Thymine DNA glycosylase can rapidly excise 5-formylcytosine and 5-carboxylcytosine: potential implications for active demethylation of CpG sites

Thymine DNA glycosylase (TDG) excises T from G·T mispairs and is thought to initiate base excision repair (BER) of deaminated 5-methylcytosine (mC). Recent studies show that TDG, including its glycosylase activity, is essential for active DNA demethylation and embryonic development. These and other findings suggest that active demethylation could involve mC deamination by a deaminase, giving a G·T mispair followed by TDG-initiated BER. An alternative proposal is that demethylation could involve iterative oxidation of mC to 5-hydroxymethylcytosine (hmC) and then to 5-formylcytosine (fC) and 5-carboxylcytosine (caC), mediated by a Tet (ten eleven translocation) enzyme, with conversion of caC to C by a putative decarboxylase. Our previous studies suggest that TDG could excise fC and caC from DNA, which could provide another potential demethylation mechanism. We show here that TDG rapidly removes fC, with higher activity than for G·T mispairs, and has substantial caC excision activity, yet it cannot remove hmC. TDG excision of fC and caC, oxidation products of mC, is consistent with its strong specificity for excising bases from a CpG context. Our findings reveal a remarkable new aspect of specificity for TDG, inform its catalytic mechanism, and suggest that TDG could protect against fC-induced mutagenesis. The results also suggest a new potential mechanism for active DNA demethylation, involving TDG excision of Tet-produced fC (or caC) and subsequent BER. Such a mechanism obviates the need for a decarboxylase and is consistent with findings that TDG glycosylase activity is essential for active demethylation and embryonic development, as are mechanisms involving TDG excision of deaminated mC or hmC.     

Dynamics of 5-carboxylcytosine during hepatic differentiation: Potential general role for active demethylation by DNA repair in lineage specification

Patterns of DNA methylation (5-methylcytosine, 5mC) are rearranged during differentiation contributing to the regulation of cell type-specific gene expression. TET proteins oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Both 5fC and 5caC can be recognized and excised from DNA by thymine-DNA glycosylase (TDG) followed by the subsequent incorporation of unmodified cytosine into the abasic site via the base excision repair (BER) pathway. We previously demonstrated that 5caC accumulates during lineage specification of neural stem cells (NSCs) suggesting that such active demethylation pathway is operational in this system; however, it is still unknown if TDG/BER-dependent demethylation is used during other types of cellular differentiation. Here we analyze dynamics of the global levels of 5hmC and 5caC during differentiation of human pluripotent stem cells toward hepatic endoderm. We show that, similar to differentiating NSCs, 5caC transiently accumulates during hepatic differentiation. The levels of 5caC increase during specification of foregut, peak at the stage of hepatic endoderm commitment, and drop in differentiating cells concurrently with the onset of expression of α fetoprotein, a marker of committed hepatic progenitors. Moreover, we show that 5caC accumulates at promoter regions of several genes expressed during hepatic specification at differentiation stages corresponding to the beginning of their expression. Our data indicate that transient 5caC accumulation is a common feature of 2 different types (neural/glial and endoderm/hepatic) of cellular differentiation. This suggests that oxidation of 5mC may represent a general mechanism of rearrangement of 5mC profiles during lineage specification of somatic cells in mammals.     

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]     

Generation and replication-dependent dilution of 5fC and 5caC during mouse preimplantation development

One of the recent advances in the epigenetic field is the demonstration that the Tet family of proteins are capable of catalyzing conversion of 5-methylcytosine (5mC) of DNA to 5-hydroxymethylcytosine (5hmC). Interestingly, recent studies have shown that 5hmC can be further oxidized by Tet proteins to generate 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), which can be removed by thymine DNA glycosylase (TDG). To determine whether Tet-catalyzed conversion of 5mC to 5fC and 5caC occurs in vivo in zygotes, we generated antibodies specific for 5fC and 5caC. By immunostaining, we demonstrate that loss of 5mC in the paternal pronucleus is concurrent with the appearance of 5fC and 5caC, similar to that of 5hmC. Importantly, instead of being quickly removed through an enzyme-catalyzed process, both 5fC and 5caC exhibit replication-dependent dilution during mouse preimplantation development. These results not only demonstrate the conversion of 5mC to 5fC and 5caC in zygotes, but also indicate that both 5fC and 5caC are relatively stable and may be functional during preimplantation development. Together with previous studies, our study suggests that Tet-catalyzed conversion of 5mC to 5hmC/5fC/5caC followed by replication-dependent dilution accounts for paternal DNA demethylation during preimplantation development.     

Excision of 5-hydroxymethyluracil and 5-carboxylcytosine by the thymine DNA glycosylase domain: its structural basis and implications for active DNA demethylation

The mammalian thymine DNA glycosylase (TDG) is implicated in active DNA demethylation via the base excision repair pathway. TDG excises the mismatched base from G:X mismatches, where X is uracil, thymine or 5-hydroxymethyluracil (5hmU). These are, respectively, the deamination products of cytosine, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). In addition, TDG excises the Tet protein products 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) but not 5hmC and 5mC, when paired with a guanine. Here we present a post-reactive complex structure of the human TDG domain with a 28-base pair DNA containing a G:5hmU mismatch. TDG flips the target nucleotide from the double-stranded DNA, cleaves the N-glycosidic bond and leaves the C1′ hydrolyzed abasic sugar in the flipped state. The cleaved 5hmU base remains in a binding pocket of the enzyme. TDG allows hydrogen-bonding interactions to both T/U-based (5hmU) and C-based (5caC) modifications, thus enabling its activity on a wider range of substrates. We further show that the TDG catalytic domain has higher activity for 5caC at a lower pH (5.5) as compared to the activities at higher pH (7.5 and 8.0) and that the structurally related Escherichia coli mismatch uracil glycosylase can excise 5caC as well. We discuss several possible mechanisms, including the amino-imino tautomerization of the substrate base that may explain how TDG discriminates against 5hmC and 5mC.     

TET蛋白：一个新的DNA修饰酶家族

DNA的胞嘧啶（C）5-甲基化是一种重要的表观修饰,它参与基因调节、基因组印记、X-染色体失活、重复序列抑制和癌症发生等过程. 5-甲基胞嘧啶（5mC）可被TET (ten-eleven translocation)蛋白家族进一步转化为5-羟甲基胞嘧啶（5hmC）,该过程是DNA去甲基化的1个必要阶段. 5hmC可在活性转录基因起始位点和Polycomb抑制基因启动子延伸区域富集.TET蛋白包括3个成员TET1、TET2和TET3,均属于α-酮戊二酸和Fe²⁺依赖的双加氧酶,其催化涉及氧化过程.小鼠Tet1在胚胎干细胞发育中拥有双重作用,即促进全能因子的转录,又参与发育调节因子的抑制.人TET蛋白的破坏与造血系统肿瘤相关,如在骨髓增生性疾病/肿瘤存在频繁的TET2基因突变.TET蛋白和5hmC的研究为DNA甲基化/去甲基化及其生物学功能提供了新的视点.     

Dynamic changes of DNA epigenetic marks in mouse oocytes during natural and accelerated aging

Aging is a complex time-dependent biological process that takes place in every cell and organ, eventually leading to degenerative changes that affect normal biological functions. In the past decades, the number of older parents has increased significantly. While it is widely recognized that oocyte aging poses higher birth and reproductive risk, the exact molecular mechanisms remain largely elusive. DNA methylation of 5-cytosine (5mC) and histone modifications are among the key epigenetic mechanisms involved in critical developmental processes and have been linked to aging. However, the impact of oocyte aging on DNA demethylation pathways has not been examined. The recent discovery of Ten-Eleven-Translocation (TET) family proteins, thymine DNA glycosylase (TDG) and the demethylation intermediates 5hmC, 5fC and 5caC has provided novel clues to delineate the molecular mechanisms in DNA demethylation. In this study, we examined the cellular level of modified cytosines (5mC, 5hmC, 5fC and 5caC) and Tet/Tdg expression in oocytes obtained from natural and accelerated oocyte aging conditions. Here we show all the DNA demethylation marks are dynamically regulated in both aging conditions, which are associated with Tet3 over-expression and Tdg repression. Such an aberrant expression pattern was more profound in accelerated aging condition. The results suggest that DNA demethylation may be actively involved in oocyte aging and have implications for development of potential drug targets to rejuvenate aging oocytes.This article is part of a Directed Issue entitled: Epigenetics dynamics in development and disease.     

Deep C diving: mapping the low-abundance modifications of the DNA demethylation pathway

Two new studies imply that the reprogramming of 5-methylcytosine via TET- and TDG-family enzymes is both widespread throughout the genome and functionally significant.In the mammalian genome, the dinucleotide CpG acts as a unique signaling module that can regulate the local chromatin environment through the recruitment of specific chromatin modifying proteins [1]. Although it is thought to be context specific, the general enzymatic acquisition of methylation at CpG dinucleotides by DNA methlytransferase enzymes (DNMTs) over promoter regions tends to be associated with gene silencing events and heterochromatin formation. The maintenance of 5-methylcytosine (5mC) modification patterns has since been implicated in many important roles in normal cell function during mammalian development and disease progression [1]. Although it is widely understood how DNA can become enzymatically methylated, less is known regarding the active removal of 5mC at specific loci, aside from the potential for passive loss during cell division in the absence of DNMT activity. In 2009, a second form of DNA modification, that of 5-hydroxymethylcytosine (5hmC), was rediscovered, and enzymatic oxidation reactions (involving the ten-eleven translocation (TET) proteins) responsible for generating 5hmC from 5mC were identified [2]. Subsequent work has since identified the downstream, TET-dependent, oxidative derivatives of 5hmC, those of 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) [2]. This has led to the proposal of an active DNA demethylation cycle relying on the initial oxidation of 5mC into 5hmC, through the TET family of enzymes, before further oxidation to the 5fC and 5caC derivatives (Figure ​(Figure1a).1a). In contrast to the more abundant 5hmC modification, these lower-abundance downstream intermediates are proposed to be removed by base excision repair mechanisms that are highly reliant on the thymine DNA glycosylase (TDG) protein, ultimately resulting in the replacement of modified cytosine with non-modified cytosine.Open in a separate windowFigure 15fC and 5caC as TDG-mediated DNA demethylation intermediates. (a) The proposed cycle of DNA methylation (red arrow) and active demethylation (blue arrows). Enzymes are shown for each step along with required co-factors. (b) Visualization of the datasets derived by the two studies over the Hoxa1 and Hoxa2 genes (i) and the Igf2 gene (ii), both in wild-type (WT) and thymine DNA glycosylase (TDG) depleted/knockout mouse embryonic stem cells. 5fC data are plotted as both blue (He and colleagues [7]) and gold (Zhang and colleagues [6]) tracks, while 5caC, as reported by Zhang and colleagues [6], is displayed in red. Although both techniques profile the 5fC mark in WT and TDG depleted cells with a large degree of overlap (i), there are some regions that show technique-dependent enrichment (ii). Data have been filtered to remove background noise (reads <1 and <3 in the He and Zhang studies, respectively). Percentage GC plots (GC%) are shown in black, with Refseq predicted gene structures underneath. abs, antibodies; shTDG, TDG-depleting short hairpin RNA; TET, ten-eleven translocation.     

Simple and accurate single base resolution analysis of 5-hydroxymethylcytosine by catalytic oxidative bisulfite sequencing using micelle incarcerated oxidants

Oxidation of 5-methylcytosine (5mC) is catalyzed by ten-eleven translocation (TET) enzymes to produce 5-hydroxymethylcytosine (5hmC) and following oxidative products. The oxidized nucleotides were shown to be the intermediates for DNA demethylation, as the nucleotides are removed by base excision repair system initiated by thymine DNA glycosylase. A simple and accurate method to determine initial oxidation product 5hmC at single base resolution in genomic DNA is necessary to understand demethylation mechanism. Recently, we have developed a new catalytic oxidation reaction using micelle-incarcerated oxidants to oxidize 5hmC to form 5-formylcytosine (5fC), and subsequent bisulfite sequencing can determine the positions of 5hmC in DNA. In the present study, we described the optimization of the catalytic oxidative bisulfite sequencing (coBS-seq), and its application to the analysis of 5hmC in genomic DNA at single base resolution in a quantitative manner. As the oxidation step showed quite low damage on genomic DNA, the method allows us to down scale the sample to be analyzed.     

TET蛋白的去甲基化机制及其在调控小鼠发育过程中的作用

TET(Ten-eleven translocation)蛋白家族共有3个成员,分别为TET1、TET2和TET3,均属于α-酮戊二酸(α-KG)和Fe²⁺依赖的双加氧酶,可以将5-甲基胞嘧啶(5-methylcytosine, 5 mC)氧化为5-羟甲基胞嘧啶(5-hydroxymethylcytosine, 5 hmC)、5-甲酰基胞嘧啶(5-formylcytosine, 5 fC)及5-羧基胞嘧啶(5-carboxylcytosine, 5 caC)。研究表明,TET蛋白通过不同机制以主动或被动的方式调控DNA去甲基化,且去甲基化的活性可能受其他因子的调控。TET蛋白广泛参与哺乳动物发育过程的调节,其中在原始生殖细胞的形成、胚胎发育、干细胞多能性及神经和脑发育等方面发挥了重要作用。TET蛋白生物功能的发现为表观遗传学研究开辟了全新的研究领域,而且相关研究结果对拓展生命科学研究具有重要意义。文章综述了TET蛋白家族的结构、去甲基化分子机制及在小鼠发育过程中的作用,为深入了解TET蛋白的功能提供理论基础。     

Hydroxyl-radical-induced oxidation of 5-methylcytosine in isolated and cellular DNA

The methylation and oxidative demethylation of cytosine in CpG dinucleotides plays a critical role in the regulation of genes during cell differentiation, embryogenesis and carcinogenesis. Despite its low abundance, 5-methylcytosine (5mC) is a hotspot for mutations in mammalian cells. Here, we measured five oxidation products of 5mC together with the analogous products of cytosine and thymine in DNA exposed to ionizing radiation in oxygenated aqueous solution. The products can be divided into those that arise from hydroxyl radical (•OH) addition at the 5,6-double bond of 5mC (glycol, hydantoin and imidazolidine products) and those that arise from H-atom abstraction from the methyl group of 5mC including 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC). Based on the analysis of these products, we show that the total damage at 5mC is about 2-fold greater than that at C in identical sequences. The formation of hydantoin products of 5mC is favored, compared to analogous reactions of thymine and cytosine, which favor the formation of glycol products. The distribution of oxidation products is sequence dependent in specific ODN duplexes. In the case of 5mC, the formation of 5hmC and 5fC represents about half of the total of •OH-induced oxidation products of 5mC. Several products of thymine, cytosine, 5mC, as well as 8-oxo-7,8-dihydroguanine (8oxoG), were also estimated in irradiated cells.     

Structural Insights into the Mechanism of Base Excision by MBD4

DNA glycosylases remove damaged or modified nucleobases by cleaving the N-glycosyl bond and the correct nucleotide is restored through subsequent base excision repair. In addition to excising threatening lesions, DNA glycosylases contribute to epigenetic regulation by mediating DNA demethylation and perform other important functions. However, the catalytic mechanism remains poorly defined for many glycosylases, including MBD4 (methyl-CpG binding domain IV), a member of the helix-hairpin-helix (HhH) superfamily. MBD4 excises thymine from G·T mispairs, suppressing mutations caused by deamination of 5-methylcytosine, and it removes uracil and modified uracils (e.g., 5-hydroxymethyluracil) mispaired with guanine. To investigate the mechanism of MBD4 we solved high-resolution structures of enzyme-DNA complexes at three stages of catalysis. Using a non-cleavable substrate analog, 2′-deoxy-pseudouridine, we determined the first structure of an enzyme-substrate complex for wild-type MBD4, which confirms interactions that mediate lesion recognition and suggests that a catalytic Asp, highly conserved in HhH enzymes, binds the putative nucleophilic water molecule and stabilizes the transition state. Observation that mutating the Asp (to Gly) reduces activity by 2700-fold indicates an important role in catalysis, but probably not one as the nucleophile in a double-displacement reaction, as previously suggested. Consistent with direct-displacement hydrolysis, a structure of the enzyme-product complex indicates a reaction leading to inversion of configuration. A structure with DNA containing 1-azadeoxyribose models a potential oxacarbenium-ion intermediate and suggests the Asp could facilitate migration of the electrophile towards the nucleophilic water. Finally, the structures provide detailed snapshots of the HhH motif, informing how these ubiquitous metal-binding elements mediate DNA binding.     

表观遗传新标志物5hmC的研究进展

5-羟甲基胞嘧啶(5-hydroxymethylcytosine,5hmC)作为表观遗传的新标志物,已引起人们的极大兴趣.5hmC由TET家族酶催化氧化5-甲基胞嘧啶(5-methylcytosine,5mC)产生,被称为高等生物基因组DNA的"第六碱基".5hmC不仅可以影响基因组结构及功能,还在早期胚胎发育中发挥重要的作用.本文综述了5hmC的代谢通路、生物学功能、在基因组的分布及分析方法的研究进展.