Publications by authors named "Tatsuo Fukagawa"

115 Publications

CENP-C Phosphorylation by CDK1 .

Bio Protoc 2021 Jan 5;11(1):e3879. Epub 2021 Jan 5.

Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan.

Accurate chromosome segregation during mitosis requires the kinetochore, a large protein complex, which makes a linkage between chromosomes and spindle microtubes. An essential kinetochore component, CENP-C, is phosphorylated by Cyclin-B-Cyclin dependent kinase 1 (CDK1) that is a master kinase for mitotic progression, promoting proper kinetochore assembly during mitosis. Here, we describe an CDK1 kinase assay to detect CENP-C phosphorylation using Phos-tag SDS-PAGE without radiolabeled ATP. Our protocol has advantages in ease and safety over conventional phosphorylation assays using [γ-P]-ATP, which has potential hazards despite their better sensitivity. The protocol described here can be applicable to other kinases and be also useful for analysis of phospho-sites in substrates .
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http://dx.doi.org/10.21769/BioProtoc.3879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7952944PMC
January 2021

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

Cryo-EM structure of the CENP-A nucleosome in complex with phosphorylated CENP-C.

EMBO J 2021 Mar 19;40(5):e105671. Epub 2021 Jan 19.

Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan.

The CENP-A nucleosome is a key structure for kinetochore assembly. Once the CENP-A nucleosome is established in the centromere, additional proteins recognize the CENP-A nucleosome to form a kinetochore. CENP-C and CENP-N are CENP-A binding proteins. We previously demonstrated that vertebrate CENP-C binding to the CENP-A nucleosome is regulated by CDK1-mediated CENP-C phosphorylation. However, it is still unknown how the phosphorylation of CENP-C regulates its binding to CENP-A. It is also not completely understood how and whether CENP-C and CENP-N act together on the CENP-A nucleosome. Here, using cryo-electron microscopy (cryo-EM) in combination with biochemical approaches, we reveal a stable CENP-A nucleosome-binding mode of CENP-C through unique regions. The chicken CENP-C structure bound to the CENP-A nucleosome is stabilized by an intramolecular link through the phosphorylated CENP-C residue. The stable CENP-A-CENP-C complex excludes CENP-N from the CENP-A nucleosome. These findings provide mechanistic insights into the dynamic kinetochore assembly regulated by CDK1-mediated CENP-C phosphorylation.
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http://dx.doi.org/10.15252/embj.2020105671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7917552PMC
March 2021

Bridgin connects the outer kinetochore to centromeric chromatin.

Nat Commun 2021 01 8;12(1):146. Epub 2021 Jan 8.

Molecular Mycology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Center for Advanced Scientific Research (JNCASR), Bangalore, India, 560064.

The microtubule-binding outer kinetochore is coupled to centromeric chromatin through CENP-C, CENP-T, and CENP-U linker pathways originating from the constitutive centromere associated network (CCAN) of the inner kinetochore. Here, we demonstrate the recurrent loss of most CCAN components, including certain kinetochore linkers during the evolution of the fungal phylum of Basidiomycota. By kinetochore interactome analyses in a model basidiomycete and human pathogen Cryptococcus neoformans, a forkhead-associated domain containing protein "bridgin" was identified as a kinetochore component along with other predicted kinetochore proteins. In vivo and in vitro functional analyses of bridgin reveal its ability to connect the outer kinetochore with centromeric chromatin to ensure accurate chromosome segregation. Unlike established CCAN-based linkers, bridgin is recruited at the outer kinetochore establishing its role as a distinct family of kinetochore proteins. Presence of bridgin homologs in non-fungal lineages suggests an ancient divergent strategy exists to bridge the outer kinetochore with centromeric chromatin.
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http://dx.doi.org/10.1038/s41467-020-20161-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7794384PMC
January 2021

Essentiality of CENP-A Depends on Its Binding Mode to HJURP.

Cell Rep 2020 Nov;33(7):108388

Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan. Electronic address:

CENP-A incorporation is critical for centromere specification and is mediated by the chaperone HJURP. The CENP-A-targeting domain (CATD) of CENP-A specifically binds to HJURP, and this binding is conserved. However, the binding interface of CENP-A-HJURP is yet to be understood. Here, we identify the critical residues for chicken CENP-A or HJURP. The A59Q mutation in the α1-helix of chicken CENP-A causes CENP-A mis-incorporation and subsequent cell death, whereas the corresponding mutation in human CENP-A does not. We also find that W53 of HJURP, which is a contact site of A59 in CENP-A, is also essential in chicken cells. Our comprehensive analyses reveal that the affinities of HJURP to CATD differ between chickens and humans. However, the introduction of two arginine residues to the chicken HJURP αA-helix suppresses CENP-A mis-incorporation in chicken cells expressing CENP-A. Our data explain the mechanisms and evolution of CENP-A essentiality by the CENP-A-HJURP interaction.
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http://dx.doi.org/10.1016/j.celrep.2020.108388DOI Listing
November 2020

A super-sensitive auxin-inducible degron system with an engineered auxin-TIR1 pair.

Nucleic Acids Res 2020 10;48(18):e108

Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan.

The auxin-inducible degron (AID) system enables rapid depletion of target proteins within the cell by applying the natural auxin IAA. The AID system is useful for investigating the physiological functions of essential proteins; however, this system generally requires high dose of auxin to achieve effective depletion in vertebrate cells. Here, we describe a super-sensitive AID system that incorporates the synthetic auxin derivative 5-Ad-IAA and its high-affinity-binding partner OsTIR1F74A. The super-sensitive AID system enabled more than a 1000-fold reduction of the AID inducer concentrations in chicken DT40 cells. To apply this system to various mammalian cell lines including cancer cells containing multiple sets of chromosomes, we utilized a single-step method where CRISPR/Cas9-based gene knockout is combined with insertion of a pAID plasmid. The single-step method coupled with the super-sensitive AID system enables us to easily and rapidly generate AID-based conditional knockout cells in a wide range of vertebrate cell lines. Our improved method that incorporates the super-sensitive AID system and the single-step method provides a powerful tool for elucidating the roles of essential genes.
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http://dx.doi.org/10.1093/nar/gkaa748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7544234PMC
October 2020

H3K9me3 maintenance on a human artificial chromosome is required for segregation but not centromere epigenetic memory.

J Cell Sci 2020 07 24;133(14). Epub 2020 Jul 24.

Wellcome Trust Centre for Cell Biology, Edinburgh, UK

Most eukaryotic centromeres are located within heterochromatic regions. Paradoxically, heterochromatin can also antagonize centromere formation, and some centromeres lack it altogether. In order to investigate the importance of heterochromatin at centromeres, we used epigenetic engineering of a synthetic alphoid human artificial chromosome (HAC), to which chimeric proteins can be targeted. By tethering the JMJD2D demethylase (also known as KDM4D), we removed heterochromatin mark H3K9me3 (histone 3 lysine 9 trimethylation) specifically from the HAC centromere. This caused no short-term defects, but long-term tethering reduced HAC centromere protein levels and triggered HAC mis-segregation. However, centromeric CENP-A was maintained at a reduced level. Furthermore, HAC centromere function was compatible with an alternative low-H3K9me3, high-H3K27me3 chromatin signature, as long as residual levels of H3K9me3 remained. When JMJD2D was released from the HAC, H3K9me3 levels recovered over several days back to initial levels along with CENP-A and CENP-C centromere levels, and mitotic segregation fidelity. Our results suggest that a minimal level of heterochromatin is required to stabilize mitotic centromere function but not for maintaining centromere epigenetic memory, and that a homeostatic pathway maintains heterochromatin at centromeres.This article has an associated First Person interview with the first authors of the paper.
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http://dx.doi.org/10.1242/jcs.242610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7390644PMC
July 2020

CENP-50 is required for papilloma development in the two-stage skin carcinogenesis model.

Cancer Sci 2020 Aug 6;111(8):2850-2860. Epub 2020 Jul 6.

Department of Carcinogenesis Research, Division of Experimental Animal Research, Chiba Cancer Center Research Institute, Chiba, Japan.

CENP-50/U is a component of the CENP-O complex (CENP-O/P/Q/R/U) and localizes to the centromere throughout the cell cycle. Aberrant expression of CENP-50/U has been reported in many types of cancers. However, as Cenp-50/U-deficient mice die during early embryogenesis, its functions remain poorly understood in vivo. To investigate the role of Cenp-50/U in skin carcinogenesis, we generated Cenp-50/U conditional knockout (K14Cre -Cenp-50/U ) mice and subjected them to the 7,12-dimethylbenz(a)anthracene (DMBA)/terephthalic acid (TPA) chemical carcinogenesis protocol. As a result, early-stage papillomas decreased in Cenp-50/U-deficient mice. In contrast, Cenp-50/U-deficient mice demonstrated almost the same carcinoma incidence as control mice. Furthermore, mRNA expression analysis using DMBA/TPA-induced papillomas and carcinomas revealed that Cenp-50/U expression levels in papillomas were significantly higher than in carcinomas. These results suggest that Cenp-50/U functions mainly in early papilloma development and it has little effect on malignant conversion.
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http://dx.doi.org/10.1111/cas.14533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7419024PMC
August 2020

Dynamics of kinetochore structure and its regulations during mitotic progression.

Cell Mol Life Sci 2020 Aug 12;77(15):2981-2995. Epub 2020 Feb 12.

Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.

Faithful chromosome segregation during mitosis in eukaryotes requires attachment of the kinetochore, a large protein complex assembled on the centromere of each chromosome, to the spindle microtubules. The kinetochore is a structural interface for the microtubule attachment and provides molecular surveillance mechanisms that monitor and ensure the precise microtubule attachment as well, including error correction and spindle assembly checkpoint. During mitotic progression, the kinetochore undergoes dynamic morphological changes that are observable through electron microscopy as well as through fluorescence microscopy. These structural changes might be associated with the kinetochore function. In this review, we summarize how the dynamics of kinetochore morphology are associated with its functions and discuss recent findings on the switching of protein interaction networks in the kinetochore during cell cycle progression.
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http://dx.doi.org/10.1007/s00018-020-03472-4DOI Listing
August 2020

Artificial generation of centromeres and kinetochores to understand their structure and function.

Exp Cell Res 2020 04 6;389(2):111898. Epub 2020 Feb 6.

Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, 565-0871, Japan. Electronic address:

The centromere is an essential genomic region that provides the surface to form the kinetochore, which binds to the spindle microtubes to mediate chromosome segregation during mitosis and meiosis. Centromeres of most organisms possess highly repetitive sequences, making it difficult to study these loci. However, an unusual centromere called a "neocentromere," which does not contain repetitive sequences, was discovered in a patient and can be generated experimentally. Recent advances in genome biology techniques allow us to analyze centromeric chromatin using neocentromeres. In addition to neocentromeres, artificial kinetochores have been generated on non-centromeric loci, using protein tethering systems. These are powerful tools to understand the mechanism of the centromere specification and kinetochore assembly. In this review, we introduce recent studies utilizing the neocentromeres and artificial kinetochores and discuss current problems in centromere biology.
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http://dx.doi.org/10.1016/j.yexcr.2020.111898DOI Listing
April 2020

Cryo-EM Structures of Centromeric Tri-nucleosomes Containing a Central CENP-A Nucleosome.

Structure 2020 01 8;28(1):44-53.e4. Epub 2019 Nov 8.

Laboratory of Chromatin Structure and Function, Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan; Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan; Graduate School of Advanced Science & Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan. Electronic address:

The histone H3 variant CENP-A is a crucial epigenetic marker for centromere specification. CENP-A forms a characteristic nucleosome and dictates the higher-order configuration of centromeric chromatin. However, little is known about how the CENP-A nucleosome affects the architecture of centromeric chromatin. In this study, we reconstituted tri-nucleosomes mimicking a centromeric nucleosome arrangement containing the CENP-A nucleosome, and determined their 3D structures by cryoelectron microscopy. The H3-CENP-A-H3 tri-nucleosomes adopt an untwisted architecture, with an outward-facing linker DNA path between nucleosomes. This is distinct from the H3-H3-H3 tri-nucleosome architecture, with an inward-facing DNA path. Intriguingly, the untwisted architecture may allow the CENP-A nucleosome to be exposed to the solvent in the condensed chromatin model. These results provide a structural basis for understanding the 3D configuration of CENP-A-containing chromatin, and may explain how centromeric proteins can specifically target the CENP-A nucleosomes buried in robust amounts of H3 nucleosomes in centromeres.
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http://dx.doi.org/10.1016/j.str.2019.10.016DOI Listing
January 2020

CDK1-mediated CENP-C phosphorylation modulates CENP-A binding and mitotic kinetochore localization.

J Cell Biol 2019 12 1;218(12):4042-4062. Epub 2019 Nov 1.

Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan

The kinetochore is essential for faithful chromosome segregation during mitosis. To form a functional kinetochore, constitutive centromere-associated network (CCAN) proteins are assembled on the centromere chromatin that contains the centromere-specific histone CENP-A. CENP-C, a CCAN protein, directly interacts with the CENP-A nucleosome to nucleate the kinetochore structure. As CENP-C is a hub protein for kinetochore assembly, it is critical to address how the CENP-A-CENP-C interaction is regulated during cell cycle progression. To address this question, we investigated the CENP-C C-terminal region, including a conserved CENP-A-binding motif, in both chicken and human cells and found that CDK1-mediated phosphorylation of CENP-C facilitates its binding to CENP-A in vitro and in vivo. We observed that CENP-A binding is involved in CENP-C kinetochore localization during mitosis. We also demonstrate that the CENP-A-CENP-C interaction is critical for long-term viability in human RPE-1 cells. These results provide deeper insights into protein-interaction network plasticity in centromere proteins during cell cycle progression.
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http://dx.doi.org/10.1083/jcb.201907006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6891089PMC
December 2019

Centromere maintenance during DNA replication.

Nat Cell Biol 2019 06;21(6):669-671

Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan.

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http://dx.doi.org/10.1038/s41556-019-0335-0DOI Listing
June 2019

Where is the right path heading from the centromere to spindle microtubules?

Cell Cycle 2019 06 20;18(11):1199-1211. Epub 2019 May 20.

a Graduate School of Frontier Biosciences , Osaka University , Suita , Japan.

The kinetochore is a large protein complex that ensures accurate chromosome segregation during mitosis by connecting the centromere and spindle microtubules. One of the kinetochore sub-complexes, the constitutive centromere-associated network (CCAN), associates with the centromere and recruits another sub-complex, the KMN (KNL1, Mis12, and Ndc80 complexes) network (KMN), which binds to spindle microtubules. The CCAN-KMN interaction is mediated by two parallel pathways (CENP-C- and CENP-T-pathways) in the kinetochore, which bridge the centromere and microtubules. Here, we discuss dynamic protein-interaction changes in the two pathways that couple the centromere with spindle microtubules during mitotic progression.
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http://dx.doi.org/10.1080/15384101.2019.1617008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6592241PMC
June 2019

Live imaging of marked chromosome regions reveals their dynamic resolution and compaction in mitosis.

J Cell Biol 2019 05 11;218(5):1531-1552. Epub 2019 Mar 11.

Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, UK

When human cells enter mitosis, chromosomes undergo substantial changes in their organization to resolve sister chromatids and compact chromosomes. To comprehend the timing and coordination of these events, we need to evaluate the progression of both sister chromatid resolution and chromosome compaction in one assay. Here we achieved this by analyzing changes in configuration of marked chromosome regions over time, with high spatial and temporal resolution. This assay showed that sister chromatids cycle between nonresolved and partially resolved states with an interval of a few minutes during G2 phase before completing full resolution in prophase. Cohesins and WAPL antagonistically regulate sister chromatid resolution in late G2 and prophase while local enrichment of cohesin on chromosomes prevents precocious sister chromatid resolution. Moreover, our assay allowed quantitative evaluation of condensin II and I activities, which differentially promote sister chromatid resolution and chromosome compaction, respectively. Our assay reveals novel aspects of dynamics in mitotic chromosome resolution and compaction that were previously obscure in global chromosome assays.
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http://dx.doi.org/10.1083/jcb.201807125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6504890PMC
May 2019

The CENP-A centromere targeting domain facilitates H4K20 monomethylation in the nucleosome by structural polymorphism.

Nat Commun 2019 02 4;10(1):576. Epub 2019 Feb 4.

Laboratory of Chromatin Structure and Function, Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan.

Centromeric nucleosomes are composed of the centromere-specific histone H3 variant CENP-A and the core histones H2A, H2B, and H4. To establish a functional kinetochore, histone H4 lysine-20 (H4K20) must be monomethylated, but the underlying mechanism has remained enigmatic. To provide structural insights into H4K20 methylation, we here solve the crystal structure of a nucleosome containing an H3.1-CENP-A chimera, H3.1, which has a CENP-A centromere targeting domain and preserves essential CENP-A functions in vivo. Compared to the canonical H3.1 nucleosome, the H3.1 nucleosome exhibits conformational changes in the H4 N-terminal tail leading to a relocation of H4K20. In particular, the H4 N-terminal tail interacts with glutamine-76 and aspartate-77 of canonical H3.1 while these interactions are cancelled in the presence of the CENP-A-specific residues valine-76 and lysine-77. Mutations of valine-76 and lysine-77 impair H4K20 monomethylation both in vitro and in vivo. These findings suggest that a CENP-A-mediated structural polymorphism may explain the preferential H4K20 monomethylation in centromeric nucleosomes.
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http://dx.doi.org/10.1038/s41467-019-08314-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6362020PMC
February 2019

Publisher Correction: Multiple phosphorylations control recruitment of the KMN network onto kinetochores.

Nat Cell Biol 2018 Dec;20(12):1434

Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.

In the version of this Article originally published, the 'ON' and 'OFF' labels in panel c of Fig. 6 were incorrect. For the Tet treated cells (+Tet) in both image panels, CENP-T should have been 'OFF' and CENP-T Δ90 should have been 'ON'. For the cells untreated with Tet (-Tet) in both graph panels, CENP-T Δ90 should have been 'ON'. This has now been amended.
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http://dx.doi.org/10.1038/s41556-018-0252-7DOI Listing
December 2018

Multiple phosphorylations control recruitment of the KMN network onto kinetochores.

Nat Cell Biol 2018 12 12;20(12):1378-1388. Epub 2018 Nov 12.

Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.

To establish a functional kinetochore, the constitutive centromere-associated network (CCAN) forms a foundation on the centromere and recruits the KMN network, which directly binds to spindle microtubules. The CENP-C and CENP-T pathways in the CCAN recruit the KMN network to kinetochores, independently. The CENP-C pathway has been considered the major scaffold for the KMN network in vertebrate CCAN. However, we demonstrate that it is mainly the CENP-T pathway that recruits the KMN network onto the kinetochores and that CENP-T-KMN interactions are essential in chicken DT40 cells. By contrast, less Ndc80 binds to the CENP-C pathway in mitosis and the Mis12-CENP-C association is decreased during mitotic progression, which is consistent with the finding that the Mis12 complex-CENP-C binding is dispensable for cell viability. Furthermore, we find that multiple phosphoregulations of CENP-T and the Mis12 complex make the CENP-T pathway dominant. These results provide key insights into kinetochore dynamics during mitotic progression.
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http://dx.doi.org/10.1038/s41556-018-0230-0DOI Listing
December 2018

3D genomic architecture reveals that neocentromeres associate with heterochromatin regions.

J Cell Biol 2019 01 5;218(1):134-149. Epub 2018 Nov 5.

Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan

The centromere is an important genomic locus for chromosomal segregation. Although the centromere is specified by sequence-independent epigenetic mechanisms in most organisms, it is usually composed of highly repetitive sequences, which associate with heterochromatin. We have previously generated various chicken DT40 cell lines containing differently positioned neocentromeres, which do not contain repetitive sequences and do not associate with heterochromatin. In this study, we performed systematic 4C analysis using three cell lines containing differently positioned neocentromeres to identify neocentromere-associated regions at the 3D level. This analysis reveals that these neocentromeres commonly associate with specific heterochromatin-rich regions, which were distantly located from neocentromeres. In addition, we demonstrate that centromeric chromatin adopts a compact structure, and centromere clustering also occurs in vertebrate interphase nuclei. Interestingly, the occurrence of centromere-heterochromatin associations depend on CENP-H, but not CENP-C. Our analyses provide an insight into understanding the 3D architecture of the genome, including the centromeres.
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http://dx.doi.org/10.1083/jcb.201805003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314543PMC
January 2019

Kinetochore assembly and disassembly during mitotic entry and exit.

Curr Opin Cell Biol 2018 06 21;52:73-81. Epub 2018 Feb 21.

Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan. Electronic address:

Faithful chromosome segregation during mitosis in eukaryotes requires a large protein complex, kinetochore, formed on the centromere of each chromosome, to attach to spindle microtubules. Among the kinetochore proteins, Constitutive Centromere-Associated Network (CCAN) and KMN-network proteins form the base of the vertebrate kinetochore architecture. The CCAN proteins constitutively localize to the centromere throughout the cell cycle, whereas KMN-network proteins are recruited to the CCAN only during mitosis. Recent studies in cellular and structural biology, as well as biochemical reconstitutions, have revealed that mitotic phosphorylation of kinetochore proteins has critical roles in kinetochore organization. Here, we discuss the molecular processes of kinetochore assembly during mitotic entry and its disassembly during mitotic exit.
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http://dx.doi.org/10.1016/j.ceb.2018.02.005DOI Listing
June 2018

Site-Specific Cleavage by Topoisomerase 2: A Mark of the Core Centromere.

Int J Mol Sci 2018 Feb 10;19(2). Epub 2018 Feb 10.

Department of Genetics, University of Cambridge, Downing St, Cambridge CB2 3EH, UK.

In addition to its roles in transcription and replication, topoisomerase 2 (topo 2) is crucial in shaping mitotic chromosomes and in ensuring the orderly separation of sister chromatids. As well as its recruitment throughout the length of the mitotic chromosome, topo 2 accumulates at the primary constriction. Here, following cohesin release, the enzymatic activity of topo 2 acts to remove residual sister catenations. Intriguingly, topo 2 does not bind and cleave all sites in the genome equally; one preferred site of cleavage is within the core centromere. Discrete topo 2-centromeric cleavage sites have been identified in α-satellite DNA arrays of active human centromeres and in the centromere regions of some protozoans. In this study, we show that topo 2 cleavage sites are also a feature of the centromere in , the metazoan and in another vertebrate species, (chicken). In vertebrates, we show that this site-specific cleavage is diminished by depletion of CENP-I, an essential constitutive centromere protein. The presence, within the core centromere of a wide range of eukaryotes, of precise sites hypersensitive to topo 2 cleavage suggests that these mark a fundamental and conserved aspect of this functional domain, such as a non-canonical secondary structure.
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http://dx.doi.org/10.3390/ijms19020534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5855756PMC
February 2018

Critical Foundation of the Kinetochore: The Constitutive Centromere-Associated Network (CCAN).

Prog Mol Subcell Biol 2017;56:29-57

Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, 565-0871, Japan.

The kinetochore is a large protein complex, which is assembled at the centromere of a chromosome to ensure faithful chromosome segregation during M-phase. The centromere in most eukaryotes is epigenetically specified by DNA sequence-independent mechanisms. The constitutive centromere-associated network (CCAN) is a subcomplex in the kinetochore that localizes to the centromere throughout the cell cycle. The CCAN has interfaces bound to the centromeric chromatin and the spindle microtubule-binding complex; therefore, it functions as a foundation of kinetochore formation. Here, we summarize recent progress in our understanding of the structure and organization of the CCAN. We also discuss an additional role of the CCAN in the maintenance of centromere position and dynamic reorganization of the CCAN.
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http://dx.doi.org/10.1007/978-3-319-58592-5_2DOI Listing
May 2019

CENP-R acts bilaterally as a tumor suppressor and as an oncogene in the two-stage skin carcinogenesis model.

Cancer Sci 2017 Nov 30;108(11):2142-2148. Epub 2017 Aug 30.

Department of Carcinogenesis Research, Division of Experimental Animal Research, Chiba Cancer Center Research Institute, Chiba, Japan.

CENP-R is a component of the CENP-O complex, including CENP-O, CENP-P, CENP-Q, CENP-R, and CENP-U and is constitutively localized to kinetochores throughout the cell cycle in vertebrates. CENP-R-deficient chicken DT40 cells are viable and show a very minor effect on mitosis. To investigate the functional roles of CENP-R in vivo, we generated CENP-R-deficient mice (Cenp-r ). Mice heterozygous or homozygous for Cenp-r null mutation are viable and healthy, with no apparent defect in growth and morphology, indicating Cenp-r is not essential for normal development. Accordingly, to investigate the role of the Cenp-r gene in skin carcinogenesis, we subjected Cenp-r mice to the 7,12-dimethylbenz(a)anthracene (DMBA)/TPA chemical carcinogenesis protocol and monitored tumor development. As a result, Cenp-r mice initially developed significantly more papillomas than control wild-type mice. However, papillomas in Cenp-r mice showed a decrease of proliferative cells and an increase of apoptotic cells. As a result, they did not grow bigger and some papillomas showed substantial regression. Furthermore, papillomas in Cenp-r mice showed lower frequency of malignant conversion to squamous cell carcinomas. These results indicate Cenp-r functions bilaterally in cancer development: during early developmental stages, Cenp-r functions as a tumor suppressor, but during the expansion and progression of papillomas it functions as a tumor-promoting factor.
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http://dx.doi.org/10.1111/cas.13348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5665765PMC
November 2017

Association of M18BP1/KNL2 with CENP-A Nucleosome Is Essential for Centromere Formation in Non-mammalian Vertebrates.

Dev Cell 2017 07;42(2):181-189.e3

Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan. Electronic address:

Centromeres are specified and maintained by sequence-independent epigenetic mechanisms through the incorporation of CENP-A into centromeres. Given that CENP-A incorporation requires the Mis18 complex to be in the centromere region, it is necessary to precisely understand how the Mis18 complex localizes to the centromere region. Here, we showed that centromere localization of the Mis18 complex depends on CENP-A, but not CENP-C or CENP-T, in chicken DT40 cells. Furthermore, we demonstrated that M18BP1/KNL2, a member of the Mis18 complex, contained the CENP-C-like motif in chicken and other vertebrates, which is essential for centromere localization and M18BP1/KNL2 function in DT40 cells. We also showed that in vitro reconstituted CENP-A nucleosome, but not H3 nucleosome, bound to the CENP-C-like motif containing M18BP1/KNL2. Based on these results, we conclude that M18BP1/KNL2 is essential for centromere formation through direct binding to CENP-A nucleosome in non-mammalian vertebrates. This explains how new CENP-A recognizes the centromere position.
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http://dx.doi.org/10.1016/j.devcel.2017.06.019DOI Listing
July 2017

Critical histone post-translational modifications for centromere function and propagation.

Authors:
Tatsuo Fukagawa

Cell Cycle 2017 Jul 9;16(13):1259-1265. Epub 2017 Jun 9.

a Graduate School of Frontier Biosciences , Osaka University , Suita , Osaka , Japan.

The centromere is a critical genomic region that enables faithful chromosome segregation during mitosis, and must be distinguishable from other genomic regions to facilitate establishment of the kinetochore. The centromere-specific histone H3-variant CENP-A forms a special nucleosome that functions as a marker for centromere specification. In addition to the CENP-A nucleosomes, there are additional H3 nucleosomes that have been identified in centromeres, both of which are predicted to exhibit specific features. It is likely that the composite organization of CENP-A and H3 nucleosomes contributes to the formation of centromere-specific chromatin, termed 'centrochromatin'. Recent studies suggest that centrochromatin has specific histone modifications that mediate centromere specification and kinetochore assembly. We use chicken non-repetitive centromeres as a model of centromeric activities to characterize functional features of centrochromatin. This review discusses our recent progress, and that of various other research groups, in elucidating the functional roles of histone modifications in centrochromatin.
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http://dx.doi.org/10.1080/15384101.2017.1325044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5531634PMC
July 2017

An efficient method to generate conditional knockout cell lines for essential genes by combination of auxin-inducible degron tag and CRISPR/Cas9.

Chromosome Res 2017 10 6;25(3-4):253-260. Epub 2017 Jun 6.

Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, 565-0871, Japan.

Generation of cells with a loss-of-function mutation in a gene (knockout cells) is a valuable technique for studying the function of a given gene product. However, if the product of the target gene is essential for cell viability, conditional knockout cell lines must be generated. Recently, as gene editing technology using CRISPR/Cas9 has developed, it has become possible to produce conditional knockout cell lines using this technique. However, to obtain final conditional knockout cell lines, it is necessary to perform several experiments with multiple complicated steps. In this paper, we introduce an easy and efficient method to generate conditional knockout cell lines based on combining auxin-inducible degron (AID) technology with CRISPR/Cas9 gene editing. Our method only requires performing a single transfection and is therefore an easy and rapid method to obtain a conditional knockout cell line.
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http://dx.doi.org/10.1007/s10577-017-9559-7DOI Listing
October 2017

Stepwise unfolding supports a subunit model for vertebrate kinetochores.

Proc Natl Acad Sci U S A 2017 03 6;114(12):3133-3138. Epub 2017 Mar 6.

Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, Scotland, United Kingdom;

During cell division, interactions between microtubules and chromosomes are mediated by the kinetochore, a proteinaceous structure located at the primary constriction of chromosomes. In addition to the centromere histone centromere protein A (CENP-A), 15 other members of the constitutive centromere associated network (CCAN) participate in the formation of a chromatin-associated scaffold that supports kinetochore structure. We performed a targeted screen analyzing unfolded centrochromatin from CENP-depleted chromosomes. Our results revealed that CENP-C and CENP-S are critical for the stable folding of mitotic kinetochore chromatin. Multipeak fitting algorithms revealed the presence of an organized pattern of centrochromatin packing consistent with arrangement of CENP-A-containing nucleosomes into up to five chromatin "subunits"-each containing roughly 20-30 nucleosomes. These subunits could be either layers of a boustrophedon or small loops of centromeric chromatin.
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http://dx.doi.org/10.1073/pnas.1614145114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5373359PMC
March 2017

Constitutive centromere-associated network controls centromere drift in vertebrate cells.

J Cell Biol 2017 Jan 9;216(1):101-113. Epub 2016 Dec 9.

Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan

Centromeres are specified by sequence-independent epigenetic mechanisms, and the centromere position may drift at each cell cycle, but once this position is specified, it may not be frequently moved. Currently, it is unclear whether the centromere position is stable. To address this question, we systematically analyzed the position of nonrepetitive centromeres in 21 independent clones isolated from a laboratory stock of chicken DT40 cells using chromatin immunoprecipitation combined with massive parallel sequencing analysis with anti-CENP-A antibody. We demonstrated that the centromere position varies among the clones, suggesting that centromere drift occurs during cell proliferation. However, when we analyzed this position in the subclones obtained from one isolated clone, the position was found to be relatively stable. Interestingly, the centromere drift was shown to occur frequently in CENP-U- and CENP-S-deficient cells. Based on these results, we suggest that the centromere position can change after many cell divisions, but this drift is suppressed in short-term cultures, and the complete centromere structure contributes to the suppression of the centromere drift.
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http://dx.doi.org/10.1083/jcb.201605001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5223601PMC
January 2017

Acetylation of histone H4 lysine 5 and 12 is required for CENP-A deposition into centromeres.

Nat Commun 2016 11 4;7:13465. Epub 2016 Nov 4.

Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan.

Centromeres are specified epigenetically through the deposition of the centromere-specific histone H3 variant CENP-A. However, how additional epigenetic features are involved in centromere specification is unknown. Here, we find that histone H4 Lys5 and Lys12 acetylation (H4K5ac and H4K12ac) primarily occur within the pre-nucleosomal CENP-A-H4-HJURP (CENP-A chaperone) complex, before centromere deposition. We show that H4K5ac and H4K12ac are mediated by the RbAp46/48-Hat1 complex and that RbAp48-deficient DT40 cells fail to recruit HJURP to centromeres and do not incorporate new CENP-A at centromeres. However, C-terminally-truncated HJURP, that does not bind CENP-A, does localize to centromeres in RbAp48-deficient cells. Acetylation-dead H4 mutations cause mis-localization of the CENP-A-H4 complex to non-centromeric chromatin. Crucially, CENP-A with acetylation-mimetic H4 was assembled specifically into centromeres even in RbAp48-deficient DT40 cells. We conclude that H4K5ac and H4K12ac, mediated by RbAp46/48, facilitates efficient CENP-A deposition into centromeres.
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http://dx.doi.org/10.1038/ncomms13465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5097169PMC
November 2016

Chromatin determinants of the inner-centromere rely on replication factors with functions that impart cohesion.

Oncotarget 2016 10;7(42):67934-67947

IFOM, The FIRC Institute for Molecular Oncology Foundation, Milan, Italy.

Replication fork-associated factors promote genome integrity and protect against cancer. Mutations in the DDX11 helicase and the ESCO2 acetyltransferase also cause related developmental disorders classified as cohesinopathies. Here we generated vertebrate model cell lines of these disorders and cohesinopathies-related genes. We found that vertebrate DDX11 and Tim-Tipin are individually needed to compensate for ESCO2 loss in chromosome segregation, with DDX11 also playing complementary roles with ESCO2 in centromeric cohesion. Our study reveals that overt centromeric cohesion loss does not necessarily precede chromosome missegregation, while both these problems correlate with, and possibly originate from, inner-centromere defects involving reduced phosphorylation of histone H3T3 (pH3T3) in the region. Interestingly, the mitotic pH3T3 mark was defective in all analyzed replication-related mutants with functions in cohesion. The results pinpoint mitotic pH3T3 as a postreplicative chromatin mark that is sensitive to replication stress and conducts with different kinetics to robust centromeric cohesion and correct chromosome segregation.
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http://dx.doi.org/10.18632/oncotarget.11982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5356530PMC
October 2016