利用ChIP-seq/双荧光素酶报告基因技术验证PPARγ2的靶向基因MYH7  

Verification of the PPARγ2 Target Gene MYH7 by ChIP-seq Com-bined with Dual-luciferase Assay

作　　者：张仙玉 左中[1] 毛敏[1] Zhang Xianyu;Zuo Zhong;Mao Min(Department of Cardiology,The First Affiliated Hospital of Chongqing Medical University,Chongqing,400016;Key Laboratory of Ministry of Education for Translational Medicine in Major Metabolic Diseases,Chongqing Medical University,Chongqing,400016)

机构地区：[1]重庆医科大学附属第一医院心血管内科,重庆400016 [2]教育部重大代谢性疾病转化医学重点实验室,重庆医科大学,重庆400016

出　　处：《基因组学与应用生物学》2022年第1期223-230,共8页Genomics and Applied Biology

基　　金：重庆市自然科学基金面上项目(cstc2019jcyj-msxmX0433);重庆市渝中区科委课题(20130131)共同资助。

摘　　要：为验证2型过氧化物酶体增殖激活受体γ(peroxisome proliferator-activated receptorγ_(2),PPARγ_(2))对编码β-肌球蛋白重链(β-myosin heavy chain,β-MHC)基因MYH7的靶向调控机制及其结合位点,本研究将pTT5空载体和pTT5-3flag-PPARγ_(2)质粒分别转染小鼠心肌细胞(mouse cardiac myocytes,MCM),RT-qPCR和Western blot检测MYH7和PPARγ_(2)的表达情况;利用染色质免疫共沉淀技术(chromatin immunoprecipita-tion,ChIP)特异性富集并纯化MCM中可与PPARγ_(2)结合的DNA片段,通过高通量测序技术(high-throughput sequencing,HiSeq)检测和分析可直接与PPARγ_(2)结合的基因。采用生物信息学软件预测PPARγ_(2)结合于靶标基因MYH7的启动子区,并将预测到的转录因子结合位点和相应的突变位点分别插入pgl3-basic载体的萤火虫荧光素酶基因上游,构建野生型和突变型荧光素酶报告质粒,再分别与pcDNA3.1载体、pcDNA3.1-PPARγ_(2)质粒和海肾荧光素酶基因载体pRL-TK共转染293T细胞,检测荧光素酶相对活性。实验结果表明:ChIP-seq初步验证了MYH7是PPARγ_(2)直接调控的可能靶向基因;经基因测序验证MYH7野生型和突变型荧光素酶质粒(pgl3-basic-MYH7-WT、pgl3-basic-MYH7-mut)构建成功;双荧光素酶报告基因检测显示,与MYH7-WT+pcDNA3.1组的相对荧光素酶活(0.366±0.021)相比,MYH7-WT+pcDNA3.1-PPARγ_(2)组的相对荧光素酶活性(2.477±0.212)增强,差异具有统计学意义(P<0.001);与MYH7-WT+pcDNA3.1-PPARγ_(2)的相对荧光素酶活(2.477±0.212)相比,MYH7-mut+pcDNA3.1-PPARγ_(2)组的相对荧光素酶活性(2.677±0.201)无明显变化(P>0.05)。本研究通过ChIP-seq/双荧光素酶报告基因技术初步验证PPARγ_(2)能够直接结合MYH7的启动子区域并靶向正调控MYH7基因的表达。To clarify the targeted regulation mechanism and binding sites of PPARγ_(2)(peroxisome proliferator-activated receptorγ_(2))on the MYH7 gene encodingβ-myosin heavy chain(β-MHC).In this study,the pTT5 basic vector and pTT5-3 flag-PPARγ_(2)plasmid were transfected into mouse cardiac myocytes(MCM),respectively,and the expression of MYH7 and PPARγ_(2)was detected by RT-qPCR and Western blot.Chromatin immunoprecipitation(ChIP)was used to specifically enrich and purify DNA fragments binding to PPARγ_(2)in the MCM,and high-throughput sequencing(HiSeq)was used to detect and analyze genes that can directly bind to PPARγ_(2).Relevant bioinformatics software was used to predict PPARγ_(2)binding to the promoter of the MYH7 gene,and inserted the predicted transcription factor binding site and corresponding mutation site into the upstream of the luciferase gene in the pgl3-basic vector,respectively.Wild and mutant types of dual-luciferase reporter plasmid were constructed.Meanwhile,they were co-transfected into 293 T cells with pcDNA3.1 vector,pcDNA3.1-PPARγ_(2)plasmid and renilla luciferase gene vector pRL-TK.Afterwards,the relative luciferase activity was detected.The results showed that ChIP-seq preliminarily verified that MYH7 was a possible target gene directly regulated by PPARγ_(2).The wild and mutant types of dual-luciferase reporter plasmids(pgl3-basic-MYH7-WT and pgl3-basic-MYH7-mut)were successfully built that identified by gene sequencing.Double fluorescence detection results showed that the relative luciferase activity of MYH7-WT+pcDNA3.1-PPARγ_(2)(2.477±0.212)was higher than that of MYH7-WT+pcDNA3.1(0.366±0.021),and the difference was statistically significant(P<0.001).There was no difference in the relative luciferase activity between groups MYH7-WT+pcDNA3.1-PPARγ_(2)(2.477±0.212)and MYH7-mut+pcDNA3.1-PPARγ_(2)(2.677±0.201)(P>0.05).In this study,ChIP-seq and dual-luciferase assay technique preliminarily confirm that PPARγ_(2)can directly bind to the promoter region of MYH7 and have a regulator

关 键 词：2型过氧化物酶体增殖激活受体γ 肌球蛋白重链 染色质免疫共沉淀 高通量测序 双荧光素酶报告基因技术 

分 类 号：Q78[生物学—分子生物学]