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。     

DNA甲基化及其对植物发育的调控

DNA甲基化属于一种表观遗传修饰,主要发生在CpG双核苷酸序列中的胞嘧啶上,是在DNA甲基转移酶催化下,以S-腺苷甲硫氨酸为甲基供体,将甲基转移到胞嘧啶上,生成5-甲基胞嘧啶的一种反应。DNA甲基化在植物生长过程中具有极其重要的作用。综述了植物DNA甲基化的特征、调控机制,及其对植物基因表达影响的研究进展。     

DNA主动去甲基化的Science之路

DNA甲基化是一种非常重要的表观遗传调控方式,在基因印迹、X染色体失活、转座子与外源DNA的沉默及组织特异性基因的中发挥着重要的作用。在哺乳动物的配子发生过程及从受精到着床的早期胚胎发育阶段,基因组DNA发生大规模的主动去甲基化。但去甲基化的分子机制一直是表观遗传领域的谜题。2009年,Anjana Rao及其同事发现一种DNA双氧化酶TET蛋白能够将5-甲基胞嘧啶氧化成5-羟甲基胞嘧啶,这为DNA去甲基化的机制研究开拓了新的思路。在此基础上,徐国良实验室展开了深入研究,发现TET蛋白能够进一步将5-羟甲基胞嘧啶氧化成5-羧基胞嘧啶,并发现糖苷酶TDG能够特异性地识别并切除DNA中的5-羧基胞嘧啶,进而启动碱基切除修复途径完成DNA去甲基化,从而提出了氧化作用与碱基切除修复途径协同介导的DNA主动去甲基化机制。     

哺乳动物DNA甲基化修饰的动态调控机制

DNA甲基化是生命体最主要的表观遗传修饰之一。哺乳动物DNA甲基化主要发生在胞嘧啶第五位碳原子上,称为5-甲基胞嘧啶(5-methylcytosine,5m C)。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,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。现结合国内外同行研究进展,介绍课题组近年来对DNA甲基化修饰酶的结构与功能研究。     

环境因素对DNA甲基化的影响

在哺乳动物中, DNA甲基化是指在DNA 甲基转移酶(DNA-methyl transferase, DNMT)的作用下, 以S-腺苷甲硫氨酸提供甲基供体, 将其甲基转移到脱氧胞嘧啶环第5位碳原子形成甲基化脱氧胞嘧啶的共价修饰。DNA甲基化改变组蛋白和DNA之间的相互作用, 使染色质构象发生改变从而影响基因的表达, 总体来说DNA甲基化水平与基因的表达呈负相关。越来越多的报道证实, 环境因素可以影响表观遗传修饰, 其并没有涉及遗传信息的改变, 所以在一定范围内可以解释表型变化。文章围绕环境因素(温度、营养供给、异常化学因子、早期环境刺激和辐射等)对DNA甲基化产生的影响进行综述, 这些影响包括亲代和子代DNA甲基化的改变及子代行为和表型变化等方面, 以期进一步阐释环境因素与基因互作的关系。     

RNA介导的植物DNA甲基化研究进展

RNA介导的DNA甲基化作用(RNA-directed DNA Methylation,RdDM)是首次在植物中发现的基因组表观修饰现象,RdDM通过RNA-DNA序列相互作用直接导致DNA甲基化。植物中的RdDM和siRNA介导的mRNA降解现象,都是通过RNA使序列特异性基因发生沉默,它们对于植物的染色体重排、抵御病毒感染、基因表达调控和发育的许多过程起到了非常重要的作用。在植物中有很多的文献报道RdDM现象,但是对于其具体调控机理还不是很清楚。这里对RNA介导的植物DNA甲基化的基本特征进行了简要概述,主要对RdDM机理的研究进展进行了综述,其中包括RdDM过程中的DNA甲基转移酶的种类及其作用机理,DNA甲基化与染色质修饰之间的关系,以及与RdDM相关的重要蛋白质的研究等。在植物中,转录和转录后水平都可能发生RdDM,诱发基因沉默,前者常涉及靶基因启动子的甲基化,后者则牵涉到编码区的甲基化。RdDM的发生依赖于RNAi途径中相似的siRNA和酶,如DCL3、RdR2、SDE4和AGO4。植物中至少含有三类DNA甲基转移酶DRM1/2、MET1和CMT3,其作用部位是与RNA同源的DNA区域中的所有胞嘧啶,而组蛋白H3第九位赖氨酸的甲基化影响着胞嘧啶的甲基化。     

DNA甲基化在中枢神经系统发育中的研究进展

DNA甲基化是最早被发现的表观遗传修饰之一。近年来,大量的研究显示DNA甲基化在中枢神经系统(CNS)发育中发挥了重要作用。不同种类的DNA甲基转移酶(Dnmt)和DNA甲基结合蛋白(MBD)在CNS发育的不同阶段发挥不同的作用。DNA甲基化促进神经干细胞向神经元方向分化,抑制其向胶质细胞分化。Dnmt和MBD主要在神经元中表达,而在胶质细胞不表达或表达较少。DNA甲基化调节神经发生和突触的形成,参与学习记忆。星型胶质细胞的标志物GFAP去甲基化促进早期神经上皮分化为星型胶质细胞。少突胶质细胞相关基因MAG和Sox10等也受甲基化的调节。本文主要从以上方面综述了DNA甲基化在中枢神经系统发育中的作用。     

综述: DNA甲基化在动物胚胎和生殖细胞发育过程中的重编程

表观遗传信息DNA甲基化在动物的发育、细胞分化和器官形成过程中,起着至关重要的作用．近期,关于DNA甲基化在脊椎动物胚胎发育和生殖细胞发育过程重编程的研究取得了重要的进展．发现斑马鱼的早期胚胎完整地继承了精子的DNA甲基化图谱,而哺乳动物的早期胚胎和原始生殖细胞发育过程则经历了整体去甲基化并重新建立甲基化图谱的过程,但胚胎发育过程中基因的印迹区未发生DNA去甲基化,而生殖细胞发育过程中印迹区的甲基化修饰被消除．     

TET家族DNA羟化酶与5-hmC在肿瘤中的作用机制的研究进展

DNA甲基化失调引起基因表达异常是表观遗传学的一个显著特点。目前已知,由DNA甲基转移酶（DNA methyltransferases,DMNTs）催化DNA甲基化,其酶基因突变或表达异常引起DNA甲基化水平的改变。近期研究发现了一种DNA去甲基化酶--TET（Ten-Eleventranslocation）家族DNA羟化酶,能通过多种途径催化5-甲基胞嘧啶（5．methylcytosine,5-mC）去甲基化,从而调控DNA基化的平衡。5-羟甲基胞嘧啶（5-hydroxymethylcytosine,5-hmC）作为DNA去甲基化多重步骤中重要的中间产物,其水平在肿瘤的发生和发展时期发生显著变化。该文从TET家族蛋白展开,介绍TET蛋白的结构、功能及作用机制以及多种人类肿瘤中丁E丁家族基因与5-hmC水平的相关性及其对肿瘤发生发展、诊断预后等临床意义的研究进展。     

DNA羟甲基化在小鼠胚胎干细胞中的研究进展

简要总结DNA羟甲基化在小鼠胚胎干细胞(mouse embryonic stem cells,mESC)中的最新研究进展.DNA甲基化(DNAmethylation)影响染色质的结构与功能,在发育与疾病发生过程中具有重要作用.2009年Tahiliani等发现TET1可以催化甲基化胞嘧啶(5-methylcytosine,5mC)氧化为羟甲基化胞嘧啶(5-hydroxymethylcytosine,5hmC).DNA羟甲基化(DNAhydroxymethylation)被认为是调节DNA甲基化的一种重要方式,成为了表观遗传学的研究热点之一.     

Uhrf1 and Dnmt1 are required for development and maintenance of the zebrafish lens

DNA methylation is one of the key mechanisms underlying the epigenetic regulation of gene expression. During DNA replication, the methylation pattern of the parent strand is maintained on the replicated strand through the action of Dnmt1 (DNA Methyltransferase 1). In mammals, Dnmt1 is recruited to hemimethylated replication foci by Uhrf1 (Ubiquitin-like, Containing PHD and RING Finger Domains 1). Here we show that Uhrf1 is required for DNA methylation in vivo during zebrafish embryogenesis. Due in part to the early embryonic lethality of Dnmt1 and Uhrf1 knockout mice, roles for these proteins during lens development have yet to be reported. We show that zebrafish mutants in uhrf1 and dnmt1 have defects in lens development and maintenance. uhrf1 and dnmt1 are expressed in the lens epithelium, and in the absence of Uhrf1 or of catalytically active Dnmt1, lens epithelial cells have altered gene expression and reduced proliferation in both mutant backgrounds. This is correlated with a wave of apoptosis in the epithelial layer, which is followed by apoptosis and unraveling of secondary lens fibers. Despite these disruptions in the lens fiber region, lens fibers express appropriate differentiation markers. The results of lens transplant experiments demonstrate that Uhrf1 and Dnmt1 functions are required lens-autonomously, but perhaps not cell-autonomously, during lens development in zebrafish. These data provide the first evidence that Uhrf1 and Dnmt1 function is required for vertebrate lens development and maintenance.     

Epigenetic regulation of left–right asymmetry by DNA methylation

DNA methylation is a major epigenetic modification; however, the precise role of DNA methylation in vertebrate development is still not fully understood. Here, we show that DNA methylation is essential for the establishment of the left–right (LR) asymmetric body plan during vertebrate embryogenesis. Perturbation of DNA methylation by depletion of DNA methyltransferase 1 (dnmt1) or dnmt3bb.1 in zebrafish embryos leads to defects in dorsal forerunner cell (DFC) specification or collective migration, laterality organ malformation, and disruption of LR patterning. Knockdown of dnmt1 in Xenopus embryos also causes similar defects. Mechanistically, loss of dnmt1 function induces hypomethylation of the lefty2 gene enhancer and promotes lefty2 expression, which consequently represses Nodal signaling in zebrafish embryos. We also show that Dnmt3bb.1 regulates collective DFC migration through cadherin 1 (Cdh1). Taken together, our data uncover dynamic DNA methylation as an epigenetic mechanism to control LR determination during early embryogenesis in vertebrates.     

DNA methylation is a primary mechanism for silencing postmigratory primordial germ cell genes in both germ cell and somatic cell lineages

DNA methylation is necessary for the silencing of endogenous retrotransposons and the maintenance of monoallelic gene expression at imprinted loci and on the X chromosome. Dynamic changes in DNA methylation occur during the initial stages of primordial germ cell development; however, all consequences of this epigenetic reprogramming are not understood. DNA demethylation in postmigratory primordial germ cells coincides with erasure of genomic imprints and reactivation of the inactive X chromosome, as well as ongoing germ cell differentiation events. To investigate a possible role for DNA methylation changes in germ cell differentiation, we have studied several marker genes that initiate expression at this time. Here, we show that the postmigratory germ cell-specific genes Mvh, Dazl and Scp3 are demethylated in germ cells, but not in somatic cells. Premature loss of genomic methylation in Dnmt1 mutant embryos leads to early expression of these genes as well as GCNA1, a widely used germ cell marker. In addition, GCNA1 is ectopically expressed by somatic cells in Dnmt1 mutants. These results provide in vivo evidence that postmigratory germ cell-specific genes are silenced by DNA methylation in both premigratory germ cells and somatic cells. This is the first example of ectopic gene activation in Dnmt1 mutant mice and suggests that dynamic changes in DNA methylation regulate tissue-specific gene expression of a set of primordial germ cell-specific genes.     

MPTP诱导的帕金森病小鼠模型黑质脑组织DNA甲基化研究

为研究DNA甲基化在帕金森病发病机制中的作用,本研究用环境毒素1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)连续腹腔给药诱导小鼠帕金森病(Parkison's disease,PD)模型,应用ELISA检测小鼠黑质脑组织总体甲基化水平,应用实时荧光定量PCR方法检测DNA甲基转移酶表达水平,探讨MPTP诱导的小鼠PD模型黑质部位是否存在DNA甲基化异常.进一步应用甲基化DNA免疫共沉淀结合DNA甲基化芯片方法,构建MPTP诱导的小鼠PD模型黑质脑组织DNA甲基化谱,并寻找DNA甲基化修饰异常的PD相关基因对其进行验证.结果表明,模型组小鼠黑质脑组织DNA总体甲基化水平较对照组显著降低,Dnmt1的表达水平显著增高.利用DNA甲基化芯片在全基因组内筛选出甲基化差异修饰位点共48个,涉及44个基因,这些甲基化差异基因参与信号转导、分子转运、转录调控、发育、细胞分化、凋亡调控、氧化应激、蛋白质降解等生物学过程.在甲基化差异修饰基因中,对Uchl1基因及Arih2基因进行了甲基化水平以及表达水平的验证.结果表明,模型组小鼠黑质脑组织Uchl1启动子区域甲基化水平较对照组增高,m RNA及蛋白质表达水平降低,Arih2启动子区域甲基化水平较对照组降低,m RNA及蛋白质表达水平增高.实验结果进一步证实,DNA甲基化修饰异常在帕金森病发病机制中有重要作用,环境因素(如MPTP)可以通过改变DNA甲基化修饰参与帕金森病的发生发展.     

Variant Histone H2afv reprograms DNA methylation during early zebrafish development

The DNA methylome is re-patterned during discrete phases of vertebrate development. In zebrafish, there are 2 waves of global DNA demethylation and re-methylation: the first occurs before gastrulation when the parental methylome is changed to the zygotic pattern and the second occurs after formation of the embryonic body axis, during organ specification. The occupancy of the histone variant H2A.Z and regions of DNA methylation are generally anti-correlated, and it has been proposed that H2A.Z restricts the boundaries of highly methylated regions. While many studies have described the dynamics of methylome changes during early zebrafish development, the factors involved in establishing the DNA methylation landscape in zebrafish embryos have not been identified. We test the hypothesis that the zebrafish ortholog of H2A.Z (H2afv) restricts DNA methylation during development. We find that, in control embryos, bulk genome methylation decreases after gastrulation, with a nadir at the bud stage, and peaks during mid-somitogenesis; by 24 hours post -fertilization, total DNA methylation levels return to those detected in gastrula. Early zebrafish embryos depleted of H2afv have significantly more bulk DNA methylation during somitogenesis, suggesting that H2afv limits methylation during this stage of development. H2afv deficient embryos are small, with multisystemic abnormalities. Genetic interaction experiments demonstrate that these phenotypes are suppressed by depletion of DNA methyltransferase 1 (Dnmt1). This work demonstrates that H2afv is essential for global DNA methylation reprogramming during early vertebrate development and that embryonic development requires crosstalk between H2afv and Dnmt1.     

Aberrant DNA methylation in 5'regions of DNA methyltransferase genes in aborted bovine clones

High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning.It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation.DNA methylation is established and maintained by DNA methyltransferases(DNMTs),therefore,it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs.Since DNA methylation can strongly inhibit gene expression,aberrant DNA methylation of DNMT genes may disturb gene expression.But presently,it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos.In our study,we analyzed methylation patterns of the 5' regions of four DNMT genes including Dnmt3a,Dnmt3b,Dnmtl and Dnmt2 in four aborted bovine clones.Using bisulfite sequencing method,we found that 3 out of 4 aborted bovine clones(AF1,AF2 and AF3)showed either hypermethylation or hypomethylation in the 5' regions of Dnmt3a and Dnmt3b.indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed.However,the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized (IVF)fetuses.Besides,we found that tle 5'regions of Dnmtl and Dnmt2 were nearly completely unmethylated in all normal adults.IVF fetuses,sperm and aborted clones.Together,our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5' regions is probably associated with the high abortion of bovine clones.     

Novel splice variants associated with one of the zebrafish <Emphasis Type="Italic">dnmt3</Emphasis>genes

Background  

DNA methylation and the methyltransferases are known to be important in vertebrate development and this may be particularly true for the Dnmt3 family of enzymes because they are thought to be the de novo methyltransferases. Mammals have three Dnmt3 genes; Dnmt3a, Dnmt3b, and Dnmt3L, two of which encode active enzymes and one of which produces an inactive but necessary cofactor. However, due to multiple promoter use and alternative splicing there are actually a number of dnmt3 isoforms present. Six different dnmt3 genes have recently been identified in zebrafish.