title: Dependence of radiation-induced H2AX phosphorylation on histone methylation: evidence from the chromatin immunoprecipitation assay.
abstract: OBJECTIVE
  To evaluate ionizing radiation (IR)-induced DNA damage response within euchromatic and heterochromatic regions.
  
  
  METHODS
  Chromatin immunoprecipitation (ChIP) and immunofluorescence analysis were used to explore the distribution of phosphorylated H2AX (γH2AX).
  
  
  RESULTS
  ChlP experiments after IR at 30 and 60 Gy showed by a factor of 1.28 (1.08-1.53, 95% confidence interval) higher γH2AX signal at 45 min after IR in histone H3 trimethylated lysine 4 (H3K4me3) compared to lysine 9 (H3K9me3) enriched chromatin fragments. Halving the radiation dose from 60-30 Gy led to a reduction of γH2AX signal by a factor of 0.49 (0.37-0.64), independent of the chromatin region. Repair incubation for 240 min led to a decrease of the γH2AX signal by a factor of 0.55 (0.45-0.67) in both regions. The fraction of H3K9me3 was determined with immunofluorescent microscopy to be 30.5 ± 3.8% of the whole chromatin. The fraction of γH2AX foci within H3K9me3 regions was shown to be 12.9 ± 0.4% and 13.9 ± 0.6% at 45 min and 4 h after 0.5 Gy, respectively, and thus by a factor of about 2.2 lower than the fraction expected from an isotropic distribution.
  
  
  CONCLUSIONS
  These data strengthen the dependence of IR-induced DNA damage response on the chromatin region.
authors.affiliation: Department of Radiotherapy, University Hospital Essen , Essen , Germany.
authors.forename: Ali
authors.initials: A
authors.lastname: Sak
authors.forename: Dennis
authors.initials: D
authors.lastname: Kübler
authors.forename: Kristina
authors.initials: K
authors.lastname: Bannik
authors.forename: Michael
authors.initials: M
authors.lastname: Groneberg
authors.forename: Martin
authors.initials: M
authors.lastname: Stuschke
mesh_terms: D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D053903:DNA Breaks, Double-Stranded; D006657:Histones / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D008745:Methylation; D008856:Microscopy, Fluorescence; D010766:Phosphorylation
chemical_list: C468783:H2AX protein, human; D006657:Histones
keywords: ChIP; DNA double-strand breaks; H2AX; euchromatin; heterochromatin; histone methylation; radiation