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 - Radiation Biology Center
Department of Mutagenesis (Division of Chromatin Regulatory Network)
The purpose of our research is to clarify the role of chromatin remodeling, which is required for the DNA metabolisms such as transcription, DNA replication, and DNA repair. In particular, we focus on the molecular mechanisms by which histone modifier complexes regulate the histone eviction as chromatin remodeling machinery upon DNA damage induced by ionizing radiation. We use the protein complex purification system in combination with genetic analysis to determine the relationship between histone eviction and DNA damage signaling pathway. Our goal is to understand how histone eviction activates DNA damage signaling pathways and functions as an anti-cancer signaling.

 
 
Research and Education
Eukaryotic genome is tightly packed into the chromatin, a hierarchically organized complex of DNA, histone and nonhistone proteins. This packing represents a common obstacle for most of the DNA functions. Concerning transcription, covalent modifications of core histone N-termini and ATP-dependent nucleosome remodeling plays a role in regulation of gene expression in the chromatin context. On the other hand, role of these chromatin modifications in other aspects of DNA metabolism, especially DNA repair, remains largely unexplored. We have shown that TIP60 histone acetylase complex involved in DNA repair and apoptosis. However, it remains unknown how TIP60 complex involve in DNA repair. To better understand the mechanism of TIP60 complex in DNA repair, TIP60 complex purifies from chromatin soluble fraction after exposure of ionizing radiation. As a result, the TIP60 complex is associated with chromatin including histone H2AX after DNA damage. Histone H2AX, histone H2A variant, is phosphorylated at the site of DNA double-strand breaks (DSBs). We found that TIP60 acetylates histone H2AX. By acetylating H2AX, TIP60 stimulates its ubiquitination by the ubiquitin-conjugating enzyme UBC13. Furthermore, we revealed that the acetylation-dependent ubiquitination by the TIP60-UBC13 complex leads to the eviction of H2AX from damaged chromatin using live cell imaging and UV laser micro-irradiation. Since ubiquitination of H2AX is required for the recruitment of DNA repair and checkpoint proteins at the damage site, TIP60/UBC13-dependent eviction of H2AX may function as a molecular link between DNA damage sensing and chromatin remodeling. We are now investigating the role of the eviction of H2AX upon DNA damage in chromatin remodeling and checkpoint activation.


Laboratory of Chromatin Regulatory Network,
Department of Mutagenesis,
Radiation Biology Center
Associate
 Professor


Tsuyoshi Ikura, D.D.S., Ph.D.
TEL +81-75-753-7556
FAX +81-75-753-7564
e-mail ikurathouse.rbc.kyoto-u.ac.jp
Model for the role of TIP60 and UBC13 in DNA damage response. The TIP60 complex is recruited to the damaged chromatin immediately after induction of DSBs and acetylates H2AX. UBC13 is recruited and/or the activity of UBC13 is modulated by TIP60, facilitates poly-ubiquitination of H2AX, resulting in histone H2AX eviction for the repair of DSBs.
The strategy fro the TIP60 complex purification
Nuclear extracts were prepared from HeLa cells stably expressing Flag-HA tagged TP60. Affinity purification by using ant-Flag and anti-HA antibody was performed. Purified TIP60 complex was analyzed by SDS-PAGE and then the components of the TIP60 complex were determined by Mass Spectrometry analysis.
SDS-PAGE analysis of the TIP60 complex by silver staining.
Recent Publications
1. Ikura, T., Ogryzko, V V., Grigoriev, M., Groisman, R., Wang, J., Horikoshi, M., Scully, R., Qin, J., Nakatani, Y. Involvement of the TIP60 Histone Acetylase Complex in DNA repair and apoptosis (2000). Cell. 102: 463-473.
2. Fuchs, M., Gerber, J., Drapkin, R., Sif, S., Ikura, T., Ogryzko, V., Lane, WS., Nakatani, Y., Livingston, DM. The p400 complex is an essential E1A transformation target (2001).Cell. 106: 297-307.
3. Ikura, T., Tashiro, S., Kakino, A., Shima, H., Jacob, N., Amunugama, R., Yoder, K., Izumi, S., Kuraoka, I., Tanaka, K., Kimura, H., Ikura, M., Nishikubo, S., Ito, T., Muto, A., Miyagawa, K., Takeda, S., Fishel, R., Igarashi, K., Kamiya, K. DNA damage-dependent acetylation and ubiquitination of H2AX enhances chromatin dynamics (2007). Mol. Cell. Biol. 27: 7028-40.
4. Dohi, Y., Ikura, T., Hoshikawa, Y., Katoh, Y., Ota, K., Nakanome, A., Muto, A., Omura, S., Ohta, T., Ito, A., Yoshida, M., Noda, T., Igarashi, K. Bach1 inhibits oxidative stress-induced cellular senescence by impeding p53 function on chromatin (2008). Nat. Struct. Mol. Biol. 15. 1246-54.