Sak Ali, Kübler Dennis, Bannik Kristina, Groneberg Michael, Stuschke Martin
Department of Radiotherapy, University Hospital Essen , Essen , Germany.
Int J Radiat Biol. 2015 Apr;91(4):346-53. doi: 10.3109/09553002.2015.997895. Epub 2015 Jan 29.
To evaluate ionizing radiation (IR)-induced DNA damage response within euchromatic and heterochromatic regions.
Chromatin immunoprecipitation (ChIP) and immunofluorescence analysis were used to explore the distribution of phosphorylated H2AX (γH2AX).
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.
These data strengthen the dependence of IR-induced DNA damage response on the chromatin region.
评估电离辐射（IR）诱导的常染色质和异染色质区域内的DNA损伤反应。
采用染色质免疫沉淀（ChIP）和免疫荧光分析来探究磷酸化H2AX（γH2AX）的分布。
在30 Gy和60 Gy的IR照射后的ChIP实验显示，与富含赖氨酸9（H3K9me3）的染色质片段相比，在组蛋白H3三甲基化赖氨酸4（H3K4me3）富集的染色质片段中，IR照射后45分钟时γH2AX信号高1.28倍（1.08 - 1.53，95%置信区间）。将辐射剂量从60 Gy减半至30 Gy导致γH2AX信号降低0.49倍（0.37 - 0.64），与染色质区域无关。修复孵育240分钟导致两个区域的γH2AX信号均降低0.55倍（0.45 - 0.67）。通过免疫荧光显微镜测定，H3K9me3在整个染色质中的比例为30.5±3.8%。在0.5 Gy照射后45分钟和4小时时，H3K9me3区域内γH2AX焦点的比例分别为12.9±0.4%和13.9±0.6%，因此比各向同性分布预期的比例低约2.2倍。
这些数据强化了IR诱导的DNA损伤反应对染色质区域的依赖性。
