計測と再構築による生細胞内クロマチンダイナミクスの高次元的理解

2014 Fiscal Year Annual Research Report

• Project Area: Dynamic chromatin structure and function
• Project/Area Number: 25116005
• Research Institution: Tokyo Institute of Technology
• Principal Investigator: 木村 宏 東京工業大学, 生命理工学研究科, 教授 (30241392)
• Co-Investigator(Kenkyū-buntansha): 山縣 一夫 大阪大学, 微生物病研究所, 特任准教授 (10361312)
• Project Period (FY): 2013-06-28 – 2018-03-31
• Keywords: 発生・分化 / ゲノム / 細胞・組織 / バイオテクノロジー / シグナル伝達 / 遺伝子発現制御 / クロマチン / エピジェネティクス

Outline of Annual Research Achievements

本研究は、クロマチン動態の正確な「計測」と再構成ポリヌクレオソームを用いた「再構築」を行い、生細胞や生物個体におけるクロマチンの動的変化とその機能発現における意義を明らかにすることを目的として行っている。本年度は、転写活性化とヒストン修飾の動態に関する研究を主に行った。
活性化型RNAポリメラーゼII（RNAP2）の指標となるリン酸化修飾をFabLEM法により検出し、RNAP2による転写の開始と伸長のキネティクスを生細胞で計測した。転写の開始と伸長時に見られるRNAP2の最大サブユニットのC末端領域（CTD）のセリン残基のリン酸化に修飾に特異的なモノクローナル抗体を作成し、それらの蛍光標識Fabを生細胞計測に用いた。モデル系として、ステロイドホルモンの一種であるグルココルチコイドで誘導される遺伝子アレイの転写活性化を計測した。これらの計測データなどを元に、転写活性化のキネティクパラメータを求めた結果、転写開始の効率が10％程度であるのに対して、開始から伸長への移行は80%以上の高効率で起こることがわかった。さらに、転写活性化のキネティクスにヒストン修飾がどのように影響を与えるのかを調べるたところ、ヒストンH3K27のアセチル化（H3K27ac）がホルモン誘導前から遺伝子アレイ上に濃縮されていることが明らかになった。H3K27acのレベルと転写活性化の関係を解析した結果、ホルモン刺激前にH3K27acレベルが高い細胞では転写因子の遺伝子アレイへの集積とRNAポリメラーゼIIの転写開始から伸長への移行が促進されることが分かった。H3K27acによる転写制御には、アセチル化結合ドメインを持つアセチル化酵素であるp300と巨大転写伸長複合体を介してRNAポリメラーゼIIの伸長反応を促進すると考えられた。

Current Status of Research Progress

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

Reason

FabLEM法を用いて、転写活性化におけるヒストン修飾の役割を明らかにした意義は大きいと考えている。

Strategy for Future Research Activity

これまでの研究体制を継続し、培養細胞を用いた系で詳細な解析を行いつつ、動物個体を用いた系で発生や分化に伴うクロマチン動態を明らかにしていく。また、次年度以降は「再構成」系にも取り組む。

Research Products

• [Journal Article] Phenotype Specific Analyses Reveal Distinct Regulatory Mechanism for Chronically Activated p53. (2015)
  • Author(s): Kirschner K, Samarajiwa SA, Cairns JM, Menon S, Pérez-Mancera PA, Tomimatsu K, Bermejo-Rodriguez C, Ito Y, Chandra T, Narita M, Lyons SK, Lynch AG, Kimura H, Ohbayashi T, Tavaré S, Narita M.
  • Journal Title: PLoS Genet Volume: 11 Pages: e1005053
  • DOI: 10.1371/journal.pgen.1005053.
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Stella preserves maternal chromosome integrity by inhibiting 5hmC-induced γH2AX accumulation. (2015)
  • Author(s): Nakatani T, Yamagata K, Kimura T, Oda M, Nakashima H, Hori M, Sekita Y, Arakawa T, Nakamura T, Nakano T.
  • Journal Title: EMBO Rep Volume: 17 Pages: e201439427
• [Journal Article] H3K36 Trimethylation-Mediated Epigenetic Regulation is Activated by Bam and Promotes Germ Cell Differentiation During Early Oogenesis in Drosophila. (2015)
  • Author(s): Mukai M, Hira S, Nakamura K, Nakamura S, Kimura H, Sato M, Kobayashi S.
  • Journal Title: Biol Open Volume: 4 Pages: 119-124
  • DOI: 10.1242/bio.201410850.
  • Peer Reviewed / Open Access
• [Journal Article] Incorporation of histone H3.1 suppresses the lineage potential of skeletal muscle. (2015)
  • Author(s): Harada A, Maehara K, Sato Y, Konno D, Tachibana T, Kimura H, Ohkawa Y.
  • Journal Title: Nucleic Acids Res Volume: 43 Pages: 775-786
  • DOI: 10.1093/nar/gku1346.
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Coordinated expression of H3K9 histone methyltransferases during tooth development in mice. (2015)
  • Author(s): Kamiunten T, Ideno H, Shimada A, Nakamura Y, Kimura H, Nakashima K, Nifuji A.
  • Journal Title: Histochem Cell Biol Volume: 143 Pages: 259-266
  • DOI: 10.1007/s00418-014-1284-0.
  • Peer Reviewed
• [Journal Article] Regulation of RNA polymerase II activation by histone acetylation in single living cells. (2014)
  • Author(s): Stasevich TJ, Hayashi-Takanaka Y, Sato Y, Maehara K, Ohkawa Y, Sakata-Sogawa K, Tokunaga M, Nagase T, Nozaki N, McNally JG, Kimura H.
  • Journal Title: Nature. Volume: 516 Pages: 272-275
  • DOI: 10.1038/nature13714
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Evaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging. (2014)
  • Author(s): Hayashi-Takanaka Y, Stasevich TJ, Kurumizaka H, Nozaki N, Kimura H.
  • Journal Title: PLoS One Volume: 9 Pages: e106271
  • DOI: 10.1371/journal.pone.0106271
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] DNA methylation reader MECP2: cell type- and differentiation stage-specific protein distribution. (2014)
  • Author(s): Song C, Feodorova Y, Guy J, Peichl L, Jost KL, Kimura H, Cardoso MC, Bird A, Leonhardt H, Joffe B, Solovei I.
  • Journal Title: Epigenetics Chromatin Volume: 7 Pages: 17
  • DOI: 10.1186/1756-8935-7-17.
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Quantifying histone and RNA polymerase II post-translational modification dynamics in mother and daughter cells. (2014)
  • Author(s): Stasevich TJ, Sato Y, Nozaki N, Kimura H.
  • Journal Title: Methods Volume: 70 Pages: 77-88
  • DOI: 10.1016/j.ymeth.2014.08.002.
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Adhesion of suspension cells on a coverslip in serum-free conditions. (2014)
  • Author(s): Nakayama T, Mihara K, Kawata J, Kimura H, Saitoh H.
  • Journal Title: Anal Biochem Volume: 466 Pages: 1-3
  • DOI: 10.1016/j.ab.2014.07.023.
  • Peer Reviewed
• [Journal Article] Hierarchical molecular events driven by oocyte-specific factors lead to rapid and extensive reprogramming. (2014)
  • Author(s): Jullien J, Miyamoto K, Pasque V, Allen GE, Bradshaw CR, Garrett NJ, Halley-Stott RP, Kimura H, Ohsumi K, Gurdon JB.
  • Journal Title: Mol Cell. Volume: 55 Pages: 524-536
  • DOI: 10.1016/j.molcel.2014.06.024.
  • Peer Reviewed
• [Journal Article] Nuclear dynamics of topoisomerase IIβ reflects its catalytic activity that is regulated by binding of RNA to the C-terminal domain. (2014)
  • Author(s): Onoda A, Hosoya O, Sano K, Kiyama K, Kimura H, Kawano S, Furuta R, Miyaji M, Tsutsui K, Tsutsui KM.
  • Journal Title: Nucleic Acids Res Volume: 42 Pages: 9005-9020
  • DOI: 10.1093/nar/gku640
  • Peer Reviewed / Open Access
• [Journal Article] Improved and robust detection of cell nuclei from four dimensional fluorescence images. (2014)
  • Author(s): Bashar MK, Yamagata K, Kobayashi TJ.
  • Journal Title: PLoS One. Volume: 9 Pages: e101891
  • DOI: 10.1371/journal.pone.0101891.
  • Peer Reviewed / Open Access
• [Journal Article] Histone H4 Lys 20 monomethylation of the CENP-A nucleosome is essential for kinetochore assembly. (2014)
  • Author(s): Hori T, Shang WH, Toyoda A, Misu S, Monma N, Ikeo K, Molina O, Vargiu G, Fujiyama A, Kimura H, Earnshaw WC, Fukagawa T.
  • Journal Title: Dev Cell Volume: 29 Pages: 740-749
  • DOI: 10.1016/j.devcel.2014.05.001.
  • Peer Reviewed
• [Journal Article] Loss of histone H4K20 trimethylation predicts poor prognosis in breast cancer and is associated with invasive activity. (2014)
  • Author(s): Yokoyama Y, Matsumoto A, Hieda M, Shinchi Y, Ogihara E, Hamada M, Nishioka Y, Kimura H, Yoshidome K, Tsujimoto M, Matsuura N.
  • Journal Title: Breast Cancer Res Volume: 16 Pages: R66.
  • DOI: 10.1186/bcr3681.
  • Peer Reviewed
• [Journal Article] Nucleosomal regulation of chromatin composition and nuclear assembly revealed by histone depletion. (2014)
  • Author(s): Zierhut C, Jenness C, Kimura H, Funabiki H.
  • Journal Title: Nat Struct Mol Biol Volume: 21 Pages: 617-625
  • DOI: 10.1038/nsmb.2845.
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Heterochromatin dynamics during the differentiation process revealed by the DNA methylation reporter mouse, MethylRO. (2014)
  • Author(s): Ueda J, Maehara K, Mashiko D, Ichinose T, Yao T, Hori M, Sato Y, Kimura H, Ohkawa Y, Yamagata K.
  • Journal Title: Stem Cell Reports Volume: 2 Pages: 910-924
  • DOI: 10.1016/j.stemcr.2014.05.008
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Direct evidence for pitavastatin induced chromatin structure change in the KLF4 gene in endothelial cells. (2014)
  • Author(s): Maejima T et al.
  • Journal Title: PLoS One Volume: 9 Pages: e96005
  • DOI: 10.1371/journal.pone.0096005.
  • Peer Reviewed / Open Access
• [Journal Article] Glycolytic genes are targets of the nuclear receptor Ad4BP/SF-1. (2014)
  • Author(s): Baba T, Otake H, Sato T, Miyabayashi K, Shishido Y, Wang CY, Shima Y, Kimura H, Yagi M, Ishihara Y, Hino S, Ogawa H, Nakao M, Yamazaki T, Kang D, Ohkawa Y, Suyama M, Chung BC, Morohashi K.
  • Journal Title: Nat Commun Volume: 5 Pages: 3634
  • DOI: 10.1038/ncomms4634.
  • Peer Reviewed
• [Presentation] Visualization of Dynamics of Methylated DNA in living cell and animal. (2015)
  • Author(s): Kazuo Yamagata
  • Organizer: International Symposium on Bio-imaging and Gene Targeting Sciences in Okayama
  • Place of Presentation: 岡山
  • Year and Date: 2015-02-15
  • Invited
• [Presentation] メチローマウスで動くメチル化DNAを追う (2015)
  • Author(s): 山縣一夫
  • Organizer: "平成26年度基礎生物学研究所重点共同利用研究
  • Place of Presentation: 岡崎
  • Year and Date: 2015-01-19
  • Invited
• [Presentation] Tracking functional chromatin components in living cells and organisms (2014)
  • Author(s): Hiroshi Kimura
  • Organizer: 4D Nucleome meeting
  • Place of Presentation: 広島
  • Year and Date: 2014-12-17 – 2014-12-19
  • Invited
• [Presentation] Tracking epigenetic modifications and transcription activity in living cells and organisms (2014)
  • Author(s): Hiroshi Kimura
  • Organizer: 8th International Symposium on Nanomedicine
  • Place of Presentation: 松山
  • Year and Date: 2014-12-04 – 2014-12-06
  • Invited
• [Presentation] 初期胚の高精細イメージングで見えてくるもの (2014)
  • Author(s): 山縣一夫
  • Organizer: 第59回本生殖医学会学術講演会
  • Place of Presentation: 東京
  • Year and Date: 2014-12-03 – 2014-12-05
  • Invited
• [Presentation] 発生過程や刺激に応答した転写活性化とヒストン修飾の動態 (2014)
  • Author(s): 木村 宏、スタセビッチ ティモシー、佐藤優子
  • Organizer: 第37回日本分子生物学会年会
  • Place of Presentation: 横浜
  • Year and Date: 2014-11-25 – 2014-11-27
  • Invited
• [Presentation] Regulation of Transcription Cycle by Histone Modifications in Living Cells (2014)
  • Author(s): Hiroshi Kimura
  • Organizer: The International Symposium on Transcription Cycle 2014
  • Place of Presentation: 東京
  • Year and Date: 2014-11-24
  • Invited
• [Presentation] Histone modification and transcription dynamics in living cells and organisms (2014)
  • Author(s): Hiroshi Kimura
  • Organizer: The 9th 3R Symposium
  • Place of Presentation: 御殿場
  • Year and Date: 2014-11-17 – 2014-11-21
  • Invited
• [Presentation] メチローで 動くメチル化ＤＮＡを追う (2014)
  • Author(s): 山縣一夫
  • Organizer: 第87回日本生化学会大会
  • Place of Presentation: 京都
  • Year and Date: 2014-10-15 – 2014-10-18
  • Invited
• [Presentation] ヒストンとＲＮＡポリメラーゼの翻訳後修飾の生細胞・生体内計測 (2014)
  • Author(s): 木村 宏
  • Organizer: 第52回日本生物物理学会年会
  • Place of Presentation: 札幌
  • Year and Date: 2014-09-25 – 2014-09-27
  • Invited
• [Presentation] 発生・分化に伴うヒストン修飾のダイナミクス (2014)
  • Author(s): 木村 宏
  • Organizer: 第56回歯科基礎医学会学術大会
  • Place of Presentation: 福岡
  • Year and Date: 2014-09-25 – 2014-09-27
  • Invited
• [Presentation] ヒストン修飾翻訳後修飾と転写の生細胞動態 (2014)
  • Author(s): 木村 宏
  • Organizer: 第14回蛋白質科学会年会
  • Place of Presentation: 横浜
  • Year and Date: 2014-06-25 – 2014-06-27
  • Invited
• [Presentation] ライブセルイメージングで「卵子の質」を定量化する (2014)
  • Author(s): 山縣一夫
  • Organizer: 第66回日本細胞生物学会大会
  • Place of Presentation: 奈良
  • Year and Date: 2014-06-11 – 2014-06-13
  • Invited
• [Presentation] Quantification of embryo quality by live-cell imaging (2014)
  • Author(s): Kazuo Yamagata
  • Organizer: 47th Annual Meeting of the Japanese Society of Developmental Biologists
  • Place of Presentation: 名古屋
  • Year and Date: 2014-05-27 – 2014-05-30
  • Invited
• [Presentation] ライブセルイメージングで「卵子の質」を評価する (2014)
  • Author(s): 山縣一夫
  • Organizer: 第55回日本卵子学会
  • Place of Presentation: 神戸
  • Year and Date: 2014-05-17 – 2014-05-18
  • Invited
• [Remarks] 東京工業大学大学院生命理工学研究科 木村宏研究室
  • URL: http://kimura-lab.bio.titech.ac.jp/
• [Remarks] 東京工業大学大学院生命理工学研究科 木村宏研究室
  • URL: http://www.bio.titech.ac.jp/laboratory/kimura-kajikawa/kimura/index.html