Molecular Mechanism for cellular senescence and immortalization

• Project/Area Number: 13043024
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Kyoto University
• Principal Investigator: SHINKAI Yoichi Kyoto University, Institute for Vims Research, Professor, ウイルス研究所, 教授 (20211972)
• Co-Investigator(Kenkyū-buntansha): TACHIBANA Makoto Kyoto University, Institute for Virus Research, Associate Professor, ウイルス研究所, 助教授 (80303915)
• Project Period (FY): 2001 – 2005
• Project Status: Completed (Fiscal Year 2005)
• Budget Amount: ¥65,300,000 (Direct Cost: ¥65,300,000); Fiscal Year 2005: ¥12,500,000 (Direct Cost: ¥12,500,000); Fiscal Year 2004: ¥12,200,000 (Direct Cost: ¥12,200,000); Fiscal Year 2003: ¥13,400,000 (Direct Cost: ¥13,400,000); Fiscal Year 2002: ¥14,000,000 (Direct Cost: ¥14,000,000); Fiscal Year 2001: ¥13,200,000 (Direct Cost: ¥13,200,000)
• Keywords: telomere / telomerase / telomere binding protein / TRF 1 / cell cycle / DNA rep air / chromosomal instability / TRF2 / TIN2 / 細胞増殖 / 染色体の安定性 / DNA障害チェック機構 / テロメアーゼ / 染色体 / 老化 / 不死化

Research Abstract

I. The ends of chromosomes, telomeres cohsist of the tandem repeat arrays of TTAGGG sequences and various telomeric proteins by forming a large complex which play an important role to protect the chromosomal ends from DNA damage response. TRF1 and TRF2 directly bind to double-stranded telomeric DNA as a homodimer. Another telomeric protein, TIN2 interacts with both TRF1 and TRF2 and stabilizes them on telomere. In addition, TIN2 interacts with PTOP which tethers single-stranded telomeric DNA binding protein, POT1 to telomere. We have established mouse TRF1 conditional knockout ES cells and demonstrated that TRF1 plays critical roles for the maintenance of the "functional" telomere structure and organizes the localization of these telomeric proteins on telomere. In the new study, we further describe functional interaction of TRF1 and other telomere proteins, especially on regulation of H2AX phosphorylation (H2AX) and cell growth.
First, we generated chicken and mouse fusion TRF1(cmTRF1), N-terminal domain is derived from chicken TRF1 and C-terminal is mouse, because it was found that chicken TRF1 is not associated with mouse Tin2. The cmTRF1 protein was not associated with mTin2 and didn't rescue any TRF 1-deficient phenotypes. Next, full-length or truncated TIN2 and PTOP were fused with cmTRF1 and expressed into the TRF1 conditional KO ES cells. All the TIN2-or PTOP-cmTRF1 fusion molecules excepting the one which did not rescue POT1 of telomere localization, suppressed DH2AX accumulation on telomeres. Taken collectively, we suggest that POT1 arid/or PTOP are critical for regulation of DH2AX on telomeres. However, none of TIN2-or PTOP-cmTRFl suppressed abnormal telomere signals of TRF1-deficient ES cells. This data suggests that TRF1 possesses at least two distinct roles for "functional telomere", one is TRF 1-dependent and the other is dependent on TRF 1-associating telomere molecules.
II. Some immortal cells use the alternative lengthening of telomeres (ALT) pathway to maintain their telomeres instead of telomerase. Previous studies revealed that homologous recombination (HR) contributes to the ALT pathway. To further elucidate molecular mechanisms, we inactivated Rad54 involved in HR, in mouse ALT embryonic stem (ES) cells. Although Rad54-deficient ALT ES cells showed radiosensitivity in line with expectation, cell growth and telomeres were maintained for more than 200 cell divisions. Furthermore, although MMC-stimulated sister chromatid exchange (SCE) was suppressed in the Rad54-deficient ALT ES cells, ALT-associated telomere SCE was not affected. This is the first genetic evidence that mouse Rad54 is dispensable for the ALT pathway.

Research Products

• [Journal Article] Rad54 is dispensable for the ALT pathway. (2006)
  • Author(s): Akiyama, K., Yusa, K., Hashimoto, H., Poonepalli, A., Hande, M.P., Kakazu, N., Takeda, J., Tachibana M., Shinkai, Y.
  • Journal Title: Genes to Cells 11 Pages: 1305-1315
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Rad54 is dispensable for the ALT pathway. (2006)
  • Author(s): Akiyama, K.Yusa, K.Hashimoto, H.Poonepalli, A.Hande, M.P.Kakazu, N.Takeda, J.Tachibana, M., Shinkai, Y.
  • Journal Title: Genes to Cells 11 Pages: 1305-1315
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Genetic and epigenetic properties of mouse male germline stem cells during long-term culture. (2005)
  • Author(s): Kanatsu-Shinohara, M., Ogonuki, N., Iwano, T., Lee, J., Kazuki, Y., Inoue, K., Miki, H., Takehashi, M., Toyokuni, S., Shinkai, Y., Oshimura, M., Shino, F., Ogura, A., Shinohara, T.
  • Journal Title: Development 132 Pages: 4155-4163
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Genetic and epigenetic properties of mouse male germline stem cells during long-term culture. (2005)
  • Author(s): Kanatsu-Shinohara, M.Ogonuki, N., Iwano, T., Lee, J., Kazuki, Y., Inoue, K., Miki, H., Takehashi, M., Toyokuni, S., Shinkai, Y., Oshimura, M., Ishino, F., Ogura, A., Shinohara, T.
  • Journal Title: Development 132 Pages: 4155-4163
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Importance of TRF1 for functional telomere structure. (2004)
  • Author(s): Iwano, T., Tachibana, M., Reth, M., Shinkai, Y.
  • Journal Title: J. Biol. Chem. 279 Pages: 1442-1448
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Importance of TRF1 for fun ctional telomere structure. (2004)
  • Author(s): Iwano, T.Tachibana, M.Reth, M., Shinkai, Y.
  • Journal Title: J. Biol. Chem. 279 Pages: 1442-1448
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Free Full Text G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis. (2002)
  • Author(s): Tachibana M, Sugimoto K, Nozaki M, Ueda J, Ohta T, Ohki M, Fukuda M, Takeda N, Niida H, Kato H, Shinkai Y
  • Journal Title: Genes Dev. 16 Pages: 1779-1791
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Free Full Text G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis. (2002)
  • Author(s): Tachibana M, Sugimoto K, Nozaki M, Ueda J, Ohta T, Ohki M, Fukuda M, Takeda N, Niida H, Kato H, Shinkai Y.
  • Journal Title: Genes Dev. 16 Pages: 1779-1791
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Set domain-containing protein, G9a, is a novel lysine-preferring mammalian histone methyltransferase with hyperactivity and specific selectivity to lysines 9 and 27 of histone H3. (2001)
  • Author(s): Tachibana M, Sugimoto K, Fukushima T, Shinkai Y
  • Journal Title: J. Mol. Chem. 276 Pages: 25309-25317
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Set domain-containing protein, G9a, is a novel lysine-preferring mammalian histone methyltransferase with hyperactivity and specific selectivity to lysines 9 and 27 of histone H3. (2001)
  • Author(s): Tachibana M, Sugimoto K, Fukushima T, Shinkai Y.
  • Journal Title: J. Mol. Chem. 276 Pages: 25309-25317
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Tomohiko Iwano, Makoto Tachibana, Michael Reth, Yoichi Shinkai: "Importance of TRF1 for Functional Telomere Structure."The Journal of Biological Chemistry. 279. 1442-1448 (2004)
• [Publications] Tachibana, M., Sugimoto, K., Nozaki, M., Ueda, J., Ohta, T., Ohki, M., Fukuda, M., Takeda, N., Niida, H., Kato, H., Shinkai, Y.: "G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis"Genes and Development. 16(14). 1779-1791 (2002)
• [Publications] Tachibana, M., Sugimoto, K.: "ET-domain containing protein, C9a, is a novel lysine-preferring mammalian histone methyltransferase with hyperactivity and specific selectivity to lysines 9 and 27 of"J. Biol. Chem.. 276. 25309-25317 (2001)