title: Reorganization of damaged chromatin by the exchange of histone variant H2A.Z-2.
abstract: OBJECTIVE
  The reorganization of damaged chromatin plays an important role in the regulation of the DNA damage response. A recent study revealed the presence of 2 vertebrate H2A.Z isoforms, H2A.Z-1 and H2A.Z-2. However, the roles of the vertebrate H2A.Z isoforms are still unclear. Thus, in this study we examined the roles of the vertebrate H2A.Z isoforms in chromatin reorganization after the induction of DNA double-strand breaks (DSBs).
  
  
  METHODS
  To examine the dynamics of H2A.Z isoforms at damaged sites, we constructed GM0637 cells stably expressing each of the green fluorescent protein (GFP)-labeled H2A.Z isoforms, and performed fluorescence recovery after photobleaching (FRAP) analysis and inverted FRAP analysis in combination with microirradiation. Immunofluorescence staining using an anti-RAD51 antibody was performed to study the kinetics of RAD51 foci formation after 2-Gy irradiation of wild-type (WT), H2A.Z-1- and H2A.Z-2-deficient DT40 cells. Colony-forming assays were also performed to compare the survival rates of WT, H2A.Z-1-, and H2A.Z-2-deficient DT40 cells with control, and H2A.Z-1- and H2A.Z-2-depleted U2OS cells after irradiation.
  
  
  RESULTS
  FRAP analysis revealed that H2A.Z-2 was incorporated into damaged chromatin just after the induction of DSBs, whereas H2A.Z-1 remained essentially unchanged. Inverted FRAP analysis showed that H2A.Z-2 was released from damaged chromatin. These findings indicated that H2A.Z-2 was exchanged at DSB sites immediately after the induction of DSBs. RAD51 focus formation after ionizing irradiation was disturbed in H2A.Z-2-deficient DT40 cells but not in H2A.Z-1-deficient cells. The survival rate of H2A.Z-2-deficient cells after irradiation was lower than those of WT and H2A.Z-1- DT40 cells. Similar to DT40 cells, H2A.Z-2-depleted U2OS cells were also radiation-sensitive compared to control and H2A.Z-1-depleted cells.
  
  
  CONCLUSIONS
  We found that vertebrate H2A.Z-2 is involved in the regulation of the DNA damage response at a very early stage, via the damaged chromatin reorganization required for RAD51 focus formation.
authors: Department of Cellular Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan; Department of Radiation Oncology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan; Department of Radiation Oncology, Hiroshima Prefectural Hospital, Hiroshima, Japan.
authors: Ikuno
authors: I
authors: Nishibuchi
authors: Department of Cellular Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan.
authors: Hidekazu
authors: H
authors: Suzuki
authors: Department of Cellular Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan.
authors: Aiko
authors: A
authors: Kinomura
authors: Department of Cellular Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan.
authors: Jiying
authors: J
authors: Sun
authors: Department of Cellular Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan.
authors: Ning-Ang
authors: NA
authors: Liu
authors: Department of Cellular Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan; Research Center for Mathematics of Chromatin Live Dynamics, Hiroshima University, Hiroshima, Japan.
authors: Yasunori
authors: Y
authors: Horikoshi
authors: Department of Biochemistry, Graduate School of Medical Sciences, Tohoku University, Sendai, Japan.
authors: Hiroki
authors: H
authors: Shima
authors: Laboratory of Molecular Biology, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan.
authors: Masayuki
authors: M
authors: Kusakabe
authors: Laboratory of Molecular Biology, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan.
authors: Masahiko
authors: M
authors: Harata
authors: Department of Molecular Genetics, National Institute of Genetics and The Graduate University for Advanced Studies, Mishima, Japan.
authors: Tatsuo
authors: T
authors: Fukagawa
authors: Laboratory of Chromatin Regulatory Network, Department of Mutagenesis, Radiation Biology Center, Kyoto University, Kyoto, Japan.
authors: Tsuyoshi
authors: T
authors: Ikura
authors: Department of Cardiovascular Medicine, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
authors: Takafumi
authors: T
authors: Ishida
authors: Department of Radiation Oncology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
authors: Yasushi
authors: Y
authors: Nagata
authors: Department of Cellular Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan; Research Center for Mathematics of Chromatin Live Dynamics, Hiroshima University, Hiroshima, Japan. Electronic address: ktashiro@hiroshima-u.ac.jp.
authors: Satoshi
authors: S
authors: Tashiro
mesh_terms: D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D002843:Chromatin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003114:Colony-Forming Units Assay / Q000379:methods; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000502:physiology*; D005455:Fluorescent Antibody Technique / Q000379:methods; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020033:Protein Isoforms / Q000378:metabolism; D051135:Rad51 Recombinase / Q000378:metabolism*
chemical_list: D002843:Chromatin; D006657:Histones; D020033:Protein Isoforms; C077020:histone H2A.F-Z; C495153:RAD51 protein, human; D051135:Rad51 Recombinase