通过组蛋白变体 H2A.Z-2 的交换来重组受损的染色质。

Reorganization of damaged chromatin by the exchange of histone variant H2A.Z-2.

机构信息

Department of Cellular Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan; Department of Radiation Oncology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan; Department of Radiation Oncology, Hiroshima Prefectural Hospital, Hiroshima, Japan.

Department of Cellular Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan.

出版信息

Int J Radiat Oncol Biol Phys. 2014 Jul 15;89(4):736-44. doi: 10.1016/j.ijrobp.2014.03.031.

DOI:10.1016/j.ijrobp.2014.03.031

PMID:24969791

Abstract

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.

摘要

目的

受损染色质的重排在 DNA 损伤反应的调控中起着重要作用。最近的一项研究揭示了 2 种脊椎动物 H2A.Z 异构体 H2A.Z-1 和 H2A.Z-2 的存在。然而，脊椎动物 H2A.Z 异构体的作用仍不清楚。因此，在本研究中，我们研究了在诱导 DNA 双链断裂 (DSB) 后，脊椎动物 H2A.Z 异构体在染色质重排中的作用。

方法和材料

为了研究损伤部位 H2A.Z 异构体的动态变化，我们构建了稳定表达 GFP 标记的 H2A.Z 异构体的 GM0637 细胞，并进行了光漂白后荧光恢复 (FRAP) 分析和与微照射相结合的反转 FRAP 分析。使用抗 RAD51 抗体进行免疫荧光染色，研究野生型 (WT)、H2A.Z-1 和 H2A.Z-2 缺陷型 DT40 细胞在 2Gy 照射后 RAD51 焦点形成的动力学。还进行了集落形成测定，以比较 WT、H2A.Z-1 和 H2A.Z-2 缺陷型 DT40 细胞与对照以及 H2A.Z-1 和 H2A.Z-2 耗竭的 U2OS 细胞在照射后的存活率。

结果

FRAP 分析表明，H2A.Z-2 在 DSB 诱导后立即被掺入受损的染色质中，而 H2A.Z-1 则基本不变。反转 FRAP 分析表明，H2A.Z-2 从受损的染色质中释放出来。这些发现表明，H2A.Z-2 在 DSB 诱导后立即在 DSB 位点交换。离子照射后 RAD51 焦点的形成在 H2A.Z-2 缺陷型 DT40 细胞中受到干扰，但在 H2A.Z-1 缺陷型细胞中不受干扰。照射后 H2A.Z-2 缺陷型细胞的存活率低于 WT 和 H2A.Z-1-DT40 细胞。与 DT40 细胞类似，与对照和 H2A.Z-1 耗竭细胞相比，H2A.Z-2 耗竭的 U2OS 细胞也对辐射敏感。