Dependence of radiation-induced H2AX phosphorylation on histone methylation: Evidence from the chromatin immunoprecipitation assay. (April 2015)

• Record Type: Journal Article
• Authors: Sak, Ali; Kübler, Dennis; Bannik, Kristina; Groneberg, Michael; Stuschke, Martin
• Is Part Of: International journal of radiation biology. Volume 91:Number 4(2015:Apr.)
• Page Start: 346
• Page End: 353
• Publication Date: 2015-04
• Subjects: H2AX -- ChIP -- radiation -- histone methylation -- heterochromatin -- euchromatin -- DNA double-strand breaks; Radiation -- Physiological effect -- Periodicals; Radiobiology -- Periodicals; 571.45
• DOI: 10.3109/09553002.2015.997895
• Languages: English
• ISSNs: 0955-3002

Abstract

Abstract: 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 (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. Conclusion : These data strengthen the dependence of IR-induced DNA damage response on the chromatin region.