Publications by authors named "Hitoshi Kurumizaka"

228 Publications

Sequence-dependent nucleosome formation in trinucleotide repeats evaluated by in vivo chemical mapping.

Biochem Biophys Res Commun 2021 Apr 8;556:179-184. Epub 2021 Apr 8.

Department of Chemistry, Graduate School of Science and Engineering, Meisei University, 2-1-1 Hodokubo, Hino, Tokyo, 191-8506, Japan. Electronic address:

Trinucleotide repeat sequences (TRSs), consisting of 10 unique classes of repeats in DNA, are members of microsatellites and abundantly and non-randomly distributed in many eukaryotic genomes. The lengths of TRSs are mutable, and the expansions of several TRSs are implicated in hereditary neurological diseases. However, the underlying causes of the biased distribution and the dynamic properties of TRSs in the genome remain elusive. Here, we examined the effects of TRSs on nucleosome formation in vivo by histone H4-S47C site-directed chemical cleavages, using well-defined yeast minichromosomes in which each of the ten TRS classes resided in the central region of a positioned nucleosome. We showed that (AAT) and (ACT) act as strong nucleosome-promoting sequences, while (AGG) and (CCG) act as nucleosome-excluding sequences in vivo. The local histone binding affinity scores support the idea that nucleosome formation in TRSs, except for (AGG), is mainly determined by the affinity for the histone octamers. Overall, our study presents a framework for understanding the nucleosome-forming abilities of TRSs.
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http://dx.doi.org/10.1016/j.bbrc.2021.03.155DOI Listing
April 2021

H4K20me1 and H3K27me3 are concurrently loaded onto the inactive X chromosome but dispensable for inducing gene silencing.

EMBO Rep 2021 Mar 19;22(3):e51989. Epub 2021 Feb 19.

EMBL Heidelberg, Heidelberg, Germany.

During X chromosome inactivation (XCI), in female placental mammals, gene silencing is initiated by the Xist long non-coding RNA. Xist accumulation at the X leads to enrichment of specific chromatin marks, including PRC2-dependent H3K27me3 and SETD8-dependent H4K20me1. However, the dynamics of this process in relation to Xist RNA accumulation remains unknown as is the involvement of H4K20me1 in initiating gene silencing. To follow XCI dynamics in living cells, we developed a genetically encoded, H3K27me3-specific intracellular antibody or H3K27me3-mintbody. By combining live-cell imaging of H3K27me3, H4K20me1, the X chromosome and Xist RNA, with ChIP-seq analysis we uncover concurrent accumulation of both marks during XCI, albeit with distinct genomic distributions. Furthermore, using a Xist B and C repeat mutant, which still shows gene silencing on the X but not H3K27me3 deposition, we also find a complete lack of H4K20me1 enrichment. This demonstrates that H4K20me1 is dispensable for the initiation of gene silencing, although it may have a role in the chromatin compaction that characterises facultative heterochromatin.
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http://dx.doi.org/10.15252/embr.202051989DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7926250PMC
March 2021

Histone variant H2A.B-H2B dimers are spontaneously exchanged with canonical H2A-H2B in the nucleosome.

Commun Biol 2021 Feb 12;4(1):191. Epub 2021 Feb 12.

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

H2A.B is an evolutionarily distant histone H2A variant that accumulates on DNA repair sites, DNA replication sites, and actively transcribing regions in genomes. In cells, H2A.B exchanges rapidly in chromatin, but the mechanism has remained enigmatic. In the present study, we found that the H2A.B-H2B dimer incorporated within the nucleosome exchanges with the canonical H2A-H2B dimer without assistance from additional factors, such as histone chaperones and nucleosome remodelers. High-speed atomic force microscopy revealed that the H2A.B nucleosome, but not the canonical H2A nucleosome, transiently forms an intermediate "open conformation", in which two H2A.B-H2B dimers may be detached from the H3-H4 tetramer and bind to the DNA regions near the entry/exit sites. Mutational analyses revealed that the H2A.B C-terminal region is responsible for the adoption of the open conformation and the H2A.B-H2B exchange in the nucleosome. These findings provide mechanistic insights into the histone exchange of the H2A.B nucleosome.
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http://dx.doi.org/10.1038/s42003-021-01707-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7881002PMC
February 2021

Structural basis of nucleosomal histone H4 lysine 20 methylation by SET8 methyltransferase.

Life Sci Alliance 2021 04 11;4(4). Epub 2021 Feb 11.

Laboratory of Chromatin Structure and Function, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan

SET8 is solely responsible for histone H4 lysine-20 (H4K20) monomethylation, which preferentially occurs in nucleosomal H4. However, the underlying mechanism by which SET8 specifically promotes the H4K20 monomethylation in the nucleosome has not been elucidated. Here, we report the cryo-EM structures of the human SET8-nucleosome complexes with histone H3 and the centromeric H3 variant, CENP-A. Surprisingly, we found that the overall cryo-EM structures of the SET8-nucleosome complexes are substantially different from the previous crystal structure models. In the complexes with H3 and CENP-A nucleosomes, SET8 specifically binds the nucleosomal acidic patch via an arginine anchor, composed of the Arg188 and Arg192 residues. Mutational analyses revealed that the interaction between the SET8 arginine anchor and the nucleosomal acidic patch plays an essential role in the H4K20 monomethylation activity. These results provide the groundwork for understanding the mechanism by which SET8 specifically accomplishes the H4K20 monomethylation in the nucleosome.
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http://dx.doi.org/10.26508/lsa.202000919DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7893823PMC
April 2021

Live-cell epigenome manipulation by synthetic histone acetylation catalyst system.

Proc Natl Acad Sci U S A 2021 Jan;118(4)

Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan;

Chemical modifications of histones, such as lysine acetylation and ubiquitination, play pivotal roles in epigenetic regulation of gene expression. Methods to alter the epigenome thus hold promise as tools for elucidating epigenetic mechanisms and as therapeutics. However, an entirely chemical method to introduce histone modifications in living cells without genetic manipulation is unprecedented. Here, we developed a chemical catalyst, PEG-LANA-DSSMe 11, that binds with nucleosome's acidic patch and promotes regioselective, synthetic histone acetylation at H2BK120 in living cells. The size of polyethylene glycol in the catalyst was a critical determinant for its in-cell metabolic stability, binding affinity to histones, and high activity. The synthetic acetylation promoted by 11 without genetic manipulation competed with and suppressed physiological H2B ubiquitination, a mark regulating chromatin functions, such as transcription and DNA damage response. Thus, the chemical catalyst will be a useful tool to manipulate epigenome for unraveling epigenetic mechanisms in living cells.
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http://dx.doi.org/10.1073/pnas.2019554118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848698PMC
January 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

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

Acetylation-modulated communication between the H3 N-terminal tail domain and the intrinsically disordered H1 C-terminal domain.

Nucleic Acids Res 2020 11;48(20):11510-11520

Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY 14642, USA.

Linker histones (H1s) are key structural components of the chromatin of higher eukaryotes. However, the mechanisms by which the intrinsically disordered linker histone carboxy-terminal domain (H1 CTD) influences chromatin structure and gene regulation remain unclear. We previously demonstrated that the CTD of H1.0 undergoes a significant condensation (reduction of end-to-end distance) upon binding to nucleosomes, consistent with a transition to an ordered structure or ensemble of structures. Here, we show that deletion of the H3 N-terminal tail or the installation of acetylation mimics or bona fide acetylation within H3 N-terminal tail alters the condensation of the nucleosome-bound H1 CTD. Additionally, we present evidence that the H3 N-tail influences H1 CTD condensation through direct protein-protein interaction, rather than alterations in linker DNA trajectory. These results support an emerging hypothesis wherein the H1 CTD serves as a nexus for signaling in the nucleosome.
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http://dx.doi.org/10.1093/nar/gkaa949DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7672455PMC
November 2020

Contributions of Histone Variants in Nucleosome Structure and Function.

J Mol Biol 2021 Mar 14;433(6):166678. Epub 2020 Oct 14.

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

Chromatin compacts genomic DNA in eukaryotes. The primary chromatin unit is the nucleosome core particle, composed of four pairs of the core histones, H2A, H2B, H3, and H4, and 145-147 base pairs of DNA. Since replication, recombination, repair, and transcription take place in chromatin, the structure and dynamics of the nucleosome must be versatile. These nucleosome characteristics underlie the epigenetic regulation of genomic DNA. In higher eukaryotes, many histone variants have been identified as non-allelic isoforms, which confer nucleosome diversity. In this article, we review the manifold types of nucleosomes produced by histone variants, which play important roles in the epigenetic regulation of chromatin.
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http://dx.doi.org/10.1016/j.jmb.2020.10.012DOI Listing
March 2021

Structural basis for the inhibition of cGAS by nucleosomes.

Science 2020 10 10;370(6515):455-458. Epub 2020 Sep 10.

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

The cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) senses invasion of pathogenic DNA and stimulates inflammatory signaling, autophagy, and apoptosis. Organization of host DNA into nucleosomes was proposed to limit cGAS autoinduction, but the underlying mechanism was unknown. Here, we report the structural basis for this inhibition. In the cryo-electron microscopy structure of the human cGAS-nucleosome core particle (NCP) complex, two cGAS monomers bridge two NCPs by binding the acidic patch of the histone H2A-H2B dimer and nucleosomal DNA. In this configuration, all three known cGAS DNA binding sites, required for cGAS activation, are repurposed or become inaccessible, and cGAS dimerization, another prerequisite for activation, is inhibited. Mutating key residues linking cGAS and the acidic patch alleviates nucleosomal inhibition. This study establishes a structural framework for why cGAS is silenced on chromatinized self-DNA.
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http://dx.doi.org/10.1126/science.abd0237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7584773PMC
October 2020

Interaction of the pioneer transcription factor GATA3 with nucleosomes.

Nat Commun 2020 08 18;11(1):4136. Epub 2020 Aug 18.

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

During cellular reprogramming, the pioneer transcription factor GATA3 binds chromatin, and in a context-dependent manner directs local chromatin remodeling and enhancer formation. Here, we use high-resolution nucleosome mapping in human cells to explore the impact of the position of GATA motifs on the surface of nucleosomes on productive enhancer formation, finding productivity correlates with binding sites located near the nucleosomal dyad axis. Biochemical experiments with model nucleosomes demonstrate sufficiently stable transcription factor-nucleosome interaction to empower cryo-electron microscopy structure determination of the complex at 3.15 Å resolution. The GATA3 zinc fingers efficiently bind their target 5'-GAT-3' sequences in the nucleosome when they are located in solvent accessible, consecutive major grooves without significant changes in nucleosome structure. Analysis of genomic loci bound by GATA3 during reprogramming suggests a correlation of recognition motif sequence and spacing that may distinguish productivity of new enhancer formation.
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http://dx.doi.org/10.1038/s41467-020-17959-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7434886PMC
August 2020

Chromatin integration labeling for mapping DNA-binding proteins and modifications with low input.

Nat Protoc 2020 10 17;15(10):3334-3360. Epub 2020 Aug 17.

Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Japan.

Cell identity is determined by the selective activation or silencing of specific genes via transcription factor binding and epigenetic modifications on the genome. Chromatin immunoprecipitation (ChIP) has been the standard technique for mapping the sites of transcription factor binding and histone modification. Recently, alternative methods to ChIP have been developed for addressing the increasing demands for low-input epigenomic profiling. Chromatin integration labeling (ChIL) followed by sequencing (ChIL-seq) has been demonstrated to be particularly useful for epigenomic profiling of low-input samples or even single cells because the technique amplifies the target genomic sequence before cell lysis. After labeling the target protein or modification in situ with an oligonucleotide-conjugated antibody (ChIL probe), the nearby genome sequence is amplified by Tn5 transposase-mediated transposition followed by T7 RNA polymerase-mediated transcription. ChIL-seq enables the detection of the antibody target localization under a fluorescence microscope and at the genomic level. Here we describe the detailed protocol of ChIL-seq with assessment methods for the key steps, including ChIL probe reaction, transposition, in situ transcription and sequencing library preparation. The protocol usually takes 3 d to prepare the sequencing library, including overnight incubations for the ChIL probe reaction and in situ transcription. The ChIL probe can be separately prepared and stored for several months, and its preparation and evaluation protocols are also documented in detail. An optional analysis for multiple targets (multitarget ChIL-seq) is also described. We anticipate that the protocol presented here will make the ChIL technique more widely accessible for analyzing precious samples and facilitate further applications.
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http://dx.doi.org/10.1038/s41596-020-0375-8DOI Listing
October 2020

Acetylated histone H4 tail enhances histone H3 tail acetylation by altering their mutual dynamics in the nucleosome.

Proc Natl Acad Sci U S A 2020 08 3;117(33):19661-19663. Epub 2020 Aug 3.

Graduate School of Medical Life Science, Yokohama City University, Yokohama 230-0045, Japan;

The structural unit of eukaryotic chromatin is a nucleosome, comprising two histone H2A-H2B heterodimers and one histone (H3-H4) tetramer, wrapped around by ∼146 bp of DNA. The N-terminal flexible histone tails stick out from the histone core and have extensive posttranslational modifications, causing epigenetic changes of chromatin. Although crystal and cryogenic electron microscopy structures of nucleosomes are available, the flexible tail structures remain elusive. Using NMR, we have examined the dynamics of histone H3 tails in nucleosomes containing unmodified and tetra-acetylated H4 tails. In unmodified nucleosome, the H3 tail adopts a dynamic equilibrium structure between DNA-contact and reduced-contact states. In acetylated H4 nucleosome, however, the H3 tail equilibrium shifts to a mainly DNA-contact state with a minor reduced-contact state. The acetylated H4 tail is dynamically released from its own DNA-contact state to a reduced-contact state, while the H3 tail DNA-contact state becomes major. Notably, H3 K14 in the acetylated H4 nucleosome is much more accessible to acetyltransferase Gcn5 relative to unmodified nucleosome, possibly due to the formation of a favorable H3 tail conformation for Gcn5. In summary, each histone tail adopts a characteristic dynamic state but regulates one other, probably creating a histone tail network even on a nucleosome.
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http://dx.doi.org/10.1073/pnas.2010506117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7443954PMC
August 2020

Linker DNA and histone contributions in nucleosome binding by p53.

J Biochem 2020 Dec;168(6):669-675

Laboratory of Chromatin Structure and Function, Institute for Quantitative Biosciences.

The tumour suppressor protein p53 regulates various genes involved in cell-cycle arrest, apoptosis and DNA repair in response to cellular stress, and apparently functions as a pioneer transcription factor. The pioneer transcription factors can bind nucleosomal DNA, where many transcription factors are largely restricted. However, the mechanisms by which p53 recognizes the nucleosomal DNA are poorly understood. In the present study, we found that p53 requires linker DNAs for the efficient formation of p53-nucleosome complexes. p53 forms an additional specific complex with the nucleosome, when the p53 binding sequence is located around the entry/exit region of the nucleosomal DNA. We also showed that p53 directly binds to the histone H3-H4 complex via its N-terminal 1-93 amino acid region. These results shed light on the mechanism of nucleosome recognition by p53.
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http://dx.doi.org/10.1093/jb/mvaa081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7763433PMC
December 2020

Nucleosome binding by the pioneer transcription factor OCT4.

Sci Rep 2020 07 16;10(1):11832. Epub 2020 Jul 16.

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

Transcription factor binding to genomic DNA is generally prevented by nucleosome formation, in which the DNA is tightly wrapped around the histone octamer. In contrast, pioneer transcription factors efficiently bind their target DNA sequences within the nucleosome. OCT4 has been identified as a pioneer transcription factor required for stem cell pluripotency. To study the nucleosome binding by OCT4, we prepared human OCT4 as a recombinant protein, and biochemically analyzed its interactions with the nucleosome containing a natural OCT4 target, the LIN28B distal enhancer DNA sequence, which contains three potential OCT4 target sequences. By a combination of chemical mapping and cryo-electron microscopy single-particle analysis, we mapped the positions of the three target sequences within the nucleosome. A mutational analysis revealed that OCT4 preferentially binds its target DNA sequence located near the entry/exit site of the nucleosome. Crosslinking mass spectrometry consistently showed that OCT4 binds the nucleosome in the proximity of the histone H3 N-terminal region, which is close to the entry/exit site of the nucleosome. We also found that the linker histone H1 competes with OCT4 for the nucleosome binding. These findings provide important information for understanding the molecular mechanism by which OCT4 binds its target DNA in chromatin.
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http://dx.doi.org/10.1038/s41598-020-68850-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7367260PMC
July 2020

Integral approach to biomacromolecular structure by analytical-ultracentrifugation and small-angle scattering.

Commun Biol 2020 Jun 8;3(1):294. Epub 2020 Jun 8.

Institute for Integrated Radiation and Nuclear Science, Kyoto University, 2-1010 Asashironishi, Kumatori, Sennan-gun, Osaka, 590-0494, Japan.

Currently, a sample for small-angle scattering (SAS) is usually highly purified and looks monodispersed: The Guinier plot of its SAS intensity shows a fine straight line. However, it could include the slight aggregates which make the experimental SAS profile different from the monodispersed one. A concerted method with analytical-ultracentrifugation (AUC) and SAS, named as AUC-SAS, offers the precise scattering intensity of a concerned biomacromolecule in solution even with aggregates as well that of a complex under an association-dissociation equilibrium. AUC-SAS overcomes an aggregation problem which has been an obstacle for SAS analysis and, furthermore, has a potential to lead to a structural analysis for a general multi-component system.
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http://dx.doi.org/10.1038/s42003-020-1011-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7280208PMC
June 2020

The N-terminal and C-terminal halves of histone H2A.Z independently function in nucleosome positioning and stability.

Genes Cells 2020 Aug 22;25(8):538-546. Epub 2020 Jul 22.

Laboratory of Chromatin Structure and Function, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan.

Nucleosome positioning and stability affect gene regulation in eukaryotic chromatin. Histone H2A.Z is an evolutionally conserved histone variant that forms mobile and unstable nucleosomes in vivo and in vitro. In the present study, we reconstituted nucleosomes containing human H2A.Z.1 mutants, in which the N-terminal or C-terminal half of H2A.Z.1 was replaced by the corresponding canonical H2A region. We found that the N-terminal portion of H2A.Z.1 is involved in flexible nucleosome positioning, whereas the C-terminal portion leads to weak H2A.Z.1-H2B association in the nucleosome. These results indicate that the N-terminal and C-terminal portions are independently responsible for the H2A.Z.1 nucleosome characteristics.
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http://dx.doi.org/10.1111/gtc.12791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496805PMC
August 2020

Improved Methods for Preparing the Telomere Tethering Complex Bqt1-Bqt2 for Structural Studies.

Protein J 2020 04;39(2):174-181

Department of Chemistry, Graduate School of Science and Engineering, Meisei University, 2-1-1 Hodokubo, Hino-shi, Tokyo, 191-8506, Japan.

In eukaryotes, chromosome ends (telomeres) are tethered to the inner nuclear membrane. During the early stages of meiosis, telomeres move along the nuclear membrane and gather near the spindle-pole body, resulting in a bouquet-like arrangement of chromosomes. This chromosomal configuration appears to be widely conserved among eukaryotes, and is assumed to play an important role in the normal progression of meiosis, by mediating the proper pairing of homologous chromosomes. In fission yeast, the Bqt1-Bqt2 protein complex plays a key role in tethering the telomere to the inner nuclear membrane. However, the structural details of the complex required to clarify how telomeres are gathered near the spindle-pole body remain enigmatic. Previously, we devised a preparation procedure for the Schizosaccharomyces japonicus Bqt1-Bqt2 complex, in which a SUMO tag was fused to the N-terminus of the Bqt1 protein. This allowed us to purify the Bqt1-Bqt2 complex from the soluble fraction. In the present study, we found that a maltose-binding protein homolog, Athe_0614, served as a better fusion partner than the SUMO protein, resulting in the marked increase in the solubility of the Bqt1-Bqt2 complex. The Athe_0614 fusion partner may open up new avenues for X-ray crystallographic analyses of the structure of the Bqt1-Bqt2 complex.
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http://dx.doi.org/10.1007/s10930-020-09887-zDOI Listing
April 2020

Nucleosome destabilization by nuclear non-coding RNAs.

Commun Biol 2020 02 11;3(1):60. Epub 2020 Feb 11.

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

In the nucleus, genomic DNA is wrapped around histone octamers to form nucleosomes. In principle, nucleosomes are substantial barriers to transcriptional activities. Nuclear non-coding RNAs (ncRNAs) are proposed to function in chromatin conformation modulation and transcriptional regulation. However, it remains unclear how ncRNAs affect the nucleosome structure. Eleanors are clusters of ncRNAs that accumulate around the estrogen receptor-α (ESR1) gene locus in long-term estrogen deprivation (LTED) breast cancer cells, and markedly enhance the transcription of the ESR1 gene. Here we detected nucleosome depletion around the transcription site of Eleanor2, the most highly expressed Eleanor in the LTED cells. We found that the purified Eleanor2 RNA fragment drastically destabilized the nucleosome in vitro. This activity was also exerted by other ncRNAs, but not by poly(U) RNA or DNA. The RNA-mediated nucleosome destabilization may be a common feature among natural nuclear RNAs, and may function in transcription regulation in chromatin.
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http://dx.doi.org/10.1038/s42003-020-0784-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012929PMC
February 2020

Native Mass Spectrometry of Protein and DNA Complexes Prepared in Nonvolatile Buffers.

J Am Soc Mass Spectrom 2020 Mar 31;31(3):711-718. Epub 2020 Jan 31.

Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.

Inorganic salts and nonvolatile-buffer components affect the structure and stability of proteins, and some protein complexes are unable to maintain their function and structure without them. However, it is well-known that these components cause suppression of analyte ionization during the electrospray ionization process. Thus, to establish appropriate methods for observation of the intact ions of protein and DNA complexes by native mass spectrometry (native MS) in the presence of nonvolatile buffer components, we herein examined the effect of ammonium acetate addition to a model homotetramer protein, alcohol dehydrogenase (ADH), which was prepared in a range of nonvolatile buffers, including Tris-HCl, phosphate, and HEPES buffers. Furthermore, native MS of nucleosome core particle (NCP), a large protein-DNA complex, prepared in nonvolatile buffer, was also examined. Intact ADH and NCP ions could be observed upon the addition of ammonium acetate, but NCP does not require as high of a concentration of ammonium acetate as ADH. Well-resolved peaks with different charge numbers could be observed for NCP prepared in Tris-HCl by addition of a lower amount of ammonium acetate than for ADH. This suggests that the effects of additives on native MS of biomolecular complexes can vary depending on the intramolecular interactions present. More specifically, NCP is stabilized mainly by electrostatic interactions, whereas the ADH tetramer depends on the presence of hydrophobic interactions between the four subunits. The results presented herein therefore are expected to contribute to structural biology studies of unstable protein-DNA complexes that are formed transiently during the transcription process.
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http://dx.doi.org/10.1021/jasms.9b00145DOI Listing
March 2020

Structural Studies of Overlapping Dinucleosomes in Solution.

Biophys J 2020 05 18;118(9):2209-2219. Epub 2019 Dec 18.

Institute for Quantum Life Science, National Institutes for Quantum and Radiological Science and Technology, Kizugawa, Japan. Electronic address:

An overlapping dinucleosome (OLDN) is a structure composed of one hexasome and one octasome and appears to be formed through nucleosome collision promoted by nucleosome remodeling factor(s). In this study, the solution structure of the OLDN was investigated through the integration of small-angle x-ray and neutron scattering (SAXS and SANS, respectively), computer modeling, and molecular dynamics simulations. Starting from the crystal structure, we generated a conformational ensemble based on normal mode analysis and searched for the conformations that reproduced the SAXS and SANS scattering curves well. We found that inclusion of histone tails, which are not observed in the crystal structure, greatly improved model quality. The obtained structural models suggest that OLDNs adopt a variety of conformations stabilized by histone tails situated at the interface between the hexasome and octasome, simultaneously binding to both the hexasomal and octasomal DNA. In addition, our models define a possible direction for the conformational changes or dynamics, which may provide important information that furthers our understanding of the role of chromatin dynamics in gene regulation.
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http://dx.doi.org/10.1016/j.bpj.2019.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7202943PMC
May 2020

Biochemical and structural analyses of the nucleosome containing human histone H2A.J.

J Biochem 2020 Apr;167(4):419-427

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

Histone H2A.J, a histone H2A variant conserved in mammals, may function in the expression of genes related to inflammation and cell proliferation. In the present study, we purified the human histone H2A.J variant and found that H2A.J is efficiently incorporated into the nucleosome in vitro. H2A.J formed the stable nucleosome, which accommodated the DNA ends. Mutations in the H2A.J-specific residues did not affect the nucleosome stability, although the mutation of the H2A.J Ala40 residue, which is conserved in some members of the canonical H2A class, reduced the nucleosome stability. Consistently, the crystal structure of the H2A.J nucleosome revealed that the H2A.J-specific residues, including the Ala40 residue, did not affect the nucleosome structure. These results provide basic information for understanding the function of the H2A.J nucleosome.
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http://dx.doi.org/10.1093/jb/mvz109DOI Listing
April 2020

Transcription through the nucleosome.

Curr Opin Struct Biol 2020 04 29;61:42-49. Epub 2019 Nov 29.

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

Eukaryotic genomic DNA is wrapped around the histone octamer and forms the nucleosome, which is the basic unit of chromatin. Nevertheless, eukaryotic RNA polymerases transcribe the DNA that is tightly bound to the histone core in the nucleosome. For transcription to proceed on the nucleosome, the RNA polymerases must overcome the tight contacts between histones and DNA during transcription elongation. Here, we review the structural and biochemical studies of the transcription mechanism on the nucleosome, and focus on recent information to understand how RNA polymerases transcribe the genomic DNA in chromatin.
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http://dx.doi.org/10.1016/j.sbi.2019.10.007DOI Listing
April 2020

Incorporation and influence of Leishmania histone H3 in chromatin.

Nucleic Acids Res 2019 12;47(22):11637-11648

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

Immunopathologies caused by Leishmania cause severe human morbidity and mortality. This protozoan parasite invades and persists inside host cells, resulting in disease development. Leishmania modifies the epigenomic status of the host cells, thus probably averting the host cell defense mechanism. To accomplish this, Leishmania may change the host cell chromatin structure. However, the mechanism by which the parasite changes the host cell chromatin has not been characterized. In the present study, we found that ectopically produced Leishmania histone H3, LmaH3, which mimics the secreted LmaH3 in infected cells, is incorporated into chromatin in human cells. A crystallographic analysis revealed that LmaH3 forms nucleosomes with human histones H2A, H2B and H4. We found that LmaH3 was less stably incorporated into the nucleosome, as compared to human H3.1. Consistently, we observed that LmaH3-H4 association was remarkably weakened. Mutational analyses revealed that the specific LmaH3 Trp35, Gln57 and Met98 residues, which correspond to the H3.1 Tyr41, Arg63 and Phe104 residues, might be responsible for the instability of the LmaH3 nucleosome. Nucleosomes containing LmaH3 resisted the Mg2+-mediated compaction of the chromatin fiber. These distinct physical characteristics of LmaH3 support the possibility that histones secreted by parasites during infection may modulate the host chromatin structure.
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http://dx.doi.org/10.1093/nar/gkz1040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145708PMC
December 2019

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

Structure determination of the nucleosome core particle by selenium SAD phasing.

Acta Crystallogr D Struct Biol 2019 Oct 24;75(Pt 10):930-936. Epub 2019 Sep 24.

Department of Chemistry, Graduate School of Science and Engineering, Meisei University, 2-1-1 Hodokubo, Hino-shi, Tokyo 191-8506, Japan.

The eukaryotic genome is compacted inside the nucleus of the cell in the form called chromatin. The fundamental unit of chromatin is the nucleosome, which contains four types of histones (H3, H4, H2A and H2B) and approximately 150 base pairs of DNA wrapped around the histone complex. The structure of the nucleosome is highly conserved across several eukaryotic species, and molecular replacement has been the primary phasing method used to solve nucleosome structures by X-ray crystallography. However, there is currently no simple, widely applicable experimental phasing method for the nucleosome. In the present study, it is demonstrated that selenomethionine-incorporated histones H3, H2A and H2B can be reconstituted into nucleosomes and crystallized for structural determination. Unexpectedly, it was found that the nucleosome can be phased with a relatively small number of Se atoms. The structures of nucleosome core particles containing 12 and 16 Se atoms were solved by SAD phasing at 2.5 and 2.4 Å resolution, respectively. The present study demonstrates a simple method for determining nucleosome structures by experimental phasing, which may be particularly useful for noncanonical structures that cannot be solved by molecular replacement.
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http://dx.doi.org/10.1107/S2059798319012713DOI Listing
October 2019

A novel mode of DnaA-DnaA interaction promotes ADP dissociation for reactivation of replication initiation activity.

Nucleic Acids Res 2019 12;47(21):11209-11224

Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan.

ATP-DnaA is temporally increased to initiate replication during the cell cycle. Two chromosomal loci, DARS (DnaA-reactivating sequences) 1 and 2, promote ATP-DnaA production by nucleotide exchange of ADP-DnaA for timely initiation. ADP-DnaA complexes are constructed on DARS1 and DARS2, bearing a cluster of three DnaA-binding sequences (DnaA boxes I-III), promoting ADP dissociation. Although DnaA has an AAA+ domain, which ordinarily directs construction of oligomers in a head-to-tail manner, DnaA boxes I and II are oriented oppositely. In this study, we constructed a structural model of a head-to-head dimer of DnaA AAA+ domains, and analyzed residues residing on the interface of the model dimer. Gln208 was specifically required for DARS-dependent ADP dissociation in vitro, and in vivo analysis yielded consistent results. Additionally, ADP release from DnaA protomers bound to DnaA boxes I and II was dependent on Gln208 of the DnaA protomers, and DnaA box III-bound DnaA did not release ADP nor require Gln208 for ADP dissociation by DARS-DnaA complexes. Based on these and other findings, we propose a model for DARS-DnaA complex dynamics during ADP dissociation, and provide novel insight into the regulatory mechanisms of DnaA and the interaction modes of AAA+ domains.
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http://dx.doi.org/10.1093/nar/gkz795DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868365PMC
December 2019

Structural transition of the nucleosome during chromatin remodeling and transcription.

Curr Opin Struct Biol 2019 12 29;59:107-114. Epub 2019 Aug 29.

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

In eukaryotes, the nucleosome is the basic unit of chromatin. Since the genomic DNA is tightly wrapped around the histone octamer in the nucleosome, its function is severely restricted in chromatin. To overcome the nucleosome barrier, the nucleosome structure must dynamically change during genomic DNA functions. Recent structural studies revealed that chromatin remodelers and RNA polymerase II drastically alter the nucleosome structures during the chromatin remodeling and transcription processes. These results provide important information for understanding how genes are properly regulated in eukaryotes. Here, we review the recent structural studies of nucleosome versatility and dynamics during chromatin remodeling and transcription by RNA polymerase II.
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http://dx.doi.org/10.1016/j.sbi.2019.07.011DOI Listing
December 2019