Elucidation of the role of chromatin structure on genome instability using genome editing technique

• Project/Area Number: 25281019
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Partial Multi-year Fund
• Section: 一般
• Research Field: Risk sciences of radiation and chemicals
• Research Institution: Osaka University
• Principal Investigator: Fujiwara Tomoko 大阪大学, 医学(系)研究科(研究院), 助教 (70402922)
• Co-Investigator(Renkei-kenkyūsha): Todo Takeshi 大阪大学, 大学院医学系研究科, 教授 (90163948)
• Project Period (FY): 2013-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥17,420,000 (Direct Cost: ¥13,400,000、Indirect Cost: ¥4,020,000); Fiscal Year 2015: ¥5,330,000 (Direct Cost: ¥4,100,000、Indirect Cost: ¥1,230,000); Fiscal Year 2014: ¥5,330,000 (Direct Cost: ¥4,100,000、Indirect Cost: ¥1,230,000); Fiscal Year 2013: ¥6,760,000 (Direct Cost: ¥5,200,000、Indirect Cost: ¥1,560,000)
• Keywords: メダカ / 損傷メモリー / 遺伝的不安定性 / マイクロサテライト不安定性 / 環境ストレス / エピジェネティック変化 / クロマチン構造変化 / ゲノム編集技術

Outline of Final Research Achievements

Genetic instability induced by radiation or chemical mutagens is an interesting phenomenon and plays an important role on carcinogenesis, which appear through long latency period. The factor based under these phenomena is considered to "damage memory", however its entity is not clear. Based on several reports, we hypothesized an epigenetic events such as DNA and histone modification significantly contribute to the "damage memory". In this study, to test the hypothesis we have established a system for detecting the genetic instability. For inducing double strand breaks (DSBs) we used CRISPR, by which we can introduce DSBs at any genomic sequence we want and for the target of genetic instability that on microsatellite was tested. In addition, we have constructed BAC vector containing the entire genome region of ATR or DNA-PKcs gene for the rescue of each mutant, respectively.

Research Products

• [Journal Article] Viable neuronopathic Gaucher disease model in medaka (Oryzias latipes) displays axonal accumulation of alpha-synuclein (2015)
  • Author(s): Norihito Uemura, Masato Koike, Satoshi Ansai, Masato Kinoshita, Tomoko Ishikawa-Fujiwara, Hideaki Matsui, Kiyoshi Naruse, Naoaki Sakamoto, Yasuo Uchiyama, Takeshi Todo, Shunichi Takeda, Hodaka Yamakado, Ryosuke Takahashi
  • Journal Title: PLoS Genetics Volume: 11 Issue: 4 Pages: 1-14
  • DOI: 10.1371/journal.pgen.1005065
  • Peer Reviewed / Open Access
• [Journal Article] Loss of follicle-stimulating hormone receptor function causes masculinization and sippression of ovarian development in genetically female medaka. (2014)
  • Author(s): Murozumi N, Nakashima R, Hirai T, Kamei Y, Ishikawa-Fujiwara T, Todo T, Kitano T.
  • Journal Title: Endocrinology Volume: 155(8) Issue: 8 Pages: 3136-45
  • DOI: 10.1210/en.2013-2060
  • Peer Reviewed / Open Access
• [Journal Article] The Cryptochrome/Photolyase Family in aquatic organisms. (2014)
  • Author(s): Oliveri P, et al.
  • Journal Title: Mar Genomics Volume: pii: S1874-7787(14) Pages: 22-1
  • DOI: 10.1016/j.margen.2014.02.001
  • Peer Reviewed / Open Access
• [Journal Article] Human papillomavirus and p53 mutations in head and neck squamous cell carcinoma among Japanese population. (2014)
  • Author(s): Maruyama H, Yasui T, Ishikawa-Fujiwara T, Morii E, Yamamoto Y, Yoshii T, Takenaka Y, Nakahara S, Todo T, Hongyo T, Inohara H.
  • Journal Title: Cancer Sci. Volume: 105(4) Issue: 4 Pages: 409-417
  • DOI: 10.1111/cas.12369
  • Peer Reviewed / Open Access
• [Journal Article] p53-Dependent suppression of genome instability in germ cells. (2014)
  • Author(s): Otozai S, Ishikawa-Fujiwara T, Oda S, Kamei Y, Ryo H, Sato A, Nomura T, Mitani H, Tsujimura T, Inohara H, Todo T.
  • Journal Title: Mutat Res, Volume: 760 Pages: 24-32
  • DOI: 10.1016/j.mrfmmm.2013.12.004
  • Peer Reviewed
• [Journal Article] Circadian and Circalunar Clock Interactions in a Marine Annelid. (2013)
  • Author(s): Zantke J, et al.
  • Journal Title: Cell Rep. Volume: 5(1) Issue: 1 Pages: 99-113
  • DOI: 10.1016/j.celrep.2013.08.031
  • Peer Reviewed
• [Journal Article] ATF6α/β-mediated adjustment of ER chaperone levels is essential for development of the notochord in medaka fish. (2013)
  • Author(s): Ishikawa T, Okada T, Ishikawa-Fujiwara T, Todo T, Kamei Y, Shigenobu S, Tanaka M, Saito TL, Yoshimura J, Morishita S, Toyoda A, Sakaki Y, Taniguchi Y, Takeda S, Mori K.
  • Journal Title: Mol Biol Cell Volume: 24(9) Issue: 9 Pages: 1387-1395
  • DOI: 10.1091/mbc.e12-11-0830
  • Peer Reviewed
• [Presentation] Targeted Inactivation of Oryzias latipes Rev1 Gene (2016)
  • Author(s): Tomoko I Fujiwara, Yoshihiro Fujikawa, Kumi Nakamura, Takeshi Todo
  • Organizer: International Meeting on Aquatic Model Organisms for Human Disease and Toxicology Research
  • Place of Presentation: 愛知
  • Year and Date: 2016-03-18
  • Int'l Joint Research
• [Presentation] Comprehensive establishment of TLS polymerase mutants using TALENs in Medaka fish. (2016)
  • Author(s): Fujikawa Y, Ishikawa-Fujiawara T, Sakuma T, Yamamoto T, Todo T
  • Organizer: International Meeting on Aquatic Model Organisms for Human Disease and Toxicology Research
  • Place of Presentation: 愛知
  • Year and Date: 2016-03-18
  • Int'l Joint Research
• [Presentation] TALENsを利用したメダカTLSポリメラーゼ遺伝子群変異体の網羅的作製 (2015)
  • Author(s): 藤川芳宏、藤原（石川）智子、佐久間哲史、山本卓、藤堂剛
  • Organizer: 日本環境変異原学会第44回大会
  • Place of Presentation: 福岡
  • Year and Date: 2015-11-27
• [Presentation] Targeted inactivation of translesion DNA synthesis polymerase genes in Medaka fish using TALENs. (2015)
  • Author(s): Fujikawa Y, Ishikawa-Fujiawara T, Sakuma T, Yamamoto T, Todo T
  • Organizer: Conference on Transposition and Genome Engineering 2015
  • Place of Presentation: 奈良
  • Year and Date: 2015-11-17
  • Int'l Joint Research
• [Presentation] Establishment of translesion DNA synthesis polymerase gene mutants in Medaka fish using TALENs. (2015)
  • Author(s): Fujikawa Y, Ishikawa-Fujiawara T, Sakuma T, Yamamoto T, Todo T
  • Organizer: The 7th Asia and Oceania Conference on Photobiology
  • Place of Presentation: Taipei
  • Year and Date: 2015-11-15
  • Int'l Joint Research
• [Presentation] TALENsによるメダカTLS polymerase 遺伝子破壊 (2015)
  • Author(s): 藤川芳宏、藤原（石川）智子、佐久間哲史、山本卓、藤堂剛
  • Organizer: 第１回放射線ワークショップ
  • Place of Presentation: 富山
  • Year and Date: 2015-10-16
• [Presentation] ゲノム編集技術を用いた紫外線感受性変異体の作成 (2015)
  • Author(s): 藤原（石川）智子、藤川芳宏、白石絵吏、藤堂剛
  • Organizer: 第２１回小型魚類研究会
  • Place of Presentation: 大阪
  • Year and Date: 2015-09-19
• [Presentation] ゲノム編集技術(TALENs)を用いた損傷乗越えDNA合成ポリメラーゼ(Rev3L, Rev)変異体メダカの作製 (2015)
  • Author(s): 藤川芳宏、藤原（石川）智子、佐久間哲史、山本卓、藤堂剛
  • Organizer: 第２１回小型魚類研究会
  • Place of Presentation: 大阪
  • Year and Date: 2015-09-19
• [Presentation] メダカにおけるゲノム編集技術（TALENs）を用いた損傷乗り越えDNA合成ポリメラーゼ（Rev3L、Rev7）変異体の作製 (2015)
  • Author(s): 藤川芳宏、藤原（石川）智子、佐久間哲史、山本卓、藤堂剛
  • Organizer: 変異機構研究会・第28回夏の学校
  • Place of Presentation: 愛知
  • Year and Date: 2015-07-25
• [Presentation] モデル生物を使った紫外線感受性解析系の確立 (2015)
  • Author(s): 藤原（石川）智子
  • Organizer: 第３７回日本光医学・光生物学会
  • Place of Presentation: 宮崎
  • Year and Date: 2015-07-17
  • Invited
• [Presentation] メダカにおける新規cryptochromeの機能解析 (2015)
  • Author(s): 藤原（石川）智子、藤川芳宏、白石絵吏、藤堂剛
  • Organizer: 第３７回日本光医学・光生物学会
  • Place of Presentation: 宮崎
  • Year and Date: 2015-07-17
• [Presentation] ゲノム編集技術TALENを利用したメダカのRev3L, Rev7遺伝子変異体の作製 (2015)
  • Author(s): 藤川芳宏、藤原（石川）智子、佐久間哲史、山本卓、藤堂剛
  • Organizer: 第３７回日本光医学・光生物学会
  • Place of Presentation: 宮崎
  • Year and Date: 2015-07-17
• [Presentation] Targeted Inactivation of Rev1 Gene (2015)
  • Author(s): Tomoko I Fujiwara, Yoshihiro Fujikawa, Kumi Nakamura, Takeshi Todo
  • Organizer: The 15th International Congress of Radiation Research
  • Place of Presentation: Kyoto
  • Year and Date: 2015-05-25
  • Int'l Joint Research
• [Presentation] p53-Dependent Suppression of Genome Instability in Germ Cells (2015)
  • Author(s): Tomoko Ishikawa-Fujiwara, Shinji Otozai, Shoji Oda, Hiroshi Mitani, Takeshi Todo
  • Organizer: The 15th International Congress of Radiation Research
  • Place of Presentation: Kyoto
  • Year and Date: 2015-05-25
  • Int'l Joint Research
• [Presentation] Genome Editing in Medaka: Targeted Inactivation of TLS polymerase genes using TALENs. (2015)
  • Author(s): Fujikawa Y, Ishikawa-Fujiawara T, Nakamura K, Sakuma T, Yamamoto T, Todo T
  • Organizer: The 15th International Congress of Radiation Research
  • Place of Presentation: Kyoto
  • Year and Date: 2015-05-25
  • Int'l Joint Research
• [Presentation] In vivo and In vitro Analysis of Testis-ova Formation due to the Irradiation in p53 De¬cient Medaka Testis (2015)
  • Author(s): Kento Nagata, Ayaka Nishitani, Takako Yasuda, Tomomi Asaka, Shoji Oda, Yutaka Suzuki, Tomoko I Fujiwara, Takeshi Todo, Hiroshi Mitani
  • Organizer: The 15th International Congress of Radiation Research
  • Place of Presentation: Kyoto
  • Year and Date: 2015-05-25
  • Int'l Joint Research
• [Presentation] Molecular mechanism of mutagenesis in Medaka fish (2015)
  • Author(s): Tomoko Ishikawa-Fujiwara, Yoshihiro Fujikawa, Eri Shiraishi, Takeshi Todo
  • Organizer: International Symposium on Genome Science 2015
  • Place of Presentation: 東京・一橋講堂
  • Year and Date: 2015-01-20 – 2015-01-21
• [Presentation] CRISPR/Cas9システムによるメダカ光回復酵素遺伝子変異体の樹立 (2014)
  • Author(s): 藤原（石川） 智子、藤川 芳宏、藤堂 剛
  • Organizer: 第57回日本放射線影響学会
  • Place of Presentation: 鹿児島・鹿児島県民交流センター
  • Year and Date: 2014-10-01 – 2014-10-03
• [Presentation] メダカ生殖細胞における放射線誘発遺伝的不安定性の解析 (2014)
  • Author(s): 藤原（石川） 智子、音在 信治、尾田 正二、三谷 啓志、猪原 秀典、藤堂 剛
  • Organizer: 第57回日本放射線影響学会
  • Place of Presentation: 鹿児島・鹿児島県民交流センター
  • Year and Date: 2014-10-01 – 2014-10-03
• [Presentation] TALENs によるメダカにおける TLS ポリメラーゼ遺伝子群変異体の網羅的作製 (2014)
  • Author(s): 藤川 芳宏、藤原（石川） 智子、佐久間 哲史、山本 卓、藤堂 剛
  • Organizer: 第57回日本放射線影響学会
  • Place of Presentation: 鹿児島・鹿児島県民交流センター
  • Year and Date: 2014-10-01 – 2014-10-03
• [Presentation] Functional conversion of (6-4) photolyase and CPD photolyase (2014)
  • Author(s): Daichi Yamada, Junpei Yamamoto, Tomoko Ishikawa, Tomohiro Suzuki, I M. Mahaputra Wijaya, Tatsuya Iwata, Elizabeth D. Getzoff, Takeshi Todo, Shigenori Iwai and Hideki Kandori
  • Organizer: 第52回日本生物物理学会年会
  • Place of Presentation: 北海道・札幌コンベンションセンター
  • Year and Date: 2014-09-25 – 2014-09-27
• [Presentation] ゲノム編集技術：CRISPR/Cas9システムによるメダカ光回復酵素遺伝子変異体の樹立 (2014)
  • Author(s): 藤原（石川） 智子、藤川 芳宏、藤堂 剛
  • Organizer: 第18回日本光生物学協会年会
  • Place of Presentation: 大阪・大阪市立大学学術総合センター
  • Year and Date: 2014-08-22 – 2014-08-23
• [Presentation] TALENsを利用した遺伝子破壊によるメダカの損傷乗り越えDNA合成ポリメラーゼ遺伝子変異体の作成 (2014)
  • Author(s): 藤川 芳宏、藤原（石川） 智子、佐久間 哲史、山本 卓、藤堂 剛
  • Organizer: 第18回日本光生物学協会年会
  • Place of Presentation: 大阪・大阪市立大学学術総合センター
  • Year and Date: 2014-08-22 – 2014-08-23
• [Presentation] 変異導入による(6-4)光回復酵素からCPD光回復酵素への機能転換 (2014)
  • Author(s): 山田 大智、山元 淳平、.I. M. Mahaputra Wijaya、岩田 達也、鈴木智大、石川 智子、Elizabeth D. Getzoff、藤堂 剛、岩井 成憲、神取 秀樹
  • Organizer: 第18回日本光生物学協会年会
  • Place of Presentation: 大阪・大阪市立大学学術総合センター
  • Year and Date: 2014-08-22 – 2014-08-23
• [Presentation] CRISPR/Cas9システムによるメダカ光回復酵素遺伝子変異体の樹立 (2014)
  • Author(s): 藤原（石川） 智子、 藤川 芳宏、 藤堂 剛
  • Organizer: 第36回光医学光生物学会
  • Place of Presentation: 大阪・大阪大学医学部学友会館
  • Year and Date: 2014-07-25 – 2014-07-26
• [Presentation] メダカにおけるゲノム編集技術(TALENs)を用いた損傷乗り越えDNA合成ポリメラーゼ遺伝子群変異体の作製 (2014)
  • Author(s): 藤川 芳宏、藤原（石川） 智子、佐久間 哲史、山本 卓、藤堂 剛
  • Organizer: 第36回光医学光生物学会
  • Place of Presentation: 大阪・大阪大学医学部学友会館
  • Year and Date: 2014-07-25 – 2014-07-26
• [Presentation] メダカにおける人工ヌクレアーゼ（TALEN）を用いた損傷乗り越えポリメラーゼ変異体の作製 (2013)
  • Author(s): 藤川芳宏、藤原（石川）智子、佐久間哲史、山本卓、藤堂剛
  • Organizer: 日本環境変異原学会第42回大会
  • Place of Presentation: 岡山市
• [Presentation] Targeted Inactivation of Rev1 gene (2013)
  • Author(s): Tomoko Ishikawa-Fujiwara, Kumi Nakamura, Yoshihiro Fujikawa, Takeshi Todo
  • Organizer: The 6th Asia and Oceania Conference on Photobiology
  • Place of Presentation: Sydney, Australia
• [Presentation] The molecular structure and damaged-DNA repair activity affected by the chromophore binding (2013)
  • Author(s): Kazunori Zikihara, Kohei Kasakawa, Tomoko Ishikawa, Kristin Tessmar-Raible, Satoru Tokutomi, Takeshi Todo
  • Organizer: The 6th Asia and Oceania Conference on Photobiology
  • Place of Presentation: Sydney, Australia
• [Presentation] Targeted Inactivation of DNA photolyase family genes using CRISPR system (2013)
  • Author(s): Tomoko Ishikawa-Fujiwara, Kumi Nakamura, Yoshihiro Fujikawa, Takeshi Todo
  • Organizer: The 6th Asia and Oceania Conference on Photobiology
  • Place of Presentation: Sydney, Australia
• [Presentation] ゲノム編集技術（CRISPR）を利用したメダカ光回復酵素遺伝子変異体の作製 (2013)
  • Author(s): 藤原 石川 智子、藤川 芳宏、中村 公美、藤堂 剛
  • Organizer: 日本放射線影響学会第56回大会
  • Place of Presentation: 青森市
• [Presentation] ゲノム編集技術（TALEN）を用いたメダカにおけるTLS ポリメラーゼ変異体の網羅的作製 (2013)
  • Author(s): 藤川 芳宏、藤原（石川） 智子、中村 公美、佐久間 哲史、山本 卓、藤堂 剛
  • Organizer: 日本放射線影響学会第56回大会
  • Place of Presentation: 青森市
• [Presentation] ゲノム編集技術TALEN を用いた特異的欠損部位変異の作製方法の樹立；メダカRev1 BRCT ドメイン欠損変異体作製 (2013)
  • Author(s): 中村 公美、藤原（石川） 智子、佐久間 哲史、山本 卓、藤堂 剛
  • Organizer: 日本放射線影響学会第56回大会
  • Place of Presentation: 青森市
• [Presentation] DNA-PK 自己リン酸化異常時におけるp53のDSB 修復経路選択への関わり (2013)
  • Author(s): 大西 壽子、漆原 佑介、藤原 智子、尾田 正二、藤堂 剛、三谷 啓志
  • Organizer: 日本放射線影響学会第56回大会
  • Place of Presentation: 青森市
• [Presentation] Genome editing in medaka: targeted inactivation of DNA photolyase family genes using CRISPR system (2013)
  • Author(s): Tomoko Ishikawa-Fujiwara, Yoshihiro Fujikawa, Kumi Nakamura, Takeshi Todo
  • Organizer: 18th Japanese Medaka and Zebrafish Meeting
  • Place of Presentation: 仙台市
• [Presentation] Genome editing in medaka: targeted inactivation of TLS polymerase genes usingTALENs (2013)
  • Author(s): Yoshihiro Fujikawa, Tomoko Ishikawa-Fujiwara, Kumi Nakamura, Tetsushi Sakuma, Takashi Yamamoto, Takeshi Todo
  • Organizer: 18th Japanese Medaka and Zebrafish Meeting
  • Place of Presentation: 仙台市
• [Presentation] Genome editing in medaka: targeted inactivation of Rev1 gene by TALEN (2013)
  • Author(s): Kumi Nakamura, Yoshihiro Fujikawa, Tomoko Ishikawa-Fujiwara, Tetsushi Sakuma, Takashi Yamamoto, Takeshi Todo
  • Organizer: 18th Japanese Medaka and Zebrafish Meeting
  • Place of Presentation: 仙台市
• [Presentation] ATF6α/β-mediated adjustment of ER chaperone levels is essential for development of the notochord in medaka fish (2013)
  • Author(s): Tokiro Ishikawa, Tetsuya Okada, Tomoko Ishikawa-Fujiwara, Takeshi Todo, Yasuhiro Kamei, Shuji Shigenobu, Minoru Tanaka, Taro L. Saito, Jun Yoshimura, Shinichi Morishita, Atsushi Toyoda, Yoshiyuki Sakaki, Yoshihito Taniguchi, Shunichi Takeda, Kazutoshi Mori
  • Organizer: 18th Japanese Medaka and Zebrafish Meeting
  • Place of Presentation: 仙台市
• [Book] 光老化科学の最前線 第11章「DNA光化学と概日リズム」 (2015)
  • Author(s): 藤原（石川）智子、藤堂剛
  • Total Pages: 301
  • Publisher: シーエムシー出版