载脂蛋白B mRNA编辑酶催化亚基3A的表达及其DNA去甲基化功能鉴定
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摘要
目的真核细胞内,DNA甲基化调控在转座子沉默、基因表达调控、甚至病毒感染过程中起关键作用,其中HIV-1启动子的甲基化修饰在病毒潜伏感染中扮演重要角色。为明确载脂蛋白B mRNA编辑酶催化亚基3A(APOBEC3A,A3A)对HIV-1启动子5'LTR上CpG岛甲基化程度的调节作用,本研究构建并筛选出A3A异构体A3A Isotype a(A3Aa)和A3A Isotype b(A3A-b)高表达真核载体,分析其去甲基化功能。方法从THP-1细胞mRNA中扩增得到A3A-a和A3A-b的c DNA,分别克隆至真核表达载体pASIB-HA和pCAGGS-HA。转染HEK-293T细胞,蛋白印迹法检测A3A-a和A3A-b的蛋白表达,并筛选出A3A-a和A3A-b的高表达载体。接着将A3A-a和A3A-b高表达载体与实验室构建并修饰的HIV-1启动子甲基化载体p-me LTR-EGFP共转染HEK-293T,检测指示蛋白EGFP在细胞水平中的表达,评价A3A-a和A3A-b的去甲基化调节作用。结果 PCR结果、酶切鉴定和测序结果均表明A3A真核表达载体pASIB-HA-A3A1-a、pCAGGS-HA-A3A2-a、pASIB-HA-A3A1-b、pCAGGS-HA-A3A2-b构建成功。经过筛选,pCAGGS-HA-A3A2-a和pCAGGS-HA-A3A2-b中A3A蛋白表达量最高,并用于后续实验。甲基化载体共转染实验显示,相较于对照A3G转染组,A3A-a和A3A-b组中EGFP的表达量明显上调。结论本研究在细胞水平上证实A3A分子Isotype a和Isotype b均可识别DNA中5-甲基化胞嘧啶,催化HIV-1启动子CpG岛去甲基化,为后续研究A3A对HIV潜伏感染调控奠定了基础。
Objective In eukaryotes, DNA methylation is involved in transposon silencing, gene regulation and viral infection, within which methylation of HIV-1 promoter plays an important role in HIV-1 latent infection. In order to determine the demethylation effect of APOBEC3 A(A3 A) on CpG island of HIV-1 5'LTR promoter, the eukaryotic vectors expressing A3 A isotype a and A3 A isotype b were constructed and selected, and A3 A mediated DNA-demethylation was further characterized. Methods The c DNA of A3 A-a and A3 A-b were amplified from THP-1 mRNA and cloned into eukaryotic expression vectors pASIB-HA and pCAGGS-HA. The HEK-293 T cells were transfected, and the protein expression of A3 A-a and A3 A-b was detected by Western blot. The A3 A-a and A3 A-b highly expressive vectors were screened and were then,the A3 A-a and A3 A-b co-transfected with the HIV-1 methylated promoter p-me LTR-EGFP, which was constructed and modified by our laboratory, into the HEK-293 T, and the expression of EGFP was detected.Results The results of PCR, enzymatic identification and sequencing showed that the A3 A eukaryotic expression vector pASIB-HA-A3 A1-a, pCAGGS-HA-A3 A2-a, pASIB-HA-A3 A1-b, pCAGGS-HA-A3 A2-b were successfully constructed. After screening, A3 A proteins were highly expressed in pCAGGS-HA-A3 A2-a and pCAGGS-HA-A3 A2-b, which could be used in following experiments. The co-transfection experiment showed that in comparison with A3 G transfected control group, the fluorescence intensity and expression level of EGFP in A3 A-a and A3 A-b groups were significantly increased. Conclusion This study indicated that both A3 A isotype a and isotype b could recognize the 5-methylcytosine and catalyze the demethylation of CpG island in a HIV-1 promoter, providing a function for further study on the role of A3 A in HIV latency infection.
Objective In eukaryotes, DNA methylation is involved in transposon silencing, gene regulation and viral infection, within which methylation of HIV-1 promoter plays an important role in HIV-1 latent infection. In order to determine the demethylation effect of APOBEC3 A(A3 A) on CpG island of HIV-1 5'LTR promoter, the eukaryotic vectors expressing A3 A isotype a and A3 A isotype b were constructed and selected, and A3 A mediated DNA-demethylation was further characterized. Methods The c DNA of A3 A-a and A3 A-b were amplified from THP-1 mRNA and cloned into eukaryotic expression vectors pASIB-HA and pCAGGS-HA. The HEK-293 T cells were transfected, and the protein expression of A3 A-a and A3 A-b was detected by Western blot. The A3 A-a and A3 A-b highly expressive vectors were screened and were then,the A3 A-a and A3 A-b co-transfected with the HIV-1 methylated promoter p-me LTR-EGFP, which was constructed and modified by our laboratory, into the HEK-293 T, and the expression of EGFP was detected.Results The results of PCR, enzymatic identification and sequencing showed that the A3 A eukaryotic expression vector pASIB-HA-A3 A1-a, pCAGGS-HA-A3 A2-a, pASIB-HA-A3 A1-b, pCAGGS-HA-A3 A2-b were successfully constructed. After screening, A3 A proteins were highly expressed in pCAGGS-HA-A3 A2-a and pCAGGS-HA-A3 A2-b, which could be used in following experiments. The co-transfection experiment showed that in comparison with A3 G transfected control group, the fluorescence intensity and expression level of EGFP in A3 A-a and A3 A-b groups were significantly increased. Conclusion This study indicated that both A3 A isotype a and isotype b could recognize the 5-methylcytosine and catalyze the demethylation of CpG island in a HIV-1 promoter, providing a function for further study on the role of A3 A in HIV latency infection.
引文
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[19]刘彦礼,于亚楠,杨坤,等.两种真核启动子体内外转录活性比较研究[J].中国细胞生物学学报,2015,37(10):1370—1376
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[23]Chen H,Lilley CE,Yu Q,et al.APOBEC3A is a potent inhibitor of adeno-associated virus and retrotransposons[J].Current Biology:CB,2006,16(5):480—485
[2]Guo JU,Su Y,Zhong C,et al.Hydroxylation of 5-methylcytosine by TET1 promotes active DNA demethylation in the adult brain[J].Cell,2011,145(3):423—434
[3]Siliciano JD,Kajdas J,Finzi D,et al.Long-term followup studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+T cells[J].Nature Medicine,2003,9(6):727—728
[4]Kumar A,Darcis G,Van Lint C,et al.Epigenetic control of HIV-1 post integration latency:implications for therapy[J].Clinical Epigenetics,2015,7(1):103
[5]Stenglein MD,Burns MB,Li M,et al.APOBEC3proteins mediate the clearance of foreign DNA from human cells[J].Nature Structural&Molecular Biology,2010,17(2):222—229
[6]Bulliard Y,Narvaiza I,Bertero A,et al.Structure-function analyses point to a polynucleotide-accommodating groove essential for APOBEC3A restriction activities[J].Journal of Virology,2011,85(4):1765—1776
[7]Landry S,Narvaiza I,Linfesty DC,et al.APOBEC3A can activate the DNA damage response and cause cellcycle arrest[J].EMBO Reports,2011,12(5):444—450
[8]Wu X,Zhang Y.TET-mediated a ctive DNA demethylation:mechanism,function and beyond[J].Nature Reviews Genetics,2017,18(9):517—534
[9]Gehring M,Reik W,Henikoff S.DNA demethylation by DNA repair[J].Trends in Genetics,2009,25(2):82—90
[10]Morgan HD,Dean W,Coker HA,et al.Activationinduced cytidine deaminase deaminates 5-methylcytosine in DNA and is expressed in pluripotent tissues:implications for epigenetic reprogramming[J].The Journal of Biological Chemistry,2004,279(50):52353—52360
[11]Bransteitter R,Pham P,Scharff MD,et al.Activationinduced cytidine deaminase deaminates deoxycytidine on single-stranded DNA but requires the action of RNase[J].Proceedings of the National Academy of Sciences of the United States of America,2003,100(7):4102—4107
[12]Larijani M,Frieder D,Sonbuchner TM,et al.Methylation protects cytidines from AID-mediated deamination[J].Molecular Immunology,2005,42(5):599—604
[13]Popp C,Dean W,Feng S,et al.Genome-wide erasure of DNA methylation in mouse primordial germ cells is affected by AID deficiency[J].Nature,2010,463(7284):1101—1105
[14]Bhutani N,Brady JJ,Damian M,et al.Reprogramming towards pluripotency requires AID-dependent DNA demethylation[J].Nature,2010,463(7284):1042—1047
[15]Wijesinghe P,Bhagwat AS.Efficient deamination of5-methylcytosines in DNA by human APOBEC3A,but not by AID or APOBEC3G[J].Nucleic Acids Research,2012,40(18):9206—9217
[16]Ito F,Fu Y,Kao SA,et al.Family-wide comparative analysis of cytidine and methylcytidine deamination by eleven human APOBEC proteins[J].Journal of Molecular Biology,2017,429(12):1787—1799
[17]Harris RS,Liddament MT.Retroviral restriction by APOBEC proteins[J].Nature Reviews.Immunology,2004,4(11):868—877
[18]吴小霞.APOBEC3A的功能研究新进展[J].中国艾滋病性病,2016,22(6):481—484
[19]刘彦礼,于亚楠,杨坤,等.两种真核启动子体内外转录活性比较研究[J].中国细胞生物学学报,2015,37(10):1370—1376
[20]Conticello SG.The AID/APOBEC family of nucleic acid mutators[J].Genome Biology,2008,9(6):229
[21]Wedekind JE,Dance GS,Sowden MP,et al.Messenger RNA editing in mammals:new members of the APOBEC family seeking roles in the family business[J].Trends in Genetics,2003,19(4):207—216
[22]Jarmuz A,Chester A,Bayliss J,et al.An anthropoid-specific locus of orphan C to U RNA-editing enzymes on chromosome 22[J].Genomics,2002,79(3):285—296
[23]Chen H,Lilley CE,Yu Q,et al.APOBEC3A is a potent inhibitor of adeno-associated virus and retrotransposons[J].Current Biology:CB,2006,16(5):480—485