2025/01/09 更新

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写真a

ハラダ アキヒト
原田 哲仁
HARADA AKIHITO
所属
医学研究院 先端医療医学部門 教授
システム生命科学府 システム生命科学専攻(併任)
医学系学府 医科学専攻(併任)
職名
教授
連絡先
メールアドレス
電話番号
0926424534
外部リンク

学位

  • 農学

研究テーマ・研究キーワード

  • 研究テーマ: 生殖細胞におけるヒストンバリアントによるゲノムマーキング機構の解明

    研究キーワード: 生殖細胞、細胞分化、エピジェネティクス、ヒストンバリアント、転写

    研究期間: 2016年4月

  • 研究テーマ: 骨格筋分化をモデルとした細胞運命決定機構の解明

    研究キーワード: 骨格筋、細胞分化、エピジェネティクス、ヒストンバリアント、転写

    研究期間: 2016年4月

  • 研究テーマ: 骨格筋分化における新規ヒストンH3バリアントH3mm7の遺伝子選択機構の解明

    研究キーワード: 骨格筋、細胞分化、エピジェネティクス、ヒストンバリアント、転写

    研究期間: 2015年4月

受賞

  • 公益財団法人福岡県すこやか健康事業団平成28年度がん研究助成金優秀賞

    2017年1月   公益財団法人福岡県すこやか健康事業団   発がんを誘発するヒストンH3バリアントの変異部位の同定とその誘発メカニズムを明らかにすることを目的としエピジェネティック破綻により引き起こされた筋肉腫の解析を進める。具体的には、横紋筋肉腫をモデルとし、正常組織とがん組織における各H3バリアントのゲノム領域への取り込み、またそれらが形成するクロマチン構造を明らかにし、ヒストンバリアントの破綻によるがん化メカニズムを解明する。

論文

  • Chromatin integration labeling for mapping DNA-binding proteins and modifications with low input. 査読 国際誌

    †Tetsuya Handa, †Akihito Harada, †Kazumitsu Maehara, Shoko Sato, Masaru Nakao, Naoki Goto, Hitoshi Kurumizaka, Yasuyuki Ohkawa, Hiroshi Kimura

    Nature Protocols   15 ( 10 )   3334 - 3360   2020年10月

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    担当区分:筆頭著者   記述言語:日本語   掲載種別:研究論文(学術雑誌)  

    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.

    DOI: 10.1038/s41596-020-0375-8.

  • A chromatin integration labelling method enables epigenomic profiling with lower input 査読

    Akihito Harada, Kazumitsu Maehara, Tetsuya Handa, Yasuhiro Arimura, Jumpei Nogami, Yoko Hayashi-Takanaka, Katsuhiko Shirahige, Hitoshi Kurumizaka, Hiroshi Kimura, Yasuyuki Ohkawa

    Nature Cell Biology   21 ( 2 )   287 - 296   2019年2月

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Chromatin plays a crucial role in gene regulation, and chromatin immunoprecipitation followed by sequencing (ChIP–seq) has been the standard technique for examining protein–DNA interactions across the whole genome. However, it is difficult to obtain epigenomic information from limited numbers of cells by ChIP–seq because of sample loss during chromatin preparation and inefficient immunoprecipitation. In this study, we established an immunoprecipitation-free epigenomic profiling method named chromatin integration labelling (ChIL), which enables the amplification of genomic sequences closely associated with the target molecules before cell lysis. Using ChIL followed by sequencing (ChIL–seq), we reliably detected the distributions of histone modifications and DNA-binding factors in 100–1,000 cells. In addition, ChIL–seq successfully detected genomic regions associated with histone marks at the single-cell level. Thus, ChIL–seq offers an alternative method to ChIP–seq for epigenomic profiling using small numbers of cells, in particular, those attached to culture plates and after immunofluorescence.

    DOI: 10.1038/s41556-018-0248-3

  • Histone H3.3 sub-variant H3mm7 is required for normal skeletal muscle regeneration 査読

    Akihito Harada, Kazumitsu Maehara, Yusuke Ono, Hiroyuki Taguchi, Kiyoshi Yoshioka, Yasuo Kitajima, Yan Xie, Yuko Sato, Takeshi Iwasaki, Jumpei Nogami, Seiji Okada, Tetsuro Komatsu, Yuichiro Semba, Tatsuya Takemoto, Hiroshi Kimura, Hitoshi Kurumizaka, Yasuyuki Ohkawa

    Nature Communications   9 ( 1 )   2018年12月

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Regulation of gene expression requires selective incorporation of histone H3 variant H3.3 into chromatin. Histone H3.3 has several subsidiary variants but their functions are unclear. Here we characterize the function of histone H3.3 sub-variant, H3mm7, which is expressed in skeletal muscle satellite cells. H3mm7 knockout mice demonstrate an essential role of H3mm7 in skeletal muscle regeneration. Chromatin analysis reveals that H3mm7 facilitates transcription by forming an open chromatin structure around promoter regions including those of myogenic genes. The crystal structure of the nucleosome containing H3mm7 reveals that, unlike the S57 residue of other H3 proteins, the H3mm7-specific A57 residue cannot form a hydrogen bond with the R40 residue of the cognate H4 molecule. Consequently, the H3mm7 nucleosome is unstable in vitro and exhibited higher mobility in vivo compared with the H3.3 nucleosome. We conclude that the unstable H3mm7 nucleosome may be required for proper skeletal muscle differentiation.

    DOI: 10.1038/s41467-018-03845-1

  • Temporal regulation of chromatin during myoblast differentiation 査読

    Akihito Harada, Yasuyuki Ohkawa, Anthony N. Imbalzano

    Seminars in Cell and Developmental Biology   72   77 - 86   2017年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    The commitment to and execution of differentiation programmes involves a significant change in gene expression in the precursor cell to facilitate development of the mature cell type. In addition to being regulated by lineage-determining and auxiliary transcription factors that drive these changes, the structural status of the chromatin has a considerable impact on the transcriptional competence of differentiation-specific genes, which is clearly demonstrated by the large number of cofactors and the extraordinary complex mechanisms by which these genes become activated. The terminal differentiation of myoblasts to myotubes and mature skeletal muscle is an excellent system to illustrate these points. The MyoD family of closely related, lineage-determining transcription factors directs, largely through targeting to chromatin, a cascade of cooperating transcription factors and enzymes that incorporate or remove variant histones, post-translationally modify histones, and alter nucleosome structure and positioning via energy released by ATP hydrolysis. The coordinated action of these transcription factors and enzymes prevents expression of differentiation-specific genes in myoblasts and facilitates the transition of these genes from transcriptionally repressed to activated during the differentiation process. Regulation is achieved in both a temporal as well as spatial manner, as at least some of these factors and enzymes affect local chromatin structure at myogenic gene regulatory sequences as well as higher-order genome organization. Here we discuss the transition of genes that promote myoblast differentiation from the silenced to the activated state with an emphasis on the changes that occur to individual histones and the chromatin structure present at these loci.

    DOI: 10.1016/j.semcdb.2017.10.022

  • Testis-Specific Histone Variant H3t Gene Is Essential for Entry into Spermatogenesis 査読

    Jun Ueda, Akihito Harada, Takashi Urahama, Shinichi Machida, Kazumitsu Maehara, Masashi Hada, Yoshinori Makino, Jumpei Nogami, Naoki Horikoshi, Akihisa Osakabe, Hiroyuki Taguchi, Hiroki Tanaka, Hiroaki Tachiwana, Tatsuma Yao, Minami Yamada, Takashi Iwamoto, Ayako Isotani, Masahito Ikawa, Taro Tachibana, Yuki Okada, Hiroshi Kimura, Yasuyuki Ohkawa, Hitoshi Kurumizaka, Kazuo Yamagata

    Cell Reports   18 ( 3 )   593 - 600   2017年1月

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Cellular differentiation is associated with dynamic chromatin remodeling in establishing a cell-type-specific epigenomic landscape. Here, we find that mouse testis-specific and replication-dependent histone H3 variant H3t is essential for very early stages of spermatogenesis. H3t gene deficiency leads to azoospermia because of the loss of haploid germ cells. When differentiating spermatogonia emerge in normal spermatogenesis, H3t appears and replaces the canonical H3 proteins. Structural and biochemical analyses reveal that H3t-containing nucleosomes are more flexible than the canonical nucleosomes. Thus, by incorporating H3t into the genome during spermatogonial differentiation, male germ cells are able to enter meiosis and beyond.

    DOI: 10.1016/j.celrep.2016.12.065

  • Tissue-specific expression of histone H3 variants diversified after species separation 査読

    Kazumitsu Maehara, Akihito Harada, Yuko Sato, Masaki Matsumoto, Keiichi Nakayama, Hiroshi Kimura, Yasuyuki Ohkawa

    Epigenetics and Chromatin   8 ( 1 )   2015年9月

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Background: The selective incorporation of appropriate histone variants into chromatin is critical for the regulation of genome function. Although many histone variants have been identified, a complete list has not been compiled. Results: We screened mouse, rat and human genomes by in silico hybridization using canonical histone sequences. In the mouse genome, we identified 14 uncharacterized H3 genes, among which 13 are similar to H3.3 and do not have human or rat counterparts, and one is similar to human testis-specific H3 variant, H3T/H3.4, and had a rat paralog. Although some of these genes were previously annotated as pseudogenes, their tissue-specific expression was confirmed by sequencing the 3′-UTR regions of the transcripts. Certain new variants were also detected at the protein level by mass spectrometry. When expressed as GFP-tagged versions in mouse C2C12 cells, some variants were stably incorporated into chromatin and the genome-wide distributions of most variants were similar to that of H3.3. Moreover, forced expression of H3 variants in chromatin resulted in alternate gene expression patterns after cell differentiation. Conclusions: We comprehensively identified and characterized novel mouse H3 variant genes that encoded highly conserved amino acid sequences compared to known histone H3. We speculated that the diversity of H3 variants acquired after species separation played a role in regulating tissue-specific gene expression in individual species. Their biological relevance and evolutionary aspect involving pseudogene diversification will be addressed by further functional analysis.

    DOI: 10.1186/s13072-015-0027-3

  • Spatial re-organization of myogenic regulatory sequences temporally controls gene expression 査読 国際共著

    Akihito Harada, Chandrashekara Mallappa, Seiji Okada, John T. Butler, Stephen P. Baker, Jeanne B. Lawrence, Yasuyuki Ohkawa, Anthony N. Imbalzano

    Nucleic Acids Research   43 ( 4 )   2008 - 2021   2015年2月

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

    During skeletal muscle differentiation, the activation of some tissue-specific genes occurs immediately while others are delayed. The molecular basis controlling temporal gene regulation is poorly understood. We show that the regulatory sequences, but not other regions of genes expressed at late times of myogenesis, are in close physical proximity in differentiating embryonic tissue and in differentiating culture cells, despite these genes being located on different chromosomes. Formation of these inter-chromosomal interactions requires the lineage-determinant MyoD and functional Brg1, the ATPase subunit of SWI/SNF chromatin remodeling enzymes. Ectopic expression of myogenin and a specific Mef2 isoform induced myogenic differentiation without activating endogenous MyoD expression. Under these conditions, the regulatory sequences of late gene loci were not in close proximity, and these genes were prematurely activated. The data indicate that the spatial organization of late genes contributes to temporal regulation of myogenic transcription by restricting late gene expression during the early stages of myogenesis.

    DOI: 10.1093/nar/gkv046

  • Incorporation of histone H3.1 suppresses the lineage potential of skeletal muscle 査読

    Akihito Harada, Kazumitsu Maehara, Yuko Sato, Daijiro Konno, Taro Tachibana, Hiroshi Kimura, Yasuyuki Ohkawa

    Nucleic Acids Research   43 ( 2 )   775 - 786   2015年1月

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Lineage potential is triggered by lineage-specific transcription factors in association with changes in the chromatin structure. Histone H3.3 variant is thought to play an important role in the regulation of lineage-specific genes. To elucidate the function of H3.3 in myogenic differentiation, we forced the expression of GFP-H3.1 to alter the balance between H3.1 and H3.3 in mouse C2C12 cells that could be differentiated into myotubes. GFP-H3.1 replaced H3.3 in the regulatory regions of skeletal muscle (SKM) genes and induced a decrease of H3K4 trimethylation (H3K4me3) and increase of H3K27 trimethylation (H3K27me3). Similar results were obtained by H3.3 knockdown. In contrast, MyoD-dependent H3.3 incorporation into SKM genes in fibroblasts induced an increase of H3K4me3 and H3K27me3. In mouse embryos, a bivalent modification of H3K4me3 and H3K27me3 was formed on H3.3-incorporated SKM genes before embryonic skeletal muscle differentiation. These results suggest that lineage potential is established through a selective incorporation of specific H3 variants that governs the balance of histone modifications.

    DOI: 10.1093/nar/gku1346

  • Chd2 interacts with H3.3 to determine myogenic cell fate 査読

    Akihito Harada, Seiji Okada, Daijiro Konno, Jun Odawara, Tomohiko Yoshimi, Saori Yoshimura, Hiromi Kumamaru, Hirokazu Saiwai, Toshiaki Tsubota, Hitoshi Kurumizaka, Koichi Akashi, Taro Tachibana, Anthony N. Imbalzano, Yasuyuki Ohkawa

    EMBO Journal   31 ( 13 )   2994 - 3007   2012年7月

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Cell differentiation is mediated by lineage-determining transcription factors. We show that chromodomain helicase DNA-binding domain 2 (Chd2), a SNF2 chromatin remodelling enzyme family member, interacts with MyoD and myogenic gene regulatory sequences to specifically mark these loci via deposition of the histone variant H3.3 prior to cell differentiation. Directed and genome-wide analysis of endogenous H3.3 incorporation demonstrates that knockdown of Chd2 prevents H3.3 deposition at differentiation-dependent, but not housekeeping, genes and inhibits myogenic gene activation. The data indicate that MyoD determines cell fate and facilitates differentiation-dependent gene expression through Chd2-dependent deposition of H3.3 at myogenic loci prior to differentiation.

    DOI: 10.1038/emboj.2012.136

  • The anti-tumor effect of trifluridine via induction of aberrant mitosis is unaffected by mutations modulating p53 activity

    Wakasa, T; Nonaka, K; Harada, A; Ohkawa, Y; Kikutake, C; Suyama, M; Kobunai, T; Tsunekuni, K; Matsuoka, K; Kataoka, Y; Ochiiwa, H; Miyadera, K; Sagara, T; Oki, E; Ohdo, S; Maehara, Y; Iimori, M; Kitao, H

    CELL DEATH DISCOVERY   10 ( 1 )   307   2024年7月   ISSN:2058-7716 eISSN:2058-7716

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    記述言語:英語   出版者・発行元:Cell Death Discovery  

    The fluorinated thymidine analog trifluridine (FTD) is a chemotherapeutic drug commonly used to treat cancer; however, the mechanism by which FTD induces cytotoxicity is not fully understood. In addition, the effect of gain-of-function (GOF) missense mutations of the TP53 gene (encoding p53), which promote cancer progression and chemotherapeutic drug resistance, on the chemotherapeutic efficacy of FTD is unclear. Here, we revealed the mechanisms by which FTD-induced aberrant mitosis and contributed to cytotoxicity in both p53-null and p53-GOF missense mutant cells. In p53-null mutant cells, FTD-induced DNA double-stranded breaks, single-stranded DNA accumulation, and the associated DNA damage responses during the G2 phase. Nevertheless, FTD-induced DNA damage and the related responses were not sufficient to trigger strict G2/M checkpoint arrest. Thus, these features were carried over into mitosis, resulting in chromosome breaks and bridges, and subsequent cytokinesis failure. Improper mitotic exit eventually led to cell apoptosis, caused by the accumulation of extensive DNA damage and the presence of micronuclei encapsulated in the disrupted nuclear envelope. Upon FTD treatment, the behavior of the p53-GOF-missense mutant, isogenic cell lines, generated by CRISPR/Cas9 genome editing, was similar to that of p53-null mutant cells. Thus, our data suggest that FTD treatment overrode the effect on gene expression induced by p53-GOF mutants and exerted its anti-tumor activity in a manner that was independent of the p53 function.

    DOI: 10.1038/s41420-024-02083-3

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  • Plasma cell differentiation is regulated by the expression of histone variant H3.3 国際誌

    Saito, Y; Harada, A; Ushijima, M; Tanaka, K; Higuchi, R; Baba, A; Murakami, D; Nutt, SL; Nakagawa, T; Ohkawa, Y; Baba, Y

    NATURE COMMUNICATIONS   15 ( 1 )   5004 - 5004   2024年6月   eISSN:2041-1723

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Nature Communications  

    The differentiation of B cells into plasma cells is associated with substantial transcriptional and epigenetic remodeling. H3.3 histone variant marks active chromatin via replication-independent nucleosome assembly. However, its role in plasma cell development remains elusive. Herein, we show that during plasma cell differentiation, H3.3 is downregulated, and the deposition of H3.3 and chromatin accessibility are dynamically changed. Blockade of H3.3 downregulation by enforced H3.3 expression impairs plasma cell differentiation in an H3.3-specific sequence-dependent manner. Mechanistically, enforced H3.3 expression inhibits the upregulation of plasma cell-associated genes such as Irf4, Prdm1, and Xbp1 and maintains the expression of B cell-associated genes, Pax5, Bach2, and Bcl6. Concomitantly, sustained H3.3 expression prevents the structure of chromatin accessibility characteristic for plasma cells. Our findings suggest that appropriate H3.3 expression and deposition control plasma cell differentiation.

    DOI: 10.1038/s41467-024-49375-x

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  • Precise immunofluorescence canceling for highly multiplexed imaging to capture specific cell states 査読

    Tomimatsu, K; Fujii, T; Bise, R; Hosoda, K; Taniguchi, Y; Ochiai, H; Ohishi, H; Ando, K; Minami, R; Tanaka, K; Tachibana, T; Mori, S; Harada, A; Maehara, K; Nagasaki, M; Uchida, S; Kimura, H; Narita, M; Ohkawa, Y

    NATURE COMMUNICATIONS   15 ( 1 )   3657   2024年5月   eISSN:2041-1723

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    記述言語:英語   出版者・発行元:Nature Communications  

    Cell states are regulated by the response of signaling pathways to receptor ligand-binding and intercellular interactions. High-resolution imaging has been attempted to explore the dynamics of these processes and, recently, multiplexed imaging has profiled cell states by achieving a comprehensive acquisition of spatial protein information from cells. However, the specificity of antibodies is still compromised when visualizing activated signals. Here, we develop Precise Emission Canceling Antibodies (PECAbs) that have cleavable fluorescent labeling. PECAbs enable high-specificity sequential imaging using hundreds of antibodies, allowing for reconstruction of the spatiotemporal dynamics of signaling pathways. Additionally, combining this approach with seq-smFISH can effectively classify cells and identify their signal activation states in human tissue. Overall, the PECAb system can serve as a comprehensive platform for analyzing complex cell processes.

    DOI: 10.1038/s41467-024-47989-9

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  • Production of a Monoclonal Antibody for Histone H2b Isoform H2b3b 査読 国際誌

    Egashira S., Tachibana T., Nakamura M., Ohkawa Y., Harada A.

    Monoclonal Antibodies in Immunodiagnosis and Immunotherapy   43 ( 2 )   75 - 80   2024年4月

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    担当区分:最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Monoclonal Antibodies in Immunodiagnosis and Immunotherapy  

    H2b3b is one of the histone H2b isoforms that differs from canonical H2b by five to six amino acids. Previously, we identified H3t as the testis-specific histone H3 variant located in histone cluster 3, which is also the site of H2b3b. In this study, we produced monoclonal antibodies against H2b3b, using the iliac rat lymph node method for rat antibody and the immunochamber method for rabbit antibody. Immunoblot analysis confirmed that our antibodies could specifically discriminate between H2b3b and canonical H2b. Moreover, immunostaining revealed colocalization with a testicular stem cell marker, Plzf, but not with a meiotic marker, Sycp. This indicated that H2b3b is expressed in spermatogenic cells before meiosis. Our monoclonal antibodies enable further studies to reveal specific functions of H2b3b during spermatogenesis. We also hope that the established method will lead to the production of antibodies that can identify other H2b isoforms.

    DOI: 10.1089/mab.2023.0025

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  • Monoclonal Antibody Rat 2F11 and Rabbit A3 Against Anti-H2b3b 査読 国際誌

    Egashira S., Tachibana T., Nakamura M., Ohkawa Y., Harada A.

    Monoclonal Antibodies in Immunodiagnosis and Immunotherapy   43 ( 2 )   81 - 82   2024年4月

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    担当区分:最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Monoclonal Antibodies in Immunodiagnosis and Immunotherapy  

    DOI: 10.1089/mab.2024.0005

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  • 特集 クロマチンによる転写制御機構の最前線 Ⅴ.クロマチンと転写制御に関する新技術 クロマチン構造における転写制御の理解に向けた技術開発

    原田 哲仁, 富松 航佑, 武 千湄, 大川 恭行

    生体の科学   74 ( 3 )   255 - 259   2023年6月   ISSN:03709531 eISSN:18835503

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    出版者・発行元:株式会社医学書院  

    DOI: 10.11477/mf.2425201685

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  • FOXK1 promotes nonalcoholic fatty liver disease by mediating mTORC1-dependent inhibition of hepatic fatty acid oxidation 査読 国際誌

    Fujinuma, S; Nakatsumi, H; Shimizu, H; Sugiyama, S; Harada, A; Goya, T; Tanaka, M; Kohjima, M; Takahashi, M; Izumi, Y; Yagi, M; Kang, D; Kaneko, M; Shigeta, M; Bamba, T; Ohkawa, Y; Nakayama, KI

    CELL REPORTS   42 ( 5 )   112530 - 112530   2023年5月   ISSN:2211-1247

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    記述言語:その他   掲載種別:研究論文(学術雑誌)   出版者・発行元:Cell Reports  

    Nonalcoholic fatty liver disease (NAFLD) is a chronic metabolic disorder caused by overnutrition and can lead to nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). The transcription factor Forkhead box K1 (FOXK1) is implicated in regulation of lipid metabolism downstream of mechanistic target of rapamycin complex 1 (mTORC1), but its role in NAFLD-NASH pathogenesis is understudied. Here, we show that FOXK1 mediates nutrient-dependent suppression of lipid catabolism in the liver. Hepatocyte-specific deletion of Foxk1 in mice fed a NASH-inducing diet ameliorates not only hepatic steatosis but also associated inflammation, fibrosis, and tumorigenesis, resulting in improved survival. Genome-wide transcriptomic and chromatin immunoprecipitation analyses identify several lipid metabolism-related genes, including Ppara, as direct targets of FOXK1 in the liver. Our results suggest that FOXK1 plays a key role in the regulation of hepatic lipid metabolism and that its inhibition is a promising therapeutic strategy for NAFLD-NASH, as well as for HCC.

    DOI: 10.1016/j.celrep.2023.112530

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  • Photo-isolation chemistry for high-resolution and deep spatial transcriptome with mouse tissue sections 査読 国際誌

    Honda, M; Kimura, R; Harada, A; Maehara, K; Tanaka, K; Ohkawa, Y; Oki, S

    STAR PROTOCOLS   3 ( 2 )   101346 - 101346   2022年6月   ISSN:2666-1667

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:STAR Protocols  

    Photo-isolation chemistry (PIC) enables isolation of transcriptome information from locally defined areas by photo-irradiation. Here, we present an optimized PIC protocol for formalin-fixed frozen and paraffin mouse sections and fresh-frozen mouse sections. We describe tissue section preparation and permeabilization, followed by in situ reverse transcription using photo-caged primers. We then detail immunostaining and UV-mediated uncaging to the target areas, followed by linear amplification of uncaged cDNAs, library preparation, and quantification. This protocol can be applied to various animal tissue types. For complete details on the use and execution of this protocol, please refer to Honda et al. (2021).

    DOI: 10.1016/j.xpro.2022.101346

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  • Uterus-specific transcriptional regulation underlies eggshell pigment production in Japanese quail 査読 国際誌

    Ishishita, S; Kitahara, S; Takahashi, M; Iwasaki, S; Tatsumoto, S; Hara, I; Kaneko, Y; Kinoshita, K; Yamaguchi, K; Harada, A; Ohmori, Y; Ohkawa, Y; Go, Y; Shigenobu, S; Matsuda, Y; Suzuki, T

    PLOS ONE   17 ( 3 )   e0265008   2022年3月   ISSN:1932-6203

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PLoS ONE  

    The precursor of heme, protoporphyrin IX (PPIX), accumulates abundantly in the uteri of birds, such as Japanese quail, Coturnix japonica, which has brown-speckled eggshells; however, the molecular basis of PPIX production in the uterus remains largely unknown. Here, we investigated the cause of low PPIX production in a classical Japanese quail mutant exhibiting white eggshells by comparing its gene expression in the uterus with that of the wild type using transcriptome analysis. We also performed genetic linkage analysis to identify the causative genomic region of the white eggshell phenotype. We found that 11 genes, including 5’-aminolevulinate synthase 1 (ALAS1) and hephaestin-like 1 (HEPHL1), were specifically upregulated in the wild-type uterus and downregulated in the mutant. We mapped the 172 kb candidate genomic region on chromosome 6, which contains several genes, including a part of the paired-like homeodomain 3 (PITX3), which encodes a transcription factor. ALAS1, HEPHL1, and PITX3 were expressed in the apical cells of the luminal epithelium and lamina propria cells of the uterine mucosa of the wild-type quail, while their expression levels were downregulated in the cells of the mutant quail. Biochemical analysis using uterine homogenates indicated that the restricted availability of 5’-aminolevu-linic acid is the main cause of low PPIX production. These results suggest that uterus-specific transcriptional regulation of heme-biosynthesis-related genes is an evolutionarily acquired mechanism of eggshell pigment production in Japanese quail. Based on these findings, we discussed the molecular basis of PPIX production in the uteri of Japanese quails.

    DOI: 10.1371/journal.pone.0265008

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  • High-depth spatial transcriptome analysis by photo-isolation chemistry 査読 国際誌

    Mizuki Honda, Shinya Oki, Ryuichi Kimura, Akihito Harada, Kazumitsu Maehara, Kaori Tanaka, Chikara Meno, Yasuyuki Ohkawa

    Nature Communications   12 ( 1 )   2021年12月

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    記述言語:その他   掲載種別:研究論文(学術雑誌)  

    <title>Abstract</title>In multicellular organisms, expression profiling in spatially defined regions is crucial to elucidate cell interactions and functions. Here, we establish a transcriptome profiling method coupled with photo-isolation chemistry (PIC) that allows the determination of expression profiles specifically from photo-irradiated regions of interest. PIC uses photo-caged oligodeoxynucleotides for in situ reverse transcription. PIC transcriptome analysis detects genes specifically expressed in small distinct areas of the mouse embryo. Photo-irradiation of single cells demonstrated that approximately 8,000 genes were detected with 7 × 104 unique read counts. Furthermore, PIC transcriptome analysis is applicable to the subcellular and subnuclear microstructures (stress granules and nuclear speckles, respectively), where hundreds of genes can be detected as being specifically localised. The spatial density of the read counts is higher than 100 per square micrometre. Thus, PIC enables high-depth transcriptome profiles to be determined from limited regions up to subcellular and subnuclear resolutions.

    DOI: 10.1038/s41467-021-24691-8

  • Recent advances in single-cell epigenomics. 査読 国際誌

    Akihito Harada, Hiroshi Kimura, Yasuyuki Ohkawa

    Current opinion in structural biology   71   116 - 122   2021年12月

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

    The analysis of gene expression regulation, or the epigenome analysis, at the single-cell level is at the forefront of genomics research. To elucidate the mechanisms that regulate gene expression, chromatin immunoprecipitation has been conventionally used for determining the binding sites of DNA-binding proteins, such as histones and transcription factors. Now several new approaches have been emerged to reveal epigenome states at the single-cell level. Instead of using immunoprecipitation of fragmented chromatin, in situ reactions using cells or nuclei, combining with transposase tagging and other methods, have enabled single-cell analysis. Furthermore, single-cell multiomics techniques to simultaneously profiling transcriptome and open chromatin or histone modification have been developed. These single-cell analyses have the potential to identify different cell types in a cell population and reveal the dynamic changes of gene regulation, although those technologies have not yet reached a level for general application.

    DOI: 10.1016/j.sbi.2021.06.010

  • Chromatin structure-dependent histone incorporation revealed by a genome-wide deposition assay. 査読 国際誌

    Hiroaki Tachiwana, Mariko Dacher, Kazumitsu Maehara, Akihito Harada, Yosuke Seto, Ryohei Katayama, Yasuyuki Ohkawa, Hiroshi Kimura, Hitoshi Kurumizaka, Noriko Saitoh

    eLife   10   2021年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    In eukaryotes, histone variant distribution within the genome is the key epigenetic feature. To understand how each histone variant is targeted to the genome, we developed a new method, the RhIP (Reconstituted histone complex Incorporation into chromatin of Permeabilized cell) assay, in which epitope-tagged histone complexes are introduced into permeabilized cells and incorporated into their chromatin. Using this method, we found that H3.1 and H3.3 were incorporated into chromatin in replication-dependent and -independent manners, respectively. We further found that the incorporation of histones H2A and H2A.Z mainly occurred at less condensed chromatin (open), suggesting that condensed chromatin (closed) is a barrier for histone incorporation. To overcome this barrier, H2A, but not H2A.Z, uses a replication-coupled deposition mechanism. Our study revealed that the combination of chromatin structure and DNA replication dictates the differential histone deposition to maintain the epigenetic chromatin states.

    DOI: 10.7554/eLife.66290

  • Transcriptome analysis of gene expression changes upon enzymatic dissociation in skeletal myoblasts. 査読 国際誌

    Atsuko Miyawaki-Kuwakado, Qianmei Wu, Akihito Harada, Kosuke Tomimatsu, Takeru Fujii, Kazumitsu Maehara, Yasuyuki Ohkawa

    Genes to cells : devoted to molecular & cellular mechanisms   2021年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Single-cell RNA-sequencing analysis is one of the most effective tools for understanding specific cellular states. The use of single cells or pooled cells in RNA-seq analysis requires the isolation of cells from a tissue or culture. Although trypsin or more recently cold-active protease (CAP) has been used for cell dissociation, the extent to which the gene expression changes are suppressed has not been clarified. To this end, we conducted detailed profiling of the enzyme-dependent gene expression changes in mouse skeletal muscle progenitor cells, focusing on the enzyme treatment time, amount, and temperature. We found that the genes whose expression was changed by the enzyme treatment could be classified in a time-dependent manner, and that there were genes whose expression was changed independently of the enzyme treatment time, amount, and temperature. This study will be useful as reference data for genes that should be excluded or considered for RNA-seq analysis using enzyme isolation methods.

    DOI: 10.1111/gtc.12870

  • Targeted inhibition of EPAS1-driven IL-31 production by a small-molecule compound 査読 国際誌

    Yasuhisa Kamikaseda, Takehito Uruno, Kazufumi Kunimura, Akihito Harada, Kuniko Saiki, Kounosuke Oisaki, Daiji Sakata, Takeshi Nakahara, Makiko Kido-Nakahara, Motomu Kanai, Seiji Nakamura, Yasuyuki Ohkawa, Masutaka Furue, Yoshinori Fukui.

    The Journal of Allergy and Clinical Immunology   2021年4月

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    記述言語:日本語   掲載種別:研究論文(学術雑誌)  

    Background: IL-31 is a major pruritogen associated with atopic dermatitis (AD). Although a specific antibody for IL-31 receptor has been shown to alleviate pruritus in patients with AD, therapeutic approaches to inhibition of IL-31 production remain unexploited. IL-31 production by TH cells critically depends on the transcription factor EPAS1, which mediates IL31 promoter activation in collaboration with SP1.

    Objective: We aimed at developing small-molecule inhibitors that selectively block IL-31 production by TH cells.

    Methods: We generated the reporter cell line that inducibly expressed EPAS1 in the presence of doxycycline to mediate Il31 promoter activation, and we screened 9600 chemical compounds. The selected compounds were further examined by using TH cells from a spontaneous mouse model of AD and TH cells from patients with AD.

    Results: We have identified 4-(2-(4-isopropylbenzylidene)hydrazineyl)benzoic acid (IPHBA) as an inhibitor of IL31 induction. Although IPHBA did not affect nonspecific T-cell proliferation, IPHBA inhibited antigen-induced IL-31 production by TH cells from both an AD mouse model and patients with AD without affecting other cytokine production and hypoxic responses. In line with this, itch responses induced by adoptive transfer of IL-31-producing TH cells were attenuated when mice were orally treated with IPHBA. Mechanistically, IPHBA inhibited the association between EPAS1 and SP1, resulting in defective recruitment of both transcription factors to the specific sites of the IL31 promoter. We also determined the structure-activity relationship of IPHBA by synthesizing and analyzing 201 analogous compounds.

    Conclusion: IPHBA could be a potential drug leading to inhibition of EPAS1-driven IL-31 production.

    Keywords: Atopic dermatitis; EPAS1; IL-31; SP1; T(H) cells; small-molecule compounds.

    Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.jaci.2021.03.029

  • Genome-wide analysis of chromatin structure changes upon MyoD binding in proliferative myoblasts during the cell cycle. 査読 国際誌

    Qianmei Wu, Takeru Fujii, Akihito Harada, Kosuke Tomimatsu, Atsuko Miyawaki-Kuwakado, Masatoshi Fujita, Kazumitsu Maehara, Yasuyuki Ohkawa

    Journal of biochemistry   2021年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    MyoD, a myogenic differentiation protein, has been studied for its critical role in skeletal muscle differentiation. MyoD-expressing myoblasts have a potency to be differentiated with proliferation of ectopic cells. However, little is known about the effect on chromatin structure of MyoD binding in proliferative myoblasts. In this study, we evaluated the chromatin structure around MyoD-bound genome regions during the cell cycle by chromatin immunoprecipitation sequencing. Genome-wide analysis of histone modifications was performed in proliferative mouse C2C12 myoblasts during three phases (G1, S, G2/M) of the cell cycle. We found that MyoD-bound genome regions had elevated levels of active histone modifications, such as H3K4me1/2/3, and H3K27ac, compared with MyoD-unbound genome regions during the cell cycle. We also demonstrated that the elevated H3K4me2/3 modification level was maintained during the cell cycle, whereas the H3K27ac and H3K4me1 modification levels decreased to the same level as MyoD-unbound genome regions during the later phases. Immunoblot analysis revealed that MyoD abundance was high in the G1 phase then decreased in the S and G2/M phases. Our results suggest that MyoD binding formed selective epigenetic memories with H3K4me2/3 during the cell cycle in addition to myogenic gene induction via active chromatin formation coupled with transcription.

    DOI: 10.1093/jb/mvab001

  • Modeling population size independent tissue epigenomes by ChIL-seq with single-thin sections 査読 国際誌

    Kazumitsu Maehara, Kosuke Tomimatsu, Akihito Harada, Kaori Tanaka, Shoko Sato, Seiji Okada, Tetsuya Handa, Hitoshi Kurumizaka, Hiroshi Kimura, Yasuyuki Ohkawa

    Molecular Systems Biology   2020年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    <title>Abstract</title>Recent advances in omics studies have enabled analysis at the single-cell level; however, methods for analyzing the whole cell of large organs and tissues remain challenging. Here, we developed a method named tsChIL to understand the diverse cellular dynamics at the tissue level using high-depth epigenomic data. tsChIL allowed the analysis of a single tissue section and could reproducibly acquire epigenomic profiles from several types of tissues, based on the distribution of target epigenomic states, tissue morphology, and number of cells. The proposed method enabled the independent evaluation of changes in cell populations and gene activation of cells in regenerating skeletal muscle tissues, using a statistical model of RNA polymerase II distribution on gene loci. Thus, the integrative analysis by tsChIL can elucidate <italic>in vivo</italic> cell-type dynamics of tissues.

    DOI: 10.1101/2020.12.18.423434

  • Subnuclear gene positioning through lamina association affects copper tolerance 査読 国際誌

    Yuki Sakamoto, Mayuko Sato, Yoshikatsu Sato, Akihito Harada, Takamasa Suzuki, Chieko Goto, Kentaro Tamura, Kiminori Toyooka, Hiroshi Kimura, Yasuyuki Ohkawa, Ikuko Hara-Nishimura, Shingo Takagi, Sachihiro Matsunaga

    Nature Communications   11 ( 1 )   2020年12月

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    記述言語:その他   掲載種別:研究論文(学術雑誌)  

    <title>Abstract</title>The nuclear lamina plays an important role in the regulation of chromatin organization and gene positioning in animals. CROWDED NUCLEI (CRWN) is a strong candidate for the plant nuclear lamina protein in <italic>Arabidopsis thaliana</italic> but its biological function was largely unknown. Here, we show that CRWNs localize at the nuclear lamina and build the meshwork structure. Fluorescence in situ hybridization and RNA-seq analyses revealed that CRWNs regulate chromatin distribution and gene expression. More than 2000 differentially expressed genes were identified in the <italic>crwn1crwn4</italic> double mutant. Copper-associated (<italic>CA</italic>) genes that form a gene cluster on chromosome 5 were among the downregulated genes in the double mutant exhibiting low tolerance to excess copper. Our analyses showed this low tolerance to copper was associated with the suppression of <italic>CA</italic> gene expression and that CRWN1 interacts with the <italic>CA</italic> gene locus, enabling the locus to localize at the nuclear lamina under excess copper conditions.

    DOI: 10.1038/s41467-020-19621-z

  • Genome-wide kinetic properties of transcriptional bursting in mouse embryonic stem cells. 査読 国際誌

    Hiroshi Ochiai, Tetsutaro Hayashi, Mana Umeda, Mika Yoshimura, Akihito Harada, Yukiko Shimizu, Kenta Nakano, Noriko Saitoh, Zhe Liu, Takashi Yamamoto, Tadashi Okamura, Yasuyuki Ohkawa, Hiroshi Kimura, Itoshi Nikaido

    Science advances   6 ( 25 )   eaaz6699   2020年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Transcriptional bursting is the stochastic activation and inactivation of promoters, contributing to cell-to-cell heterogeneity in gene expression. However, the mechanism underlying the regulation of transcriptional bursting kinetics (burst size and frequency) in mammalian cells remains elusive. In this study, we performed single-cell RNA sequencing to analyze the intrinsic noise and mRNA levels for elucidating the transcriptional bursting kinetics in mouse embryonic stem cells. Informatics analyses and functional assays revealed that transcriptional bursting kinetics was regulated by a combination of promoter- and gene body-binding proteins, including the polycomb repressive complex 2 and transcription elongation factors. Furthermore, large-scale CRISPR-Cas9-based screening identified that the Akt/MAPK signaling pathway regulated bursting kinetics by modulating transcription elongation efficiency. These results uncovered the key molecular mechanisms underlying transcriptional bursting and cell-to-cell gene expression noise in mammalian cells.

    DOI: 10.1126/sciadv.aaz6699

  • Chromatin-bound CRM1 recruits SET-Nup214 and NPM1c onto HOX clusters causing aberrant HOX expression in leukemia cells 査読

    Masahiro Oka, Sonoko Mura, Mayumi Otani, Yoichi Miyamoto, Jumpei Nogami, Kazumitsu Maehara, Akihito Harada, Taro Tachibana, Yoshihiro Yoneda, Yasuyuki Ohkawa

    eLife   8   2019年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    We previously demonstrated that CRM1, a major nuclear export factor, accumulates at Hox cluster regions to recruit nucleoporin-fusion protein Nup98HoxA9, resulting in robust activation of Hox genes (Oka et al., 2016). However, whether this phenomenon is general to other leukemogenic proteins remains unknown. Here, we show that two other leukemogenic proteins, nucleoporin-fusion SET-Nup214 and the NPM1 mutant, NPM1c, which contains a nuclear export signal (NES) at its C-terminus and is one of the most frequent mutations in acute myeloid leukemia, are recruited to the HOX cluster region via chromatin-bound CRM1, leading to HOX gene activation in human leukemia cells. Furthermore, we demonstrate that this mechanism is highly sensitive to a CRM1 inhibitor in leukemia cell line. Together, these findings indicate that CRM1 acts as a key molecule that connects leukemogenic proteins to aberrant HOX gene regulation either via nucleoporin-CRM1 interaction (for SET-Nup214) or NES-CRM1 interaction (for NPM1c).

    DOI: 10.7554/eLife.46667

  • Sustained expression of HeyL is critical for the proliferation of muscle stem cells in overloaded muscle 査読

    Sumiaki Fukuda, Akihiro Kaneshige, Takayuki Kaji, Yu Taro Noguchi, Yusei Takemoto, Lidan Zhang, Kazutake Tsujikawa, Hiroki Kokubo, Akiyoshi Uezumi, Kazumitsu Maehara, Akihito Harada, Yasuyuki Ohkawa, So Ichiro Fukada

    eLife   8   2019年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    In overloaded and regenerating muscle, the generation of new myonuclei depends on muscle satellite cells (MuSCs). Because MuSC behaviors in these two environments have not been considered separately, MuSC behaviors in overloaded muscle remain unexamined. Here, we show that most MuSCs in overloaded muscle, unlike MuSCs in regenerating muscle, proliferate in the absence of MyoD expression. Mechanistically, MuSCs in overloaded muscle sustain the expression of Heyl, a Notch effector gene, to suppress MyoD expression, which allows effective MuSC proliferation on myofibers and beneath the basal lamina. Although Heyl-knockout mice show no impairment in an injury model, in a hypertrophy model, their muscles harbor fewer new MuSCderived myonuclei due to increased MyoD expression and diminished proliferation, which ultimately causes blunted hypertrophy. Our results show that sustained HeyL expression is critical for MuSC proliferation specifically in overloaded muscle, and thus indicate that the MuSCproliferation mechanism differs in overloaded and regenerating muscle.

    DOI: 10.7554/eLife.48284

  • Biochemical analysis of nucleosome targeting by Tn5 transposase 査読

    Shoko Sato, Yasuhiro Arimura, Tomoya Kujirai, Akihito Harada, Kazumitsu Maehara, Jumpei Nogami, Yasuyuki Ohkawa, Hitoshi Kurumizaka

    Open Biology   9 ( 8 )   2019年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Tn5 transposase is a bacterial enzyme that integrates a DNA fragment into genomic DNA, and is used as a tool for detecting nucleosome-free regions of genomic DNA in eukaryotes. However, in chromatin, the DNA targeting by Tn5 transposase has remained unclear. In the present study, we reconstituted well-positioned 601 dinucleosomes, in which two nucleosomes are connected with a linker DNA, and studied the DNA integration sites in the dinucleosomes by Tn5 transposase in vitro. We found that Tn5 transposase preferentially targets near the entry–exit DNA regions within the nucleosome. Tn5 transposase minimally cleaved the dinucleosome without a linker DNA, indicating that the linker DNA between two nucleosomes is important for the Tn5 transposase activity. In the presence of a 30 base-pair linker DNA, Tn5 transposase targets the middle of the linker DNA, in addition to the entry–exit sites of the nucleosome. Intriguingly, this Tn5-targeting characteristic is conserved in a dinucleosome substrate with a different DNA sequence from the 601 sequence. Therefore, the Tn5-targeting preference in the nucleosomal templates reported here provides important information for the interpretation of Tn5 transposase-based genomics methods, such as ATAC-seq.

    DOI: 10.1098/rsob.190116

  • Cell-autonomous and redundant roles of Hey1 and HeyL in muscle stem cells HeyL requires HeS1 to bind diverse DNA sites 査読

    Yu Taro Noguchi, Miki Nakamura, Nobumasa Hino, Jumpei Nogami, Sayaka Tsuji, Takahiko Sato, Lidan Zhang, Kazutake Tsujikawa, Toru Tanaka, Kohei Izawa, Yoshiaki Okada, Takefumi Doi, Hiroki Kokubo, Akihito Harada, Akiyoshi Uezumi, Manfred Gessler, Yasuyuki Ohkawa, So Ichiro Fukada

    Development (Cambridge)   146 ( 4 )   2019年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    The undifferentiated state of muscle stem (satellite) cells (MuSCs) is maintained by the canonical Notch pathway. Although three bHLH transcriptional factors, Hey1, HeyL and Hes1, are considered to be potential effectors of the Notch pathway exerting anti-myogenic effects, neither HeyL nor Hes1 inhibits myogenic differentiation of myogenic cell lines. Furthermore, whether these factors work redundantly or cooperatively is unknown. Here, we showed cell-autonomous functions of Hey1 and HeyL in MuSCs using conditional and genetic null mice. Analysis of cultured MuSCs revealed anti-myogenic activity of both HeyL and Hes1. We found that HeyL forms heterodimeric complexes with Hes1 in living cells. Moreover, our ChIP-seq experiments demonstrated that, compared with HeyL alone, the HeyL-Hes1 heterodimer binds with high affinity to specific sites in the chromatin, including the binding sites of Hey1. Finally, analyses of myogenin promoter activity showed that HeyL and Hes1 act synergistically to suppress myogenic differentiation. Collectively, these results suggest that HeyL and Hey1 function redundantly in MuSCs, and that HeyL requires Hes1 for effective DNA binding and biological activity.

    DOI: 10.1242/dev.163618

  • Locomotor Training Increases Synaptic Structure With High NGL-2 Expression After Spinal Cord Hemisection 査読

    Kazu Kobayakawa, Kyleigh Alexis DePetro, Hui Zhong, Bau Pham, Masamitsu Hara, Akihito Harada, Jumpei Nogami, Yasuyuki Ohkawa, V. Reggie Edgerton

    Neurorehabilitation and Neural Repair   2019年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Background. We previously demonstrated that step training leads to reorganization of neuronal networks in the lumbar spinal cord of rodents after a hemisection (HX) injury and step training, including increases excitability of spinally evoked potentials in hindlimb motor neurons. Methods. In this study, we investigated changes in RNA expression and synapse number using RNA-Seq and immunohistochemistry of the lumbar spinal cord 23 days after a mid-thoracic HX in rats with and without post-HX step training. Results. Gene Ontology (GO) term clustering demonstrated that expression levels of 36 synapse-related genes were increased in trained compared with nontrained rats. Many synaptic genes were upregulated in trained rats, but Lrrc4 (coding NGL-2) was the most highly expressed in the lumbar spinal cord caudal to the HX lesion. Trained rats also had a higher number of NGL-2/synaptophysin synaptic puncta in the lumbar ventral horn. Conclusions. Our findings demonstrate clear activity-dependent regulation of synapse-related gene expression post-HX. This effect is consistent with the concept that activity-dependent phenomena can provide a mechanistic drive for epigenetic neuronal group selection in the shaping of the reorganization of synaptic networks to learn the locomotion task being trained after spinal cord injury.

    DOI: 10.1177/1545968319829456

  • Roles of histone H3.5 in human spermatogenesis and spermatogenic disorders 査読

    K. Shiraishi, A. Shindo, Akihito Harada, H. Kurumizaka, H. Kimura, Yasuyuki Ohkawa, H. Matsuyama

    Andrology   6 ( 1 )   158 - 165   2018年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Histone H3.5 (H3.5) is a newly identified histone variant highly expressed in the human testis. We have reported the crystal structure, instability of the H3.5 nucleosome and accumulation around transcription start sites, mainly in primary spermatocytes, but its role in human spermatogenesis remains poorly understood. Testicular biopsy specimens from 30 men (mean age: 35 years) with non-obstructive azoospermia (NOA) who underwent microdissection testicular sperm extraction and 23 men with obstructive azoospermia (OA) were included. An H3.5-specific mouse monoclonal antibody recognizing an H3.5-specific synthetic peptide was generated, and immunohistological staining for H3.5 and proliferating cell nuclear antigen (PCNA) was performed on Bouin's solution-fixed sections. Expression and localization of H3.5 were compared with patient background, germinal stage, and PCNA expression. In testes of patients with normal spermatogenesis, differentially expressed H3.5 was specifically localized in either spermatogonia or preleptotene/leptotene-stage primary spermatocytes, especially during germinal stages VI–X. In NOA testes, mRNA expression of H3.5 (H3F3C) was significantly reduced compared with other H3 histone family members, and expression of H3.5 was significantly lower than that in OA. Additionally, the number of H3.5-positive germ cells was higher in hypospermatogenesis or late maturation arrest than in early maturation arrest in NOA testes (p < 0.01). A significant positive correlation was observed between H3.5 and PCNA expression (p < 0.05) but not TUNEL-positive cells, and expression of H3.5 was enhanced after hCG-based salvage hormonal therapy. Different from other testis-specific histones, which are often expressed during the histone-to-protamine transition during meiosis, H3.5 was expressed mainly in immature germ cells. H3.5 may play roles in DNA synthesis, but not apoptosis, and its expression is regulated by gonadotropins, indicating that such epigenetic regulations are important in normal spermatogenesis and spermatogenic disorders.

    DOI: 10.1111/andr.12438

  • Sensitive detection of fluorescence in western blotting by merging images 査読

    Yukari Kondo, Shinichiro Higa, Takeshi Iwasaki, Tomoya Matsumoto, Kazumitsu Maehara, Akihito Harada, Yoshihiro Baba, Masatoshi Fujita, Yasuyuki Ohkawa

    PLoS One   13 ( 1 )   2018年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    The western blotting technique is widely used to analyze protein expression levels and protein molecular weight. The chemiluminescence method is mainly used for detection due to its high sensitivity and ease of manipulation, but it is unsuitable for detailed analyses because it cannot be used to detect multiple proteins simultaneously. Recently, more attention has been paid to the fluorescence detection method because it is more quantitative and is suitable for the detection of multiple proteins simultaneously. However, fluorescence detection can be limited by poor image resolution and low detection sensitivity. Here, we describe a method to detect fluorescence in western blots using fluorescence microscopy to obtain high-resolution images. In this method, filters and fluorescent dyes are optimized to enhance detection sensitivity to a level similar to that of the chemiluminescence method.

    DOI: 10.1371/journal.pone.0191532

  • Chd2 regulates chromatin for proper gene expression toward differentiation in mouse embryonic stem cells 査読

    Yuichiro Semba, Akihito Harada, Kazumitsu Maehara, Shinya Oki, Chikara Meno, Jun Ueda, Kazuo Yamagata, Atsushi Suzuki, Mitsuho Onimaru, Jumpei Nogami, Seiji Okada, Koichi Akashi, Yasuyuki Ohkawa

    Nucleic Acids Research   45 ( 15 )   8758 - 8772   2017年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Chromatin reorganization is necessary for pluripotent stem cells, including embryonic stem cells (ESCs), to acquire lineage potential. However, it remains unclear how ESCs maintain their characteristic chromatin state for appropriate gene expression upon differentiation. Here, we demonstrate that chromodomain helicase DNA-binding domain 2 (Chd2) is required to maintain the differentiation potential of mouse ESCs. Chd2-depleted ESCs showed suppressed expression of developmentally regulated genes upon differentiation and subsequent differentiation defects without affecting gene expression in the undifferentiated state. Furthermore, chromatin immunoprecipitation followed by sequencing revealed alterations in the nucleosome occupancy of the histone variant H3.3 for developmentally regulated genes in Chd2-depleted ESCs, which in turn led to elevated trimethylation of the histone H3 lysine 27. These results suggest that Chd2 is essential in preventing suppressive chromatin formation for developmentally regulated genes and determines subsequent effects on developmental processes in the undifferentiated state.

    DOI: 10.1093/nar/gkx475

  • Crystal Structure and Characterization of Novel Human Histone H3 Variants, H3.6, H3.7, and H3.8 査読

    Hiroyuki Taguchi, Yan Xie, Naoki Horikoshi, Kazumitsu Maehara, Akihito Harada, Jumpei Nogami, Koichi Sato, Yasuhiro Arimura, Akihisa Osakabe, Tomoya Kujirai, Takeshi Iwasaki, Yuichiro Semba, Taro Tachibana, Hiroshi Kimura, Yasuyuki Ohkawa, Hitoshi Kurumizaka

    Biochemistry   56 ( 16 )   2184 - 2196   2017年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Non-allelic histone variants are considered as epigenetic factors that regulate genomic DNA functions in eukaryotic chromosomes. In this study, we identified three new human histone H3 variants (named H3.6, H3.7, and H3.8), which were previously annotated as pseudogenes. H3.6 and H3.8 conserve the H3.3-specific amino acid residues, but H3.7 shares the specific amino acid residues with H3.1. We successfully reconstituted the nucleosome containing H3.6 in vitro and determined its crystal structure. In the H3.6 nucleosome, the H3.6-specific Val62 residue hydrophobically contacts the cognate H4 molecule, but its contact area is smaller than that of the corresponding H3.3 Ile62 residue. The thermal stability assay revealed that the H3.6 nucleosome is substantially unstable, as compared to the H3.3 nucleosome. Interestingly, mutational analysis demonstrated that the H3.6 Val62 residue is fully responsible for the H3.6 nucleosome instability, probably because of the weakened hydrophobic interaction with H4. We also reconstituted the nucleosome containing H3.8, but its thermal stability was quite low. In contrast, purified H3.7 failed to form nucleosomes in vitro. The identification and characterization of these novel human histone H3 variants provide important new insights into understanding the epigenetic regulation of the human genome.

    DOI: 10.1021/acs.biochem.6b01098

  • ROLES OF HISTONE H3.5 IN HUMAN SPERMATOGENESIS AND SPERMATOGENIC DISORDERS 査読

    Koji Shiraishi, Aya Shindo, Akihito Harada, Yasuyuki Ohkawa, Hitoshi Kurumizaka, Hiroshi Kimura, Hideyasu Matsuyama

    JOURNAL OF UROLOGY   197 ( 4 )   E85 - E85   2017年4月

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    記述言語:英語  

    DOI: 10.1016/j.juro.2017.02.277

  • Periostin Promotes Scar Formation through the Interaction between Pericytes and Infiltrating Monocytes/Macrophages after Spinal Cord Injury 査読

    Kazuya Yokota, Kazu Kobayakawa, Takeyuki Saito, Masamitsu Hara, Ken Kijima, Yasuyuki Ohkawa, Akihito Harada, Ken Okazaki, Kohei Ishihara, Shigeo Yoshida, Akira Kudo, Yukihide Iwamoto, Seiji Okada

    American Journal of Pathology   187 ( 3 )   639 - 653   2017年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Scar formation is a prominent pathological feature of traumatic central nervous system (CNS) injury, which has long been implicated as a major impediment to the CNS regeneration. However, the factors affecting such scar formation remain to be elucidated. We herein demonstrate that the extracellular matrix protein periostin (POSTN) is a key player in scar formation after traumatic spinal cord injury (SCI). Using high-throughput RNA sequencing data sets, we found that the genes involved in the extracellular region, such as POSTN, were significantly expressed in the injured spinal cord. The expression of POSTN peaked at 7 days after SCI, predominantly in the scar-forming pericytes. Notably, we found that genetic deletion of POSTN in mice reduced scar formation at the lesion site by suppressing the proliferation of the pericytes. Conversely, we found that recombinant POSTN promoted the migration capacity of the monocytes/macrophages and increased the expression of tumor necrosis factor-α from the monocytes/macrophages in vitro, which facilitated the proliferation of pericytes. Furthermore, we revealed that the pharmacological blockade of POSTN suppressed scar formation and improved the long-term functional outcome after SCI. Our findings suggest a potential mechanism whereby POSTN regulates the scar formation after SCI and provide significant evidence that POSTN is a promising therapeutic target for CNS injury.

    DOI: 10.1016/j.ajpath.2016.11.010

  • The requirement of Mettl3-promoted MyoD mRNA maintenance in proliferative myoblasts for skeletal muscle differentiation 査読

    Kensuke Kudou, Tetsuro Komatsu, Jumpei Nogami, Kazumitsu Maehara, Akihito Harada, Hiroshi Saeki, Eiji Oki, Yoshihiko Maehara, Yasuyuki Ohkawa

    Open Biology   7 ( 9 )   2017年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Myogenic progenitor/stem cells retain their skeletal muscle differentiation potential by maintaining myogenic transcription factors such as MyoD. However, the mechanism of how MyoD expression is maintained in proliferative progenitor cells has not been elucidated. Here, we found that MyoD expression was reduced at the mRNA level by cell cycle arrest in S and G2 phases, which in turn led to the absence of skeletal muscle differentiation. The reduction of MyoD mRNA was correlated with the reduced expression of factors regulating RNA metabolism, including methyltransferase like 3 (Mettl3), which induces N6-methyladenosine (m6A) modifications of RNA. Knockdown of Mettl3 revealed that MyoD RNA was specifically downregulated and that this was caused by a decrease in processed, but not unprocessed, mRNA. Potential m6A modification sites were profiled by m6A sequencing and identified within the 50 untranslated region (UTR) of MyoD mRNA. Deletion of the 50 UTR revealed that it has a role in MyoD mRNA processing. These data showed that Mettl3 is required for MyoD mRNA expression in proliferative myoblasts.

    DOI: 10.1098/rsob.170119

  • Identification of immunoglobulin gene sequences from a small read number of mRNA-seq using hybridomas 査読

    Yuki Kuniyoshi, Kazumitsu Maehara, Takeshi Iwasaki, Masayasu Hayashi, Yuichiro Semba, Masatoshi Fujita, Yuko Sato, Hiroshi Kimura, Akihito Harada, Yasuyuki Ohkawa

    PLoS One   11 ( 10 )   2016年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Identification of immunoglobulin genes in hybridomas is essential for producing antibodies for research and clinical applications. A couple of methods such as RACE and degenerative PCR have been developed for determination of the Igh and Igl/Igk coding sequences (CDSs) but it has been difficult to process a number of hybridomas both with accuracy and rapidness. Here, we propose a new strategy for antibody sequence determination by mRNA-seq of hybridomas. We demonstrated that hybridomas highly expressed the Igh and Igl/Igk genes and that de novo transcriptome assembly using mRNA-seq data enabled identification of the CDS of both Igh and Igl/Igk accurately. Furthermore, we estimated that only 30,000 sequenced reads are required to identify immunoglobulin sequences from four different hybridoma clones. Thus, our approach would facilitate determining variable CDSs drastically.

    DOI: 10.1371/journal.pone.0165473

  • Histone H4 lysine 20 acetylation is associated with gene repression in human cells 査読

    Jun Ya Kaimori, Kazumitsu Maehara, Yoko Hayashi-Takanaka, Akihito Harada, Masafumi Fukuda, Satoko Yamamoto, Naotsugu Ichimaru, Takashi Umehara, Shigeyuki Yokoyama, Ryo Matsuda, Tsuyoshi Ikura, Koji Nagao, Chikashi Obuse, Naohito Nozaki, Shiro Takahara, Toshifumi Takao, Yasuyuki Ohkawa, Hiroshi Kimura, Yoshitaka Isaka

    Scientific Reports   6   2016年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Histone acetylation is generally associated with gene activation and chromatin decondensation. Recent mass spectrometry analysis has revealed that histone H4 lysine 20, a major methylation site, can also be acetylated. To understand the function of H4 lysine 20 acetylation (H4K20ac), we have developed a specific monoclonal antibody and performed ChIP-seq analysis using HeLa-S3 cells. H4K20ac was enriched around the transcription start sites (TSSs) of minimally expressed genes and in the gene body of expressed genes, in contrast to most histone acetylation being enriched around the TSSs of expressed genes. The distribution of H4K20ac showed little correlation with known histone modifications, including histone H3 methylations. A motif search in H4K20ac-enriched sequences, together with transcription factor binding profiles based on ENCODE ChIP-seq data, revealed that most transcription activators are excluded from H4K20ac-enriched genes and a transcription repressor NRSF/REST co-localized with H4K20ac. These results suggest that H4K20ac is a unique acetylation mark associated with gene repression.

    DOI: 10.1038/srep24318

  • Chd5 Regulates MuERV-L/MERVL Expression in Mouse Embryonic Stem Cells Via H3K27me3 Modification and Histone H3.1/H3.2 査読

    Masayasu Hayashi, Kazumitsu Maehara, Akihito Harada, Yuichiro Semba, Kensuke Kudo, Hidehisa Takahashi, Shinya Oki, Chikara Meno, Kenji Ichiyanagi, Koichi Akashi, Yasuyuki Ohkawa

    Journal of Cellular Biochemistry   117 ( 3 )   780 - 792   2016年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Chd5 is an essential factor for neuronal differentiation and spermatogenesis and is a known tumor suppressor. H3K27me3 and H3K4un are modifications recognized by Chd5; however, it remains unclear how Chd5 remodels chromatin structure. We completely disrupted the Chd5 locus using the CRISPR-Cas9 system to generate a 52 kbp long deletion and analyzed Chd5 function in mouse embryonic stem cells. Our findings show that Chd5 represses murine endogenous retrovirus-L (MuERV-L/MERVL), an endogenous retrovirus-derived retrotransposon, by regulating H3K27me3 and H3.1/H3.2 function. J. Cell. Biochem. 117: 780-792, 2016.

    DOI: 10.1002/jcb.25368

  • Histone H3.5 forms an unstable nucleosome and accumulates around transcription start sites in human testis 査読

    Takashi Urahama, Akihito Harada, Kazumitsu Maehara, Naoki Horikoshi, Koichi Sato, Yuko Sato, Koji Shiraishi, Norihiro Sugino, Akihisa Osakabe, Hiroaki Tachiwana, Wataru Kagawa, Hiroshi Kimura, Yasuyuki Ohkawa, Hitoshi Kurumizaka

    Epigenetics and Chromatin   9 ( 1 )   2016年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Background: Human histone H3.5 is a non-allelic H3 variant evolutionally derived from H3.3. The H3.5 mRNA is highly expressed in human testis. However, the function of H3.5 has remained poorly understood. Results: We found that the H3.5 nucleosome is less stable than the H3.3 nucleosome. The crystal structure of the H3.5 nucleosome showed that the H3.5-specific Leu103 residue, which corresponds to the H3.3 Phe104 residue, reduces the hydrophobic interaction with histone H4. Mutational analyses revealed that the H3.5-specific Leu103 residue is responsible for the instability of the H3.5 nucleosome, both in vitro and in living cells. The H3.5 protein was present in human seminiferous tubules, but little to none was found in mature sperm. A chromatin immunoprecipitation coupled with sequencing analysis revealed that H3.5 accumulated around transcription start sites (TSSs) in testicular cells. Conclusions: We performed comprehensive studies of H3.5, and found the instability of the H3.5 nucleosome and the accumulation of H3.5 protein around TSSs in human testis. The unstable H3.5 nucleosome may function in the chromatin dynamics around the TSSs, during spermatogenesis.

    DOI: 10.1186/s13072-016-0051-y

  • Distribution of histone H4 modifications as revealed by a panel of specific monoclonal antibodies 査読

    Yoko Hayashi-Takanaka, Kazumitsu Maehara, Akihito Harada, Takashi Umehara, Shigeyuki Yokoyama, Chikashi Obuse, Yasuyuki Ohkawa, Naohito Nozaki, Hiroshi Kimura

    Chromosome Research   23 ( 4 )   753 - 766   2015年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Post-translational histone modifications play a critical role in genome functions such as epigenetic gene regulation and genome maintenance. The tail of the histone H4 N-terminus contains several amino acids that can be acetylated and methylated. Some of these modifications are known to undergo drastic changes during the cell cycle. In this study, we generated a panel of mouse monoclonal antibodies against histone H4 modifications, including acetylation at K5, K8, K12, and K16, and different levels of methylation at K20. Their specificity was evaluated by ELISA and immunoblotting using synthetic peptide and recombinant proteins that harbor specific modifications or amino acid substitutions. Immunofluorescence confirmed the characteristic distributions of target modifications. An H4K5 acetylation (H4K5ac)-specific antibody CMA405 reacted with K5ac only when the neighboring K8 was unacetylated. This unique feature allowed us to detect newly assembled H4, which is diacetylated at K5 and K12, and distinguish it from hyperacetylated H4, where K5 and K8 are both acetylated. Chromatin immunoprecipiation combined with deep sequencing (ChIP-seq) revealed that acetylation of both H4K8 and H4K16 were enriched around transcription start sites. These extensively characterized and highly specific antibodies will be useful for future epigenetics and epigenome studies.

    DOI: 10.1007/s10577-015-9486-4

  • Relationship between the risk for a shrimp allergy and freshness or cooking 査読

    Masakatsu Usui, Akihito Harada, Shinya Yasumoto, Yoshimasa Sugiura, Anri Nishidai, Maria Ikarashi, Honami Takaba, Taiko Miyasaki, Hiroyuki Azakami, Masakazu Kondo

    Bioscience, Biotechnology and Biochemistry   79 ( 10 )   1698 - 1701   2015年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Tropomyosins are defined as risk factors for shrimp allergy. However, their concentration in different preparations has not been clarified. We quantified the tropomyosin concentration in shrimp meat, which was cooked using several methods or was stored under various conditions. The results demonstrated that shrimp meat from various preparations and storage conditions maintained tropomyosin concentrations that were sufficient to cause food allergies.

    DOI: 10.1080/09168451.2015.1045830

  • Establishment of neutralizing rat monoclonal antibodies for fibroblast growth factor-2 査読

    Masako Tanaka, Maki Yamaguchi, Masayuki Shiota, Yukiko Kawamoto, Katsuyuki Takahashi, Azusa Inagaki, Mayuko Osada-Oka, Akihito Harada, Hideki Wanibuchi, Yasukatsu Izumi, Katsuyuki Miura, Hiroshi Iwao, Yasuyuki Ohkawa

    Monoclonal Antibodies in Immunodiagnosis and Immunotherapy   33 ( 4 )   261 - 269   2014年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Fibroblast growth factor-2 (FGF-2) plays a critical role in endothelial survival, proliferation, and angiogenesis and is localized on the cell membrane by binding to heparan sulfate proteoglycans. Here we established a neutralizing monoclonal antibody, 1B9B9, against FGF-2 using the rat medial iliac lymph node method. 1B9B9 blocked the binding of FGF-2 to its receptor, inhibiting FGF-2-induced proliferation and corresponding downstream signaling in endothelial cells. Treatment of human umbilical vein endothelial cells with 1B9B9 reduced the basal phosphorylation levels of Akt and MAPK. Furthermore, continued treatment with 1B9B9 induced cell death by apoptosis. Compared with FGF-2 knockdown, 1B9B9 significantly reduced cell survival. In addition, the combination of FGF-2 siRNA and 1B9B9 showed a synergistic effect. The data indicate that 1B9B9 established by the rat iliac lymph node method is a fully compatible neutralizing antibody.

    DOI: 10.1089/mab.2013.0085

  • Hsc70 contributes to cancer cell survival by preventing Rab1A degradation under stress conditions 査読

    Masako Tanaka, Saya Mun, Akihito Harada, Yasuyuki Ohkawa, Azusa Inagaki, Soichi Sano, Katsuyuki Takahashi, Yasukatsu Izumi, Mayuko Osada-Oka, Hideki Wanibuchi, Masayo Yamagata, Tokihito Yukimura, Katsuyuki Miura, Masayuki Shiota, Hiroshi Iwao

    PLoS One   9 ( 5 )   2014年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Heat shock cognate protein 70 (Hsc70) acts as a molecular chaperone for the maintenance of intracellular proteins, which allows cancer cells to survive under proteotoxic stress. We attempted to use Hsc70 to identify key molecules in cancer cell survival. Here, we performed mass-spectrometry-based proteomics analysis utilizing affinity purification with anti-Hsc70 antibodies; as a result, 83 differentially expressed proteins were identified under stress conditions. This result implies that there was a change in the proteins with which Hsc70 interacted in response to stress. Among the proteins identified under both serum-depleted and 5-fluorouracil-treated conditions, Rab1A was identified as an essential molecule for cancer cell survival. Hsc70 interacted with Rab1A in a chaperone-dependent manner. In addition, Hsc70 knockdown decreased the level of Rab1A and increased the level of its ubiquitination under stress conditions, suggesting that Hsc70 prevented the degradation of Rab1A denatured by stress exposure. We also found that Rab1A knockdown induced cell death by inhibition of autophagosome formation. Rab1A may therefore contribute to overcoming proteotoxic insults, which allows cancer cells to survive under stress conditions. Analysis of Hsc70 interactors provided insight into changes of intracellular status. We expect further study of the Hsc70 interactome to provide a more comprehensive understanding of cancer cell physiology.

    DOI: 10.1371/journal.pone.0096785

  • Production of a monoclonal antibody for C/EBPβ The subnuclear localization of C/EBPβ in mouse L929 cells 査読

    Akihito Harada, Etsuko Okazaki, Seiji Okada, Taro Tachibana, Yasuyuki Ohkawa

    Monoclonal Antibodies in Immunodiagnosis and Immunotherapy   33 ( 1 )   34 - 37   2014年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    The CCAAT/enhancer-binding protein (C/EBP)β belongs to the C/EBP family of proteins that possesses a basic leucine zipper DNA-binding domain. These proteins bind DNA by dimerization and play a role in the transcriptional regulation of various cells. There are six different types of C/EBPs, and some form isoforms through the use of alternative translation initiation sites. The functional analysis of the C/EBP family is therefore difficult to achieve. Here we report on the production of specific monoclonal antibodies against mouse C/EBPβ using a rat medial iliac lymph node method. Immunoblotting using C/EBPβ monoclonal antibodies identified two types of isoforms, while immunostaining revealed a subnuclear localization for C/EBPβ. Use of this antibody should contribute to the further elucidation of the transcriptional regulatory function of C/EBPβ.

    DOI: 10.1089/mab.2013.0069

  • Generation of a monoclonal antibody for INI1/hSNF5/BAF47 査読

    Akihito Harada, Masayasu Hayashi, Yuuki Kuniyoshi, Yuichiro Semba, Satoko Sugahara, Taro Tachibana, Yasuyuki Ohkawa, Masatoshi Fujita

    Monoclonal Antibodies in Immunodiagnosis and Immunotherapy   33 ( 1 )   49 - 51   2014年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    INI1/hSNF5/BAF47, which has an SNF5 domain, belongs to the SWI/SNF family. This family is known as ATP-dependent regulators of gene expression by remodeling chromatin structure during cell differentiation. However, the detailed function of INI1/hSNF5/BAF47 is unclear. Here we report the generation of a specific monoclonal antibody for INI1/hSNF5/BAF47 by the mouse iliac lymph node method. The obtained antibody recognized two isoforms of INI1/hSNF5/BAF47 in immunoblotting and precisely recognized the nuclear localization of INI1/hSNF5/BAF47 in immunostaining. This antibody can contribute to further elucidation of the mechanisms of gene expression regulation by INI1/hSNF5/BAF47 during cell differentiation.

    DOI: 10.1089/mab.2013.0065

  • Contribution of structural reversibility to the heat stability of the tropomyosin shrimp allergen 査読

    Masakatsu Usui, Akihito Harada, Takayuki Ishimaru, Emiri Sakumichi, Fumihiko Saratani, Chiho Sato-Minami, Hiroyuki Azakami, Taiko Miyasaki, Ken'ichi Hanaoka

    Bioscience, Biotechnology and Biochemistry   77 ( 5 )   948 - 953   2013年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Tropomyosins are common heat-stable crustacean allergens. However, their heat stability and their effects on antigenicity have not been clarified. We purified tropomyosin in this study from raw kuruma prawns (Marsupenaeus japonicus) without heat processing. SDS-PAGE of the purified protein showed a band at approximately 35 kDa that cross-reacted with IgE from the serum of a shrimp-allergic patient, identifying it as Pen j 1. The circular dichroism spectrum of native Pen j 1 revealed the common α-helical structure of tropomyosins which easily collapsed upon heating to 80 °C. However, there were no insoluble aggregates after heating, and the protein regained its native CD spectral pattern after cooling to 25 °C. There was no significant difference in total IgG production between mice sensitized with native and heated Pen j 1. These results suggest that heat-denatured Pen j 1 refolded upon cooling and maintained its antigenicity following the heat treatment.

    DOI: 10.1271/bbb.120887

  • A co-localization model of paired ChIP-seq data using a large ENCODE data set enables comparison of multiple samples 査読

    Kazumitsu Maehara, Jun Odawara, Akihito Harada, Tomohiko Yoshimi, Koji Nagao, Chikashi Obuse, Koichi Akashi, Taro Tachibana, Toshio Sakata, Yasuyuki Ohkawa

    Nucleic Acids Research   41 ( 1 )   54 - 62   2013年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Deep sequencing approaches, such as chromatin immunoprecipitation by sequencing (ChIP-seq), have been successful in detecting transcription factor-binding sites and histone modification in the whole genome. An approach for comparing two different ChIP-seq data would be beneficial for predicting unknown functions of a factor. We propose a model to represent co-localization of two different ChIP-seq data. We showed that a meaningful overlapping signal and a meaningless background signal can be separated by this model. We applied this model to compare ChIP-seq data of RNA polymerase II C-terminal domain (CTD) serine 2 phosphorylation with a large amount of peak-called data, including ChIP-seq and other deep sequencing data in the Encyclopedia of DNA Elements (ENCODE) project, and then extracted factors that were related to RNA polymerase II CTD serine 2 in HeLa cells. We further analyzed RNA polymerase II CTD serine 7 phosphorylation, of which their function is still unclear in HeLa cells. Our results were characterized by the similarity of localization for transcription factor/histone modification in the ENCODE data set, and this suggests that our model is appropriate for understanding ChIP-seq data for factors where their function is unknown.

    DOI: 10.1093/nar/gks1010

  • Structural reversibility contributes the heat stability of shrimp allergen tropomyosin 査読

    M. Usui, A. Harada, T. Ishimaru, E. Sakumichi, F. Saratani, C. Sato, H. Azakami, T. Miyasaki, K. Hanaoka

    Bioscience, Biotechnology, and Biochemistry   77 ( 5 )   2012年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

  • The classification of mRNA expression levels by the phosphorylation state of RNAPII CTD based on a combined genome-wide approach 査読

    Jun Odawara, Akihito Harada, Tomohiko Yoshimi, Kazumitsu Maehara, Taro Tachibana, Seiji Okada, Koichi Akashi, Yasuyuki Ohkawa

    BMC Genomics   12   2011年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Background: Cellular function is regulated by the balance of stringently regulated amounts of mRNA. Previous reports revealed that RNA polymerase II (RNAPII), which transcribes mRNA, can be classified into the pausing state and the active transcription state according to the phosphorylation state of RPB1, the catalytic subunit of RNAPII. However, genome-wide association between mRNA expression level and the phosphorylation state of RNAPII is unclear. While the functional importance of pausing genes is clear, such as in mouse Embryonic Stem cells for differentiation, understanding this association is critical for distinguishing pausing genes from active transcribing genes in expression profiling data, such as microarrays and RNAseq. Therefore, we examined the correlation between the phosphorylation of RNAPII and mRNA expression levels using a combined analysis by ChIPseq and RNAseq.Results: We first performed a precise quantitative measurement of mRNA by performing an optimized calculation in RNAseq. We then visualized the recruitment of various phosphorylated RNAPIIs, such as Ser2P and Ser5P. A combined analysis using optimized RNAseq and ChIPseq for phosphorylated RNAPII revealed that mRNA levels correlate with the various phosphorylation states of RNAPII.Conclusions: We demonstrated that the amount of mRNA is precisely reflected by the phased phosphorylation of Ser2 and Ser5. In particular, even the most "pausing" genes, for which only Ser5 is phosphorylated, were detectable at a certain level of mRNA. Our analysis indicated that the complexity of quantitative regulation of mRNA levels could be classified into three categories according to the phosphorylation state of RNAPII.

    DOI: 10.1186/1471-2164-12-516

  • Generation of a rat monoclonal antibody specific for Hsp72 査読

    Masako Tanaka, Masayuki Shiota, Seiji Okada, Akihito Harada, Jun Odawara, Saya Mun, Hiroshi Iwao, Yasuyuki Ohkawa

    Monoclonal Antibodies in Immunodiagnosis and Immunotherapy   30 ( 4 )   397 - 400   2011年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    The heat shock protein 70 (Hsp70) family members function as ATP-dependent molecular chaperones that assist in the folding of newly synthesized polypeptides and in the refolding of misfolded/aggregated proteins. These heat shock proteins comprise at least eight sets of molecular groups that share high homology, but differ from each other in their expression level and subcellular localization. Hsp72, which is also known as Hsp70 and Hsp70-1, is localized mainly in the cytoplasm but is also found in the nucleus. Stress-induced Hsp72 functions as a chaperone enabling the cells to cope with harmful aggregations of denatured proteins during and following stress. The difference in the function of Hsp72 from that of other Hsp70 members, however, remains unclear. We report the establishment of a monoclonal antibody specific for Hsp72 using the rat medial iliac lymph node method. Immunoblot analysis revealed that our monoclonal antibody against Hsp72 specifically identified the 65 kDa protein. Immunocytochemical staining also revealed that Hsp72 localized in the cytoplasm and nucleus, and aggregated in the nucleus in response to heat stress. This MAb against Hsp72 will allow for further studies to elucidate the mechanism by which Hsp72 is localized in the cell in response to stress stimuli, and aid in the identification of specific interacting molecules.

    DOI: 10.1089/hyb.2011.0015

  • Flow cytometric sorting of neuronal and glial nuclei from central nervous system tissue 査読

    Seiji Okada, Hirokazu Saiwai, Hiromi Kumamaru, Kensuke Kubota, Akihito Harada, Masahiro Yamaguchi, Yukihide Iwamoto, Yasuyuki Ohkawa

    Journal of Cellular Physiology   226 ( 2 )   552 - 558   2011年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Due to the complex cellular heterogeneity of the central nervous system (CNS), it is relatively difficult to reliably obtain molecular descriptions with cell-type specificity. In particular, comparative analysis of epigenetic regulation or molecular profiles is hampered by the lack of adequate methodology for selective purification of defined cell populations from CNS tissue. Here, we developed a direct purification strategy of neural nuclei from CNS tissue based on fluorescence-activated cell sorting (FACS). We successfully fractionated nuclei from complex tissues such as brain, spinal cord, liver, kidney, and skeletal muscle extruded mechanically or chemically, and fractionated nuclei were structurally maintained and contained nucleoproteins and nuclear DNA/RNA. We collected sufficient numbers of nuclei from neurons and oligodendrocytes using FACS with immunolabeling for nucleoproteins or from genetically labeled transgenic mice. In addition, the use of Fab fragments isolated from papain antibody digests, which effectively enriched the specialized cell populations, significantly enhanced the immunolabeling efficacy. This methodology can be applied to a wide variety of heterogeneous tissues and is crucial for understanding the cell-specific information about chromatin dynamics, nucleoproteins, protein-DNA/RNA interactions, and transcriptomes retained in the nucleus, such as non-coding RNAs.

    DOI: 10.1002/jcp.22365

  • Generation of a rat monoclonal antibody specific for heat shock cognate protein 70 査読

    Masayuki Shiota, Hirokazu Saiwai, Saya Mun, Akihito Harada, Seiji Okada, Jun Odawara, Masako Tanaka, Hiroshi Iwao, Yasuyuki Ohkawa

    Monoclonal Antibodies in Immunodiagnosis and Immunotherapy   29 ( 5 )   453 - 456   2010年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Human heat shock cognate protein 70 (Hsc70), also known as Hsp73 and Hsp70-8, is a molecular chaperone. The human Hsp70 family comprises at least eight different molecular groups with strong homology. Among them, Hsc70 and Hsp72 share 86% homology. Both Hsp72 and Hsc70 localize in the cell cytoplasm and the nucleus. While Hsp72 expression is enhanced by stress, Hsc70 is constitutively expressed, suggesting that Hsc70 is critically involved in cell functions other than the stress response. Hsc70 has cell-specific and tissue-specific functions, such as cellular signaling, but its functions are not well understood. To further study the functions of Hsc70, we established a monoclonal antibody specific for Hsc70 using a rat medial iliac lymph node method. Immunoblot analysis with this antibody revealed that it specifically recognizes Hsc70. Immunocytochemical staining using this newly established antibody revealed that Hsc70 localizes predominantly in the cytoplasm in unstressed cells, whereas oxidative stress produced by H2O 2 induces Hsc70 to translocate into the nucleus. This monoclonal antibody will be useful for further studies of Hsc70, including changes in its intracellular location, binding molecules, and functions.

    DOI: 10.1089/hyb.2010.0024

  • Monoclonal antibody specific for Dhx9/NDHII/RHA 査読

    Manato Kotani, Akihito Harada, Jun Odawara, Masayuki Azuma, Seiji Okada, Yuko Nishiyama, Mako Nakamura, Taro Tachibana, Yasuyuki Ohkawa

    Monoclonal Antibodies in Immunodiagnosis and Immunotherapy   29 ( 3 )   259 - 261   2010年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Dhx9/NDHII/RHA is a member of the DEAH family of proteins, which possess a double-stranded RNA-binding domain (dsRBD) and a helicase domain. The DEAH protein family plays a critical role in RNA metabolism. DEAH family members function as ATP-dependent RNA helicases and regulation of transcription. In the present study, we report the establishment of a monoclonal antibody specific for Dhx9 using the rat medial iliac lymph node method. Immunoblot analysis using our antibody against Dhx9 detected full-length Dhx9. In addition, immunocytochemical staining using our antibody against Dhx9 revealed the nuclear localization of Dhx9. This monoclonal antibody against Dhx9 will allow for further detailed studies of Dhx9 expression.

    DOI: 10.1089/hyb.2009.0107

  • Rat monoclonal antibody specific for MyoD 査読

    Akihito Harada, Yasuyuki Ohkawa, Shinpei Ao, Jun Odawara, Seiji Okada, Masayuki Azuma, Yuko Nishiyama, Mako Nakamura, Taro Tachibana

    Monoclonal Antibodies in Immunodiagnosis and Immunotherapy   29 ( 3 )   255 - 258   2010年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Myogenic determination 1 (MyoD) is a myogenic regulatory factor (MRF) possessing a basic domain and a helix-loop-helix domain. MRFs play a critical role in myoblast fate and terminal differentiation. MyoD is a transcriptional factor that induces transcription by binding with gene regulatory factors expressed in skeletal muscle. As a master gene, MyoD also determines skeletal muscle differentiation. In this study, we established a monoclonal antibody specific for MyoD using the rat medial iliac lymph node method. Immunoblot analysis revealed that our monoclonal antibody against MyoD could identify full-length MyoD. Moreover, immunocytochemical staining revealed a change in the expression of MyoD at the skeletal muscle differentiation stage. This monoclonal antibody against MyoD allows for further studies to elucidate the mechanism by which MyoD influences skeletal muscle differentiation.

    DOI: 10.1089/hyb.2009.0117

  • Rat monoclonal antibody specific for the chromatin remodeling factor, CHD1 査読

    Saori Yoshimura, Akihito Harada, Jun Odawara, Masayuki Azuma, Seiji Okada, Mako Nakamura, Yasuyuki Ohkawa, Taro Tachibana

    Monoclonal Antibodies in Immunodiagnosis and Immunotherapy   29 ( 3 )   237 - 240   2010年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    CHD1 is a subfamily member of the CHD family, which possesses a chromodomain, a helicase domain, and a DNA-binding domain. The CHD family regulates gene expression by contributing to ATP-dependent chromatin remodeling. CHD1 exists in the transcriptionally active region and alters the chromatin structure. Little is known about the function of endogenous CHD1, however, and studies have been hindered by the lack of an antibody specific for CHD1 in mammals. In the present study, we established a monoclonal antibody specifically against CHD1 using the rat medial iliac lymph node method. Immunoblot analysis using our monoclonal antibody showed specific binding to CHD1, allowing us to identify the deduced full-length CHD1. In addition, cell immunostaining clearly revealed the nuclear localization of CHD1. This monoclonal antibody will be useful for further analysis of CHD1 function in mammals.

    DOI: 10.1089/hyb.2009.0106

  • Generation of a rat monoclonal antibody specific for Chd2 査読

    Akihito Harada, Saori Yoshimura, Jun Odawara, Masayuki Azuma, Seiji Okada, Mako Nakamura, Taro Tachibana, Yasuyuki Ohkawa

    Monoclonal Antibodies in Immunodiagnosis and Immunotherapy   29 ( 2 )   173 - 177   2010年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    CHD2 is a member of the CHD family that contains chromodomain, helicase domain as well as DNA-binding domain. The CHD family is involved in gene expression and transcription by ATP-dependent chromatin remodeling. Analysis of mutant mouse revealed that CHD2 is involved in development as well as hematopoiesis, which suggests the involvement of CHD2 in gene expression. However, CHD2 has not yet been analyzed biochemically as there is no specific antibody against it. Here, we report on the establishment of specific monoclonal antibody (MAb) against CHD2 utilizing a rat medial iliac lymph node method. Through cell immunostaining utilizing established MAb to CHD2, we confirmed that CHD2 was localized in euchromatin. Additionally, IP-Western revealed that the expression level of full-length CHD2 did not change during the differentiation stage. Additionally, a specific signal was confirmed around 95 kDa at the undifferentiated stage. This clearly indicated that CHD2 was involved in specific gene expression at this stage. Thus, this antibody can contribute to elucidating the function of CHD2 in cell expression.

    DOI: 10.1089/hyb.2009.0090

  • Production of a rat monoclonal antibody specific for Myf5 査読

    Akihito Harada, Seiji Okada, Jun Odawara, Hiromi Kumamaru, Hirokazu Saiwai, Mayumi Aoki, Mako Nakamura, Yuko Nishiyama, Yasuyuki Ohkawa

    Monoclonal Antibodies in Immunodiagnosis and Immunotherapy   29 ( 1 )   59 - 62   2010年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Myogenic regulatory factors (MRFs) are transcription factors that possess a characteristic basic helix-loop-helix domain. Myf5, MyoD, MRF4, and myogenin are well-known MRF family members that activate muscle-specific genes during differentiation. Myf5 is expressed first among MRFs at the very early phase and plays an important role in myoblast specificity and cell proliferation. Myf5 shares high homology with MyoD, and therefore some commercial Myf5 antibodies are cross-reactive for Myf5 and MyoD. To allow for detailed studies of the function of Myf5, we generated a monoclonal antibody specific for Myf5 utilizing a rat medial iliac lymph node method. Immunoblot analysis using our monoclonal antibody enabled us to identify Myf5 protein from rat myoblast L6E9 cell extract. Moreover, cell immunostaining revealed the nuclear localization of Myf5 in the L6E9 cells. This monoclonal antibody against Myf5 will allow us to perform further detailed studies of Myf5 and Myf5 function.

    DOI: 10.1089/hyb.2009.0066

  • The LTB4-BLT1 axis mediates neutrophil infiltration and secondary injury in experimental spinal cord injury 査読

    Hirokazu Saiwai, Yasuyuki Ohkawa, Hisakata Yamada, Hiromi Kumamaru, Akihito Harada, Hideyuki Okano, Takehiko Yokomizo, Yukihide Iwamoto, Seiji Okada

    American Journal of Pathology   176 ( 5 )   2352 - 2366   2010年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Traumatic injury in the central nervous system induces inflammation; however, the role of this inflammation is controversial. Precise analysis of the inflammatory cells is important to gain a better understanding of the inflammatory machinery in response to neural injury. Here, we demonstrated that leukotriene B4 plays a significant role in mediating leukocyte infiltration after spinal cord injury. Using flow cytometry, we revealed that neutrophil and monocyte/macrophage infiltration peaked 12 hours after injury and was significantly suppressed in leukotriene B4 receptor 1 knockout mice. Similar findings were observed in mice treated with a leukotriene B4 receptor antagonist. Further, by isolating each inflammatory cell subset with a cell sorter, and performing quantitative reverse transcription-PCR, we demonstrated the individual contributions of more highly expressed subsets, ie, interleukins 6 and 1β, tumor necrosis factor-α, and FasL, to the inflammatory reaction and neural apoptosis. Inhibition of leukotriene B4 suppressed leukocyte infiltration after injury, thereby attenuating the inflammatory reaction, sparing the white matter, and reducing neural apoptosis, as well as inducing better functional recovery. These findings are the first to demonstrate that leukotriene B4 is involved in the pathogenesis of spinal cord injury through the amplification of leukocyte infiltration, and provide a potential therapeutic strategy for traumatic spinal cord injury.

    DOI: 10.2353/ajpath.2010.090839

  • Generation of a rat monoclonal antibody specific for Pax7 査読

    Akihito Harada, Seiji Okada, Hirokazu Saiwai, Mayumi Aoki, Mako Nakamura, Yasuyuki Ohkawa

    Monoclonal Antibodies in Immunodiagnosis and Immunotherapy   28 ( 6 )   451 - 453   2009年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Pax7 is a nuclear localization protein, well known as a member of the paired box family. It is expressed at a very early stage of muscle differentiation and is also found in muscle satellite cells that are recognized as muscle stem cells. Pax7 is also recognized as a tumor cell marker since it is greatly expressed in various types of tumor cells. Pax7 has homology among other paired family members and is not easy to distinguish one from the others. In this study, we report on the establishment of monoclonal antibodies (MAb) against Pax7 using a rat medial iliac lymph node method. The quality of the antibody was examined by immunoblotting analysis. It was confirmed that the antibody can specifically recognize the Pax7 protein. It was also revealed that the MAb antibody successfully recognizes the nuclear localized Pax7 protein in Ewing's sarcoma cells by immunocytochemistry. The antibody can clearly show the regions of euchromatin and heterochromatin where hoechst is positive.

    DOI: 10.1089/hyb.2009.0039

  • Production of a rat monoclonal antibody against Brg1 査読

    Yasuyuki Ohkawa, Akihito Harada, Mako Nakamura, Saori Yoshimura, Taro Tachibana

    Monoclonal Antibodies in Immunodiagnosis and Immunotherapy   28 ( 6 )   463 - 466   2009年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Brm-related gene-1 (Brg1) is a catalytic subunit of the SWI/SNF chromatin remodeling enzyme complex that has ATPase activity. This complex facilitates chromatin remodeling for gene expression by utilizing energy for ATP hydrolysis. It is well known that the SWI/SNF chromatin remodeling enzyme complex is essential for cell differentiation, cell cycle regulation, and embryogenesis. Here we report the establishment of a hybridoma cell line for producing an antibody against Brg1 subunit by the rat medial iliac lymph node method. Immunoblot analysis showed that our antibody can specifically recognize Brg1. It was revealed by immunocytochemistry that Brg1 is located in euchromatin of C2C12 myoblast nuclei. These data suggested this antibody is useful for analyzing molecular function of Brg1 protein in cells.

    DOI: 10.1089/hyb.2009.0041

  • Generation of a rat monoclonal antibody specific for Brm 査読

    Seiji Okada, Akihito Harada, Hirokazu Saiwai, Mako Nakamura, Yasuyuki Ohkawa

    Monoclonal Antibodies in Immunodiagnosis and Immunotherapy   28 ( 6 )   455 - 458   2009年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Brm is a subunit of the SWI/SNF complex that has a ATPase activity. It is well known that the complex plays a major role in cell processes, such as proliferation, differentiation, and DNA repair of cells. Here we report the production of monoclonal antibody (1H7A10) against Brm by rat medial iliac lymph node method. Immunoblot analysis with the antibody revealed the specific recognition of Brm and increase of Brm protein level in skeletal muscle differentiation. Immunocytochemistry analysis shows nuclear localization in myoblast C2C12 and involvement of transcription in the late stages of differentiation.

    DOI: 10.1089/hyb.2009.0044

  • Amyloid fibril formation of hen lysozyme depends on the instability of the C-helix (88-99) 査読

    Akihito Harada, Hiroyuki Azakami, Akio Kato

    Bioscience, Biotechnology and Biochemistry   72 ( 6 )   1523 - 1530   2008年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Stable and unstable mutant lysozymes in long helices B and C were constructed to evaluate the effect of the helices on amyloid fibril formation at pH 2. Stable mutant N27D and unstable mutant K33D in the B-helix did not change in amyloid fibril formation. In contrast, stable mutant N93D and unstable mutant K97D in the C-helix showed big differences in behavior as to amyloid fibril formation. Stable mutant N93D showed a longer lag phase of aggregation and suppressed the amyloid fibril formation, whereas unstable mutant K97D showed a shorter lag phase of aggregation and accelerated amyloid fibril formation. These results suggest that the long C-helix is involved mainly in the α-helix to β-sheet transition during amyloid formation of lysozyme.

    DOI: 10.1271/bbb.80032

  • Relationship between the stability of hen egg-white lysozymes mutated at sites designed to interact with α-helix dipoles and their secretion amounts in yeast 査読

    Akihito Harada, Hiroshi Yagi, Akira Saito, Hiroyuki Azakami, Akio Kato

    Bioscience, Biotechnology and Biochemistry   71 ( 12 )   2952 - 2961   2007年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    The positively charged lysine at the C-terminals of three long α-helices (5-15, 25-35, and 88-99) was replaced with alanine (K13A, K33A, K97A) or aspartic acid (K13D, K33D, K97D) in hen lysozyme by genetic engineering. The denaturation transition point (Tm) and Gibbs energy change ΔG of the mutant lysozymes decreased remarkably, suggesting that the positive charge at the C-terminals of helices is involved in the stabilization of the helix dipole. On the other hand, the non-charged asparagine at the N-terminal of the long α-helices (25-35 and 88-99) was replaced with negatively charged aspartic acid (N27D and N93D). The Tm and ΔG of N27D increased, suggesting that the dipole moment of the N-terminal of the helices is diminished by replacement with negatively charged amino acid strengthening the stability of the helices. The stabilities of those hen egg white lysozymes mutated at the N- or Cterminal sites of the three long α-helices were related with their secretion amounts in yeast (Pichia pastoris). The secretion amounts of these mutant lysozymes in yeast were closely correlated with their stability.

    DOI: 10.1271/bbb.70354

  • Characterization of recombinant amyloidogenic chicken cystatin mutant I66Q expressed in yeast 査読

    Jianwei He, Youtao Song, Nobuhiro Ueyama, Akihito Harada, Hiroyuki Azakami, Akio Kato

    Journal of Biochemistry   137 ( 4 )   477 - 485   2005年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Amyloidogenic chicken cystatin mutant I66Q (cC I66Q) was successfully secreted by yeasts Pichia pastoris and Saccharomyces cerevisiae. The soluble monomer and dimer forms of amyloidogenic cC I66Q were found in the culture medium, while large amounts of insoluble aggregate and polymeric form cC I66Q besides the monomer and dimer forms were secreted into the culture medium. The amyloidogenic cC I66Q showed a comparable circular dichroism spectrum to that of the wild cystatin, and the monomer form exhibited a similar level of inhibitory activity toward papain, but the dimmer form did not. During storage of amyloidogenic cC I66Q under physiological and acidic conditions, typical binding with Congo red and thioflavin T, and the formation of amyloid fibrils were observed, whereas the characteristic of similar amyloidosis was hardly detected for the wild recombinant cystatin.

    DOI: 10.1093/jb/mvi064

  • Effect of EPS1 gene deletion in Saccharomyces cerevisiae on the secretion of foreign proteins which have disulfide bridges 査読

    Jianwei He, Takashi Sakamoto, Youtao Song, Akira Saito, Akihito Harada, Hiroyuki Azakami, Akio Kato

    FEBS Letters   579 ( 11 )   2277 - 2283   2005年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Both amyloid-prone cystatin and unstable mutant C94A lysozyme were secreted in wild-type and Δeps1 Saccharomyces cerevisiae cells. Amyloid-prone cystatin secreted at much higher level in Δeps1 cells than that in wild-type yeast. In parallel, the secretion amount of disulfide bond disrupted mutant C94A lysozyme greatly increased in Δeps1 cells although that was apparently low in wild-type yeast cells compared with the secretion amount of wild-type lysozyme. It is interesting that neither the unstable mutant C94A lysozyme nor amyloid-prone cystatin secreted in Δeps1 cells maintained their specific activities. These observations lead to the supposition that yeast cells deficient for the protein disulfide isomerase-family-member EPS1 locus secrete more of labile disulfide-containing model proteins.

    DOI: 10.1016/j.febslet.2005.03.019

▼全件表示

講演・口頭発表等

  • High-throughput single cell epigenomic profiling using chromatin integration labelling-sequence 国際会議

    Akihito Harada, @Kazumitsu Maehara, @Yasuyuki Ohkawa

    Keystone Symposia on Molecular and Cellular Biology  2019年3月 

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    開催年月日: 2019年3月

    記述言語:英語   会議種別:口頭発表(一般)  

    開催地:Banff, Alberta (Canada)   国名:カナダ  

    Analysis of chromatin states is regularly performed in development and disease research of multicellular organisms. The standard method of epigenomic analysis has been chromatin immunoprecipitation followed by sequencing (ChIP-seq) which is a powerful technique capable of unveiling protein-DNA interactions on the whole genome. There has been a recent development of the method allowing sequencing from single cells, which could facilitate the analysis of clinical samples as well as rare cell populations such as stem cells. However, the single-cell ChIP protocol employs microfluidic devices and hence still requires a great number of cells. Recently, we have established a new immunoprecipitation-free epigenomic profiling method based on immunostaining named chromatin integration labeling followed by sequencing (ChIL-seq). While ChIL-seq was demonstrated to detect histone modifications at the single-cell level, the current published protocol is not cost-effective for high-throughput assays because all the reactions need to be performed separately. We report on our progress on the further development of single-cell ChIL-seq at high-throughput.

  • Functional analysis of novel histone variant H3mm13 in skeletal muscle to Histone variants 国際会議

    Harada A

    EMBO Workshop Histone Variants  2017年6月 

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    開催年月日: 2017年9月

    記述言語:英語  

    開催地:BioMedical Center (BMC)(Germany)   国名:ドイツ連邦共和国  

  • 細胞状態変化を計測するマルチオミクス技術開発 招待

    原田 哲仁、富松 航佑

    第46回日本分子生物学会  2023年12月 

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    開催年月日: 2023年12月 - 2024年12月

    記述言語:日本語  

    開催地:神戸ポートアイランド   国名:日本国  

  • 単一細胞マルチエピゲノム解析による分化過程のクロマチンダイナミクスの可視化 招待

    原田哲仁、藤井健、前原一満、木村宏、大川恭行

    第46回日本分子生物学会  2023年12月 

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    開催年月日: 2023年12月

    記述言語:日本語   会議種別:シンポジウム・ワークショップ パネル(公募)  

    開催地:神戸ポートアイランド   国名:日本国  

  • High-throughput single cell multi-epigenomic profiling using chromatin integration labelling-sequence 国際会議

    Akihito Harada

    Cell Symposia The Conceptual Power of Single Cell Biology  2023年8月 

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    開催年月日: 2023年8月 - 2024年8月

    記述言語:日本語  

    開催地:Paradise Point Resort, San Diego, CA, USA   国名:アメリカ合衆国  

  • 細胞状態変化を計測するためのマルチオミクス技術開発 招待

    原田哲仁、富松航佑

    第16回日本エピジェネティクス研究会年会  2023年6月 

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    開催年月日: 2023年6月

    記述言語:日本語  

    開催地:学術総合センター 一橋講堂   国名:日本国  

  • 単一細胞エピゲノム解析を用いたクロマチンマッピングによるゲノム機能の解明 招待

    原⽥ 哲仁、藤井 健、前原 ⼀満、⽊村 宏、⼤川 恭⾏

    第45回日本分子生物学会  2022年6月 

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    開催年月日: 2022年11月 - 2022年12月

    記述言語:日本語   会議種別:シンポジウム・ワークショップ パネル(公募)  

    国名:日本国  

  • 単一細胞エピゲノム解析によるゲノム動作原理の理解

    原田 哲仁

    第15回日本エピジェネティクス研究会  2022年6月 

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    開催年月日: 2022年6月

    記述言語:日本語   会議種別:シンポジウム・ワークショップ パネル(公募)  

    開催地:九州大学医学部百年講堂   国名:日本国  

    a

  • Epigenomic lineage tracing toward the understanding acquisition of tissue-specific gene expression profile 招待

    Akihito Harada

    理研生命医科学研究センター(IMS)  2021年9月 

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    開催年月日: 2022年6月

    記述言語:英語   会議種別:口頭発表(一般)  

    開催地:オンライン   国名:日本国  

  • 組織特異的な遺伝子発現機構の解明 招待

    原田哲仁

    筑波大学TARAセミナー  2022年4月 

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    開催年月日: 2022年4月 - 2023年4月

    記述言語:日本語   会議種別:口頭発表(一般)  

    開催地:オンライン   国名:日本国  

  • ChIL法による単一細胞プロファイリング 招待

    Akihito Harada

    第45回日本分子生物学会年会  2021年12月 

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    開催年月日: 2021年12月

    記述言語:日本語   会議種別:シンポジウム・ワークショップ パネル(公募)  

    国名:日本国  

  • 単一細胞解析の最前線 招待

    原田哲仁

    山口大学年次セミナー  2020年12月 

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    開催年月日: 2020年12月

    記述言語:日本語   会議種別:口頭発表(一般)  

    国名:日本国  

  • ChIL法による単一細胞エピゲノムプロファイリング

    原田哲仁、前原一満、半田 哲也、木村 宏、大川 恭行

    第43回日本分子生物学会  2020年12月 

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    開催年月日: 2020年12月

    記述言語:日本語   会議種別:シンポジウム・ワークショップ パネル(公募)  

    開催地:オンライン   国名:日本国  

    Analysis of chromatin states is regularly performed in development and disease research of multicellular organisms. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) has become the standard technology for genome-wide analysisof chromatin status in cells or tissues. However, ChIP-seq analysis in a small n umber of cells, including single cells, is still difficult to analyze due to low recover chromatin preparation. Recently, we have established a immunoprecipitation-free epigenomic profiling method based on immunostaining named chromatin integration labeling followed by sequencing (ChIL-seq). ChIL-seq can be started from a single cell on a well of a multi-well plate, and the level and spatial distr ibution of the target in the same cell can be analyzed by fluorescence microscop y. Furthermore, using combinatorial indexing methods, we demonstrate to detect h istone modifications such as H3K4me3 at a thousand of cells each single cell. In this talk, we will report on the progress of our research towards high-throughp ut of ChIL-seq at the single cell level.

  • ヒストンH3バリアントの選択的取り込みによる組織特異的な遺伝子発現制御

    原田 哲仁, 小松 哲郎, 前原 一満, 近藤 友佳理, 田中 かおり, 桑門 温子, 佐藤 優子, 木村 宏, 林 克彦, 小野 悠介, 竹本 龍也, 胡桃坂 仁志, 大川 恭行

    第47回日本分子生物学会年会  2019年12月 

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    開催年月日: 2019年12月

    記述言語:日本語  

    開催地:福岡国際会議場   国名:日本国  

  • 単一細胞multi-omicsによりシンギュラリティ一細胞同定技術の開発

    原田哲仁

    新学術領域研究「シンギュラリティ生物学」第2回領域会議  2019年5月 

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    開催年月日: 2019年12月

    記述言語:日本語  

    開催地:淡路夢舞台国際会議場   国名:日本国  

  • ChIL法を用いたエピゲノムおよび転写量を同時計測する技術開発

    原田哲仁、前原一満、半田哲也、木村宏、大川恭行

    新学術領域研究「クロマチン潜在能」第2回班会議  2019年6月 

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    開催年月日: 2019年12月

    記述言語:日本語  

    開催地:ホテル竹島   国名:日本国  

  • Analysis of genomic structures responsible for tissue-specific gene expression

    原田哲仁

    CREST・さきがけ「ゲノム合成」第2回 合同キックオフ及び領域会議  2019年11月 

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    開催年月日: 2019年12月

    記述言語:日本語  

    開催地:プラザヴェルデ沼津   国名:日本国  

  • 極微量サンプルからのクロマチン修飾解析を可能にするChIL-seqの紹介 招待

    原田哲仁

    トランスオミクス医学研究拠点ネットワーク形成事業 技術講習会  2019年3月 

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    開催年月日: 2019年3月

    記述言語:日本語   会議種別:口頭発表(一般)  

    開催地:東京医科歯科大学 M&Dタワー11階 大学院講義室3   国名:日本国  

  • 生殖細胞におけるヒストンバリアントによるゲノムマーキング機構の解明

    原田 哲仁

    新学術領域 生殖細胞のエピゲノムダイナミクスとその制御 取りまとめ公開シンポジウム  2018年12月 

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    開催年月日: 2018年12月

    記述言語:日本語   会議種別:シンポジウム・ワークショップ パネル(公募)  

    開催地:京都教育文化センター   国名:日本国  

  • 非免疫沈降法ChILT法による少数細胞のエピゲノム解析

    原田哲仁、半田哲也、野上順平、有村泰宏、関根慧、中尾勝、藤田理沙、前原一満、胡桃坂仁志、木村宏、大川恭行

    2017年度生命科学系学会合同年次大会  2017年12月 

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    開催年月日: 2017年12月

    記述言語:日本語   会議種別:シンポジウム・ワークショップ パネル(公募)  

    開催地:神戸ポートアイランド   国名:日本国  

  • 生殖細胞におけるヒストンバリアントによるゲノムマーキング機構の解明

    原田哲仁

    生殖細胞のエピゲノムダイナミクスとその制御 第5回公開シンポジウム  2017年11月 

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    開催年月日: 2017年11月

    記述言語:日本語   会議種別:シンポジウム・ワークショップ パネル(公募)  

    開催地:つくばノバホール   国名:日本国  

  • 急性期の筋萎縮過程における遺伝子発現変動解析

    原田 哲仁, 岩崎 健, 横田 和也, 岡田 誠司, 大川 恭行

    日本農芸化学会2017年度大会  2017年3月 

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    開催年月日: 2017年3月

    記述言語:日本語   会議種別:口頭発表(一般)  

    開催地:京都女子大(京都府)   国名:日本国  

  • Functional switching of histone H3 variants in myogenesis 招待

    原田 哲仁

    第4回ヒストンバリアント研究会  2017年2月 

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    開催年月日: 2017年2月

    記述言語:日本語   会議種別:口頭発表(一般)  

    開催地:東北大学青葉山新キャンパス 青葉山コモンズ (宮城県仙台市)   国名:日本国  

  • エピジェネティック破綻により引き起こされた筋肉腫の解析 招待

    原田 哲仁

    公益財団法人福岡県すこやか健康事業団平成28年度がん研究助成金優秀賞受賞講演  2017年1月 

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    開催年月日: 2017年1月

    記述言語:日本語   会議種別:口頭発表(一般)  

    開催地:西鉄グランドホテル(福岡県福岡市)   国名:日本国  

  • ヒストンH3バリアントの発現変動は筋萎縮過程の初期イベントである

    原田 哲仁, 岩崎 健, 横田 和也, 岡田 誠司, 大川 恭行

    第39回日本分子生物学会年会  2016年11月 

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    開催年月日: 2016年11月 - 2016年12月

    記述言語:日本語  

    開催地:パシフィコ横浜(神奈川県横浜市)   国名:日本国  

  • Histone H3.3 sub-variant H3mm7 is required for normal skeletal muscle regeneration. 国際会議

    Harada A

    1st München-Japan Mini Symposium “Chromatin Structure and Function”  2017年9月 

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    開催年月日: 2016年9月 - 2017年9月

    記述言語:英語  

    開催地:Helmholtz Zentrum münchen(Germany)   国名:ドイツ連邦共和国  

  • 骨格筋分化におけるクロマチン構造解析-トランスクリプトミクスで理解する骨格筋形成 招待

    原田 哲仁

    日本農芸化学会中四国支部 支部創立15周年記念 第24回若手シンポジウム  2016年7月 

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    開催年月日: 2016年7月

    記述言語:日本語   会議種別:口頭発表(一般)  

    開催地:水産大学校 (山口県下関市)   国名:日本国  

  • 生殖細胞の品質の定量に向けて 光を用いた空間トランスクリプトーム解析

    木村 龍一, 本田 瑞季, 原田 哲仁, 前原 一満, 田中 かおり, 大川 恭行, 沖 真弥

    日本生化学会大会プログラム・講演要旨集  2023年10月  (公社)日本生化学会

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    記述言語:英語  

  • 抗SARS-CoV-2免疫反応および抗体産生株 ヒストン変異体H3.3の発現が形質細胞の分化を制御する(Anti SARS-CoV-2 immune responses and Antibody producers Histone variant H3.3 expression controls the plasma cell differentiation)

    Saito Yuichi, Baba Yoshihiro, Ohkawa Yasuyuki, Harada Akihito

    日本免疫学会総会・学術集会記録  2022年11月  (NPO)日本免疫学会

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    記述言語:英語  

  • 形質細胞の分化はヒストン変異体H3.3の発現によって制御される(Plasma cell differentiation is regulated by the expression of histone variant H3.3)

    Saito Yuichi, Murakami Daisuke, Baba Yoshihiro, Harada Akihito, Ohkawa Yasuyuki, Takashi Nakagawa

    日本耳鼻咽喉科頭頸部外科学会会報  2024年4月  (一社)日本耳鼻咽喉科頭頸部外科学会

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    記述言語:英語  

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MISC

  • クロマチン構造における転写制御の理解に向けた技術開発

    原田哲仁, 富松航佑, 武 千湄, 大川恭行

    生体の科学   2023年6月

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    記述言語:日本語   掲載種別:記事・総説・解説・論説等(学術雑誌)  

  • 1細胞エピゲノム

    原田 哲仁, 大川 恭行

    月刊「細胞」   2023年6月

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    記述言語:日本語   掲載種別:記事・総説・解説・論説等(学術雑誌)  

  • 【1細胞解析技術】1細胞エピゲノム

    原田 哲仁, 大川 恭行

    細胞   55 ( 7 )   372 - 375   2023年6月   ISSN:1346-7557

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    記述言語:日本語   出版者・発行元:(株)ニュー・サイエンス社  

    ヒトのほぼすべての種類の細胞は同一のゲノムを共有している。この同一のゲノムを持つ異なる種類の細胞の特性と機能は細胞のエピゲノムによって決定される。1細胞RNA-seqによって細胞集団の不均一性が明らかとなり,エピゲノムについても1細胞レベルの解析が求められるようになっている。1細胞エピゲノムは,ここ10年あまりで著しく進歩し,我々研究者はバルク法では見えなかった細胞間の不均一性や希少細胞集団の特性評価などの問題に取り組むことができるようになった。ここでは,個々の細胞レベルでの高解像度エピゲノム解析技術に向けた取り組みについて,代表的な技術を紹介する。(著者抄録)

  • 【クロマチンによる転写制御機構の最前線】クロマチンと転写制御に関する新技術 クロマチン構造における転写制御の理解に向けた技術開発

    原田 哲仁, 富松 航佑, 武 千び, 大川 恭行

    生体の科学   74 ( 3 )   255 - 259   2023年6月   ISSN:0370-9531

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    記述言語:日本語   出版者・発行元:(公財)金原一郎記念医学医療振興財団  

    <文献概要>ヒトのほぼすべての種類の細胞は同一のゲノムを持つ。この同一のゲノムを持つ異なる種類の細胞の特性と機能は,遺伝子が存在するクロマチン構造の転写における選択性により支配されている。したがって,転写はRNAポリメラーゼIIによりゲノムDNAからRNAが合成されるプロセスという側面に加えて,異なるエピゲノム状態を判別し,適切な遺伝子発現を選択する過程と言える。近年の1細胞解析技術の高感度化,高深度化に伴い,細胞の型のみならず,細胞状態変化まで遺伝子発現の総体であるトランスクリプトームで区別可能になりつつある。一方で,これら細胞の状態変化が可塑的な範囲か,あるいは細胞型として不可逆的な変化を示したのかは判定が困難である。改めて,クロマチンレベルの遺伝子発現解析の重要性が高まっていると言えよう。本稿では,転写の場であるクロマチン構造変化と転写を測定する試みについて代表的な技術を紹介したい。

  • 少数細胞エピゲノム解析技術の開発

    原田 哲仁, 大川 恭行

    生化学 テクニカルノート   2021年6月

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    記述言語:日本語   掲載種別:記事・総説・解説・論説等(学術雑誌)  

  • 1細胞エピゲノム解析技術開発の最前線 査読

    大川 恭行,原田 哲仁,前原 一満

    医歯薬出版株式会社 週刊医学のあゆみ 2021年 Vol278No.10:912-917   2021年3月

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    記述言語:日本語   掲載種別:記事・総説・解説・論説等(学術雑誌)  

  • 骨格筋研究のための最先端解析技術

    原田 哲仁,大川 恭行

    羊土社 実験医学 2020年10月号 Vol.38 No.16:2686-2692   2020年9月

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    記述言語:日本語   掲載種別:記事・総説・解説・論説等(学術雑誌)  

  • シングルセルでのエピゲノム情報の計測技術

    原田 哲仁, 大川 恭行

    羊土社 実験医学 2020年 Vol.38   2020年4月

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    記述言語:日本語   掲載種別:記事・総説・解説・論説等(学術雑誌)  

  • クロマチン挿入標識法(ChIL)による単一細胞エピゲノム解析

    原田 哲仁,大川 恭行

    羊土社 実験医学 2019年増刊号Vol.37   2019年12月

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    記述言語:日本語   掲載種別:記事・総説・解説・論説等(学術雑誌)  

  • 発生起源の違いに着目した身体内骨格筋の不均一性と筋再性制御の違い

    吉岡 潔志, 瀬古 大暉, 北嶋 康雄, 土屋 吉史, 野上 順平, 原田 哲仁, 大川 恭行, 岡崎 成弘, 米倉 暁彦, 千葉 恒, 大場 誠悟, 住田 吉慶, 朝比奈 泉, 小野 悠介

    日本筋学会学術集会プログラム・抄録集   2018年8月

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    記述言語:日本語  

    発生起源の違いに着目した身体内骨格筋の不均一性と筋再性制御の違い

  • Temporal regulation of chromatin during myoblast differentiation.

    Harada A, Ohkawa Y, Imbalzano AN

    Semin Cell Dev Biol   2017年12月

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    記述言語:英語   掲載種別:記事・総説・解説・論説等(学術雑誌)  

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所属学協会

  • 日本分子生物学会

  • 日本農芸化学会

  • 日本エピジェネティクス研究会

  • 日本農芸化学会

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  • 日本分子生物学会

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学術貢献活動

  • 組織委員

    第15回日本エピジェネティクス研究会年会  ( 九州大学医学部百年講堂 ) 2022年6月

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    種別:大会・シンポジウム等 

共同研究・競争的資金等の研究課題

  • 細胞競合における細胞間相互作用を計測するための空間オミクス技術開発

    2023年6月 - 2025年6月

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    担当区分:研究代表者 

  • 精子形成における体細胞型H3から精子型H3T/tへのヒストン置換の意義の解明

    2023年4月 - 2026年3月

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    担当区分:研究代表者 

  • 精子形成における体細胞型H3から精子型H3T/tへのヒストン置換の意義の解明

    研究課題/領域番号:23H02394  2023年 - 2025年

    日本学術振興会  科学研究費助成事業  基盤研究(B)

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    担当区分:研究代表者  資金種別:科研費

  • 精子形成における体細胞型H3から精子型H3T/tへのヒストン置換の意義の解明

    研究課題/領域番号:23K27087  2023年 - 2025年

    日本学術振興会  科学研究費助成事業  基盤研究(B)

    原田 哲仁

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    資金種別:科研費

    精子形成過程では、「次世代へ伝播される遺伝情報」を形成するためクロマチン構造の再構成が起こる。この過程で起こる精子形成固有の現象が、体細胞型のヒストンから精巣特異的ヒストンへの置換である。研究代表者らは、最近、独自のエピゲノム解析からヒストンH3tは体細胞型 ヒストンH3.1/2と分化初期段階で入れ代わることを示唆する結果を得た。つまり、精子形成過程ではH3tが精子形成を進める特別なゲノム構造 を作り出している可能性が高い。そこで、本研究では、独自のエピゲノム解析技術と空間オミクス技術を駆使し、体細胞型ヒストンから精子特 異的ヒストンバリアントH3tへの置換の意義の解明に迫る。

    CiNii Research

  • 精子形成における体細胞型H3から精子型H3T/tへのヒストン置換の意義の解明

    研究課題/領域番号:23H02394  2023年 - 2025年

    日本学術振興会  科学研究費助成事業  基盤研究(B)

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    資金種別:科研費

  • 無精子症における精巣内内分泌環境に着目した単一細胞トランスクリプトーム解析

    2022年4月 - 2025年3月

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    担当区分:研究分担者 

  • 無精子症における精巣内内分泌環境に着目した単一細胞トランスクリプトーム解析

    研究課題/領域番号:22K09475  2022年 - 2024年

    日本学術振興会  科学研究費助成事業  基盤研究(C)

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    担当区分:研究分担者  資金種別:科研費

  • 無精子症における精巣内内分泌環境に着目した単一細胞トランスクリプトーム解析

    研究課題/領域番号:22K09475  2022年 - 2024年

    日本学術振興会  科学研究費助成事業  基盤研究(B)

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    資金種別:科研費

  • 競合的コミュニケーションから迫る多細胞生命システムの自律性

    研究課題/領域番号:21H05283  2021年 - 2025年

    日本学術振興会  科学研究費助成事業  基盤研究(B)

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    資金種別:科研費

  • 競合的コミュニケーションから迫る多細胞生命システムの自律性

    研究課題/領域番号:21H05283  2021年 - 2025年

    日本学術振興会・文部科学省  科学研究費助成事業  学術変革領域研究(A)

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    担当区分:研究分担者  資金種別:科研費

  • 細胞競合における細胞間相互作用を計測するための空間オミクス技術開発

    研究課題/領域番号:21H05292  2021年 - 2025年

    日本学術振興会・文部科学省  科学研究費助成事業  学術変革領域研究(A)

    原田 哲仁

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    担当区分:研究代表者  資金種別:科研費

    細胞競合は細胞間の相互作用を介した細胞排除現象であり、多細胞生命システムが自身の構造や機能を最適化する「自律性」を生み出す要の一つであると考えられる。細胞競合のプロセスを完全に理解するには、、細胞集団内の細胞間にわずかな性質の差を細胞間の時空間的な相互作用と遺伝子発現・シグナル変化を単一細胞レベルで定量的に解析する必要がある。そこで本研究では、これまでに開発した独自の空間オミクス解析技術を基盤として、細胞競合における細胞間相互作用を計測するための新たな空間マルチオミクス技術の開発と独自空間オミクス技術を駆使し、本領域内の細胞競合の分子基盤と多細胞生命システムの自律性生成原理の理解に迫る。

    CiNii Research

  • 革新的Perturb-ChIL法を用いた網羅的なクロマチン構造解析研究

    研究課題/領域番号:21H00430  2021年 - 2022年

    日本学術振興会・文部科学省  科学研究費助成事業  新学術領域研究

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    担当区分:研究代表者  資金種別:科研費

  • 位置情報を保持したシンギュラリティ細胞の遺伝子発現測定技術の開発

    研究課題/領域番号:21H00430  2021年

    日本学術振興会  科学研究費助成事業  基盤研究(B)

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    資金種別:科研費

  • 単一細胞解析によるヘルペスウイルス慢性感染の分子基盤の解明

    2020年 - 2022年

    国立研究開発法人 日本医療研究開発機構

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    担当区分:研究分担者  資金種別:受託研究

  • 全能性獲得機構の解明のためのmulti-omics技術の開発

    研究課題/領域番号:20H05368  2020年 - 2021年

    日本学術振興会・文部科学省  科学研究費助成事業  新学術領域研究

    原田 哲仁

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    資金種別:科研費

    全能性の獲得には、様々なエピゲノム因子が関わると考えられるが、その全貌は明らかとなっていない。このような全能性因子の解析のためには、どの因子が協調して機能しているのかを分類するための同時性を担保した解析手法が必須である。そこで、本研究では同時性を担保するクロマチン構造解析と転写産物を同時に取得する技術multi-ChIL omicsの開発を目指す。本研究では、開発したmulti-ChIL omics技術の少数細胞での適用とハイスループット化を目標とし開発を進める。

    CiNii Research

  • 組織特異的ゲノム構造の再構築技術の開発

    2019年 - 2022年

    戦略的創造研究推進事業 (文部科学省)

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    担当区分:研究代表者  資金種別:受託研究

  • 精子特異的ヒストンバリアントH3T/tを起点としたクロマチンダイナミクスの解明

    研究課題/領域番号:19H03211  2019年 - 2021年

    日本学術振興会  科学研究費助成事業  基盤研究(B)

    原田 哲仁

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    資金種別:科研費

    精子形成過程では、「次世代へ伝播される遺伝情報」を形成するためクロマチン構造の再構成が起こる。この過程で起こる精子形成固有の現象が、体細胞型のヒストンから精巣特異的ヒストンへの置換である。我々はこれまでに、精子細胞には複数の機能未知のヒストンが発現しており、そのうちヒストンH3tは精子形成の最初期に必須であることを報告し、精子形成過程ではH3tを起点に従来の知見を超えた多彩且つ複雑なヒストン置換が起こっている可能性が高いことを示した。そこで、本研究では、体細胞型ヒストンから精子特異的ヒストンバリアントH3tへの置換を起点として、精子形成過程でのクロマチン構造変換とその制御機構の解明を目指す。

    CiNii Research

  • 単一細胞遺伝子解析によるヒト造精機能障害の分子機構の解明

    研究課題/領域番号:19K09712  2019年 - 2021年

    日本学術振興会  科学研究費助成事業  基盤研究(C)

    白石 晃司, 原田 哲仁, 大川 恭行

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    資金種別:科研費

    非閉塞性無精子症に対する根本的な治療法は存在しない。In vitroヒト精細管培養による精子形成の確立に向け、ヒト造精機能障害、特にearly maturation arrest症例について顕微鏡下精巣内精子採取術時に採取された精細胞の単一細胞網羅的遺伝子解析を行う。
    1)分化・増殖が停止する細胞と正常な細胞の遺伝子発現の比較を次世代シーケンサーを用いて単一細胞レベルで行い、造精機能障害の分子生物学的メカニズムを明らかにする。
    2)Leydig細胞を含んだヒト精細管のin vitro培養を行い、培養条件の工夫により精細胞の分化・増殖を生じうるか、また培養後の精細胞の遺伝子発現を解析する。

    CiNii Research

  • 単一細胞multi-omicsによるシンギュラリティー細胞同定技術の開発

    研究課題/領域番号:19H05425  2019年 - 2020年

    日本学術振興会・文部科学省  科学研究費助成事業  新学術領域研究

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    資金種別:科研費

  • 単一細胞超高解像度解析を可能にするゲノム3次元構造解析技術の開発

    研究課題/領域番号:18K19432  2018年 - 2019年

    日本学術振興会  科学研究費助成事業  挑戦的研究(萌芽)

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    資金種別:科研費

  • エピジェネティック破綻により引き起こされた筋肉腫の解析

    2016年 - 2018年

    公益財団法人福岡県すこやか健康事業団平成28年度がん研究助成金

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    担当区分:研究代表者  資金種別:受託研究

  • 生殖細胞におけるヒストンバリアントによるゲノムマーキング機構の解明

    研究課題/領域番号:16H01219  2016年 - 2017年

    日本学術振興会・文部科学省  科学研究費助成事業  新学術領域研究

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    資金種別:科研費

  • 骨格筋分化における新規ヒストンH3バリアントH3mm7の遺伝子選択機構の解明

    研究課題/領域番号:15K18457  2015年 - 2016年

    科学研究費助成事業  若手研究(B)

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    資金種別:科研費

  • 細胞分化運命決定の遺伝子選択メカニズムの解明

    研究課題/領域番号:13J04024  2013年 - 2015年

    日本学術振興会  科学研究費助成事業  基盤研究(B)

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    資金種別:科研費

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担当授業科目

  • 生命医科学特論Ⅲ

    2024年6月 - 2024年8月   夏学期

  • Topics in medical life sciences Ⅲ

    2024年6月 - 2024年8月   夏学期

  • Topics in medical life sciences Ⅲ

    2023年6月 - 2023年8月   夏学期

  • 生命医科学特論Ⅲ

    2023年6月 - 2023年8月   夏学期

  • 生命医科学特論Ⅰ

    2022年6月 - 2022年8月   夏学期

  • 生命医科学特論Ⅰ

    2021年6月 - 2021年8月   夏学期

  • Topics in medical life sciences Ⅰ

    2021年6月 - 2021年8月   夏学期

  • 生命医科学特論I

    2020年6月 - 2020年8月   夏学期

  • 最新トピックスから学ぶ生命科学入門

    2019年10月 - 2019年12月   秋学期

  • 生命医科学特論I

    2019年4月 - 2019年6月   春学期

  • 生命医科学特論I

    2018年4月 - 2018年6月   春学期

▼全件表示

FD参加状況

  • 2024年9月   役割:参加   名称:馬出地区4部局合同男女共同参画FD/ 病院きらめきプロジェクト講演会

    主催組織:部局

  • 2024年3月   役割:参加   名称:有体物管理センターの業務および成果有体物収入の配分率の変更について

    主催組織:全学

  • 2023年8月   役割:参加   名称:令和5年度4部局合同男女共同参画FD

    主催組織:部局

  • 2022年12月   役割:参加   名称:医学系学府教育FD「医学系大学院プログラムの進化と深化をめざして」

    主催組織:部局

  • 2022年3月   役割:参加   名称:新M2Bシステムの使い方 ~新機能を中心に紹介します~(3/17)

    主催組織:全学

  • 2021年12月   役割:参加   名称:医学系学府教育FD「学術論文の購読と投稿とこれから」

    主催組織:部局

  • 2021年7月   役割:参加   名称:生体防御医学研究所FD

    主催組織:部局

  • 2021年2月   役割:参加   名称:馬出地区4部局合同男女共同参画FD

    主催組織:部局

  • 2020年11月   役割:参加   名称:馬出地区4部局合同男女共同参画FD

    主催組織:部局

  • 2020年9月   役割:参加   名称:M2B学習支援システム講習会(オンライン開催)◇初級編・中級編◇10:00~12:00

    主催組織:全学

  • 2020年8月   役割:参加   名称:【IDE大学セミナー】大学教職員の多様な働き方について

  • 2019年10月   役割:参加   名称:馬出地区4部局合同男女共同参画FD

    主催組織:部局

  • 2018年10月   役割:参加   名称:馬出地区4部局合同男女共同参画FD

    主催組織:部局

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学内運営に関わる各種委員・役職等

  • 2022年4月 - 2023年3月   学府 学生委員会

  • 2021年4月 - 2022年3月   学府 ハラスメント等防止委員会