Reorganization of Damaged Chromatin by the Exchange of Histone Variant H2A.Z-2
Reorganization of Damaged Chromatin by the Exchange of Histone Variant H2A.Z-2
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DOI：
10.1016/j.ijrobp.2014.03.031
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发表时间：
2014-07-15
影响因子：
7
通讯作者：
Tashiro, Satoshi
中科院分区：
文献类型：
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作者：
Nishibuchi, Ikuno;Suzuki, Hidekazu;Tashiro, Satoshi
目的：受损染色质的重组在DNA损伤反应的调节中发挥着重要作用。最近的一项研究揭示了 2 种脊椎动物 H2A.Z 亚型的存在：H2A.Z-1 和 H2A.Z-2。然而，脊椎动物 H2A.Z 亚型的作用仍不清楚。因此，在这项研究中，我们检查了脊椎动物 H2A.Z 异构体在诱导 DNA 双链断裂 (DSB) 后染色质重组中的作用。方法和材料：为了检查受损位点 H2A.Z 异构体的动态，我们构建了稳定表达每种绿色荧光蛋白 (GFP) 标记的 H2A.Z 异构体的 GM0637 细胞，并在光漂白 (FRAP) 后进行荧光恢复。分析和倒置 FRAP 分析与微辐照相结合。使用抗 RAD51 抗体进行免疫荧光染色，以研究野生型 (WT)、H2A.Z-1 和 H2A.Z-2 缺陷型 DT40 细胞 2 Gy 照射后 RAD51 灶形成的动力学。还进行集落形成测定，以比较辐射后 WT、H2A.Z-1- 和 H2A.Z-2- 缺陷 DT40 细胞与对照以及 H2A.Z-1- 和 H2A.Z-2 耗尽的 U2OS 细胞的存活率。 结果：FRAP 分析显示，在 DSB 诱导后，H2A.Z-2 就掺入到受损的染色质中，而 H2A.Z-1 基本上保留不变。反向 FRAP 分析显示 H2A.Z-2 从受损的染色质中释放出来。这些发现表明，在 DSB 诱导后，H2A.Z-2 立即在 DSB 位点发生交换。在 H2A.Z-2 缺陷型 DT40 细胞中，电离辐射后 RAD51 焦点的形成受到干扰，但在 H2A.Z-1 缺陷型细胞中则没有。 H2A.Z-2缺陷细胞照射后的存活率低于WT和H2A.Z-1-DT40细胞。与 DT40 细胞类似，与对照和 H2A.Z-1 耗尽的细胞相比，H2A.Z-2 耗尽的 U2OS 细胞也对辐射敏感。结论：我们发现脊椎动物 H2A.Z-2 通过 RAD51 病灶形成所需的受损染色质重组，参与极早期 DNA 损伤反应的调节。 (C) 2014 年爱思唯尔公司。
Purpose: The reorganization of damaged chromatin plays an important role in the regulation of the DNA damage response. A recent study revealed the presence of 2 vertebrate H2A.Z isoforms, H2A.Z-1 and H2A.Z-2. However, the roles of the vertebrate H2A.Z isoforms are still unclear. Thus, in this study we examined the roles of the vertebrate H2A.Z isoforms in chromatin reorganization after the induction of DNA double-strand breaks (DSBs).Methods and Materials: To examine the dynamics of H2A.Z isoforms at damaged sites, we constructed GM0637 cells stably expressing each of the green fluorescent protein (GFP)-labeled H2A.Z isoforms, and performed fluorescence recovery after photobleaching (FRAP) analysis and inverted FRAP analysis in combination with microirradiation. Immunofluorescence staining using an anti-RAD51 antibody was performed to study the kinetics of RAD51 foci formation after 2-Gy irradiation of wild-type (WT), H2A.Z-1- and H2A.Z-2-deficient DT40 cells. Colony-forming assays were also performed to compare the survival rates of WT, H2A.Z-1-, and H2A.Z-2- deficient DT40 cells with control, and H2A.Z-1- and H2A.Z-2-depleted U2OS cells after irradiation.Results: FRAP analysis revealed that H2A.Z-2 was incorporated into damaged chromatin just after the induction of DSBs, whereas H2A.Z-1 remained essentially unchanged. Inverted FRAP analysis showed that H2A.Z-2 was released from damaged chromatin. These findings indicated that H2A.Z-2 was exchanged at DSB sites immediately after the induction of DSBs. RAD51 focus formation after ionizing irradiation was disturbed in H2A.Z-2-deficient DT40 cells but not in H2A.Z-1-deficient cells. The survival rate of H2A.Z-2-deficient cells after irradiation was lower than those of WT and H2A.Z-1-DT40 cells. Similar to DT40 cells, H2A.Z-2-depleted U2OS cells were also radiation-sensitive compared to control and H2A.Z-1-depleted cells.Conclusions: We found that vertebrate H2A.Z-2 is involved in the regulation of the DNA damage response at a very early stage, via the damaged chromatin reorganization required for RAD51 focus formation. (C) 2014 Elsevier Inc.