Kei-ichiro Ishiguro

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.

A new meiosis-specific cohesin complex implicated in the cohesin code for homologous pairing

We identify a new mammalian cohesin subunit, RAD21‐like protein (RAD21L), with sequence similarity to RAD21 and REC8. RAD21L localizes along axial elements in early meiotic prophase, in a manner that is spatiotemporally different to either REC8 or RAD21. Remarkably, RAD21L and REC8 have symmetrical, mutually exclusive localization on the not‐yet‐synapsed homologues, implying that the cohesin patterning could provide a code for homologue recognition. RAD21 transiently localizes to axial elements after the dissociation of RAD21L and REC8 in late pachytene, a period of recombination repair. Further, we show that the removal of cohesins and synaptonemal complex during late meiotic prophase is promoted by Polo‐like kinase 1, which is similar to the mitotic prophase pathway.

A conserved KASH domain protein associates with telomeres, SUN1, and dynactin during mammalian meiosis

The mammalian meiosis-specific KASH protein KASH5 connects the telomere-associated SUN1 protein to the cytoplasmic force–generating mechanism involved in meiotic chromosome movement.

Phosphorylation of mammalian Sgo2 by Aurora B recruits PP2A and MCAK to centromeres

Shugoshin (Sgo) is a conserved centromeric protein. Mammalian Sgo1 collaborates with protein phosphatase 2A (PP2A) to protect mitotic cohesin from the prophase dissociation pathway. Although another shugoshin-like protein, Sgo2, is required for the centromeric protection of cohesion in germ cells, its precise molecular function remains largely elusive. We demonstrate that hSgo2 plays a dual role in chromosome congression and centromeric protection of cohesion in HeLa cells, while the latter function is exposed only in perturbed mitosis. These functions partly overlap with those of Aurora B, a kinase setting faithful chromosome segregation. Accordingly, we identified the phosphorylation of hSgo2 by Aurora B at the N-terminal coiled-coil region and the middle region, and showed that these phosphorylations separately promote binding of hSgo2 to PP2A and MCAK, factors required for centromeric protection and chromosome congression, respectively. Furthermore, these phosphorylations are essential for localizing PP2A and MCAK to centromeres. This mechanism seems applicable to germ cells as well. Thus, our study identifies Sgo2 as a hitherto unknown crucial cellular substrate of Aurora B in mammalian cells.

Footprint Analysis of the RAG Protein Recombination Signal Sequence Complex for V(D)J Type Recombination

ABSTRACT We have studied the interaction between recombination signal sequences (RSSs) and protein products of the truncated forms of recombination-activating genes (RAG) by gel mobility shift, DNase I footprinting, and methylation interference assays. Methylation interference with dimethyl sulfate demonstrated that binding was blocked by methylation in the nonamer at the second-position G residue in the bottom strand and at the sixth- and seventh-position A residues in the top strand. DNase I footprinting experiments demonstrated that RAG1 alone, or even a RAG1 homeodomain peptide, gave footprint patterns very similar to those obtained with the RAG1-RAG2 complex. In the heptamer, partial methylation interference was observed at the sixth-position A residue in the bottom strand. In DNase I footprinting, the heptamer region was weakly protected in the bottom strand by RAG1. The effects of RSS mutations on RAG binding were evaluated by DNA footprinting. Comparison of the RAG-RSS footprint data with the published Hin model confirmed the notion that sequence-specific RSS-RAG interaction takes place primarily between the Hin domain of the RAG1 protein and adjacent major and minor grooves of the nonamer DNA.

Meikin is a conserved regulator of meiosis-I-specific kinetochore function

The kinetochore is the crucial apparatus regulating chromosome segregation in mitosis and meiosis. Particularly in meiosis I, unlike in mitosis, sister kinetochores are captured by microtubules emanating from the same spindle pole (mono-orientation) and centromeric cohesion mediated by cohesin is protected in the following anaphase. Although meiotic kinetochore factors have been identified only in budding and fission yeasts, these molecules and their functions are thought to have diverged earlier. Therefore, a conserved mechanism for meiotic kinetochore regulation remains elusive. Here we have identified in mouse a meiosis-specific kinetochore factor that we termed MEIKIN, which functions in meiosis I but not in meiosis II or mitosis. MEIKIN plays a crucial role in both mono-orientation and centromeric cohesion protection, partly by stabilizing the localization of the cohesin protector shugoshin. These functions are mediated mainly by the activity of Polo-like kinase PLK1, which is enriched to kinetochores in a MEIKIN-dependent manner. Our integrative analysis indicates that the long-awaited key regulator of meiotic kinetochore function is Meikin, which is conserved from yeasts to humans.

The TRF1-binding protein TERB1 promotes chromosome movement and telomere rigidity in meiosis

During meiotic prophase, telomere-mediated chromosomal movement along the nuclear envelope is crucial for homologue pairing and synapsis. However, how telomeres are modified to mediate chromosome movement is largely elusive. Here we show that mammalian meiotic telomeres are fundamentally modified by a meiosis-specific Myb-domain protein, TERB1, that localizes at telomeres in mouse germ cells. TERB1 forms a heterocomplex with the canonical telomeric protein TRF1 and binds telomere repeat DNA. Disruption of Terb1 in mice abolishes meiotic chromosomal movement and impairs homologous pairing and synapsis, causing infertility in both sexes. TERB1 promotes telomere association with the nuclear envelope and deposition of the SUN–KASH complex, which recruits cytoplasmic motor complexes. TERB1 also binds and recruits cohesin to telomeres to develop structural rigidity, strikingly reminiscent of centromeres. Our study suggests that TERB1 acts as a central hub for the assembly of a conserved meiotic telomere complex required for chromosome movements.

Meiosis-specific cohesin mediates homolog recognition in mouse spermatocytes

During meiosis, homologous chromosome (homolog) pairing is promoted by several layers of regulation that include dynamic chromosome movement and meiotic recombination. However, the way in which homologs recognize each other remains a fundamental issue in chromosome biology. Here, we show that homolog recognition or association initiates upon entry into meiotic prophase before axis assembly and double-strand break (DSB) formation. This homolog association develops into tight pairing only during or after axis formation. Intriguingly, the ability to recognize homologs is retained in Sun1 knockout spermatocytes, in which telomere-directed chromosome movement is abolished, and this is the case even in Spo11 knockout spermatocytes, in which DSB-dependent DNA homology search is absent. Disruption of meiosis-specific cohesin RAD21L precludes the initial association of homologs as well as the subsequent pairing in spermatocytes. These findings suggest the intriguing possibility that homolog recognition is achieved primarily by searching for homology in the chromosome architecture as defined by meiosis-specific cohesin rather than in the DNA sequence itself.

Protein 600 Is a Microtubule/Endoplasmic Reticulum-Associated Protein in CNS Neurons

There is an increasing body of literature pointing to cytoskeletal proteins as spatial organizers and interactors of organelles. In this study, we identified protein 600 (p600) as a novel microtubule-associated protein (MAP) developmentally regulated in neurons. p600 exhibits the unique feature to interact with the endoplasmic reticulum (ER). Silencing of p600 by RNA interference (RNAi) destabilizes neuronal processes in young primary neurons undergoing neurite extension and containing scarce staining of the ER marker Bip. Furthermore, in utero electroporation of p600 RNAi alters neuronal migration, a process that depends on synergistic actions of microtubule dynamics and ER functions. p600-depleted migrating neurons display thin, crooked, and “zigzag” leading process with very few ER membranes. Thus, p600 constitutes the only known MAP to associate with the ER in neurons, and this interaction may impact on multiple cellular processes ranging from neuronal development to neuronal maturation and plasticity.