高転写状態解明に向けた空間マルチオミクス解析技術の開発

2021 Fiscal Year Annual Research Report

• Project Area: Ensuring integrity in gametogenesis
• Project/Area Number: 21H00232
• Research Institution: Kyushu University
• Principal Investigator: 大川 恭行 九州大学, 生体防御医学研究所, 教授 (80448430)
• Project Period (FY): 2021-04-01 – 2023-03-31
• Keywords: 高転写状態 / 始原生殖細胞 / 細胞分化制御 / トランスクリプトーム / エピゲノム

Outline of Annual Research Achievements

私たちはこれまで免疫染色をベースに、細胞・組織を非破壊的に標的タンパク質を可視化し、同時にゲノム上の位置を決定するChILSeq を開発した(Nat Cell Biol.2019)。ChILSeq では抗体反応領域周辺のゲノムDNA にTn5 トランスポザーゼを用いて選択的にT7RNA ポリメラーゼプロモータ配列を挿入し、T7RNA ポリメラーゼの添加により強制的に転写させることでゲノム情報をRNAとして獲得する。ChIL 反応により転写されたRNA はゲノムから遊離しないため、細胞の形態や組織内の位置情報を保持した状態でsmFISH により検出可能である。これまでにChILsmFISH により、NGS なしでのイメージングのみでハイスループット且つ1 細胞レベルの高解像度エピゲノム解析技術が樹立し、エピゲノムとトランスクリプトームの同時測定技術が理論上可能となった。一方で、高転写状態解析にはこれにタンパク質の発現情報が欠かせない。そこで、本研究計画では、更に発展させ、プロテオミクスまで含めたマルチオミクス技術を開発にとりくんだ。プロテオミクスの適用の為新たに開発した連続蛍光免疫染色法（seqImmuno-Fluo:以下seqIF）の実装を行い、200種を超えるプローブの作成と検証を行い、ヒト検体を用いた実データ取得を進めた。更に、これら解析を行うための自動化デバイスの開発に取り組み、共焦点レーザー顕微鏡下でデータを自動的に取得し、反応を行う新たなデバイス開発に成功している。現在論文投稿準備中である。また、トランスクリプトーム技術seqFISH+との統合を達成した。

Current Status of Research Progress

1: Research has progressed more than it was originally planned.

Reason

当初予定の開発を早めて、データ取得を進めており当初の計画以上に進展している。

Strategy for Future Research Activity

研究計画に従って進めていく。

Research Products

• [Journal Article] Direct Conversion of Human Endothelial Cells Into Liver Cancer-Forming Cells Using Nonintegrative Episomal Vectors. (2022)
  • Author(s): Takeshi Goya, Kenichi Horisawa, Miyako Udono, Yasuyuki Ohkawa, Yoshihiro Ogawa, Sayaka Sekiya, Atsushi Suzuki.
  • Journal Title: Hepatol Commun. Volume: 6 Pages: 1725-1740
  • DOI: 10.1002/hep4.1911.
  • Peer Reviewed / Open Access
• [Journal Article] Uhrf1 governs the proliferation and differentiation of muscle satellite cells. (2022)
  • Author(s): Sakai H, Sawada Y, Tokunaga N, Tanaka K, Nakagawa S, Sakakibara I, Ono Y, Fukada SI, Ohkawa Y, Kikugawa T, Saika T, Imai Y.
  • Journal Title: iScience. Volume: 25 Pages: 103928
  • DOI: 10.1016/j.isci.2022.103928.
  • Peer Reviewed / Open Access
• [Journal Article] Unusual nucleosome formation and transcriptome influence by the histone H3mm18 variant. (2022)
  • Author(s): Hirai S, Tomimatsu K, Miyawaki-Kuwakado A, Takizawa Y, Komatsu T, Tachibana T, Fukushima Y, Takeda Y, Negishi L, Kujirai T, Koyama M, Ohkawa Y, Kurumizaka H.
  • Journal Title: Nucleic Acids Res. Volume: 50 Pages: 72-91
  • DOI: 10.1093/nar/gkab1137.
  • Peer Reviewed / Open Access
• [Journal Article] Uterus-specific transcriptional regulation underlies eggshell pigment production in Japanese quail. (2022)
  • Author(s): 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.
  • Journal Title: PLoS One. Volume: 17 Pages: e0265008
  • DOI: 10.1371/journal.pone.
  • Peer Reviewed / Open Access
• [Journal Article] Live imaging of transcription sites using an elongating RNA polymerase II-specific probe. (2021)
  • Author(s): Uchino S, Ito Y, Sato Y, Handa T, Ohkawa Y, Tokunaga M, Kimura H.
  • Journal Title: J Cell Biol. Volume: 221 Pages: e202104134
  • DOI: 10.1083/jcb.202104134.
  • Peer Reviewed / Open Access
• [Journal Article] Relayed signaling between mesenchymal progenitors and muscle stem cells ensures adaptive stem cell response to increased mechanical load. (2021)
  • Author(s): Kaneshige A, Kaji T, Zhang L, Saito H, Nakamura A, Kurosawa T, Ikemoto-Uezumi M, Tsujikawa K, Seno S, Hori M, Saito Y, Matozaki T, Maehara K, Ohkawa Y, Potente M, Watanabe S, Braun T, Uezumi A, Fukada SI.
  • Journal Title: Cell Stem Cell. Volume: 29 Pages: 265-280
  • DOI: 10.1016/j.stem.2021.11.003.
  • Peer Reviewed / Open Access
• [Journal Article] Recent advances in single-cell epigenomics. (2021)
  • Author(s): Harada A, Kimura H, Ohkawa Y.
  • Journal Title: Curr Opin Struct Biol. Volume: 71 Pages: 116-122
  • DOI: 10.1016/j.sbi.
  • Peer Reviewed / Open Access
• [Journal Article] High-throughput single-cell epigenomic profiling by targeted insertion of promoters (TIP-seq). (2021)
  • Author(s): Bartlett DA, Dileep V, Handa T, Ohkawa Y, Kimura H, Henikoff S, Gilbert DM.
  • Journal Title: J Cell Biol. Volume: 220 Pages: e202103078
  • DOI: 10.1083/jcb.202103078.
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Chromatin loading of MCM hexamers is associated with di-/tri-methylation of histone H4K20 toward S phase entry. (2021)
  • Author(s): Hayashi-Takanaka Y, Hayashi Y, Hirano Y, Miyawaki-Kuwakado A, Ohkawa Y, Obuse C, Kimura H, Haraguchi T, Hiraoka Y.
  • Journal Title: Nucleic Acids Res. Volume: 49 Pages: 12152-12166
  • DOI: 10.1093/nar/gkab1068.
  • Peer Reviewed / Open Access
• [Journal Article] Discriminative feature of cells characterizes cell populations of interest by a small subset of genes. (2021)
  • Author(s): Fujii T, Maehara K, Fujita M, Ohkawa Y.
  • Journal Title: PLoS Comput Biol. Volume: 17 Pages: e1009579
  • DOI: 10.1371/journal.pcbi.1009579.
  • Peer Reviewed / Open Access
• [Journal Article] Sex differences in metabolic pathways are regulated by Pfkfb3 and Pdk4 expression in rodent muscle. (2021)
  • Author(s): Christianto A, Baba T, Takahashi F, Inui K, Inoue M, Suyama M, Ono Y, Ohkawa Y, Morohashi KI.
  • Journal Title: Commun Biol. Volume: 4 Pages: 1264
  • DOI: 10.1038/s42003-021-02790-y.
  • Peer Reviewed / Open Access
• [Journal Article] Neural stem/precursor cells dynamically change their epigenetic landscape to differentially respond to BMP signaling for fate switching during brain development. (2021)
  • Author(s): Katada S, Takouda J, Nakagawa T, Honda M, Igarashi K, Imamura T, Ohkawa Y, Sato S, Kurumizaka H, Nakashima K.
  • Journal Title: Genes Dev. Volume: 35 Pages: 1431-1444
  • DOI: 10.1101/gad.348797.
  • Peer Reviewed / Open Access
• [Journal Article] Modeling population size independent tissue epigenomes by ChIL-seq with single thin sections. (2021)
  • Author(s): Maehara K, Tomimatsu K, Harada A, Tanaka K, Sato S, Fukuoka M, Okada S, Handa T, Kurumizaka H, Saitoh N, Kimura H, Ohkawa Y.
  • Journal Title: Mol Syst Biol. Volume: 17 Pages: e10323
  • DOI: 10.15252/msb.
  • Peer Reviewed / Open Access
• [Journal Article] Genome-wide analysis of chromatin structure changes upon MyoD binding in proliferative myoblasts during the cell cycle. (2021)
  • Author(s): Wu Q, Fujii T, Harada A, Tomimatsu K, Miyawaki-Kuwakado A, Fujita M, Maehara K, Ohkawa Y.
  • Journal Title: J Biochem. Volume: 169 Pages: 653-661
  • DOI: 10.1093/jb/mvab001.
  • Peer Reviewed / Open Access
• [Journal Article] Integrated Analysis of Transcriptome and Histone Modifications in Granulosa Cells During Ovulation in Female Mice. (2021)
  • Author(s): Shirafuta Y, Tamura I, Ohkawa Y, Maekawa R, Doi-Tanaka Y, Takagi H, Mihara Y, Shinagawa M, Taketani T, Sato S, Tamura H, Sugino N.
  • Journal Title: Endocrinology. Volume: 162 Pages: bqab128
  • DOI: 10.1210/endocr/bqab128.PMID: 34171084.
  • Peer Reviewed / Open Access
• [Journal Article] An extensive and dynamic trans-omic network illustrating prominent regulatory mechanisms in response to insulin in the liver. (2021)
  • Author(s): Matsuzaki F, Uda S, Yamauchi Y, Matsumoto M, Soga T, Maehara K, Ohkawa Y, Nakayama KI, Kuroda S, Kubota H.
  • Journal Title: Cell Rep. Volume: 36 Pages: 109569
  • DOI: 10.1016/j.celrep.2021.
  • Peer Reviewed / Open Access
• [Journal Article] Targeted inhibition of EPAS1-driven IL-31 production by a small-molecule compound. (2021)
  • Author(s): Kamikaseda Y, Uruno T, Kunimura K, Harada A, Saiki K, Oisaki K, Sakata D, Nakahara T, Kido-Nakahara M, Kanai M, Nakamura S, Ohkawa Y, Furue M, Fukui Y.
  • Journal Title: J Allergy Clin Immunol. Volume: 148 Pages: 633-638
  • DOI: 10.1016/j.jaci.2021.03.029.
  • Peer Reviewed / Open Access
• [Journal Article] High-depth spatial transcriptome analysis by photo-isolation chemistry. (2021)
  • Author(s): Honda M, Oki S, Kimura R, Harada A, Maehara K, Tanaka K, Meno C, Ohkawa Y.
  • Journal Title: Nat Commun. Volume: 12 Pages: 4416
  • DOI: 10.1038/s41467-021-24691-8.
  • Peer Reviewed / Open Access
• [Journal Article] Transcriptome analysis of gene expression changes upon enzymatic dissociation in skeletal myoblasts. (2021)
  • Author(s): Miyawaki-Kuwakado A, Wu Q, Harada A, Tomimatsu K, Fujii T, Maehara K, Ohkawa Y.
  • Journal Title: Genes Cells. Volume: 26 Pages: 530-540
  • DOI: 10.1111/gtc.12870. Epub 2021 Jun 8.
  • Peer Reviewed / Open Access
• [Journal Article] Hoxa10 mediates positional memory to govern stem cell function in adult skeletal muscle. (2021)
  • Author(s): Yoshioka K, Nagahisa H, Miura F, Araki H, Kamei Y, Kitajima Y, Seko D, Nogami J, Tsuchiya Y, Okazaki N, Yonekura A, Ohba S, Sumita Y, Chiba K, Ito K, Asahina I, Ogawa Y, Ito T, Ohkawa Y, Ono Y.
  • Journal Title: Sci Adv. Volume: 7 Pages: eabd7924
  • DOI: 10.1126/sciadv.abd7924.
  • Peer Reviewed / Open Access
• [Journal Article] Totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage. (2021)
  • Author(s): Maemura M, Taketsuru H, Nakajima Y, Shao R, Kakihara A, Nogami J, Ohkawa Y, Tsukada YI.
  • Journal Title: Sci Rep. Volume: 11 Pages: 11167
  • DOI: 10.1038/s41598-021-90653-1.
  • Peer Reviewed / Open Access
• [Journal Article] Chromatin structure-dependent histone incorporation revealed by a genome-wide deposition assay. (2021)
  • Author(s): Tachiwana H, Dacher M, Maehara K, Harada A, Seto Y, Katayama R, Ohkawa Y, Kimura H, Kurumizaka H, Saitoh N.
  • Journal Title: Elife Volume: 10 Pages: e66290
  • DOI: 10.7554/eLife.66290.
  • Peer Reviewed / Open Access
• [Presentation] 骨格筋再性能を制御するクロマチン構造等その破綻 (2021)
  • Author(s): 大川恭行
  • Organizer: 第94回日本生化学会大会
  • Invited
• [Presentation] 空間マルチオミクスの展開～エピゲノム, トランスクリプトームからプロテオームの空間計測の試み～ (2021)
  • Author(s): 大川恭行
  • Organizer: がん研セミナー
  • Invited
• [Presentation] Identification and Analysis of Minor Histone Variants (2021)
  • Author(s): Yasuyuki Ohkawa
  • Organizer: EMBO Workshop Physiology and function of histone variants
• [Book] Journal of Japanese Biochemical Society (2021)
  • Author(s): 原田 哲仁，大川 恭行
  • Total Pages: -
  • Publisher: 日本生化学会
• [Book] 実験医学2021年9月号 (2021)
  • Author(s): 沖 真弥、大川 恭行
  • Total Pages: 131
  • Publisher: 羊土社
  • ISBN: 978-4-7581-2547-5
• [Book] 遺伝子医学MOOK36 (2021)
  • Author(s): 富松航佑, 大川 恭行
  • Total Pages: 218
  • Publisher: 株式会社メディカルドゥ
• [Remarks] 九州大学 トランスクリプトミクス分野ホームページ
  • URL: https://tx.bioreg.kyushu-u.ac.jp/