低温压力对ZF4细胞组蛋白修饰的影响
|
摘要
为探索低温压力对斑马鱼细胞组蛋白修饰的影响,建立并优化使用斑马鱼(Danio rerio)成纤维细胞ZF4进行染色质免疫共沉淀(chromatin immunoprecipitation, ChIP)的条件。本研究分别优化了ChIP过程中裂解细胞所使用的NP-40浓度、超声破碎的时间,确定了最佳NP-40浓度为0.2%,超声时间为20 min。利用针对β-actin启动子区域的引物,通过常规PCR初步验证ChIP实验是否成功。琼脂糖凝胶电泳结果显示, IgG组常规PCR产物基本无条带,H3K27me3组条带较暗, H3K4me3和H3K27ac组有较亮条带,初步说明实验可靠。使用正常培养(28℃)与长期低温驯化(18℃, 30 d)的斑马鱼成纤维细胞ZF4作为实验材料,通过优化之后的ChIP技术进行实验。分别使用qPCR和ChIP-qPCR检测低温驯化对肿瘤坏死因子b(tumor necrosis factor b,tnfb)基因表达以及其启动子区域H3K4me3、H3K27ac、H3K27me3的影响。qPCR结果显示tnfb基因经过低温驯化后上调,而ChIP-qPCR结果显示,tnfb基因启动子区域的H3K4me3、H3K27ac富集度也出现升高, H3K27me3没有明显变化,提示低温压力可能通过影响tnfb基因启动子区域的H3K4me3、H3K27ac水平来调控tnfb基因的表达。本研究建立并优化的ChIP实验条件可以用来研究ZF4细胞组蛋白修饰变化,为下一步探索低温压力对斑马鱼细胞全基因组组蛋白修饰的影响奠定基础。
Environmental stresses can regulate gene expression patterns by modulating histone activity. However,the effect of low-temperature stress on whole-genome histone modification in fish has not been reported. To explore the effect of low-temperature stress on histone modification in zebrafish(Danio rerio) cells, the experimental conditions for chromatin immunoprecipitation(ChIP) were established and optimized. The optimum NP-40 concentration for lysing cells was 0.2%. The optimal time of sonication with a Covaris S220 to break the chromatin was 20 min. Primers targeting the β-actin promoter region were used to perform routine PCR to verify the preliminary ChIP experiment results. Optimized experimental conditions resulted in a very low background, with nearly no IgG band in the agarose gel. Moreover, the lack of H3 K27 me3 in the β-actin promoter region, accompanied by enriched H3 K4 me3 and H3 K27 ac, was observed as predicted. Zebrafish embryonic ZF4 fibroblasts cultured under normal condition(28℃) and cold-acclimated ZF4 cells under long-term cold exposure(18℃ for30 days) were evaluated with the optimized ChIP protocol. The effect of cold acclimation on the expression of tumor necrosis factor β(tnfb) and enrichment of H3 K4 me3, H3 K27 ac, and H3 K27 me3 at the tnfb promoter was detected by qPCR and ChIP-qPCR, respectively. The qPCR and ChIP-qPCR results showed that tnfb was upregulated after cold acclimation and that H3 K4 me3 and H3 K27 ac were enriched in the promoter region of tnfb, while enrichment of H3 K27 me3 showed no significant change. This suggests that cold pressure regulates tnfb expression by affecting H3 K4 me3 and H3 K27 ac levels in the promoter region of tnfb. In conclusion, the established and optimized ChIP method can be used to study histone modification in ZF4 cells and provides a foundation for further analysis of the effect of low-temperature stress on whole-genome histone modification in zebrafish cells.
Environmental stresses can regulate gene expression patterns by modulating histone activity. However,the effect of low-temperature stress on whole-genome histone modification in fish has not been reported. To explore the effect of low-temperature stress on histone modification in zebrafish(Danio rerio) cells, the experimental conditions for chromatin immunoprecipitation(ChIP) were established and optimized. The optimum NP-40 concentration for lysing cells was 0.2%. The optimal time of sonication with a Covaris S220 to break the chromatin was 20 min. Primers targeting the β-actin promoter region were used to perform routine PCR to verify the preliminary ChIP experiment results. Optimized experimental conditions resulted in a very low background, with nearly no IgG band in the agarose gel. Moreover, the lack of H3 K27 me3 in the β-actin promoter region, accompanied by enriched H3 K4 me3 and H3 K27 ac, was observed as predicted. Zebrafish embryonic ZF4 fibroblasts cultured under normal condition(28℃) and cold-acclimated ZF4 cells under long-term cold exposure(18℃ for30 days) were evaluated with the optimized ChIP protocol. The effect of cold acclimation on the expression of tumor necrosis factor β(tnfb) and enrichment of H3 K4 me3, H3 K27 ac, and H3 K27 me3 at the tnfb promoter was detected by qPCR and ChIP-qPCR, respectively. The qPCR and ChIP-qPCR results showed that tnfb was upregulated after cold acclimation and that H3 K4 me3 and H3 K27 ac were enriched in the promoter region of tnfb, while enrichment of H3 K27 me3 showed no significant change. This suggests that cold pressure regulates tnfb expression by affecting H3 K4 me3 and H3 K27 ac levels in the promoter region of tnfb. In conclusion, the established and optimized ChIP method can be used to study histone modification in ZF4 cells and provides a foundation for further analysis of the effect of low-temperature stress on whole-genome histone modification in zebrafish cells.
引文
[1]Donaldson M R,Cooke S J,Patterson D A,et al.Cold shock and fish[J].Journal of Fish Biology,2008,73(7):1491-1530.
[2]Beitinger T L,Bennett W A,Mccauley R W.Temperature tolerances of North American freshwater fishes exposed to dynamic changes in temperature[J].Environmental Biology of Fishes,2000,58(3):237-275.
[3]López-Olmeda J F,Sánchez-Vázquez F J.Thermal biology of zebrafish(Danio Rerio)[J].Journal of Thermal Biology,2011,36(2):91-104.
[4]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.
[5]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.
[6]Long Y,Song G L,Yan J J,et al.Transcriptomic characterization of cold acclimation in larval zebrafish[J].BMC Genomics,2013,14:612.
[7]Long Y,Li L C,Li Q,et al.Transcriptomic characterization of temperature stress responses in larval zebrafish[J].PLoSONE,2012,7(5):e37209.
[8]Yang R L,Dai Z H,Chen S E,et al.MicroRNA-mediated gene regulation plays a minor role in the transcriptomic plasticity of cold-acclimated zebrafish brain tissue[J].BMC Genomics,2011,12:605.
[9]Coustham V,Li P J,Strange A,et al.Quantitative modulation of polycomb silencing underlies natural variation in vernalization[J].Science,2012,337(6094):584-587.
[10]Sheldon C C,Finnegan E J,Peacock W J,et al.Mechanisms of gene repression by vernalization in Arabidopsis[J].The Plant Journal,2009,59(3):488-498.
[11]Diallo A O,Ali-Benali M A,Badawi M,et al.Expression of vernalization responsive genes in wheat is associated with histone H3 trimethylation[J].Molecular Genetics and Genomics,2012,287(7):575-590.
[12]Gibert J M,Mouchel-Vielh E,De Castro S,et al.Phenotypic plasticity through transcriptional regulation of the evolutionary hotspot gene tan in Drosophila melanogaster[J].PLoSGenetics,2016,12(8):e1006218.
[13]Matsumoto Y,Hannigan B,Crews D.Temperature shift alters DNA methylation and histone modification patterns in gonadal aromatase(cyp19a1)gene in species with temperature-dependent sex determination[J].PLo S ONE,2016,11(11):e0167362.
[14]Pinto R,Ivaldi C,Reyes M,et al.Seasonal environmental changes regulate the expression of the histone variant macro H2A in an eurythermal fish[J].FEBS Letters,2005,579(25):5553-5558.
[15]Solomon M J,Varshavsky A.Formaldehyde-mediated DNA-protein crosslinking:a probe for in vivo chromatin structures[J].Proceedings of the National Academy of Sciences of the United States of America,1985,82(19):6470-6474.
[16]Bernstein B E,Meissner A,Lander E S.The mammalian epigenome[J].Cell,2007,128(4):669-681.
[17]Blecher-Gonen R,Barnett-Itzhaki Z,Jaitin D,et al.Highthroughput chromatin immunoprecipitation for genomewide mapping of in vivo protein-DNA interactions and epigenomic states[J].Nature Protocols,2013,8(3):539-554.
[18]Wajant H,Pfizenmaier K,Scheurich P.Tumor necrosis factor signaling[J].Cell Death and Differentiation,2003,10(1):45-65.
[19]Qi F,Song J B,Yang H S,et al.Mmp23b promotes liver development and hepatocyte proliferation through the tumor necrosis factor pathway in zebrafish[J].Hepatology,2010,52(6):2158-2166.
[20]Hayden M S,Ghosh S.Regulation of NF-κB by TNF family cytokines[J].Seminars in Immunology,2014,26(3):253-266.
[21]Wang W L,Liu W T,Gong H Y,et al.Activation of cytokine expression occurs through the TNFα/NF-κB-mediated pathway in birnavirus-infected cells[J].Fish&Shellfish Immunology,2011,31(1):10-21.
[22]Musselman C A,Lalonde M E,C?téJ,et al.Perceiving the epigenetic landscape through histone readers[J].Nature Structural Molecular Biology,2012,19(12):1218-1227.
[2]Beitinger T L,Bennett W A,Mccauley R W.Temperature tolerances of North American freshwater fishes exposed to dynamic changes in temperature[J].Environmental Biology of Fishes,2000,58(3):237-275.
[3]López-Olmeda J F,Sánchez-Vázquez F J.Thermal biology of zebrafish(Danio Rerio)[J].Journal of Thermal Biology,2011,36(2):91-104.
[4]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.
[5]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.
[6]Long Y,Song G L,Yan J J,et al.Transcriptomic characterization of cold acclimation in larval zebrafish[J].BMC Genomics,2013,14:612.
[7]Long Y,Li L C,Li Q,et al.Transcriptomic characterization of temperature stress responses in larval zebrafish[J].PLoSONE,2012,7(5):e37209.
[8]Yang R L,Dai Z H,Chen S E,et al.MicroRNA-mediated gene regulation plays a minor role in the transcriptomic plasticity of cold-acclimated zebrafish brain tissue[J].BMC Genomics,2011,12:605.
[9]Coustham V,Li P J,Strange A,et al.Quantitative modulation of polycomb silencing underlies natural variation in vernalization[J].Science,2012,337(6094):584-587.
[10]Sheldon C C,Finnegan E J,Peacock W J,et al.Mechanisms of gene repression by vernalization in Arabidopsis[J].The Plant Journal,2009,59(3):488-498.
[11]Diallo A O,Ali-Benali M A,Badawi M,et al.Expression of vernalization responsive genes in wheat is associated with histone H3 trimethylation[J].Molecular Genetics and Genomics,2012,287(7):575-590.
[12]Gibert J M,Mouchel-Vielh E,De Castro S,et al.Phenotypic plasticity through transcriptional regulation of the evolutionary hotspot gene tan in Drosophila melanogaster[J].PLoSGenetics,2016,12(8):e1006218.
[13]Matsumoto Y,Hannigan B,Crews D.Temperature shift alters DNA methylation and histone modification patterns in gonadal aromatase(cyp19a1)gene in species with temperature-dependent sex determination[J].PLo S ONE,2016,11(11):e0167362.
[14]Pinto R,Ivaldi C,Reyes M,et al.Seasonal environmental changes regulate the expression of the histone variant macro H2A in an eurythermal fish[J].FEBS Letters,2005,579(25):5553-5558.
[15]Solomon M J,Varshavsky A.Formaldehyde-mediated DNA-protein crosslinking:a probe for in vivo chromatin structures[J].Proceedings of the National Academy of Sciences of the United States of America,1985,82(19):6470-6474.
[16]Bernstein B E,Meissner A,Lander E S.The mammalian epigenome[J].Cell,2007,128(4):669-681.
[17]Blecher-Gonen R,Barnett-Itzhaki Z,Jaitin D,et al.Highthroughput chromatin immunoprecipitation for genomewide mapping of in vivo protein-DNA interactions and epigenomic states[J].Nature Protocols,2013,8(3):539-554.
[18]Wajant H,Pfizenmaier K,Scheurich P.Tumor necrosis factor signaling[J].Cell Death and Differentiation,2003,10(1):45-65.
[19]Qi F,Song J B,Yang H S,et al.Mmp23b promotes liver development and hepatocyte proliferation through the tumor necrosis factor pathway in zebrafish[J].Hepatology,2010,52(6):2158-2166.
[20]Hayden M S,Ghosh S.Regulation of NF-κB by TNF family cytokines[J].Seminars in Immunology,2014,26(3):253-266.
[21]Wang W L,Liu W T,Gong H Y,et al.Activation of cytokine expression occurs through the TNFα/NF-κB-mediated pathway in birnavirus-infected cells[J].Fish&Shellfish Immunology,2011,31(1):10-21.
[22]Musselman C A,Lalonde M E,C?téJ,et al.Perceiving the epigenetic landscape through histone readers[J].Nature Structural Molecular Biology,2012,19(12):1218-1227.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |