Cdt1-binding protein GRWD1 is a novel histone-binding protein that facilitates MCM loading through its influence on chromatin architecture

  • Nucleic Acids Res. 2015 Jul 13;43(12):5898-911. doi: 10.1093/nar/gkv509.
Nozomi Sugimoto  1 ,  Kazumitsu Maehara  2 ,  Kazumasa Yoshida  1 ,  Shuhei Yasukouchi  1 ,  Satoko Osano  3 ,  Shinya Watanabe  1 ,  Masahiro Aizawa  1 ,  Takashi Yugawa  3 ,  Tohru Kiyono  3 ,  Hitoshi Kurumizaka  4 ,  Yasuyuki Ohkawa  2 ,  Masatoshi Fujita  5
Affiliations
  • 1. Department of Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
  • 2. Faculty of Medicine, Division of Epigenetics, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
  • 3. Division of Virology, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.
  • 4. Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan.
  • 5. Department of Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan [email protected].
PMID: 25990725 DOI: 10.1093/nar/gkv509
Abstract

Efficient pre-replication complex (pre-RC) formation on chromatin templates is crucial for the maintenance of genome integrity. However, the regulation of chromatin dynamics during this process has remained elusive. We found that a conserved protein, GRWD1 (glutamate-rich WD40 repeat containing 1), binds to two representative replication origins specifically during G1 phase in a CDC6- and Cdt1-dependent manner, and that depletion of GRWD1 reduces loading of MCM but not CDC6 and Cdt1. Furthermore, chromatin immunoprecipitation coupled with high-throughput Sequencing (Seq) revealed significant genome-wide co-localization of GRWD1 with CDC6. We found that GRWD1 has histone-binding activity. To investigate the effect of GRWD1 on chromatin architecture, we used formaldehyde-assisted isolation of regulatory elements (FAIRE)-seq or FAIRE-quantitative PCR analyses, and the results suggest that GRWD1 regulates chromatin openness at specific chromatin locations. Taken together, these findings suggest that GRWD1 may be a novel histone-binding protein that regulates chromatin dynamics and MCM loading at replication origins.