DNA甲基转移酶分类、功能及其研究进展

DNA甲基化是一种在原核和真核生物基因组中常见的复制后修饰,参与体内多种重要生理过程,主要包括:调节基因表达,基因印记,维持染色体完整性以及X-染色体灭活等.依据结构和功能的不同,哺乳动物中DNA甲基转移酶(Dnmts)主要分为两大类:DNA甲基化维持酶Dnmtl以及DNA从头甲基化酶Dnmt3a、,Dnmt3b和Dnmt3L等.此外,Dnmt2也具有弱的DNA甲基转移酶活性,近年来发现它可以甲基化tRNAAsp反密码子环处38C.这些Dnmts对于哺乳动物的生长发育是十分重要的,它们的功能异常将导致胚胎发育障碍,癌症等多种疾病.因此,Dnmts可能成为一个重要的分子靶标,在疾病的治疗和预防中发挥重要作用.文章就Dnmts的分类、功能以及研究进展进行综述.     

DNA甲基转移酶

DNA甲基化在基因调节和动物发育中起着重要作用。负责DNA甲基化作用的酶尔为DNA甲基转移酶（Dnmts）。到目前为止,在哺乳动物细胞中已经鉴定了三种DNA甲基转移酶基因家族,即Dnmt1、Dnmt2和Dnmt3。鉴定和研究DNA甲基转移酶对阐明DNA甲基化机制起着关键的作用。     

真核生物的DNA甲基转移酶与DNA甲基化

真核生物的ＤＮＡ甲基化就是在ＤＮＡ的ＣｐＧ二核苷酸胞嘧啶的第 5位碳原子上加上甲基 ,催化这一过程的是ＤＮＡ甲基转移酶 (Ｄｎｍｔ)。ＤＮＡ的甲基化修饰参与基因表达调控、胚胎发育、细胞分化、基因组印迹、Ｘ染色体灭活和细胞记忆等诸多重要生物学过程[1,2 ] 。在不同组织或同一类型细胞的不同发育阶段 ,基因组ＤＮＡ上各ＣｐＧ位点甲基化状态的差异即构成基因组的ＤＮＡ甲基化谱。根据催化反应类型。可以将ＤＮＡ甲基转移酶分为三类 :第一类将腺嘌呤转化成Ｎ6 甲基腺嘌呤 ;第二类将胞嘧啶转化成Ｎ4 甲基胞嘧啶 ;第三类将胞嘧啶转化成…     

DNA从头甲基转移酶3a和3b

ＤＮＡ甲基化是真核生物基因表达调控的一种方式。通过在ＤＮＡ的ＣＧ二核苷酸胞嘧啶的第 5位碳原子上加上甲基 ,即可抑制或关闭基因表达 ,催化这一过程的是ＤＮＡ甲基转移酶 (Ｄｎｍｔ)。直到 2年前在哺乳动物中还只鉴定出一种Ｄｎｍｔ,即从人和鼠细胞中克隆出的Ｄｎｍｔ1。Ｄｎｍｔ1在体内和体外都有维持甲基化酶 (ｍａｉｎｔｅｎａｎｃｅｍｅｔｈｙｌａｓｅ)的活性 ,即按照模板的甲基化模式 ,对新生的ＤＮＡ链进行甲基化 ,将亲代的甲基化模式遗传给子代。虽然在体外 ,Ｄｎｍｔ1也能将未修饰ＤＮＡ从头甲基化(ｄｅｎｏｖｏｍｅｔｈｙｌａ…     

O^6—甲基鸟嘌呤—DNA—甲基转移酶的基因表达调控

DNA损伤修复是生命科学研究的重要课题。DNA修复在防止基因突变,保证遗传信息稳定性的过程中起着极其重要的作用。近20年来,人们通过大量研究发现细胞内0~6-甲基鸟嘌呤-DNA-甲基转移酶(O~6-metnylguanine-DNA-methyltransferase,MGMT)能专一性很强地修复DNA烷化损伤,防止细胞癌变和死亡;大量试验还证明细胞内MGMT活性的高低是决定肿瘤细胞对亚硝脲类药物产生耐药性的分子基础。详尽了解MGMT基因表达调控机理对DNA损伤修复的理论研究和克服肿瘤细胞耐药性的实践都具有重要的意义。     

哺乳动物DNA甲基化修饰的动态调控机制

DNA甲基化是最主要的表观遗传修饰之一,主要发生在胞嘧啶第五位碳原子上,称为5-甲基胞嘧啶。哺乳动物DNA甲基化由从头DNA甲基转移酶DNMT3A/3B在胚胎发育早期建立。细胞分裂过程中甲基化模式的维持由DNA甲基转移酶DNMT1实现。TET家族蛋白氧化5-甲基胞嘧啶成为5-羟甲基胞嘧啶、5-醛基胞嘧啶和5-羧基胞嘧啶,从而起始DNA的去甲基化过程。这些DNA甲基化修饰酶精确调节DNA甲基化的动态过程,在整个生命发育过程中发挥重要作用,其失调也与多种疾病发生密切相关。本文对近年来DNA甲基化修饰酶的结构与功能研究进行讨论。     

组蛋白甲基转移酶G9a在表观遗传调控中的作用

组蛋白赖氨酸甲基化在表观遗传调控中起着关键作用。组蛋白甲基转移酶G9a(又称作常染色质组蛋白赖氨酸N-甲基转移酶2(euchromatic histone-lysine N-methyltransferase 2,EHMT2))含经典的SET结构域,是常染色质主要的甲基转移酶之一,可以甲基化组蛋白H3K9、H3K27和H1bK26等。此外,G9a也可以直接甲基化一些非组蛋白,并与DNA甲基化密切相关。G9a功能紊乱可以导致胚胎发育异常、免疫系统及神经系统发育障碍、甚至癌症的发生发展。     

DNA甲基转移酶RNAi干涉载体的构建及其鉴定

DNA甲基化(DNA Methylation)是真核生物基因组最常见的DNA共价修饰形式,影响蛋白质-DNA的相互作用,在基因表达的调控上起着重要作用,RNAi(RNA interference)干涉是关闭特定基因功能的新技术,在植物功能基因组、植物发育及生理代谢途径调控等方面有着广泛应用.本文根据植物DNA甲基转移酶(DNMTs)的保守序列设计引物,首先从拟南芥总基因组DNA中克隆出DNA甲基转移酶基因保守片段,然后以此保守片段为模板扩增长度约570bp,的靶序列用于构建RNAi载体.根据RNAi作用机制,将570bp靶序列正向、反向连接到pHANNIBAL载体上,然后将带有此反向重复结构的完整OFF框连接到植物表达栽体pGreell上,经过酶切鉴定和测序分析证实DNA甲基转移酶RNAi重组表达载体构建成功.转化红豆衫细胞表明该干涉载体具有生物学功能,为研究受DNA甲基化调控的性状改良打下基础.     

甲基化修饰在细菌表观调控中的功能

为了适应复杂多变的生存环境,微生物通常需要在保证基因组序列不变的前提下不断调整胞内代谢网络。表观调控可以在不改变DNA序列的情况下对基因表达进行调控,因此成为细菌中重要的调控方式。作为一种DNA修饰,DNA甲基化修饰是生物体中最常见的表观调控工具。在本文中我们全面、深入解析了两种孤儿甲基转移酶:DNA腺嘌呤甲基转移酶(DNA adenine methyltransferase,Dam)和细胞周期调控甲基转移酶(Cell cycle-regulated methyltransferase,Ccr M)在原核生物中的表观调控功能。我们主要探讨了DNA甲基化参与的细胞生理过程包括DNA复制起始、DNA错配修复、基因表达调控、致病性和相变异等方面。同时,我们结合三维基因组研究技术基因组结构捕获(Chromosome conformation capture,3C)技术和新型DNA磷硫酰化修饰讨论了该领域的发展前景。     

Mammalian DNA methyltransferases show different subnuclear distributions

In mammalian cells, DNA methylation patterns are precisely maintained after DNA replication with defined changes occurring during development. The major DNA methyltransferase (Dnmt1) is associated with nuclear replication sites during S-phase, which is consistent with a role in maintenance methylation. The subcellular distribution of the recently discovered de novo DNA methyltransferases, Dnmt3a and Dnmt3b, was investigated by immunofluorescence and by epitope tagging. We now show that both Dnmt3a and Dnmt3b are distributed throughout the nucleoplasm but are not associated with nuclear DNA replication sites during S-phase. These results suggest that de novo methylation by Dnmt3a and Dnmt3b occurs independently of the replication process and might involve an alternative mechanism for accessing the target DNA. The different subcellular distribution of mammalian DNA methyltransferases might thus contribute to the regulation of DNA methylation.     

Application of DNA methyltransferases in targeted DNA methylation

Taxol is a valuable plant-derived drug showing activity against various cancer types. Worldwide efforts had been made to overcome the supply problem, because the supply by isolation from the bark of the slow-growing yew trees is limited. Plant cell cultures as well as chemical and biotechnological semisynthesis are processes, which are intensively investigated for the production of taxanes paclitaxel (Taxol) and docetaxel (Taxotere) in the last few years. This article provides a comparison of the current research on taxane biosynthesis and production in yew cell cultures.     

Mycoplasma CG- and GATC-specific DNA methyltransferases selectively and efficiently methylate the host genome and alter the epigenetic landscape in human cells

Aberrant DNA methylation is frequently observed in disease, including many cancer types, yet the underlying mechanisms remain unclear. Because germline and somatic mutations in the genes that are responsible for DNA methylation are infrequent in malignancies, additional mechanisms must be considered. Mycoplasmas spp., including Mycoplasma hyorhinis, efficiently colonize human cells and may serve as a vehicle for delivery of enzymatically active microbial proteins into the intracellular milieu. Here, we performed, for the first time, genome-wide and individual gene mapping of methylation marks generated by the M. hyorhinis CG- and GATC-specific DNA cytosine methyltransferases (MTases) in human cells. Our results demonstrated that, upon expression in human cells, MTases readily translocated to the cell nucleus. In the nucleus, MTases selectively and efficiently methylated the host genome at the DNA sequence sites free from pre-existing endogenous methylation, including those in a variety of cancer-associated genes. We also established that mycoplasma is widespread in colorectal cancers, suggesting that either the infection contributed to malignancy onset or, alternatively, that tumors provide a favorable environment for mycoplasma growth. In the human genome, ∼11% of GATC sites overlap with CGs (e.g., CGAT^mCG); therefore, the methylated status of these sites can be perpetuated by human DNMT1. Based on these results, we now suggest that the GATC-specific methylation represents a novel type of infection-specific epigenetic mark that originates in human cells with a previous exposure to infection. Overall, our findings unveil an entirely new panorama of interactions between the human microbiome and epigenome with a potential impact in disease etiology.