KDM6A表达载体的构建及其对组蛋白H3K27me3修饰的影响
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摘要
组蛋白甲基化是一种重要的表观修饰方式,主要参与异染色质的形成、X染色体的失活以及RNA的转录调控。其中组蛋白H3第27位赖氨酸去甲基化酶KDM6A (lysine specific demethylase 6A)具有调控胚胎发育、细胞重编程和细胞分化的功能。目前对KDM6A的研究主要集中在体外非细胞环境下的生化实验,在细胞内的研究较少,并且未见KDM6A过表达的动物模型。本研究通过基因工程手段构建了KDM6A的表达载体pCS2-KDM6A,并将其转染小鼠(Mus musculus)胎儿成纤维细胞。经qRT-PCR检测发现,转染组KDM6A的表达量显著高于对照组(P<0.05);免疫荧光和免疫印迹检测表明,过表达KDM6A可以显著降低H3K27me3的修饰(P<0.05)。本研究成功构建了pCS2-KDM6A载体,此载体为下一步制备动物模型提供了理论基础,对解析KDM6A与H3K27me3在X染色体失活、胚胎发育、细胞重编程等生物学事件的研究机理提供了基础材料。
Histone methylation is key epigenetic regulatory feature that have important function in many biological processes which include heterochromatin formation, X-chromosome inactivation and transcriptional regulation. Among them, histone H3 lysine specific demethylase 6A(KDM6A) has functions of regulating embryonic development, cell reprogramming and cell differentiation. At present, the research on KDM6 A mainly focused on experiments in a non-cellular environment, and there were few studies in cells, and no animal model of KDM6 A overexpression is observed. In the present study, total RNA of 293 T CELL was extracted and reversely transcribed into c DNA. Specific primers were designed according to human(Homo sapiens) mRNA sequence of KDM6 A gene. The coding sequences of KDM6 A gene was cloned in vitro and was constructed the overexpression vector pcs2-KDM6 A by genetic engineering. Then, the vector pcs2-KDM6 A was transfected into mouse(Mus musculus) embryonic fibroblasts via lipofectin transfection method. The gene expression patterns were detected by qRT-PCR. The q RT-PCR assay showed that the mRNA expression levelof KDM6 A in transfected group was significantly higher than control group(P<0.05). In contrast, and there was no significant difference between expression of KDM6 B in transfected group and control group.Furthermore, H3 K27 me3 expression was detected by laser confocal immunofluorescence(IF) and western blot(WB) technology respectively. These results demonstrated that the expression of H3 K27 me3 level after transfection of pCS2-KDM6 A vector both were significantly lower than control group. Taken together, the expression vector pCS2-KDM6 A was successfully constructed, and was proved to had correct enzyme activity at the cell level. The pCS2-KDM6 A vector can be used to study the function of KDM6 A in the development and related diseases both in vivo and in vitro. Moreover, an animal model of overexpression of KDM6 A can be prepared by this vector, and a basic material for analyzing the research mechanism of KDM6 A and H3 K27 me3 in biological events such as X chromosome inactivation, embryo development and cell reprogramming is provided.
Histone methylation is key epigenetic regulatory feature that have important function in many biological processes which include heterochromatin formation, X-chromosome inactivation and transcriptional regulation. Among them, histone H3 lysine specific demethylase 6A(KDM6A) has functions of regulating embryonic development, cell reprogramming and cell differentiation. At present, the research on KDM6 A mainly focused on experiments in a non-cellular environment, and there were few studies in cells, and no animal model of KDM6 A overexpression is observed. In the present study, total RNA of 293 T CELL was extracted and reversely transcribed into c DNA. Specific primers were designed according to human(Homo sapiens) mRNA sequence of KDM6 A gene. The coding sequences of KDM6 A gene was cloned in vitro and was constructed the overexpression vector pcs2-KDM6 A by genetic engineering. Then, the vector pcs2-KDM6 A was transfected into mouse(Mus musculus) embryonic fibroblasts via lipofectin transfection method. The gene expression patterns were detected by qRT-PCR. The q RT-PCR assay showed that the mRNA expression levelof KDM6 A in transfected group was significantly higher than control group(P<0.05). In contrast, and there was no significant difference between expression of KDM6 B in transfected group and control group.Furthermore, H3 K27 me3 expression was detected by laser confocal immunofluorescence(IF) and western blot(WB) technology respectively. These results demonstrated that the expression of H3 K27 me3 level after transfection of pCS2-KDM6 A vector both were significantly lower than control group. Taken together, the expression vector pCS2-KDM6 A was successfully constructed, and was proved to had correct enzyme activity at the cell level. The pCS2-KDM6 A vector can be used to study the function of KDM6 A in the development and related diseases both in vivo and in vitro. Moreover, an animal model of overexpression of KDM6 A can be prepared by this vector, and a basic material for analyzing the research mechanism of KDM6 A and H3 K27 me3 in biological events such as X chromosome inactivation, embryo development and cell reprogramming is provided.
引文
金京波.2011.含有JmjC功能域的组蛋白去甲基化酶参与DRM2介导的DNA甲基化的维持[J].农业生物技术学报,19(1):44-44.(Jin J B.2011.Involvement of a Jumonji-C containing histone demthylase in DRM2 mediated maintance of DNA methylation[J].Journal of Agricultural Biotechnology,19(1):44-44.)
王杨,王贵英.2011.X染色体失活、失活逃逸及相关疾病的研究进展[J].畜牧与饲料科学,32(1):26-27.(Wang Y,Wang G Y.2011.Research progress on X chromosome inactivation,its escaping and the related diseases[J].Animal Husbandry and Feed Science,32(1):26-27.)
王溪,朱卫国.2008.组蛋白甲基化酶及去甲基化酶的研究进展[J].癌症,27(10):1018-1025.(Wang X,Zhu W G.2008.Advances in histone methyltransferases and histone demethylases[J].Chinese Journal of Cancer,27(10):1018-1025.)
Agger K,Cloos P A,Christensen J,et al.2007.UTX and JM-JD3 are histone H3K27 demethylases involved in HOXgene regulation and development[J].Nature,449(7163):731-734.
Bai GY,Song S H,Zhang Y W,et al.2018.Kdm6a overexpression improves the development of cloned mouse embryos[J].Zygote,26(1):24-32.
Canovas S,Cibelli J B,Ross P J.2012.Jumonji domain-containing protein 3 regulates histone 3 lysine 27 methylation during bovine preimplantation development[J].Proceedings of the National Academy of Sciences,109(7):2400-2405.
Hawkins R D,Hon G C,Lee L K,et al.2010.Distinct epigenomic landscapes of pluripotent and lineage-committed human cells[J].Cell Stem Cell,6(5):479-491.
Hong S,Cho Y W,Yu L R,et al.2007.Identification of JmjCdomain-containing UTX and JMJD3 as histone H3 lysine 27 demethylases[J].Proceedings of the National Academy of Sciences,104(47):18439-18444.
Inoue A,Jiang L,Lu F,et al.2017.Maternal H3K27me3 controls DNA methylation-independent imprinting[J].Nature,547(7664):419-424.
Jiang W,Wang J,Zhang Y.2013.Histone H3K27me3 demethylases KDM6A and KDM6B modulate definitive endoderm differentiation from human ESCs by regulating WNT signaling pathway[J].Cell Research,23(1):122-130.
Lee M G,Villa R,Trojer P,et al.2007.Demethylation of H3K27 regulates polycomb recruitment and H2A ubiquitination[J].Science,318(5849):447-450.
Lee S,Lee J W,Lee S K.2012.UTX,a histone H3-lysine 27demethylase,acts as a critical switch to activate the cardiac developmental program[J].Developmental Cell,22(1):25-37.
Mansour A A,Gafni O,Weinberger L,et al.2012.The H3K27demethylase Utx regulates somatic and germ cell epigenetic reprogramming[J].Nature,488(7411):409-413.
Martin C,Zhang Y.2005.The diverse functions of histone lysine methylation[J].Nature Reviews Molecular Cell Biology,6(11):838-849.
Mikkelsen T S,Hanna J,Zhang X,et al.2008.Dissecting direct reprogramming through integrative genomic analysis[J].Nature,454(7200):49-55.
Miller S A,Mohn S E,Weinmann A S.2010.Jmjd3 and UTXplay a demethylase-independent role in chromatin remodeling to regulate T-box family member-dependen gene expression[J].Molecular Cell,40(4):594-605.
Miyake N,Mizuno S,Okamoto N,et al.2013.KDM6A poin mutations cause Kabuki syndrome[J].Human Mutation34(1):108-110.
Pan D,Huang L,Zhu L J,et al.2015.Jmjd3-mediated H3K27me3 dynamics orchestrate brown fat development and regulate white fat plasticity[J].Developmenta Cell,35(5):568-583.
Rothbart S B,Strahl B D.2014.Interpreting the language of histone and DNA modifications[J].Biochimica et Biophysica Acta,1839(8):627-643.
Seenundun S,Rampalli S,Liu Q C,et al.2010.UTX mediates demethylation of H3K27me3 at muscle-specific genes during myogenesis[J].The EMBO Journal,29(8)1401-1411.
Shi Y,Lan F,Matson C,et al.2004.Histone demethylation mediated by the nuclear amine oxidase homolog LSD1[J].Cell,119(7):941-953.
Shpargel K B,Sengoku T,Yokoyama S,et al.2012.UTX and UTY demonstrate histone demethylase independen function in mouse embryonic development[J].PLoS Genetics,8(9):e1002964.
Shpargel K B,Starmer J,Yee D,et al.2014.KDM6 demethylase independent loss of histone H3 lysine 27 trimethylation during early embryonic development[J].PLoS Genetics,10(8):e1004507.
Thieme S,Gyárfás T,Richter C,et al.2013.The histone demethylase UTX regulates stem cell migration and hematopoiesis[J].Blood,21(13):2462-2473.
van Haaften G,Dalgliesh G L,Davies H,et al.2009.Somatic mutations of the histone H3K27 demethylase gene UTXin human cancer[J].Nature Genetics,41(5):521-523.
Voigt P,Tee W W,Reinberg D.2013.A double take on bivalent promoters[J].Genes&Development,27(12):1318-1338.
Wang A H,Zare H,Mousavi K,et al.2013.The histone chaperone Spt6 coordinates histone H3K27 demethylation and myogenesis[J].The EMBO Journal,32(8):1075-1086.
Wang C,Lee J E,Cho Y W,et al.2012.UTX regulates mesoderm differentiation of embryonic stem cells independent of H3K27 demethylase activity[J].Proceedings of the National Academy of Sciences of the USA,109(38):15324-15329.
Wang S P,Tang Z,Chen C W,et al.2017.A UTX-MLL4-p300 Transcriptional regulatory network coordinately shapes active enhancer landscapes for eliciting transcription[J].Molecular Cell,67(2):308-321.
Welstead G G,Creyghton M P,Bilodeau S,et al.2012.X-linked H3K27me3 demethylase Utx is required for embryonic development in a sex-specific manner[J].Proceedings of the National Academy of Sciences of the USA,109(32):13004-13009.
Wu S C,Kallin E M,Zhang Y.2010.Role of H3K27 methylation in the regulation of lncRNA expression[J].Cell Research,20(10):1109-1116.
Yang L,Song L S,Liu X F,et al.2016.The maternal effect genes UTX and JMJD3 play contrasting roles in Mus musculus preimplantation embryo development[J].Scientific Reports,6:26711.
Zenk F,Loeser E,Schiavo R,et al.2017.Germ line-inherited H3K27me3 restricts enhancer function during maternalto-zygotic transition[J].Science,357(6347):212-216.
Zhang M,Wang F,Kou Z,et al.2009.Defective chromatin structure in somatic cell cloned mouse embryos[J].Journal of Biological Chemistry,284(37):24981-24987.
王杨,王贵英.2011.X染色体失活、失活逃逸及相关疾病的研究进展[J].畜牧与饲料科学,32(1):26-27.(Wang Y,Wang G Y.2011.Research progress on X chromosome inactivation,its escaping and the related diseases[J].Animal Husbandry and Feed Science,32(1):26-27.)
王溪,朱卫国.2008.组蛋白甲基化酶及去甲基化酶的研究进展[J].癌症,27(10):1018-1025.(Wang X,Zhu W G.2008.Advances in histone methyltransferases and histone demethylases[J].Chinese Journal of Cancer,27(10):1018-1025.)
Agger K,Cloos P A,Christensen J,et al.2007.UTX and JM-JD3 are histone H3K27 demethylases involved in HOXgene regulation and development[J].Nature,449(7163):731-734.
Bai GY,Song S H,Zhang Y W,et al.2018.Kdm6a overexpression improves the development of cloned mouse embryos[J].Zygote,26(1):24-32.
Canovas S,Cibelli J B,Ross P J.2012.Jumonji domain-containing protein 3 regulates histone 3 lysine 27 methylation during bovine preimplantation development[J].Proceedings of the National Academy of Sciences,109(7):2400-2405.
Hawkins R D,Hon G C,Lee L K,et al.2010.Distinct epigenomic landscapes of pluripotent and lineage-committed human cells[J].Cell Stem Cell,6(5):479-491.
Hong S,Cho Y W,Yu L R,et al.2007.Identification of JmjCdomain-containing UTX and JMJD3 as histone H3 lysine 27 demethylases[J].Proceedings of the National Academy of Sciences,104(47):18439-18444.
Inoue A,Jiang L,Lu F,et al.2017.Maternal H3K27me3 controls DNA methylation-independent imprinting[J].Nature,547(7664):419-424.
Jiang W,Wang J,Zhang Y.2013.Histone H3K27me3 demethylases KDM6A and KDM6B modulate definitive endoderm differentiation from human ESCs by regulating WNT signaling pathway[J].Cell Research,23(1):122-130.
Lee M G,Villa R,Trojer P,et al.2007.Demethylation of H3K27 regulates polycomb recruitment and H2A ubiquitination[J].Science,318(5849):447-450.
Lee S,Lee J W,Lee S K.2012.UTX,a histone H3-lysine 27demethylase,acts as a critical switch to activate the cardiac developmental program[J].Developmental Cell,22(1):25-37.
Mansour A A,Gafni O,Weinberger L,et al.2012.The H3K27demethylase Utx regulates somatic and germ cell epigenetic reprogramming[J].Nature,488(7411):409-413.
Martin C,Zhang Y.2005.The diverse functions of histone lysine methylation[J].Nature Reviews Molecular Cell Biology,6(11):838-849.
Mikkelsen T S,Hanna J,Zhang X,et al.2008.Dissecting direct reprogramming through integrative genomic analysis[J].Nature,454(7200):49-55.
Miller S A,Mohn S E,Weinmann A S.2010.Jmjd3 and UTXplay a demethylase-independent role in chromatin remodeling to regulate T-box family member-dependen gene expression[J].Molecular Cell,40(4):594-605.
Miyake N,Mizuno S,Okamoto N,et al.2013.KDM6A poin mutations cause Kabuki syndrome[J].Human Mutation34(1):108-110.
Pan D,Huang L,Zhu L J,et al.2015.Jmjd3-mediated H3K27me3 dynamics orchestrate brown fat development and regulate white fat plasticity[J].Developmenta Cell,35(5):568-583.
Rothbart S B,Strahl B D.2014.Interpreting the language of histone and DNA modifications[J].Biochimica et Biophysica Acta,1839(8):627-643.
Seenundun S,Rampalli S,Liu Q C,et al.2010.UTX mediates demethylation of H3K27me3 at muscle-specific genes during myogenesis[J].The EMBO Journal,29(8)1401-1411.
Shi Y,Lan F,Matson C,et al.2004.Histone demethylation mediated by the nuclear amine oxidase homolog LSD1[J].Cell,119(7):941-953.
Shpargel K B,Sengoku T,Yokoyama S,et al.2012.UTX and UTY demonstrate histone demethylase independen function in mouse embryonic development[J].PLoS Genetics,8(9):e1002964.
Shpargel K B,Starmer J,Yee D,et al.2014.KDM6 demethylase independent loss of histone H3 lysine 27 trimethylation during early embryonic development[J].PLoS Genetics,10(8):e1004507.
Thieme S,Gyárfás T,Richter C,et al.2013.The histone demethylase UTX regulates stem cell migration and hematopoiesis[J].Blood,21(13):2462-2473.
van Haaften G,Dalgliesh G L,Davies H,et al.2009.Somatic mutations of the histone H3K27 demethylase gene UTXin human cancer[J].Nature Genetics,41(5):521-523.
Voigt P,Tee W W,Reinberg D.2013.A double take on bivalent promoters[J].Genes&Development,27(12):1318-1338.
Wang A H,Zare H,Mousavi K,et al.2013.The histone chaperone Spt6 coordinates histone H3K27 demethylation and myogenesis[J].The EMBO Journal,32(8):1075-1086.
Wang C,Lee J E,Cho Y W,et al.2012.UTX regulates mesoderm differentiation of embryonic stem cells independent of H3K27 demethylase activity[J].Proceedings of the National Academy of Sciences of the USA,109(38):15324-15329.
Wang S P,Tang Z,Chen C W,et al.2017.A UTX-MLL4-p300 Transcriptional regulatory network coordinately shapes active enhancer landscapes for eliciting transcription[J].Molecular Cell,67(2):308-321.
Welstead G G,Creyghton M P,Bilodeau S,et al.2012.X-linked H3K27me3 demethylase Utx is required for embryonic development in a sex-specific manner[J].Proceedings of the National Academy of Sciences of the USA,109(32):13004-13009.
Wu S C,Kallin E M,Zhang Y.2010.Role of H3K27 methylation in the regulation of lncRNA expression[J].Cell Research,20(10):1109-1116.
Yang L,Song L S,Liu X F,et al.2016.The maternal effect genes UTX and JMJD3 play contrasting roles in Mus musculus preimplantation embryo development[J].Scientific Reports,6:26711.
Zenk F,Loeser E,Schiavo R,et al.2017.Germ line-inherited H3K27me3 restricts enhancer function during maternalto-zygotic transition[J].Science,357(6347):212-216.
Zhang M,Wang F,Kou Z,et al.2009.Defective chromatin structure in somatic cell cloned mouse embryos[J].Journal of Biological Chemistry,284(37):24981-24987.