Effects of low-temperature stress on histone modification in ZF4 cells

Abstract

PDF HTML阅读 XML下载 导出引用 引用提醒 低温压力对ZF4细胞组蛋白修饰的影响 DOI: 作者: 作者单位: 1. 水产种质资源发掘与利用教育部重点实验室, 上海海洋大学, 上海 201306;2. 水产科学国家级实验教学示范中心, 上海海洋大学, 上海 201306;3. 海洋生物科学国际联合研究中心, 中国科学技术部, 上海海洋大学, 上海 201306 作者简介: 姜蓬垒(1988-),男,博士研究生,研究方向为表观遗传学.E-mail:770163089@qq.com 通讯作者: 中图分类号: S917 基金项目: 国家自然科学基金项目（31372516，81770165）；上海市自然科学基金项目（13ZR1419500）；上海市教育委员会"东方学者"计划支持项目. Effects of low-temperature stress on histone modification in ZF4 cells Author: Affiliation: 1. Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education;Shanghai Ocean University, Shanghai 201306, China;2. National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China;3. International Research Center for Marine Biosciences at Shanghai Ocean University, Ministry of Science and Tech-nology, Shanghai 201306, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 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（）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-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，基因经过低温驯化后上调，而ChIP-qPCR结果显示，基因启动子区域的H3K4me3、H3K27ac水平来调控基因的表达。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。 Abstract: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 () 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 -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 H3K27me3 in the -actin promoter region, accompanied by enriched H3K4me3 and H3K27ac, was observed as predicted. Zebrafish embryonic ZF4 fibroblasts cultured under normal condition (28℃) and cold-acclimated ZF4 cells under long-term cold exposure (18℃ for 30 days) were evaluated with the optimized ChIP protocol. The effect of cold acclimation on the expression of tumor necrosis factor tnfb promoter was detected by qPCR and ChIP-qPCR, respectively. The qPCR and ChIP-qPCR results showed that was upregulated after cold acclimation and that H3K4me3 and H3K27ac were enriched in the promoter region of , while enrichment of H3K27me3 showed no significant change. This suggests that cold pressure regulates expression by affecting H3K4me3 and H3K27ac levels in the promoter region of . 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. 参考文献 相似文献 引证文献