摘要
【目的】探讨影响鸡长链非编码RNA(long chain noncoding RNA,lncRNA)-骨形态发生蛋白4(bone morphogenetic protein 4,BMP4)启动子转录的因素,并对调控lncRNA-BMP4特异表达的分子机制进行研究。【方法】以鸡肌肉基因组为模板,PCR扩增并克隆鸡lncRNA-BMP4的启动子区,构建lncRNA-BMP4-EFGP载体,对lncRNA-BMP4启动活性进行定性分析;通过染色体5′-末端缺失的方法和双荧光素酶系统检测筛选lncRNA-BMP4启动子核心区域。通过在线工具预测分析调控核心区域的潜在转录因子;利用点突变和双荧光素酶系统筛查真正影响lncRNA-BMP4的转录因子;通过表观修饰验证DNA甲基化、组蛋白乙酰化对lncRNA-BMP4的转录调控作用。【结果】试验成功扩增lncRNA-BMP4启动子片段1288 bp,与pEGFP-N1载体连接后转染鸡成纤维细胞系(DF-1)具有荧光表达,说明lncRNA-BMP4启动子有启动活性。染色体5′-末端缺失和双荧光素酶系统检测发现,核心启动子区域为―832~―651 bp,Jaspar数据库分析筛选到核心区域的转录因子有SOX17、CREB1及STAT1。双荧光素酶系统检测发现,STAT1可促进lncRNA-BMP4核心启动区域的活性;DNA甲基化抑制剂5′-Azacd对lncRNA-BMP4的转录活性未有任何影响,而组蛋白乙酰化抑制剂TSA可极显著提高其转录活性(P<0.01)。【结论】提示lncRNA-BMP4的转录活性受STAT1和组蛋白乙酰化的正调控,而DNA甲基化不影响其转录。研究结果为详细解析lncRNA-BMP4的功能和分子机制提供了理论依据。
【Objective】The purpose of this study was to investigate the factors affecting the transcription of long chain noncoding RNA-bone morphogenetic protein 4(lncRNA-BMP4)promoters in chickens,and study the molecular mechanisms regulating the specific expression of lncRNA-BMP4.【Method】Using the muscle genome in chickens as a template,the promoter region of lncRNA-BMP4 in chickens was amplified by PCR and cloned,the lncRNA-BMP4-EFGP vector was constructed,and the lncRNA-BMP4 promoter activity was qualitatively analyzed.The core region of the lncRNA-BMP4 promoter was screened by chromosome 5′-terminal deletion and double luciferase system detection.The potential transcriptional factors of the regulatory core region were predicted and analyzed by online tools,and the transcriptional factors that really affected lncRNA-BMP4 were screened by point mutation and dual luciferase system.The transcriptional regulation of DNA methylation and histone acetylation on lncRNA-BMP4 was verified by apparent modification.【Result】The lncRNA-BMP4 promoter fragments(1288 bp)were successfully amplified and ligated with a pEGFP-N1 vector.After transfection into chicken fibroblast cell line(DF-1),it showed fluorescent expression,indicating that lncRNA-BMP4 promoter had promotive activity.The results of chromosome 5′-terminal deletion and double luciferase system detection showed that the core promoter region was―832 to―651 bp.The transcription factors screened by Jaspar database analysis showed that the core regions were SOX17,CREB1 and STAT1.Double luciferase system showed that STAT1 could promote the activity of lncRNA-BMP4 core promoter region,DNA methylation inhibitor 5′-Azacd had no effect on lncRNA-BMP4 transcriptional activity,while histone acetylation inhibitor TSA extremely significantly increased its transcriptional activity(P<0.01).【Conclusion】This study suggested that the transcriptional activity of lncRNA-BMP4 was positively regulated by STAT1 and histone acetylation,while DNA methylation did not affect i
作者
高晓敏
周舒简
陈晨
金晶
胡菜
张晨
左其生
张亚妮
陈国宏
李碧春
GAO Xiaomin;ZHOU Shujian;CHEN Chen;JIN Jing;HU Cai;ZHANG Chen;ZUO Qisheng;ZHANG Yani;CHEN Guohong;LI Bichun(Joint Laboratory for International Agricultural and Agricultural Product Safety Research,Ministry of Education of China,College of Animal Science and Technology,Yangzhou University,Yangzhou 225009,China;College of Biotechnology,Jiangsu University of Science and Technology,Zhenjiang 212003,China)
出处
《中国畜牧兽医》
CAS
北大核心
2022年第9期3321-3332,共12页
China Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金(31872341)。
