低温对斑马鱼ZF4细胞基因组DNA甲基化水平的影响

Effect of cold stress on genomic DNA methylation in zebrafish ZF4 cells

低温压力会导致鱼类生理功能失调、机体损伤甚至死亡,鱼类会产生各种适应性变化来应对低温压力,其中涉及的表观遗传学机制越来越受到重视。为了探讨鱼类低温应激压力下的表观遗传学调控机制,本研究对斑马鱼(Daniorerio)胚胎成纤维细胞ZF4进行短期低温胁迫(18℃处理3d、5d和10℃处理3d、5d)和长期低温胁迫(18℃处理30 d),然后用具有不同甲基化敏感性的同裂酶HpaⅡ和MspⅠ对细胞基因组DNA进行酶切以监测细胞基因组DNA甲基化水平变化。结果显示短期低温胁迫下ZF4细胞生长受到抑制甚至死亡,而经过长期低温胁迫的ZF4细胞对低温压力产生了一定适应性。并且低温胁迫下DNA甲基化水平呈现动态变化,短期低温培养细胞的基因组DNA甲基化水平明显增高,但是长期低温培养细胞的DNA甲基化水平反而下降。此外,研究发现抗氧化剂N-乙酰半胱氨酸(N-acetylcysteine,NAC)或者共济失调–毛细血管扩张突变蛋白(ataxia telangiectasia mutated,ATM)抑制剂KU-55933可以抑制18℃5 d低温处理后的ZF4细胞DNA甲基化水平的增高,说明活性氧(reactive oxygen species,ROS)的产生和ATM的激活介导了DNA甲基化水平的增高。本研究结果显示,短期低温刺激下ZF4细胞ROS的产生导致DNA损伤,激活DNA损伤修复机制,进而导致基因组DNA甲基化水平上升,该研究为后期斑马鱼低温胁迫分子机制的研究奠定基础。

Cold stress causes physiological dysfunction, tissue damage, and finally death in fishes, and increasing studies have suggested that epigenetic mechanisms play essential roles in the cold stress response in fishes. Our previous study showed that cold stress induced the production of reactive oxygen species(ROS) in zebrafish Danio rerio-derived ZF4 cells in a temperature and time-dependent manner and that the genomic DNA methylation level was increased under short-term(18℃ for 5 days) cold exposure and decreased under long-term cold exposure(18℃ for 30 days). However, the relationship among DNA methylation, ROS production, and cold acclimation in fishes remains poorly understood. In the present study, zebrafish ZF4 cells were exposed to short-term(18℃ or10℃, for 3 or 5 days) and long-term(18℃ for 30 days) cold stress, and then the global DNA methylation level was detected by digestion with the methylation-sensitive enzyme Hpa II and its methylation-insensitive isoschizomer Msp I. The results showed that short-term cold stress caused remarkable growth arrest and cell death in ZF4 cells, and cold acclimation was observed under long-term cold stress. Additionally, global DNA methylation increased remarkably under short-term cold stress(P < 0.05) but decreased under long-term cold stress(P < 0.05). Co-treatment of ZF4 cells with N-acetylcysteine inhibited global DNA methylation induced by short-term cold stress(P < 0.05),suggesting that ROS affects short-term cold stress-induced global DNA methylation levels. Co-treatment of ZF4 cells with the ataxia telangiectasia mutated(ATM) inhibitor KU-55933 also inhibited the induction of global DNA methylation under short-term cold exposure, indicating the involvement of DNA damage repair pathways in this process. Our data indicate that short-term cold stress resulted in ROS production and ataxia telangiectasia mutated activation, which then up-regulated global DNA methylation in ZF4 cells. The present study improves our understanding of the role of DNA methylation under cold stress in fishes.