マウス始原生殖細胞形成を制御する分子カスケードの解明

• Project/Area Number: 15080213
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Tohoku University (2004-2007); Research Institute, Osaka Medical Center for Maternal and Child Health (2003)
• Principal Investigator: 松居 靖久 Tohoku University, 加齢医学研究所, 教授 (40241575)
• Co-Investigator(Kenkyū-buntansha): 林 克彦 大阪府立母子保健総合医療センター研究所, 病因病態部門, 研究員 (20287486)
• Project Period (FY): 2003 – 2007
• Project Status: Completed (Fiscal Year 2007)
• Budget Amount: ¥128,600,000 (Direct Cost: ¥128,600,000); Fiscal Year 2007: ¥25,900,000 (Direct Cost: ¥25,900,000); Fiscal Year 2006: ¥26,200,000 (Direct Cost: ¥26,200,000); Fiscal Year 2005: ¥25,500,000 (Direct Cost: ¥25,500,000); Fiscal Year 2004: ¥25,500,000 (Direct Cost: ¥25,500,000); Fiscal Year 2003: ¥25,500,000 (Direct Cost: ¥25,500,000)
• Keywords: 始原生殖細胞 / DNAメチル化 / 転写制御 / トランスジェニックマウス / mil-1 / ES細胞 / GFP / Oct-3 / 4 / ノックアウトマウス / 生殖細胞 / 遺伝子 / 細胞分化 / 減数分裂 / ヒストンメチルトランスフェラーゼ / DNAマイクロアレイ / インターフェロン誘導性膜蛋白質 / 転写因子

Research Abstract

本研究では、マウス始原生殖細胞の分化運命が決定される過程を制御する分子カスケードを明らかにし体細胞と生殖細胞の本質的な違いを最初に生み出す機構を解明することを目的とする。そのためこれまでに前駆細胞の時期から始原生殖細胞で特異的に発現するmil-1遺伝子を単離した。次に、この遺伝子の近傍領域にGFP遺伝子をつないだものでトランスジェニックマウスを作成し、始原生殖細胞での特異的な発現には、転写開始点から上流3kbpまでの領域が必要であることを明らかにした。
これらの結果をふまえて本年度は、始原生殖細胞と胎仔生殖巣体細胞を用いて、この発現制御領域のDNAメチル化の差異をバイサルフェート法で調べ、始原生殖細胞ではほぼ完全に脱メチル化状態であるのに対して、体細胞では高メチル化状態になっていることがわかった。次にDNAのメチル化とmil-1遺伝子の発現制御との関連を明らかにするために、mil-1遺伝子を弱く発現している未分化ES細胞を、DNAを脱メチル化する作用のある5アザシチジン存在下で培養したところ、mil-1遺伝子の発現上昇と制御領域の脱メチル化が確認された。さらに発現制御領域にルシフェラーゼ遺伝子をつないだレポーターベクターをin vitroでメチル化したものと、脱メチル化状態のものをES細胞に導入し培養後にルシフェラーゼ活性を調べたところ、脱メチル化によりレポーター活性の上昇が見られた。これらの結果から、mil-1遺伝子の始原生殖細胞特的な発現制御には制御領域のDNAのメチル化が重要な役割を果たしていることが示唆された。

Research Products

• [Journal Article] Epigenetic events in mammalian germ cell development: reprogramming and beyond. (2008)
  • Author(s): Sasaki, H.
  • Journal Title: Nat. Rev. Genet. 9 Pages: 129-140
  • Peer Reviewed
• [Journal Article] Requirement of Oct3/4 for germ cell specification. (2008)
  • Author(s): Okamura, D.
  • Journal Title: Dev. Biol. (印刷中)
  • Peer Reviewed
• [Journal Article] Epigenetic control for induction of meiosis. (2007)
  • Author(s): Matsui, Y.
  • Journal Title: Cellular and Molecular Life Science 64 Pages: 257-262
  • Peer Reviewed
• [Journal Article] A comparison study in the proteomic signature of multipotent germline stemcells, embryonic stem cells, and germline stem cells. (2007)
  • Author(s): Kurosaki, H.
  • Journal Title: Biochemical and Biophysical Research Communications 353 Pages: 259-267
  • Peer Reviewed
• [Journal Article] Cellular dynamics associated with the genome-wide epigenetic reprogramming in migrating primordial germ cells in mice. (2007)
  • Author(s): Seki, Y.
  • Journal Title: Development 134 Pages: 2627-2638
  • Peer Reviewed
• [Journal Article] A comparison study in the proteomic signiture of multipotent germline stem cells, embryonic stem cells, and germline stem cells. (2007)
  • Author(s): Kurosaki, H.
  • Journal Title: Biochemical and Biophysical Research Communications 353 Pages: 259-267
• [Journal Article] Expression of low density lipoprotein receptor-related protein 4 (Lpr4) gene in the mouse germ cells. (2006)
  • Author(s): Yamaguchi, L. Y.
  • Journal Title: Gene Expression Patterns 6 Pages: 607-612
• [Journal Article] Discordant developmental waves of angiogenesis and hemangioblasts in the early gastrulating mouse embryo. (2006)
  • Author(s): Furuta, C.
  • Journal Title: Development 133 Pages: 2771-2779
• [Journal Article] Stage-specific Importin13 activity influence meiosis of germ cells in the mouse. (2006)
  • Author(s): Yamaguchi, L. Y.
  • Journal Title: Developmental Biology 297 Pages: 350-360
• [Journal Article] Meisetz, a novel histone tri-methyltransferase, regulates meiosis-specific epigenesis. (2006)
  • Author(s): Hayashi, K.
  • Journal Title: Cell Cycle 5 Pages: 615-620
• [Journal Article] Expression of low density lipoprotein receptor-related protein 4(Lpr4) gene in the mouse germ cells. (2006)
  • Author(s): Yamaguchi, L.Y.
  • Journal Title: Gene Expression Patterns (印刷中)
• [Journal Article] Mouse epiblast changes responsiveness to BMP4 signal required for PGC formation by the functions of extraembryonic ectoderm. (2005)
  • Author(s): Okamura, D., et al.
  • Journal Title: Molecular Reproduction and Development 70 Pages: 20-29
• [Journal Article] Mechanisms of germ cell specification in mouse embryos. (2005)
  • Author(s): Matsui, M., Okamura, D.
  • Journal Title: BioEssays 27 Pages: 136-143
• [Journal Article] Extensive and orderly reprogramming of genome-wide chromatin modification associated with specification and early development of germ cells in mice. (2005)
  • Author(s): Seki.Y.et al.
  • Journal Title: Developmental Biology 278 Pages: 440-458
• [Journal Article] Canonical Wnt signaling and its antagonist regulate anterior-posterior axis polarization by guiding cell migration in mouse visceral endoderm. (2005)
  • Author(s): Kimura-Yoshida, C.
  • Journal Title: Developmental Cell 9 Pages: 639-650
• [Journal Article] A histone H3 methyltransferase controls epigenetic events required for meiotic prophase. (2005)
  • Author(s): Hayashi, K.
  • Journal Title: Nature 438 Pages: 374-378
• [Journal Article] Mouse epiblast changes responsiveness to BMP4 signal required for PGC formation by the functions of extraembryonic ectoderm. (2005)
  • Author(s): Okamua, D., et al.
  • Journal Title: Molecular Reproduction and Development 70 Pages: 20-29
• [Journal Article] Birth of mice produced by germ cell nuclear transfer. (2005)
  • Author(s): Miki, H. et al.
  • Journal Title: Genesis (印刷中)
• [Journal Article] Extensive and orderly reprogramming of genome-wide chromatin modification associated with specification and early development of germ cells in mice. (2005)
  • Author(s): Seki.Y.et al.
  • Journal Title: Developmental Biology (印刷中)
• [Journal Article] Hrp48 regulates and couples oskar mRNA localization and translational control during Drosophila oogenesis. (2004)
  • Author(s): Yano, T. et al.
  • Journal Title: Developmental Cell 6 Pages: 637-648
• [Journal Article] Regulation of expression of mouse interferon-induced transmembrane protein like gene-1. Ifitm3 (mil-1/fragilis), in germ cells. (2004)
  • Author(s): Tanaka, S.S. et al.
  • Journal Title: Developmental Dynamics 230 Pages: 651-659
• [Presentation] Epigenetic regulation of germ cell-specific gene expression. (2008)
  • Author(s): Y. Matsui
  • Organizer: 6th NIBB-EMBL Joint Meeting, Evolution of Epigenetic Regulation'
  • Place of Presentation: Heidelberg, Germany
• [Presentation] DNA demethylation regulates primordial germ cell-specific expression of mil-1 gene in mouse. (2008)
  • Author(s): K. Mochizuki
  • Organizer: 6th NIBB-EMBL Joint Meeting, Evolution of Epigenetic Regulation'
  • Place of Presentation: Heidelberg, Germany
• [Presentation] mil-1遺伝子のマウス始原生殖細胞特異的発現へのDNA脱メチル化の関与 (2007)
  • Author(s): 望月研太郎、松居靖久
  • Organizer: 第30回日本分子生物学会年会
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-12
• [Presentation] Identification of mouse primordial germ cell subpopulation, which is eliminated by apoptosis (2007)
  • Author(s): 藤村維子
  • Organizer: 第30回日本分子生物学会年会
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-12
• [Presentation] マウス始原生殖細胞の分化過程における細胞不均一性の解析 (2007)
  • Author(s): 藤村維子
  • Organizer: 第40回日本発生生物学会、第59回日本細胞生物学会合同大会
  • Place of Presentation: 福岡
  • Year and Date: 2007-05-28
• [Presentation] Essential functions of a histone methyltransferase, Mei setz(prdm9), in meiotic prophase. (2007)
  • Author(s): Y. Matsui
  • Organizer: EMBO World Workshop, 8th European Meiosis Meeting in Japan.
  • Place of Presentation: Hayama, Japan
• [Book] 加齢医学 エイジング・ファイン (2007)
  • Author(s): 松居靖久
  • Total Pages: 300
  • Publisher: 東北大学出版会
• [Book] キーワードで理解する発生・再生イラストマップ (2005)
  • Author(s): 松居靖久
  • Total Pages: 188
  • Publisher: 羊土社
• [Patent(Industrial Property Rights)] 妊孕性に関連するポリヌクレオチドおよびその利用 (2007)
  • Inventor(s): 田中宏光
  • Industrial Property Rights Holder: 国立大学法人大阪大学
  • Industrial Property Number: 2007-236215
  • Filing Date: 2007-09-12
• [Publications] Sato, S. et al.: "Erasure of methylation imprinting of lgf2r during mouse primordial germ-cell development."Molecular Reoproduction and Development. 65. 41-50 (2003)
• [Publications] Ara, T.et al.: "Impaired colonization of the gonads by primordial germ cells in mice lacking a chemokine, stromal cell-derived factor (SDF-1)."Proceedings of the National Academy of Sciences of United States of America. 100. 5319-5323 (2003)
• [Publications] Okamura, D. et al.: "Cadherin-mediated cell interaction regulate germ cell determination in mice."Development. 130. 6423-6430 (2003)
• [Publications] Tanaka, S.S: "Regulation of expression of mouse interferon-induced transmembrane protein like gene-1, mil-1/fragilis, in germ cells."Developmental Dynamics. 印刷中. (2004)