人cGAS基因启动子的克隆鉴定及功能初探
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摘要
目的:构建人环鸟苷酸-腺苷酸合成酶(cyclic guanosine monophosphate-adenosine monophosphate synthase,cGAS)基因启动子,确认其活性,进而对转录调控机制进行初步探究。方法:采用PCR方法扩增人cGAS基因5′上游1 254 bp(-1 178～+76 bp)的片段,酶切后连接到pGL3-basic质粒,以该质粒为模板合成不同长度的启动子质粒。通过双荧光素酶报告基因实验验证不同质粒在Hela细胞中的活性,利用生物信息学方法预测启动子区存在的转录因子结合位点。通过点突变实验验证潜在的转录因子结合位点。结果:人cGAS启动子质粒初步构建成功。转染后,cGAS启动子重组质粒的相对荧光素酶活性增加(P<0.05)。人cGAS近端启动子区域(-414～+76 bp)经生物信息学软件分析可能含有Sp1、CREB、USF1、RAP1、C-JUN、OCT-1等转录因子的结合位点。点突变实验证实Sp1、CREB正向调控该启动子区域。结论:人cGAS近端启动子区域(-414～+76bp)存在较强的启动子活性,该区域含有多个潜在的转录因子结合位点。转录因子Sp1、CREB调控人cGAS启动子区。
Objective:Thisstudyaimstoconstructthepromoterofhumancyclicguanosinemonophosphate-adenosinemonophosphate synthase(cGAS)gene,andexploreitspromoteractivityandtranscriptionalregulationmechanism.Methods:The1254 bp(-1178～+76 bp)fragmentof5′upstreamofhumancGASgenewasamplifiedbyPCRandsubclonedintopGL3-basicplasmid.Byaseriesof5′deletionand promoter constructions,the core region of cGAS promoter was founded. Luciferase assays were used to detect the activity of recombinant plasmids in Hela cells. Then,the transcription factor binding sites in promoter region were predicted by bioinformatics. Finally,potential transcriptionfactorbindingsiteswereverifiedbypointmutationexperiments. Resluts:Theluciferasereportergenerecombinantplasmids of cGAS gene promoter were successfully constructed. In comparison with the pGL3-basic plasmid,the relative luciferase activities of recombinant palsmids of cGAS promoter were much higher(P < 0.05). What's more,bioinformatics software predicts that the proximal promoterregionofhumancGAS(-414～+76 bp)maycontainbindingsitesoftranscriptionfactorssuchasSp1,CREB,USF1,RAP1,C-JUN and OCT-1. Sp1 and CREB positively regulated promoter region,which was confirmed by point mutation experiment. Conclusion:It is concludedthattheproximalpromotercoreregionofhumancGAShasstrongpromoteractivity,whichcontainsmanypotentialtranscription factorsbindingsites.ThetranscriptionfactorSp1 andCREBregulatesthehumancGASpromoterregion.
Objective:Thisstudyaimstoconstructthepromoterofhumancyclicguanosinemonophosphate-adenosinemonophosphate synthase(cGAS)gene,andexploreitspromoteractivityandtranscriptionalregulationmechanism.Methods:The1254 bp(-1178～+76 bp)fragmentof5′upstreamofhumancGASgenewasamplifiedbyPCRandsubclonedintopGL3-basicplasmid.Byaseriesof5′deletionand promoter constructions,the core region of cGAS promoter was founded. Luciferase assays were used to detect the activity of recombinant plasmids in Hela cells. Then,the transcription factor binding sites in promoter region were predicted by bioinformatics. Finally,potential transcriptionfactorbindingsiteswereverifiedbypointmutationexperiments. Resluts:Theluciferasereportergenerecombinantplasmids of cGAS gene promoter were successfully constructed. In comparison with the pGL3-basic plasmid,the relative luciferase activities of recombinant palsmids of cGAS promoter were much higher(P < 0.05). What's more,bioinformatics software predicts that the proximal promoterregionofhumancGAS(-414～+76 bp)maycontainbindingsitesoftranscriptionfactorssuchasSp1,CREB,USF1,RAP1,C-JUN and OCT-1. Sp1 and CREB positively regulated promoter region,which was confirmed by point mutation experiment. Conclusion:It is concludedthattheproximalpromotercoreregionofhumancGAShasstrongpromoteractivity,whichcontainsmanypotentialtranscription factorsbindingsites.ThetranscriptionfactorSp1 andCREBregulatesthehumancGASpromoterregion.
引文
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[2] Ming Z,Zeping H,Xiaolei S,et al. MAVS,cGAS,and endogenous retroviruses in T-independent B cell responses[J]. Science,2014,346(6216):1486-1492
[3] Cai X,Chiu YH,Chen ZJMC. The cGAS-cGAMP-STING pathway of cytosolic DNA sensing and signaling[J]. Mol Cell,2014,54(2):289-296
[4] Aguirre S,Fernandez-Sesma AJJoV. Collateral damage during dengue virus infection:making sense of DNA by cGAS[J]. J Virol,2017,91(14):e01081-1016
[5] Ruangkiattikul N,Nerlich A,Abdissa K,et al. cGASSTING-TBK1-IRF3/7 induced interferon-βcontributes to the clearing of non tuberculous mycobacterial infection in mice[J]. Virulence,2017,8(7):1303-1315
[6] Juven-Gershon T,Hsu JY,Theisen JW,et al. The RNA polymeraseⅡcore promoter-the gateway to transcription[J]. Curr Opin Cell Biol,2008,20(3):253-259
[7] Tong S,Yang G,Wen T,et al. A six-nucleotide insertiondeletion polymorphism in the CASP8 promoter is associated with susceptibility to multiple cancers[J]. Nat Genet,2007,39(5):605-613
[8]徐妍妍,王艳艳,徐华国,等.小鼠STING基因启动子的克隆鉴定及功能初步分析[J].南京医科大学学报(自然科学版),2016,36(7):783-787
[9] Bakhoum SF,Ngo B,Laughney AM,et al. Chromosomal instability drives metastasis through a cytosolic DNA response[J]. Nature,2018,553(7689):467-472
[10] Harding SM,Benci JL,Irianto J,et al. Mitotic progression following DNA damage enables pattern recognition within micronuclei[J]. Nature,2017,548(7668):466-470
[11] Mackenzie KJ,Carroll P,Martin CA,et al. cGAS surveillance of micronuclei links genome instability to innate immunity[J]. Nature,2017,548(7668):461-465
[12] Lijun S,Jiaxi W,Fenghe D,et al. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferonpathway[J].Science,2013,339(6121):786-791
[13] Zhou W,Whiteley AT,Cc DOM,et al. Structure of the human cGAS-DNA complex reveals enhanced control of immune surveillance[J]. Cell,2018,174(2):300-311
[14] Liu H,Zhang H,Wu X,et al. Nuclear cGAS suppresses DNA repair and promotes tumorigenesis[J]. Nature,2018,563(7729):131-136