Publications by authors named "Norihisa Shindo"

10 Publications

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Kinetochore stretching-mediated rapid silencing of the spindle-assembly checkpoint required for failsafe chromosome segregation.

Curr Biol 2021 Feb 22. Epub 2021 Feb 22.

Division of Experimental Pathology, Cancer Institute of the Japanese Foundation for Cancer Research, Tokyo, Japan. Electronic address:

The spindle-assembly checkpoint facilitates mitotic fidelity by delaying anaphase onset in response to microtubule vacancy at kinetochores. Following microtubule attachment, kinetochores receive microtubule-derived force, which causes kinetochores to undergo repetitive cycles of deformation; this phenomenon is referred to as kinetochore stretching. The nature of the forces and the relevance relating this deformation are not well understood. Here, we show that kinetochore stretching occurs within a framework of single end-on attached kinetochores, irrespective of microtubule poleward pulling force. An experimental method to conditionally interfere with the stretching allowed us to determine that kinetochore stretching comprises an essential process of checkpoint silencing by promoting PP1 phosphatase recruitment after the establishment of end-on attachments and removal of the majority of checkpoint-activating kinase Mps1 from kinetochores. Remarkably, we found that a lower frequency of kinetochore stretching largely correlates with a prolonged metaphase in cancer cell lines with chromosomal instability. Perturbation of kinetochore stretching and checkpoint silencing in chromosomally stable cells produced anaphase bridges, which can be alleviated by reducing chromosome-loaded cohesin. These observations indicate that kinetochore stretching-mediated checkpoint silencing provides an unanticipated etiology underlying chromosomal instability and underscores the importance of a rapid metaphase-to-anaphase transition in sustaining mitotic fidelity.
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http://dx.doi.org/10.1016/j.cub.2021.01.062DOI Listing
February 2021

Prolonged mitosis causes separase deregulation and chromosome nondisjunction.

Cell Rep 2021 Jan;34(3):108652

Division of Experimental Pathology, Cancer Institute of the Japanese Foundation for Cancer Research (JFCR), Ariake 3-8-31 Koto-ku, 135-8550 Tokyo, Japan. Electronic address:

During mitotic chromosome segregation, the protease separase severs cohesin between sister chromatids. A probe for separase activity has shown that separase undergoes abrupt activation shortly before anaphase onset, after being suppressed throughout metaphase; however, the relevance of this control remains unclear. Here, we report that separase activates precociously, with respect to anaphase onset, during prolonged metaphase in multiple types of cancer cell lines. The artificial extension of metaphase in chromosomally stable diploid cells leads to precocious activation and, subsequently, to chromosomal bridges in anaphase, which seems to be attributable to incomplete cohesin removal. Conversely, shortening back of a prolonged metaphase restores the activation of separase and ameliorates anaphase bridge formation. These observations suggest that retarded metaphase progression affects the separase activation profile and its enzymatic proficiency. Our findings provide an unanticipated etiology for chromosomal instability in cancers and underscore the relevance of swift mitotic transitions for fail-safe chromosome segregation.
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http://dx.doi.org/10.1016/j.celrep.2020.108652DOI Listing
January 2021

Quantitative analyses of the metaphase-to-anaphase transition reveal differential kinetic regulation for securin and cyclin B1.

Biomed Res 2018 ;39(2):75-85

Division of Experimental Pathology, Cancer Institute of the Japanese Foundation for Cancer Research (JFCR).

Separation of sister chromatids is a drastic and irreversible step in the cell cycle. The key biochemistry behind this event is the proteolysis mediated by the ubiquitin ligase called the anaphase promoting complex, or APC/C. Securin and cyclin B1 are the two established substrates for APC/C whose degradation releases separase and inactivates cyclin B1-dependent kinase 1 (cdk1), respectively, at the metaphase-to-anaphase transition. In this study, we have combined biochemical quantifications with mathematical simulations to characterize the kinetic regulation of securin and cyclin B1, in the cytoplasmic and chromosomal compartments, and found that they are differentially distributed and degraded with different rates. Modeling their interaction with separase predicted that activation timing of separase well coincides with the decline of securin-separase concentration in the cytoplasm. Notably, it also coincides with the peak of cyclin B1-separase level on chromosomes, which appeared crucial to coordinate the timing for separase activation and cdk1 inhibition. We have also conducted phosphoproteomic analysis and identified Ki67 as a chromosomal cdk1 substrate whose dephosphorylation is facilitated by cyclin B1-separase interaction in anaphase.
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http://dx.doi.org/10.2220/biomedres.39.75DOI Listing
September 2018

Separase sensor reveals dual roles for separase coordinating cohesin cleavage and cdk1 inhibition.

Dev Cell 2012 Jul;23(1):112-23

Cancer Institute of the Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo, Japan.

Complete dissociation of sister chromatid cohesion and subsequent induction of poleward movement of disjoined sisters are two essential events underlying chromosome segregation; however, how cells coordinate these two processes is not well understood. Here, we developed a fluorescence-based sensor for the protease separase that mediates cohesin cleavage. We found that separase undergoes an abrupt activation shortly before anaphase onset in the vicinity of chromosomes. This activation profile of separase depends on the abilities of two of its binding proteins, securin and cyclin B1, to inhibit its protease activity and target it to chromosomes. Subsequent to its proteolytic activation, separase then binds to and inhibits a subset of cyclin B1-cdk1, which antagonizes cdk1-mediated phosphorylation on chromosomes and facilitates poleward movement of sisters in anaphase. Therefore, by consecutively acting as a protease and a cdk1 inhibitor, separase coordinates two key processes to achieve simultaneous and abrupt separation of sister chromatids.
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http://dx.doi.org/10.1016/j.devcel.2012.06.015DOI Listing
July 2012

Polycomb group protein Ezh1 represses Nodal and maintains the left-right axis.

Dev Biol 2010 May 19;341(2):459-63. Epub 2010 Mar 19.

Department of Biology, Waseda University, 2-2 Wakamatsu-cho, Shinjuku, Tokyo 162-8480, Japan.

The left-right (LR) axis is essential for the proper function of internal organs. In mammals and fish, left-sided Nodal expression governs LR patterning. Here, we show that the Polycomb group protein Ezh1, which is highly conserved from fish to human, participates in LR patterning. Knockdown of olezh1, a medaka homologue of Ezh1, led to LR reversal of internal organs. It was shown that OLEZH1 acts in silencing the expression of Spaw (a medaka homolog of Nodal) via a previously unknown pathway. Furthermore, coimmunoprecipitation showed physical interaction of Ezh1 with FoxH1, a Nodal regulator. This represents a novel mechanism for LR patterning and implies that Ezh1 has developmental importance.
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http://dx.doi.org/10.1016/j.ydbio.2010.03.002DOI Listing
May 2010

[Histone modification: a new era of targeting epigenetics].

Authors:
Norihisa Shindo

Rinsho Ketsueki 2009 Apr;50(4):282-8

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April 2009

A histone H1 variant is required for erythrocyte maturation in medaka.

Int J Dev Biol 2008 ;52(7):887-92

Center for Advanced Life and Medical Science, Waseda University, Tokyo, Japan.

Three histone H1 variants were identified in medaka fish and their sequence characteristics were analyzed. This paper reports one of these variants, termed H1-2, because of its possible implication in erythrocyte maturation. The amino acid sequence of H1-2 was phylogenetically similar to that of other replication-dependent histones. The mRNA transcribed from the h1-2 gene, however, possessed a poly(A) tail without a stem-loop structure, indicating that H1-2 may represent a replication-independent (RI) histone. Transcripts from the h1-2 gene were largely localized in erythrocytes, and knock-down of the h1-2 gene with morpholino antisense oligos resulted in failure to develop mature erythrocytes. In the morphants, residual erythrocytes showed severely impaired nuclear compaction. Although not structurally related to chicken RI histone H5, which is required for erythrocyte maturation, H1-2 may constitute its functional counterpart. Our findings may offer evolutionary insights into the function of H1 variants.
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http://dx.doi.org/10.1387/ijdb.072568omDOI Listing
January 2009

Zebrafish Polycomb group gene ph2alpha is required for epiboly and tailbud formation acting downstream of FGF signaling.

Biochem Biophys Res Commun 2005 Mar;328(4):858-66

Department of Biology, School of Education and in Integrated Bioscience and Biomedical Engineering, Graduate School of Science and Engineering, Waseda University, 1-6-1 Nishi-Waseda, Shinjuku, Tokyo 169-8050, Japan.

We analyzed Polycomb group gene ph2alpha functionally in zebrafish embryos by a gene knock-down procedure using morpholino antisense oligos. Inhibition of ph2alpha message translation resulted in abnormal epibolic movements as well as a thick tailbud or incomplete covering of the yolk plug. At the 24hpf stage, morphants had short trunks and tails, phenotypes similar to those with disturbances in FGF signaling. Accordingly, we looked at the effects of ph2alpha expression upstream and downstream of the FGF pathway. Treatment with SU5402, an inhibitor of Fgfrs, or injection of dominant-negative Fgfr1 DNA markedly reduced ph2alpha expression in the tailbud. In addition, cells expressing mRNAs for no tail, spadetail, myoD, and papc, which are involved in FGF-related development of posterior mesoderm, were distributed abnormally. Collectively, the data argue that ph2alpha is required for epiboly and tailbud formation, acting downstream of the FGF signaling pathway.
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http://dx.doi.org/10.1016/j.bbrc.2005.01.031DOI Listing
March 2005

The ESC-E(Z) complex participates in the hedgehog signaling pathway.

Biochem Biophys Res Commun 2005 Feb;327(4):1179-87

Department of Biology, Faculty of Education and Integrated Arts and Sciences, Major in Integrated Bioscience and Biomedical Engineering, Graduate School of Science and Engineering, Waseda University, 1-6-1 Nishi-Waseda, Shinjuku-ku, Tokyo 169-8050, Japan.

Polycomb group (PcG) genes are required for stable inheritance of epigenetic states throughout development, a phenomenon termed cellular memory. In Drosophila and mice, the product of the E(z) gene, one of the PcG genes, constitutes the ESC-E(Z) complex and specifically methylates histone H3. It has been argued that this methylation sets the stage for appropriate repression of certain genes. Here, we report the isolation of a well-conserved homolog of E(z), olezh2, in medaka. Hypomorphic knock-down of olezh2 resulted in a cyclopia phenotype and markedly perturbed hedgehog signaling, consistent with our previous report on oleed, a medaka esc. We also found cyclopia in embryos treated with trichostatin A, an inhibitor of histone deacetylase, which is a transient component of the ESC-E(Z) complex. The level of tri-methylation at lysine 27 of histone H3 was substantially decreased in both olezh2 and oleed knock-down embryos, and in embryos with hedgehog signaling perturbed by forskolin. We conclude that the ESC-E(Z) complex per se participates in hedgehog signaling.
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http://dx.doi.org/10.1016/j.bbrc.2004.12.125DOI Listing
February 2005

Participation of Polycomb group gene extra sex combs in hedgehog signaling pathway.

Biochem Biophys Res Commun 2004 Oct;323(2):523-33

Department of Biology, School of Education and Major in Integrated Bioscience and Biomedical Engineering, Graduate School of Science and Engineering, Waseda University, 1-6-1 Nishi-Waseda, Shinjuku, Tokyo 169-8050, Japan.

Polycomb group (PcG) genes are required for stable inheritance of epigenetic states across cell divisions, a phenomenon termed cellular memory. PcG proteins form multimeric nuclear complex which modifies the chromatin structure of target site. Drosophila PcG gene extra sex combs (esc) and its vertebrate orthologs constitute a member of ESC-E(Z) complex, which possesses histone methyltransferase activity. Here we report isolation and characterization of medaka esc homolog, termed oleed. Hypomorphic knock-down of oleed using morpholino antisense oligonucleotides resulted in the fusion of eyes, termed cyclopia. Prechordal plate formation was not substantially impaired, but expression of hedgehog target genes was dependent on oleed, suggesting some link with hedgehog signaling. In support of this implication, histone methylation, which requires the activity of esc gene product, is increased in hedgehog stimulated mouse NIH-3T3 cells. Our data argue for the novel role of esc in hedgehog signaling and provide fundamental insight into the epigenetic mechanisms in general.
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http://dx.doi.org/10.1016/j.bbrc.2004.08.125DOI Listing
October 2004