Manova Vasilissa, Singh Satyendra K, Iliakis George
Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, Hufelandstr, 55, 45122, Essen, Germany.
Genome Integr. 2012 Aug 22;3(1):4. doi: 10.1186/2041-9414-3-4.
Mammalian cells employ at least two subpathways of non-homologous end-joining for the repair of ionizing radiation induced DNA double strand breaks: The canonical DNA-PK-dependent form of non-homologous end-joining (D-NHEJ) and an alternative, slowly operating, error-prone backup pathway (B-NHEJ). In contrast to D-NHEJ, which operates with similar efficiency throughout the cell cycle, B-NHEJ operates more efficiently in G2-phase. Notably, B-NHEJ also shows strong and as of yet unexplained dependency on growth activity and is markedly compromised in serum-deprived cells, or in cells that enter the plateau-phase of growth. The molecular mechanisms underpinning this response remain unknown. Since chromatin structure or changes in chromatin structure are prime candidate-B-NHEJ-modulators, we study here the role of chromatin hyperacetylation, either by HDAC2 knockdown or treatment with the HDAC inhibitor TSA, on the repair by B-NHEJ of IR-induced DSBs.
siRNA-mediated knockdown of HDAC2 fails to provoke histone hyperacetylation in Lig4-/- MEFs and has no detectable effect on B-NHEJ function. Treatment with TSA that inhibits multiple HDACs causes efficient, reversible chromatin hyperacetylation in Lig4-/- MEFs, as well as in human HCT116 Lig4-/- cells and the human glioma cell line M059K. The IR yield of DSBs in TSA-treated cells remains similar to that of untreated cells despite the expected chromatin relaxation. In addition, chromatin hyperacetylation leaves unchanged repair of DSBs by B-NHEJ in irradiated exponentially growing, or plateau-phase cells. Notably, under the experimental conditions employed here, chromatin hyperacetylation fails to detectably modulate B-NHEJ in M059K cells as well.
In summary, the results show that chromatin acetylation or deacetylation does not affect the kinetics of alternative NHEJ in all types of cells examined both in exponentially growing and serum deprived cultures. We conclude that parameters beyond chromatin acetylation determine B-NHEJ efficiency in the plateau-phase of growth.
哺乳动物细胞利用至少两种非同源末端连接的亚途径来修复电离辐射诱导的DNA双链断裂：经典的依赖DNA-PK的非同源末端连接形式（D-NHEJ）和一种替代的、运作缓慢且易出错的备用途径（B-NHEJ）。与在整个细胞周期中以相似效率运作的D-NHEJ不同，B-NHEJ在G2期运作更高效。值得注意的是，B-NHEJ还表现出对生长活性的强烈且尚未解释清楚的依赖性，并在血清饥饿的细胞或进入生长平台期的细胞中明显受损。支撑这种反应的分子机制仍然未知。由于染色质结构或染色质结构的变化是B-NHEJ的主要潜在调节因子，我们在此研究通过HDAC2敲低或用HDAC抑制剂TSA处理导致的染色质高度乙酰化对IR诱导的DSB的B-NHEJ修复的作用。
siRNA介导的HDAC2敲低未能在Lig4-/- MEF中引发组蛋白高度乙酰化，并且对B-NHEJ功能没有可检测到的影响。用抑制多种HDAC的TSA处理在Lig4-/- MEF以及人HCT116 Lig4-/-细胞和人胶质瘤细胞系M059K中导致高效、可逆的染色质高度乙酰化。尽管预期染色质会松弛，但TSA处理的细胞中DSB的IR产量与未处理的细胞相似。此外，染色质高度乙酰化在受辐射的指数生长期或平台期细胞中对B-NHEJ的DSB修复没有影响。值得注意的是，在此处采用的实验条件下，染色质高度乙酰化在M059K细胞中也未能检测到对B-NHEJ的调节作用。
总之，结果表明染色质乙酰化或去乙酰化在指数生长期和血清饥饿培养的所有类型细胞中均不影响替代NHEJ的动力学。我们得出结论，超出染色质乙酰化的参数决定了生长平台期的B-NHEJ效率。
