Liu Jian, Liu Qun, Wang Haijuan, Li Chunxiao, Wen Tao, An Guangyu, Qian Haili
Medical Research Center, Beijing Chao-Yang Hospital, Capital Medical University Beijing 100020 China.
State Key Laboratory of Molecular Oncology, National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College Beijing 100021 China
RSC Adv. 2018 Oct 15;8(61):35218-35225. doi: 10.1039/c8ra06907g. eCollection 2018 Oct 10.
Metastasis-associated antigen 1 (MTA1) is a chromatin modifier mediating DNA modification and gene expression. Ku70/Ku80 complex has been reported to be essential in DNA damage response. In an effort to explore the MTA1 interactome, we captured the Ku70/Ku80 complex with two specific MTA1 antibodies in a colon cancer cell line. We first validated the interaction between MTA1 and the Ku complex by co-immunoprecipitation (co-IP) analyses in cell lysate, showing that the interaction occurred mainly at the nucleus, but also existed in the cytoplasm at a lower level. We further visualized and confirmed their interaction using proximity ligation assay (PLA), which, in line with the analysis, also demonstrated a vast majority of interaction plots in the nucleus and a small number in the cytoplasm. We previously demonstrated that MTA1 distributed dynamically and periodically during the cell cycle. Here, through fluorescent colocalization, we found that MTA1 and Ku proteins colocalized well in the nucleus at interphase and moved synchronously from prophase to anaphase. Interestingly, at the time of telophase, when MTA1 was reported to re-enter the nucleus, they were separated and moved non-synchronously. Moreover, using PLA, we visualized that the interaction occurred at both interphase and mitosis. At interphase, they interacted mainly in the nucleus, but during mitosis, they interact at the periphery of chromosomes. We also showed that MTA1 correlated well with Ku in both the cancerous and normal tissues, and that they cooperated in UV-induced DNA damage response. Collectively, our data uncover a specific interaction between MTA1 and Ku complex at both the nucleus and cytoplasm, and across the whole cell cycle. We therefore propose a potential functional crosstalk between NuRD and Ku complexes, the two most fundamental function units in cells, physical interaction.
转移相关抗原1（MTA1）是一种介导DNA修饰和基因表达的染色质修饰因子。据报道，Ku70/Ku80复合物在DNA损伤反应中至关重要。为了探索MTA1相互作用组，我们在结肠癌细胞系中用两种特异性MTA1抗体捕获了Ku70/Ku80复合物。我们首先通过细胞裂解物中的免疫共沉淀（co-IP）分析验证了MTA1与Ku复合物之间的相互作用，结果表明这种相互作用主要发生在细胞核中，但在细胞质中也有较低水平的存在。我们进一步使用邻近连接分析（PLA）可视化并确认了它们的相互作用，与co-IP分析一致，该分析也表明绝大多数相互作用位点在细胞核中，少数在细胞质中。我们之前证明MTA1在细胞周期中动态且周期性地分布。在这里，通过荧光共定位，我们发现MTA1和Ku蛋白在间期细胞核中很好地共定位，并在前期到后期同步移动。有趣的是，在末期，当据报道MTA1重新进入细胞核时，它们分离并不同步移动。此外，使用PLA，我们可视化了这种相互作用在间期和有丝分裂期均发生。在间期，它们主要在细胞核中相互作用，但在有丝分裂期，它们在染色体周边相互作用。我们还表明，MTA1在癌组织和正常组织中均与Ku密切相关，并且它们在紫外线诱导的DNA损伤反应中协同作用。总体而言，我们的数据揭示了MTA1与Ku复合物在细胞核和细胞质中以及整个细胞周期中的特异性相互作用。因此，我们提出在细胞中两个最基本的功能单元NuRD和Ku复合物之间存在潜在的功能性相互作用。
