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 Demethylation by DNMT3A and DNMT3B in vitro and of Methylated Episomal DNA in Transiently Transfected Cells

The DNA methylation program in vertebrates is an essential part of the epigenetic regulatory cascade of development, cell differentiation, and progression of diseases including cancer. While the DNA methyltransferases (DNMTs) are responsible for the in vivo conversion of cytosine (C) to methylated cytosine (5mC), demethylation of 5mC on cellular DNA could be accomplished by the combined action of the ten-eleven translocation (TET) enzymes and DNA repair. Surprisingly, the mammalian DNMTs also possess active DNA demethylation activity in vitro in a Ca²⁺- and redox conditions-dependent manner, although little is known about its molecular mechanisms and occurrence in a cellular context. In this study, we have used LC-MS/MS to track down the fate of the methyl group removed from 5mC on DNA by mouse DNMT3B in vitro and found that it becomes covalently linked to the DNA methylation catalytic cysteine of the enzyme. We also show that Ca²⁺ homeostasis-dependent but TET1/TET2/TET3/TDG-independent demethylation of methylated episomal DNA by mouse DNMT3A or DNMT3B can occur in transfected human HEK 293 and mouse embryonic stem (ES) cells. Based on these results, we present a tentative working model of Ca²⁺ and redox conditions-dependent active DNA demethylation by DNMTs. Our study substantiates the potential roles of the vertebrate DNMTs as double-edged swords in DNA methylation-demethylation during Ca²⁺-dependent physiological processes.     

The Mammalian de Novo DNA Methyltransferases DNMT3A and DNMT3B Are Also DNA 5-Hydroxymethylcytosine Dehydroxymethylases

For cytosine (C) demethylation of vertebrate DNA, it is known that the TET proteins could convert 5-methyl C (5-mC) to 5-hydroxymethyl C (5-hmC). However, DNA dehydroxymethylase(s), or enzymes able to directly convert 5-hmC to C, have been elusive. We present in vitro evidence that the mammalian de novo DNA methyltransferases DNMT3A and DNMT3B, but not the maintenance enzyme DNMT1, are also redox-dependent DNA dehydroxymethylases. Significantly, intactness of the C methylation catalytic sites of these de novo enzymes is also required for their 5-hmC dehydroxymethylation activity. That DNMT3A and DNMT3B function bidirectionally both as DNA methyltransferases and as dehydroxymethylases raises intriguing and new questions regarding the structural and functional aspects of these enzymes and their regulatory roles in the dynamic modifications of the vertebrate genomes during development, carcinogenesis, and gene regulation.     

The colorful history of active DNA demethylation

Patterns of DNA cytosine methylation are subject to mitotic inheritance in both plants and vertebrates. Plants use 5-methylcytosine glycosylases and the base excision repair pathway to remove excess cytosine methylation. In mammals, active demethylation has been proposed to operate via several very different mechanisms. Two recent reports in Nature now claim that the demethylation process is initiated by the same enzymes that establish the methylation mark, the DNA methyltransferases DNMT3A and DNMT3B (Kangaspeska et al., 2008; Métivier et al., 2008).     

Biochemical characterization of maintenance DNA methyltransferase DNMT-1 from silkworm,Bombyx mori

DNA methylation is an important epigenetic mechanism involved in gene expression of vertebrates and invertebrates. In general, DNA methylation profile is established by de novo DNA methyltransferases (DNMT-3A, -3B) and maintainance DNA methyltransferase (DNMT-1). DNMT-1 has a strong substrate preference for hemimethylated DNA over the unmethylated one. Because the silkworm genome lacks an apparent homologue of de novo DNMT, it is still unclear that how silkworm chromosome establishes and maintains its DNA methylation profile. As the first step to unravel this enigma, we purified recombinant BmDNMT-1 using baculovirus expression system and characterized its DNA-binding and DNA methylation activity. We found that the BmDNMT-1 preferentially methylates hemimethylated DNA despite binding to both unmethylated and hemimethylated DNA. Interestingly, BmDNMT-1 formed a complex with DNA in the presence or absence of methyl group donor, S-Adenosylmethionine (AdoMet) and the AdoMet-dependent complex formation was facilitated by Zn²⁺ and Mn²⁺. Our results provide clear evidence that BmDNMT-1 retained the function as maintenance DNMT but its sensitivity to metal ions is different from mammalian DNMT-1.     

Enzymatic DNA oxidation: mechanisms and biological significance

DNA methylation at cytosines (5mC) is a major epigenetic modification involved in the regulation of multiple biological processes in mammals. How methylation is reversed was until recently poorly understood. The family of dioxygenases commonly known as Ten-eleven translocation (Tet) proteins are responsible for the oxidation of 5mC into three new forms, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Current models link Tet-mediated 5mC oxidation with active DNA demethylation. The higher oxidation products (5fC and 5caC) are recognized and excised by the DNA glycosylase TDG via the base excision repair pathway. Like DNA methyltransferases, Tet enzymes are important for embryonic development. We will examine the mechanism and biological significance of Tet-mediated 5mC oxidation in the context of pronuclear DNA demethylation in mouse early embryos. In contrast to its role in active demethylation in the germ cells and early embryo, a number of lines of evidence suggest that the intragenic 5hmC present in brain may act as a stable mark instead. This short review explores mechanistic aspects of TET oxidation activity, the impact Tet enzymes have on epigenome organization and their contribution to the regulation of early embryonic and neuronal development. [BMB Reports 2014; 47(11): 609-618]     

Dynamic link of DNA demethylation,DNA strand breaks and repair in mouse zygotes

In mammalian zygotes, the 5‐methyl‐cytosine (5mC) content of paternal chromosomes is rapidly changed by a yet unknown but presumably active enzymatic mechanism. Here, we describe the developmental dynamics and parental asymmetries of DNA methylation in relation to the presence of DNA strand breaks, DNA repair markers and a precise timing of zygotic DNA replication. The analysis shows that distinct pre‐replicative (active) and replicative (active and passive) phases of DNA demethylation can be observed. These phases of DNA demethylation are concomitant with the appearance of DNA strand breaks and DNA repair markers such as γH2A.X and PARP‐1, respectively. The same correlations are found in cloned embryos obtained after somatic cell nuclear transfer. Together, the data suggest that (1) DNA‐methylation reprogramming is more complex and extended as anticipated earlier and (2) the DNA demethylation, particularly the rapid loss of 5mC in paternal DNA, is likely to be linked to DNA repair mechanisms.     

TET-TDG Active DNA Demethylation at CpG and Non-CpG Sites

In mammalian genomes, cytosine methylation occurs predominantly at CG (or CpG) dinucleotide contexts. As part of dynamic epigenetic regulation, 5-methylcytosine (mC) can be erased by active DNA demethylation, whereby ten-eleven translocation (TET) enzymes catalyze the stepwise oxidation of mC to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxycytosine (caC), thymine DNA glycosylase (TDG) excises fC or caC, and base excision repair yields unmodified cytosine. In certain cell types, mC is also enriched at some non-CG (or CH) dinucleotides, however hmC is not. To provide biochemical context for the distribution of modified cytosines observed in biological systems, we systematically analyzed the activity of human TET2 and TDG for substrates in CG and CH contexts. We find that while TET2 oxidizes mC more efficiently in CG versus CH sites, this context preference can be diminished for hmC oxidation. Remarkably, TDG excision of fC and caC is only modestly dependent on CG context, contrasting its strong context dependence for thymine excision. We show that collaborative TET-TDG oxidation-excision activity is only marginally reduced for CA versus CG contexts. Our findings demonstrate that the TET-TDG-mediated demethylation pathway is not limited to CG sites and suggest a rationale for the depletion of hmCH in genomes rich in mCH.     

DNA methylation reprogramming of human cancer cells by expression of a plant 5-methylcytosine DNA glycosylase

Patterns of DNA methylation, an important epigenetic modification involved in gene silencing and development, are disrupted in cancer cells. Understanding the functional significance of aberrant methylation in tumors remains challenging, due in part to the lack of suitable tools to actively modify methylation patterns. DNA demethylation caused by mammalian DNA methyltransferase inhibitors is transient and replication-dependent, whereas that induced by TET enzymes involves oxidized 5mC derivatives that perform poorly understood regulatory functions. Unlike animals, plants possess enzymes that directly excise unoxidized 5mC from DNA, allowing restoration of unmethylated C through base excision repair. Here, we show that expression of Arabidopsis 5mC DNA glycosylase DEMETER (DME) in colon cancer cells demethylates and reactivates hypermethylated silenced loci. Interestingly, DME expression causes genome-wide changes that include both DNA methylation losses and gains, and partially restores the methylation pattern observed in normal tissue. Furthermore, such methylome reprogramming is accompanied by altered cell cycle responses and increased sensibility to anti-tumor drugs, decreased ability to form colonospheres, and tumor growth impairment in vivo. Our study shows that it is possible to reprogram a human cancer DNA methylome by expression of a plant DNA demethylase.     

DNA 5-Methylcytosine Demethylation Activities of the Mammalian DNA Methyltransferases

Methylation at the 5-position of DNA cytosine on the vertebrate genomes is accomplished by the combined catalytic actions of three DNA methyltransferases (DNMTs), the de novo enzymes DNMT3A and DNMT3B and the maintenance enzyme DNMT1. Although several metabolic routes have been suggested for demethylation of the vertebrate DNA, whether active DNA demethylase(s) exist has remained elusive. Surprisingly, we have found that the mammalian DNMTs, and likely the vertebrates DNMTs in general, can also act as Ca²⁺ ion- and redox state-dependent active DNA demethylases. This finding suggests new directions for reinvestigation of the structures and functions of these DNMTs, in particular their roles in Ca²⁺ ion-dependent biological processes, including the genome-wide/local DNA demethylation during early embryogenesis, cell differentiation, neuronal activity-regulated gene expression, and carcinogenesis.     

DNA methylation‐mediated epigenetic control

During differentiation and development cells undergo dramatic morphological and functional changes without any change in the DNA sequence. The underlying changes of gene expression patterns are established and maintained by epigenetic processes. Early mechanistic insights came from the observation that gene activity and repression states correlate with the DNA methylation level of their promoter region. DNA methylation is a postreplicative modification that occurs exclusively at the C5 position of cytosine residues (5mC) and predominantly in the context of CpG dinucleotides in vertebrate cells. Here, three major DNA methyltransferases (Dnmt1, 3a, and 3b) establish specific DNA methylation patterns during differentiation and maintain them over many cell division cycles. CpG methylation is recognized by at least three protein families that in turn recruit histone modifying and chromatin remodeling enzymes and thus translate DNA methylation into repressive chromatin structures. By now a multitude of histone modifications have been linked in various ways with DNA methylation. We will discuss some of the basic connections and the emerging complexity of these regulatory networks. J. Cell. Biochem. 108: 43–51, 2009. © 2009 Wiley‐Liss, Inc.     

极性生长的细胞中胞嘧啶甲基化的动态变化及其对GABA信号的响应

DNA胞嘧啶(C)的甲基化(5m C)在植物发育过程中具有重要的调节作用,多种环境因子如逆境胁迫、植物内/外源性因子等均会触发DNA甲基化的变化。为探讨γ-氨基丁酸(GABA)对植物发育的可能调节机制,本研究以极性生长的烟草花粉管和拟南芥根为材料,分析5m C的含量及其对GABA信号的响应。结果表明,1.0 mmol/L GABA能显著促进烟草花粉管和拟南芥根的极性生长;同时,GABA处理使烟草花粉管和拟南芥根的基因组中5m C含量显著降低、5-羟基胞嘧啶(5hm C)含量显著增加。5hm C是5m C去甲基化途径中的一个重要中间产物,本研究证实了GABA可以作为一种重要的外源信号调节DNA甲基化的动态变化。     

Mechanistic Insights into Cytosine-N3 Methylation by DNA Methyltransferase DNMT3A

Recently, it has been discovered that different DNA-(cytosine C5)-methyltransferases including DNMT3A generate low levels of 3mC [Rosic et al. (2018), Nat. Genet., 50, 452–459]. This reaction resulted in the co-evolution of DNMTs and ALKB2 DNA repair enzymes, but its mechanism remained elusive. Here, we investigated the catalytic mechanism of DNMT3A for cytosine N3 methylation. We generated several DNMT3A variants with mutated catalytic residues and measured their activities in 5mC and 3mC generation by liquid chromatography linked to tandem mass spectrometry. Our data suggest that the methylation of N3 instead of C5 is caused by an inverted binding of the flipped cytosine target base into the active-site pocket of the DNA methyltransferase, which is partially compatible with the arrangement of catalytic amino acid residues. Given that all DNA-(cytosine C5)-methyltransferases have a common catalytic mechanism, it is likely that other enzymes of this class generate 3mC following the same mechanism.     

DNA excision repair proteins and Gadd45 as molecular players for active DNA demethylation

DNA cytosine methylation represents an intrinsic modification signal of the genome that plays important roles in heritable gene silencing, heterochromatin formation and certain transgenerational epigenetic inheritance. In contrast to the process of DNA methylation that is catalyzed by specific classes of methyltransferases, molecular players underlying active DNA demethylation have long been elusive. Emerging biochemical and functional evidence suggests that active DNA demethylation in vertebrates can be mediated through DNA excision repair enzymes, similar to the well-known repair-based DNA demethylation mechanism in Arabidopsis. As key regulators, non-enzymatic Gadd45 proteins function to recruit enzymatic machineries and promote coupling of deamination, base and nucleotide-excision repair in the process of DNA demethylation. In this article, we review recent findings and discuss functional and evolutionary implications of such mechanisms underlying active DNA demethylation.     

Mammalian DNA demethylation

DNA cytosine methylation is a reversible epigenetic mark regulating gene expression. Aberrant methylation profiles are concomitant with developmental defects and cancer. Numerous studies in the past decade have identified enzymes and pathways responsible for active DNA demethylation both on a genome-wide as well as gene-specific scale. Recent findings have strengthened the idea that 5-methylcytosine oxidation catalyzed by members of the ten-eleven translocation (Tet1–3) oxygenases in conjunction with replication-coupled dilution of the conversion products causes the majority of genome-wide erasure of methylation marks during early development. In contrast, short and long patch DNA excision repair seems to be implicated mainly in gene-specific demethylation. Growth arrest and DNA damage-inducible protein 45 a (Gadd45a) regulates gene-specific demethylation within regulatory sequences of limited lengths raising the question of how such site specificity is achieved. A new study identified the protein inhibitor of growth 1 (Ing1) as a reader of the active chromatin mark histone H3 lysine 4 trimethylation (H3K4me3). Ing1 binds and directs Gadd45a to target sites, thus linking the histone code with DNA demethylation.     

Mammalian DNMTs in the male germ line DNA of Drosophila

It is controversial whether DNA methylation plays a functional role in Drosophila. We have studied testis DNA of Drosophila melanogaster Meigen, 1830 with antisera against 5-methylcytosine (5mC) and found no evidence for the presence of significant amounts of 5mC. Reactions occur only with 1 of 3 5mC antisera, but they are restricted to nuclear regions without detectable amounts of DNA. The antisera apparently cross-react with other nuclear components. If the murine de novo DNA methyltransferases, DNMT3A and DNMT3B, are expressed under the control of the spermatocyte-specific beta2-tubulin promoter in testes, DNA methylation is not increased and no effects on the fertility of the fly are seen. DNA methylation has, therefore, no functional relevance in the male germ line of Drosophila.