Publications by authors named "Kota Nagasaka"

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

Wapl repression by Pax5 promotes V gene recombination by Igh loop extrusion.

Nature 2020 08 1;584(7819):142-147. Epub 2020 Jul 1.

Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria.

Nuclear processes, such as V(D)J recombination, are orchestrated by the three-dimensional organization of chromosomes at multiple levels, including compartments and topologically associated domains (TADs) consisting of chromatin loops. TADs are formed by chromatin-loop extrusion, which depends on the loop-extrusion function of the ring-shaped cohesin complex. Conversely, the cohesin-release factor Wapl restricts loop extension. The generation of a diverse antibody repertoire, providing humoral immunity to pathogens, requires the participation of all V genes in V(D)J recombination, which depends on contraction of the 2.8-Mb-long immunoglobulin heavy chain (Igh) locus by Pax5. However, how Pax5 controls Igh contraction in pro-B cells remains unknown. Here we demonstrate that locus contraction is caused by loop extrusion across the entire Igh locus. Notably, the expression of Wapl is repressed by Pax5 specifically in pro-B and pre-B cells, facilitating extended loop extrusion by increasing the residence time of cohesin on chromatin. Pax5 mediates the transcriptional repression of Wapl through a single Pax5-binding site by recruiting the polycomb repressive complex 2 to induce bivalent chromatin at the Wapl promoter. Reduced Wapl expression causes global alterations in the chromosome architecture, indicating that the potential to recombine all V genes entails structural changes of the entire genome in pro-B cells.
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http://dx.doi.org/10.1038/s41586-020-2454-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116900PMC
August 2020

ESCO1 and CTCF enable formation of long chromatin loops by protecting cohesin from WAPL.

Elife 2020 02 17;9. Epub 2020 Feb 17.

Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria.

Eukaryotic genomes are folded into loops. It is thought that these are formed by cohesin complexes extrusion, either until loop expansion is arrested by CTCF or until cohesin is removed from DNA by WAPL. Although WAPL limits cohesin's chromatin residence time to minutes, it has been reported that some loops exist for hours. How these loops can persist is unknown. We show that during G1-phase, mammalian cells contain acetylated cohesin which binds chromatin for hours, whereas cohesin binds chromatin for minutes. Our results indicate that CTCF and the acetyltransferase ESCO1 protect a subset of cohesin complexes from WAPL, thereby enable formation of long and presumably long-lived loops, and that ESCO1, like CTCF, contributes to boundary formation in chromatin looping. Our data are consistent with a model of nested loop extrusion, in which acetylated cohesin forms stable loops between CTCF sites, demarcating the boundaries of more transient cohesin extrusion activity.
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http://dx.doi.org/10.7554/eLife.52091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054000PMC
February 2020

Absolute quantification of cohesin, CTCF and their regulators in human cells.

Elife 2019 06 17;8. Epub 2019 Jun 17.

Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria.

The organisation of mammalian genomes into loops and topologically associating domains (TADs) contributes to chromatin structure, gene expression and recombination. TADs and many loops are formed by cohesin and positioned by CTCF. In proliferating cells, cohesin also mediates sister chromatid cohesion, which is essential for chromosome segregation. Current models of chromatin folding and cohesion are based on assumptions of how many cohesin and CTCF molecules organise the genome. Here we have measured absolute copy numbers and dynamics of cohesin, CTCF, NIPBL, WAPL and sororin by mass spectrometry, fluorescence-correlation spectroscopy and fluorescence recovery after photobleaching in HeLa cells. In G1-phase, there are ~250,000 nuclear cohesin complexes, of which ~ 160,000 are chromatin-bound. Comparison with chromatin immunoprecipitation-sequencing data implies that some genomic cohesin and CTCF enrichment sites are unoccupied in single cells at any one time. We discuss the implications of these findings for how cohesin can contribute to genome organisation and cohesion.
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http://dx.doi.org/10.7554/eLife.46269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6606026PMC
June 2019

Werner syndrome helicase is a selective vulnerability of microsatellite instability-high tumor cells.

Elife 2019 03 25;8. Epub 2019 Mar 25.

Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria.

Targeted cancer therapy is based on exploiting selective dependencies of tumor cells. By leveraging recent functional screening data of cancer cell lines we identify Werner syndrome helicase (WRN) as a novel specific vulnerability of microsatellite instability-high (MSI-H) cancer cells. MSI, caused by defective mismatch repair (MMR), occurs frequently in colorectal, endometrial and gastric cancers. We demonstrate that WRN inactivation selectively impairs the viability of MSI-H but not microsatellite stable (MSS) colorectal and endometrial cancer cell lines. In MSI-H cells, WRN loss results in severe genome integrity defects. ATP-binding deficient variants of WRN fail to rescue the viability phenotype of WRN-depleted MSI-H cancer cells. Reconstitution and depletion studies indicate that WRN dependence is not attributable to acute loss of MMR gene function but might arise during sustained MMR-deficiency. Our study suggests that pharmacological inhibition of WRN helicase function represents an opportunity to develop a novel targeted therapy for MSI-H cancers.
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http://dx.doi.org/10.7554/eLife.43333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6435321PMC
March 2019

Author Correction: Sister chromatid resolution is an intrinsic part of chromosome organization in prophase.

Nat Cell Biol 2018 04;20(4):503

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

In the version of this Letter originally published, the authors omitted a citation of an early study demonstrating topoisomerase-II-dependent sister chromatid resolution. This reference has now been added to the reference list as reference number 28, and the relevant text has been amended as follows to include its citation: 'Resolution must reflect the removal of sister-sister contacts, and we show here that Topo-IIα-mediated release of DNA catenation plays a major role (Fig. 4), in agreement with previous findings, whereas, surprisingly, cohesin dissociation is not strictly required (Fig. 3).' Subsequent references have been renumbered. All online versions of the Letter have been updated to reflect this change.
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http://dx.doi.org/10.1038/s41556-018-0044-0DOI Listing
April 2018

Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins.

EMBO J 2017 12 7;36(24):3573-3599. Epub 2017 Dec 7.

Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria

Mammalian genomes are spatially organized into compartments, topologically associating domains (TADs), and loops to facilitate gene regulation and other chromosomal functions. How compartments, TADs, and loops are generated is unknown. It has been proposed that cohesin forms TADs and loops by extruding chromatin loops until it encounters CTCF, but direct evidence for this hypothesis is missing. Here, we show that cohesin suppresses compartments but is required for TADs and loops, that CTCF defines their boundaries, and that the cohesin unloading factor WAPL and its PDS5 binding partners control the length of loops. In the absence of WAPL and PDS5 proteins, cohesin forms extended loops, presumably by passing CTCF sites, accumulates in axial chromosomal positions (vermicelli), and condenses chromosomes. Unexpectedly, PDS5 proteins are also required for boundary function. These results show that cohesin has an essential genome-wide function in mediating long-range chromatin interactions and support the hypothesis that cohesin creates these by loop extrusion, until it is delayed by CTCF in a manner dependent on PDS5 proteins, or until it is released from DNA by WAPL.
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http://dx.doi.org/10.15252/embj.201798004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5730888PMC
December 2017

Sister chromatid resolution is an intrinsic part of chromosome organization in prophase.

Nat Cell Biol 2016 06 2;18(6):692-9. Epub 2016 May 2.

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

The formation of mitotic chromosomes requires both compaction of chromatin and the resolution of replicated sister chromatids. Compaction occurs during mitotic prophase and prometaphase, and in prophase relies on the activity of condensin II complexes. Exactly when and how sister chromatid resolution occurs has been largely unknown, as has its molecular requirements. Here, we established a method to visualize sister resolution by sequential replication labelling with two distinct nucleotide derivatives. Quantitative three-dimensional imaging then allowed us to measure the resolution of sister chromatids throughout mitosis by calculating their non-overlapping volume within the whole chromosome. Unexpectedly, we found that sister chromatid resolution starts already at the beginning of prophase, proceeds concomitantly with chromatin compaction and is largely completed by the end of prophase. Sister chromatid resolution was abolished by inhibition of topoisomerase IIα and by depleting or preventing mitotic activation of condensin II, whereas blocking cohesin dissociation from chromosomes had little effect. Mitotic sister chromatid resolution is thus an intrinsic part of mitotic chromosome formation in prophase that relies largely on DNA decatenation and shares the molecular requirement for condensin II with prophase compaction.
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http://dx.doi.org/10.1038/ncb3353DOI Listing
June 2016

Clarifying the role of condensin in shaping chromosomes.

Nat Cell Biol 2015 Jun;17(6):711-3

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

A major controversy in the field of chromosome research has been whether condensin is required for achieving the highly compacted state of chromatin fibres in mitosis and meiosis. Through genetic experiments in mouse oocytes, condensin is now found to be indispensable for meiotic chromosome assembly by mediating chromosome compaction and disentanglement of sister chromatids and by conferring rigidity to chromosomes.
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http://dx.doi.org/10.1038/ncb3183DOI Listing
June 2015

The initial phase of chromosome condensation requires Cdk1-mediated phosphorylation of the CAP-D3 subunit of condensin II.

Genes Dev 2011 Apr;25(8):863-74

Cancer Institute, Japanese Foundation for Cancer Research (JFCR), Tokyo 135-8550, Japan.

The cell cycle transition from interphase into mitosis is best characterized by the appearance of condensed chromosomes that become microscopically visible as thread-like structures in nuclei. Biochemically, launching the mitotic program requires the activation of the mitotic cyclin-dependent kinase Cdk1 (cyclin-dependent kinase 1), but whether and how Cdk1 triggers chromosome assembly at mitotic entry are not well understood. Here we report that mitotic chromosome assembly in prophase depends on Cdk1-mediated phosphorylation of the condensin II complex. We identified Thr 1415 of the CAP-D3 subunit as a Cdk1 phosphorylation site, which proved crucial as it was required for the Polo kinase Plk1 (Polo-like kinase 1) to localize to chromosome axes through binding to CAP-D3 and thereby hyperphosphorylate the condensin II complex. Live-cell imaging analysis of cells carrying nonphosphorylatable CAP-D3 mutants in place of endogenous protein suggested that phosphorylation of Thr 1415 is required for timely chromosome condensation during prophase, and that the Plk1-mediated phosphorylation of condensin II facilitates its ability to assemble chromosomes properly. These observations provide an explanation for how Cdk1 induces chromosome assembly in cells entering mitosis, and underscore the significance of the cooperative action of Plk1 with Cdk1.
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http://dx.doi.org/10.1101/gad.2016411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3078710PMC
April 2011