低温对斑马鱼ZF4细胞基因组DNA甲基化水平的影响
|
摘要
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.
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.
引文
[1]Singer-Sam J,Riggs A D.DNA Methylation:Molecular Biology and Biological Significance[M].New York:SpringerVerlag,1993:358-384.
[2]Attwood J T,Yung R L,Richardson B C.DNA methylation and the regulation of gene transcription[J].Cell and Molecular Life Science,2002,59(2):241-257.
[3]Lister R,Pelizzola M,Dowen R H,et al.Human DNA methylomes at base resolution show widespread epigenomic differences[J].Nature,2009,462(7271):315-322.
[4]Secco D,Wang C,Shou H X,et al.Stress induced gene expression drives transient DNA methylation changes at adjacent repetitive elements[J].eLife,2015,4:e09343.
[5]Kisliouk T,Cramer T,Meiri N.Methyl CpG level at distal part of heat-shock protein promoter HSP70 exhibits epigenetic memory for heat stress by modulating recruitment of POU2F1-associated nucleosome-remodeling deacetylase(NuRD)complex[J].Journal of Neurochemistry 2017,141(3):358-372.
[6]Wang M,Qin L M,Xie C,et al.Induced and constitutive DNA methylation in a salinity-tolerant wheat introgression line[J].Plant and Cell Physiology,2014,55(7):1354-1365.
[7]Pecinka A,Dinh H Q,Baubec T,et al.Epigenetic regulation of repetitive elements is attenuated by prolonged heat stress in Arabidopsis[J].Plant Cell,2010,22(9):3118-3129.
[8]Dai T M,LüZ C,Wang Y S,et al.Molecular characterizations of DNA methyltransferase 3 and its roles in temperature tolerance in the whitefly,Bemisia tabaci Mediterranean[J].Insect Molecular Biology,2018,27(1):123-132.
[9]Hernández J A,Ferrer M A,Jiménez A,et al.Antioxidant systems and O2-?/H2O2 production in the apoplast of pea leaves.Its relation with salt-induced necrotic lesions in minor veins[J].Plant Physiology,2001,127(3):817-831.
[10]Xu Q Q,Han B S,Luo J T,et al.Effects of cold stress on ROS production and expression of MAPK proteins in zebrafish ZF4 cells[J].Journal of Fishery Sciences of China,2016,23(4):771-776.[许琼琼,韩兵社,罗军涛,等.低温胁迫诱导斑马鱼ZF4细胞ROS及MAPK相关蛋白表达的影响[J].中国水产科学,2016,23(4):771-776].
[11]Liu H,Ouyang B,Zhang J H,et al.Differential modulation of photosynthesis,signaling,and transcriptional regulation between tolerant and sensitive tomato genotypes under cold stress[J].PLoS ONE,2012,7(11):e50785.
[12]Mortusewicz O,Schermelleh L,Walter J,et al.Recruitment of DNA methyltransferase I to DNA repair sites[J].Proceedings of the National Academy of Sciences of the United States of America,2005,102(25):8905-8909.
[13]Ding N,Bonham E M,Hannon B E,et al.Mismatch repair proteins recruit DNA methyltransferase 1 to sites of oxidative DNA damage[J].Journal of Molecular Cell Biology,2016,8(3):244-254.
[14]Berger N D,Stanley F K T,Moore S,et al.ATM-dependent pathways of chromatin remodelling and oxidative DNAdamage responses[J].Philosophical Transactions of the Royal Society of London Series B-Biological Sciences,2017,372(1731):20160283.
[15]Guo Z,Kozlov S,Lavin M F,et al.ATM activation by oxidative stress[J].Science,2010,330(6003):517-521.
[16]Howe K,Clark M D,Torroja C F,et al.The zebrafish reference genome sequence and its relationship to the human genome[J].Nature,2013,496(7446):498-503.
[17]Hu P,Liu M L,Zhang D,et al.Global identification of the genetic networks and cis-regulatory elements of the cold response in zebrafish[J].Nucleic Acids Research,2015,43(19):9198-9213.
[18]Han B S,Li W H,Chen Z Z,et al.Variation of DNA methylome of zebrafish cells under cold pressure[J].PLoS ONE,2016,11(8):e0160358
[19]Chwastek J,Jantas D,LasońW.The ATM kinase inhibitor KU-55933 provides neuroprotection against hydrogen peroxide-induced cell damage via aγH2AX/p-p53/caspase-3-independent mechanism:Inhibition of calpain and cathepsin D[J].The International Journal of Biochemistry&Cell Biology,2017,87:38-53.
[20]Ugarte F,Sousae R,Cinquin B,et al.Progressive chromatin condensation and H3K9 methylation regulate the differentiation of embryonic and hematopoietic stem cells[J].Stem Cell Reports,2015,5(5):728-740.
[21]Wang G F,Kang N,Gong H M,et al.Upregulation of uncoupling protein Ucp2 through acute cold exposure increases post-thaw sperm quality in zebrafish[J].Cryobiology,2015,71(3):464-471.
[22]Wu S M,Liu J H,Shu L H,et al.Anti-oxidative responses of zebrafish(Danio rerio)gill,liver and brain tissues upon acute cold shock[J].Comparative Biochemistry and Physiology Part A:Molecular&Integrative Physiology,2015,187:202-213.
[23]Chen S E,Yu M C,Chu X,et al.Cold-induced retrotransposition of fish LINEs[J].Journal of Genetics and Genomics,2017,44(8):385-394.
[24]Shamma A,Suzuki M,Hayashi N,et al.ATM mediates pRBfunction to control DNMT1 protein stability and DNA methylation[J].Molecular and Cellular Biology,2013,33(16):3113-3124.
[25]Russo G,Landi R,Pezone A,et al.DNA damage and Repair Modify DNA methylation and Chromatin Domain of the Targeted Locus:Mechanism of allele methylation polymorphism[J].Scientific Reports,2016,6:33222.
[2]Attwood J T,Yung R L,Richardson B C.DNA methylation and the regulation of gene transcription[J].Cell and Molecular Life Science,2002,59(2):241-257.
[3]Lister R,Pelizzola M,Dowen R H,et al.Human DNA methylomes at base resolution show widespread epigenomic differences[J].Nature,2009,462(7271):315-322.
[4]Secco D,Wang C,Shou H X,et al.Stress induced gene expression drives transient DNA methylation changes at adjacent repetitive elements[J].eLife,2015,4:e09343.
[5]Kisliouk T,Cramer T,Meiri N.Methyl CpG level at distal part of heat-shock protein promoter HSP70 exhibits epigenetic memory for heat stress by modulating recruitment of POU2F1-associated nucleosome-remodeling deacetylase(NuRD)complex[J].Journal of Neurochemistry 2017,141(3):358-372.
[6]Wang M,Qin L M,Xie C,et al.Induced and constitutive DNA methylation in a salinity-tolerant wheat introgression line[J].Plant and Cell Physiology,2014,55(7):1354-1365.
[7]Pecinka A,Dinh H Q,Baubec T,et al.Epigenetic regulation of repetitive elements is attenuated by prolonged heat stress in Arabidopsis[J].Plant Cell,2010,22(9):3118-3129.
[8]Dai T M,LüZ C,Wang Y S,et al.Molecular characterizations of DNA methyltransferase 3 and its roles in temperature tolerance in the whitefly,Bemisia tabaci Mediterranean[J].Insect Molecular Biology,2018,27(1):123-132.
[9]Hernández J A,Ferrer M A,Jiménez A,et al.Antioxidant systems and O2-?/H2O2 production in the apoplast of pea leaves.Its relation with salt-induced necrotic lesions in minor veins[J].Plant Physiology,2001,127(3):817-831.
[10]Xu Q Q,Han B S,Luo J T,et al.Effects of cold stress on ROS production and expression of MAPK proteins in zebrafish ZF4 cells[J].Journal of Fishery Sciences of China,2016,23(4):771-776.[许琼琼,韩兵社,罗军涛,等.低温胁迫诱导斑马鱼ZF4细胞ROS及MAPK相关蛋白表达的影响[J].中国水产科学,2016,23(4):771-776].
[11]Liu H,Ouyang B,Zhang J H,et al.Differential modulation of photosynthesis,signaling,and transcriptional regulation between tolerant and sensitive tomato genotypes under cold stress[J].PLoS ONE,2012,7(11):e50785.
[12]Mortusewicz O,Schermelleh L,Walter J,et al.Recruitment of DNA methyltransferase I to DNA repair sites[J].Proceedings of the National Academy of Sciences of the United States of America,2005,102(25):8905-8909.
[13]Ding N,Bonham E M,Hannon B E,et al.Mismatch repair proteins recruit DNA methyltransferase 1 to sites of oxidative DNA damage[J].Journal of Molecular Cell Biology,2016,8(3):244-254.
[14]Berger N D,Stanley F K T,Moore S,et al.ATM-dependent pathways of chromatin remodelling and oxidative DNAdamage responses[J].Philosophical Transactions of the Royal Society of London Series B-Biological Sciences,2017,372(1731):20160283.
[15]Guo Z,Kozlov S,Lavin M F,et al.ATM activation by oxidative stress[J].Science,2010,330(6003):517-521.
[16]Howe K,Clark M D,Torroja C F,et al.The zebrafish reference genome sequence and its relationship to the human genome[J].Nature,2013,496(7446):498-503.
[17]Hu P,Liu M L,Zhang D,et al.Global identification of the genetic networks and cis-regulatory elements of the cold response in zebrafish[J].Nucleic Acids Research,2015,43(19):9198-9213.
[18]Han B S,Li W H,Chen Z Z,et al.Variation of DNA methylome of zebrafish cells under cold pressure[J].PLoS ONE,2016,11(8):e0160358
[19]Chwastek J,Jantas D,LasońW.The ATM kinase inhibitor KU-55933 provides neuroprotection against hydrogen peroxide-induced cell damage via aγH2AX/p-p53/caspase-3-independent mechanism:Inhibition of calpain and cathepsin D[J].The International Journal of Biochemistry&Cell Biology,2017,87:38-53.
[20]Ugarte F,Sousae R,Cinquin B,et al.Progressive chromatin condensation and H3K9 methylation regulate the differentiation of embryonic and hematopoietic stem cells[J].Stem Cell Reports,2015,5(5):728-740.
[21]Wang G F,Kang N,Gong H M,et al.Upregulation of uncoupling protein Ucp2 through acute cold exposure increases post-thaw sperm quality in zebrafish[J].Cryobiology,2015,71(3):464-471.
[22]Wu S M,Liu J H,Shu L H,et al.Anti-oxidative responses of zebrafish(Danio rerio)gill,liver and brain tissues upon acute cold shock[J].Comparative Biochemistry and Physiology Part A:Molecular&Integrative Physiology,2015,187:202-213.
[23]Chen S E,Yu M C,Chu X,et al.Cold-induced retrotransposition of fish LINEs[J].Journal of Genetics and Genomics,2017,44(8):385-394.
[24]Shamma A,Suzuki M,Hayashi N,et al.ATM mediates pRBfunction to control DNMT1 protein stability and DNA methylation[J].Molecular and Cellular Biology,2013,33(16):3113-3124.
[25]Russo G,Landi R,Pezone A,et al.DNA damage and Repair Modify DNA methylation and Chromatin Domain of the Targeted Locus:Mechanism of allele methylation polymorphism[J].Scientific Reports,2016,6:33222.