拟南芥DNA甲基转移酶DNMT2与组蛋白去乙酰化酶家族HD2互作及功能研究
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摘要
表观遗传学是指DNA序列不发生变化,而序列上的修饰发生变化从而影响生物体的表型性状和遗传功能。DNA甲基化和组蛋白去乙酰化是经典的表观遗传学现象,在生命进程中发挥着重要的调节作用。目前,表观修饰对植物逆境胁迫应答调控的研究取得了很大进展,这方面的研究对阐明植物响应逆境胁迫的分子机制具有重要意义。本论文利用生物化学,分子生物学,遗传学分析和生物信息学等方法,对拟南芥DNA甲基转移酶DNMT2与组蛋白去乙酰化酶HD2s蛋白家族的互作以及生理生化的功能进行研究,并且通过基因芯片分析,对DNA甲基化和组蛋白去乙酰化在冷胁迫下对基因表达的调控进行了系统的分析和全面的阐述,为表观遗传学调控植物逆境耐受提供了理伦依据。研究结果如下：
1.通过共定位Co-localization分析,双分子荧光互补实验Bimolecular fluorescence complementation分析和体外免疫共沉淀pull down分析,证实DNMT2亚细胞定位在细胞核内,在体内和体外能够与组蛋白去乙酰化酶HD2s家族互作。
2. GAL4/UAS系统被用于DNMT2抑制基因表达的研究,通过DNMT2::GUS融合蛋白表达量的测定,对DNMT2基因domain的功能进行了分析,我们确定和绘制了DNMT2互作和抑制区域,靠近C端(aa177-384)是基因抑制区,可以有效的结合靶向基因并且抑制表达。而靠近N端(aal-105)可以与HD2s互作,与组蛋白去乙酰化的活性紧密相联。
3.分析了干旱、冷胁迫、盐胁迫和ABA对拟南芥DNMT2, HD2s表达的影响,结果发现冷胁迫下HD2C的表达有升高的趋势,DNMT2的表达在3小时出现了升高。
构建pEarleyGate101-35S::DNMT2,然后转化野生型拟南芥(Col-0),得到了DNMT2超表达的转基因植株,并对表型进行了观察。培养20天的野生型植物和35S::DNMT2转基因植物以及35S:://D2C(COE)转基因植物和HD2C-mut (T99)在0℃下放置不同的时间段,然后对ICE1和COR1基因表达量进行了分析,结果发现HD2C对冷应答基因具有一定的影响。DNMT2虽然没有直接的参与这个活动,但是可能是间接调控机体活动的重要因素。
4.同时在HD2C不同表达体系中：35s::HD2C转基因过表达植物(COE)和hd2c knock down突变体植物(T99),通过类ELISA反应和RT-PCR对DNMTs活性以及DNMT2表达量的分析得到了表达一致的结论,呈现了一种协同作用的关系。
5.通过免疫共沉淀Co-IP,和MALDI-ToF质谱分析,对DNMT2的潜在互作蛋白进行了分析,发现与鸟苷酸环化脱氢酶有关系,说明DNMT2可能与植物代谢途径有关。
6.对DNA甲基化抑制剂(5-aza-2'-deoxycytosine)预处理的植株,组蛋白去乙酰化抑制剂(trichostatin A)预处理的植株与对照植株进行0℃冷胁迫,处理24小时后,利用基因芯片手段,进行基因表达比对分析,并在相关的数据库进行了衍射。大量的数据分析结果显示,DNA甲基化和组蛋白去乙酰化不是完全的协同,即使是冷胁迫下,也大体呈现了拮抗的关系,只有局部是协同的,说明了这两种表观修饰在体内是动态平衡和互补的关系。
综合以上的研究结果,我们认为DNA甲基转移酶DNMT2参与了组蛋白去乙酰化酶HD2s基因家族的活动,具有抑制基因表达的功能,对植物的生长发育有调节作用。通过基因芯片对基因表达进行高通量的分析,讨论了植物DNA甲基化和组蛋白去乙酰化与抗冻的关系,我们希望对阐明表观修饰调节机理,以及为植物遗传工程提高作物的抗逆境能力提供一定的理伦依据。
Epigenetics is the study of heritable changes in gene function that occur without a change in the DNA sequence. DNA methylation and histone dacetylation are common epigenetic modification phenomena. They play important roles in eukaryotic development including the perception and transduction of the changes of internal (hormonal) and environmental signals. DNA methylation is a universal modification phenomenon presentted in the eukaryotic genome. It regulates the expression of corresponding genes through the changes of chromatin structure in plant growth and development. Histone post-translational modifications, especial acetylation and deacetylation, are other important ways of regulating eukaryotic gene expression. They are closely linked to DNA methylation. Plants contain a unique type of histone deacetylase called HD2-type. DNA methytransferase2 (DNMT2) is always deemed to be enigmatic over a long period of time, because it contains highly conserved DNA methyltransferase motifs but lacks the DNA methylation catalytic capability. DNMT2 is also deemed to the ancestor of DNA methyltransferase family because of its structure.
In this study AtDNMT2 is localized in nucleus. We have shown that AtDNMT2 can interact with AtHD2 family except AtHD2D both in vivo and in vitro and have mapped the interaction and the repression domains in AtDNMT2. Domains at C-terminal of AtDNMT2 are able to mediate repression of gene expression and the domain close to the N-terminal can interact with AtHD2C. This interaction potentially affects correlative enzyme activity. A decrease in the expression of AtDNMT2 is found in athd2c knock-out line. These results show the evidence for a possible function of AtDNMT2 that involves in histone deacetylation activity. While the activity of DNMTs presents synergistic in 35s::HD2C transgenic plants (COE) and hd2c knock down mutant plants (T99). Microarray analysis was performed to test the cold tolerance of DNA methylation inhibitors (5-aza-2'-deoxycytosine) treated plants and histone deacetylase inhibitor (trichostatin A) treated plants. The different expression of genes were analysed by bioinformatics to find correlative pathways and transcription factor. DNA methylation and histone acetylation are critical pathways on cold reponses. DNA methylation and histone acetylation is not a simple synergistic or antagonistic, to some extent, they form a complementary or balanced system.
In summary, DNMT2 can repress gene expression and involves in histone deacetylase HD2s gene family activity. DNMT2 plays important role in plant growth and development. These results have raised the likelihood that the biological roles of DNMT2 might be broader than those have been previously considered. Through exploring the mechanism of epigenetic regulation in plant cold tolerance, we hope our research might provide some promising clues for future applications in crop plants.
1.通过共定位Co-localization分析,双分子荧光互补实验Bimolecular fluorescence complementation分析和体外免疫共沉淀pull down分析,证实DNMT2亚细胞定位在细胞核内,在体内和体外能够与组蛋白去乙酰化酶HD2s家族互作。
2. GAL4/UAS系统被用于DNMT2抑制基因表达的研究,通过DNMT2::GUS融合蛋白表达量的测定,对DNMT2基因domain的功能进行了分析,我们确定和绘制了DNMT2互作和抑制区域,靠近C端(aa177-384)是基因抑制区,可以有效的结合靶向基因并且抑制表达。而靠近N端(aal-105)可以与HD2s互作,与组蛋白去乙酰化的活性紧密相联。
3.分析了干旱、冷胁迫、盐胁迫和ABA对拟南芥DNMT2, HD2s表达的影响,结果发现冷胁迫下HD2C的表达有升高的趋势,DNMT2的表达在3小时出现了升高。
构建pEarleyGate101-35S::DNMT2,然后转化野生型拟南芥(Col-0),得到了DNMT2超表达的转基因植株,并对表型进行了观察。培养20天的野生型植物和35S::DNMT2转基因植物以及35S:://D2C(COE)转基因植物和HD2C-mut (T99)在0℃下放置不同的时间段,然后对ICE1和COR1基因表达量进行了分析,结果发现HD2C对冷应答基因具有一定的影响。DNMT2虽然没有直接的参与这个活动,但是可能是间接调控机体活动的重要因素。
4.同时在HD2C不同表达体系中：35s::HD2C转基因过表达植物(COE)和hd2c knock down突变体植物(T99),通过类ELISA反应和RT-PCR对DNMTs活性以及DNMT2表达量的分析得到了表达一致的结论,呈现了一种协同作用的关系。
5.通过免疫共沉淀Co-IP,和MALDI-ToF质谱分析,对DNMT2的潜在互作蛋白进行了分析,发现与鸟苷酸环化脱氢酶有关系,说明DNMT2可能与植物代谢途径有关。
6.对DNA甲基化抑制剂(5-aza-2'-deoxycytosine)预处理的植株,组蛋白去乙酰化抑制剂(trichostatin A)预处理的植株与对照植株进行0℃冷胁迫,处理24小时后,利用基因芯片手段,进行基因表达比对分析,并在相关的数据库进行了衍射。大量的数据分析结果显示,DNA甲基化和组蛋白去乙酰化不是完全的协同,即使是冷胁迫下,也大体呈现了拮抗的关系,只有局部是协同的,说明了这两种表观修饰在体内是动态平衡和互补的关系。
综合以上的研究结果,我们认为DNA甲基转移酶DNMT2参与了组蛋白去乙酰化酶HD2s基因家族的活动,具有抑制基因表达的功能,对植物的生长发育有调节作用。通过基因芯片对基因表达进行高通量的分析,讨论了植物DNA甲基化和组蛋白去乙酰化与抗冻的关系,我们希望对阐明表观修饰调节机理,以及为植物遗传工程提高作物的抗逆境能力提供一定的理伦依据。
Epigenetics is the study of heritable changes in gene function that occur without a change in the DNA sequence. DNA methylation and histone dacetylation are common epigenetic modification phenomena. They play important roles in eukaryotic development including the perception and transduction of the changes of internal (hormonal) and environmental signals. DNA methylation is a universal modification phenomenon presentted in the eukaryotic genome. It regulates the expression of corresponding genes through the changes of chromatin structure in plant growth and development. Histone post-translational modifications, especial acetylation and deacetylation, are other important ways of regulating eukaryotic gene expression. They are closely linked to DNA methylation. Plants contain a unique type of histone deacetylase called HD2-type. DNA methytransferase2 (DNMT2) is always deemed to be enigmatic over a long period of time, because it contains highly conserved DNA methyltransferase motifs but lacks the DNA methylation catalytic capability. DNMT2 is also deemed to the ancestor of DNA methyltransferase family because of its structure.
In this study AtDNMT2 is localized in nucleus. We have shown that AtDNMT2 can interact with AtHD2 family except AtHD2D both in vivo and in vitro and have mapped the interaction and the repression domains in AtDNMT2. Domains at C-terminal of AtDNMT2 are able to mediate repression of gene expression and the domain close to the N-terminal can interact with AtHD2C. This interaction potentially affects correlative enzyme activity. A decrease in the expression of AtDNMT2 is found in athd2c knock-out line. These results show the evidence for a possible function of AtDNMT2 that involves in histone deacetylation activity. While the activity of DNMTs presents synergistic in 35s::HD2C transgenic plants (COE) and hd2c knock down mutant plants (T99). Microarray analysis was performed to test the cold tolerance of DNA methylation inhibitors (5-aza-2'-deoxycytosine) treated plants and histone deacetylase inhibitor (trichostatin A) treated plants. The different expression of genes were analysed by bioinformatics to find correlative pathways and transcription factor. DNA methylation and histone acetylation are critical pathways on cold reponses. DNA methylation and histone acetylation is not a simple synergistic or antagonistic, to some extent, they form a complementary or balanced system.
In summary, DNMT2 can repress gene expression and involves in histone deacetylase HD2s gene family activity. DNMT2 plays important role in plant growth and development. These results have raised the likelihood that the biological roles of DNMT2 might be broader than those have been previously considered. Through exploring the mechanism of epigenetic regulation in plant cold tolerance, we hope our research might provide some promising clues for future applications in crop plants.
引文
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