Dependence of radiation-induced H2AX phosphorylation on histone methylation: Evidence from the chromatin immunoprecipitation assay

Purpose: To evaluate ionizing radiation (IR)-induced DNA damage response within euchromatic and heterochromatic regions. Material and methods: Chromatin immunoprecipitation (ChIP) and immunofluorescence analysis were used to explore the distribution of phosphorylated H2AX (gamma H2AX). 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 gamma H2AX 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 gamma H2AX 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 gamma H2AX 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 gamma H2AX 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. Conclusion: These data strengthen the dependence of IR-induced DNA damage response on the chromatin region.