating from opposite poles [1]. The Aurora-B-containing chromosome passenger complex (CPC) plays a crucial role in achieving chromosome bi-orientation by correcting erroneous microtubule attachment [2]. The centromeric localization of the CPC relies largely on histone H3-T3 phosphorylation (H3-pT3), which is mediated by the mitotic histone kinase Haspin/Hrk1 [3-5]. Hrk1 localization to centromeres depends largely on the cohesin subunit Pds5 in fission yeast [5]; however, it is unknown how Pds5 regulates Hrk1 localization. Here we identify a conserved Hrk1-interacting motif (HIM) in Pds5 and a Pds5-interacting motif (PIM) in Hrk1 in fission yeast. Mutations in either motif result in the displacement of Hrk1 from centromeres. We also show that the mechanism of Pds5-dependent Hrk1 recruitment is conserved in human cells. Notably, the PIM in Haspin/Hrk1 is reminiscent of the YSR motif found in the mammalian cohesin destabilizer Wapl and stabilizer Sororin, both of which bind PDS5 [6-12]. Similarly, and through the same motifs, fission yeast Pds5 binds to Wpl1/Wapl and acetyltransferase Eso1/Eco1, in addition to Hrk1. Thus, we have identified a protein-protein interaction module in Pds5 that serves as a chromatin platform for regulating sister-chromatid cohesion and chromosome bi-orientation.

Meikin-associated polo-like kinase specifies Bub1 distribution in meiosis I

In meiosis I, sister chromatids are captured by microtubules emanating from the same pole (mono‐orientation), and centromeric cohesion is protected throughout anaphase. Shugoshin, which is localized to centromeres depending on the phosphorylation of histone H2A by Bub1 kinase, plays a central role in protecting meiotic cohesin Rec8 from separase cleavage. Another key meiotic kinetochore factor, meikin, may regulate cohesion protection, although the underlying molecular mechanisms remain elusive. Here, we show that fission yeast Moa1 (meikin), which associates stably with CENP‐C during meiosis I, recruits Plo1 (polo‐like kinase) to the kinetochores and phosphorylates Spc7 (KNL1) to accumulate Bub1. Consequently, in contrast to the transient kinetochore localization of mitotic Bub1, meiotic Bub1 persists at kinetochores until anaphase I. The meiotic Bub1 pool ensures robust Sgo1 (shugoshin) localization and cohesion protection at centromeres by cooperating with heterochromatin protein Swi6, which binds and stabilizes Sgo1. Furthermore, molecular genetic analyses show a hierarchical regulation of centromeric cohesion protection by meikin and shugoshin that is important for establishing meiosis‐specific chromosome segregation. We provide evidence that the meiosis‐specific Bub1 regulation is conserved in mouse.

Author Correction: Heterochromatin links to centromeric protection by recruiting shugoshin

An Amendment to this Letter has been published and is linked from the HTML version of this paper.