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.     

Insights into DNA hydroxymethylation in the honeybee from in-depth analyses of TET dioxygenase

In mammals, a family of TET enzymes producing oxidized forms of 5-methylcytosine (5mC) plays an important role in modulating DNA demethylation dynamics. In contrast, nothing is known about the function of a single TET orthologue present in invertebrates. Here, we show that the honeybee TET (AmTET) catalytic domain has dioxygenase activity and converts 5mC to 5-hydroxymethylcytosine (5hmC) in a HEK293T cell assay. In vivo, the levels of 5hmC are condition-dependent and relatively low, but in testes and ovaries 5hmC is present at approximately 7–10% of the total level of 5mC, which is comparable to that reported for certain mammalian cells types. AmTET is alternatively spliced and highly expressed throughout development and in adult tissues with the highest expression found in adult brains. Our findings reveal an additional level of flexible genomic modifications in the honeybee that may be important for the selection of multiple pathways controlling contrasting phenotypic outcomes in this species. In a broader context, our study extends the current, mammalian-centred attention to TET-driven DNA hydroxymethylation to an easily manageable organism with attractive and unique biology.     

A sensitive mass spectrometry method for simultaneous quantification of DNA methylation and hydroxymethylation levels in biological samples

The recent discovery of 5-hydroxymethyl-cytosine (5hmC) in embryonic stem cells and postmitotic neurons has triggered the need for quantitative measurements of both 5-methyl-cytosine (5mC) and 5hmC in the same sample. We have developed a method using liquid chromatography electrospray ionization tandem mass spectrometry with multiple reaction monitoring (LC–ESI–MS/MS–MRM) to simultaneously measure levels of 5mC and 5hmC in digested genomic DNA. This method is fast, robust, and accurate, and it is more sensitive than the current 5hmC quantitation methods such as end labeling with thin layer chromatography and radiolabeling by glycosylation. Only 50 ng of digested genomic DNA is required to measure the presence of 0.1% 5hmC in DNA from mouse embryonic stem cells. Using this procedure, we show that human induced pluripotent stem cells exhibit a dramatic increase in 5mC and 5hmC levels compared with parental fibroblast cells, suggesting a dynamic regulation of DNA methylation and hydroxymethylation during cellular reprogramming.     

The role of 5-hydroxymethylcytosine in human cancer

The patterns of DNA methylation in human cancer cells are highly abnormal and often involve the acquisition of DNA hypermethylation at hundreds or thousands of CpG islands that are usually unmethylated in normal tissues. The recent discovery of 5-hydroxymethylcytosine (5hmC) as an enzymatic oxidation product of 5-methylcytosine (5mC) has led to models and experimental data in which the hypermethylation and 5mC oxidation pathways seem to be connected. Key discoveries in this setting include the findings that several genes coding for proteins involved in the 5mC oxidation reaction are mutated in human tumors, and that a broad loss of 5hmC occurs across many types of cancer. In this review, we will summarize current knowledge and discuss models of the potential roles of 5hmC in human cancer biology.     

Loss of 5-hydroxymethylcytosine is linked to gene body hypermethylation in kidney cancer

Both 5-methylcytosine (5mC) and its oxidized form 5-hydroxymethylcytosine (5hmC) have been proposed to be involved in tumorigenesis. Because the readout of the broadly used 5mC mapping method, bisulfite sequencing (BS-seq), is the sum of 5mC and 5hmC levels, the 5mC/5hmC patterns and relationship of these two modifications remain poorly understood. By profiling real 5mC (BS-seq corrected by Tet-assisted BS-seq, TAB-seq) and 5hmC (TAB-seq) levels simultaneously at single-nucleotide resolution, we here demonstrate that there is no global loss of 5mC in kidney tumors compared with matched normal tissues. Conversely, 5hmC was globally lost in virtually all kidney tumor tissues. The 5hmC level in tumor tissues is an independent prognostic marker for kidney cancer, with lower levels of 5hmC associated with shorter overall survival. Furthermore, we demonstrated that loss of 5hmC is linked to hypermethylation in tumors compared with matched normal tissues, particularly in gene body regions. Strikingly, gene body hypermethylation was significantly associated with silencing of the tumor-related genes. Downregulation of IDH1 was identified as a mechanism underlying 5hmC loss in kidney cancer. Restoring 5hmC levels attenuated the invasion capacity of tumor cells and suppressed tumor growth in a xenograft model. Collectively, our results demonstrate that loss of 5hmC is both a prognostic marker and an oncogenic event in kidney cancer by remodeling the DNA methylation pattern.     

Lead exposure induces changes in 5-hydroxymethylcytosine clusters in CpG islands in human embryonic stem cells and umbilical cord blood

Prenatal exposure to neurotoxicants such as lead (Pb) may cause stable changes in the DNA methylation (5mC) profile of the fetal genome. However, few studies have examined its effect on the DNA de-methylation pathway, specifically the dynamic changes of the 5-hydroxymethylcytosine (5hmC) profile. Therefore, in this study, we investigate the relationship between Pb exposure and 5mC and 5hmC modifications during early development. To study the changes in the 5hmC profile, we use a novel modification of the Infinium™ HumanMethylation450 assay (Illumina, Inc.), which we named HMeDIP-450K assay, in an in vitro human embryonic stem cell model of Pb exposure. We model Pb exposure-associated 5hmC changes as clusters of correlated, adjacent CpG sites, which are co-responding to Pb. We further extend our study to look at Pb-dependent changes in high density 5hmC regions in umbilical cord blood DNA from 48 mother-infant pairs from the Early Life Exposure in Mexico to Environmental Toxicants (ELEMENT) cohort. For our study, we randomly selected umbilical cord blood from 24 male and 24 female children from the 1st and 4th quartiles of Pb levels. Our data show that Pb-associated changes in the 5hmC and 5mC profiles can be divided into sex-dependent and sex-independent categories. Interestingly, differential 5mC sites are better markers of Pb-associated sex-dependent changes compared to differential 5hmC sites. In this study we identified several 5hmC and 5mC genomic loci, which we believe might have some potential as early biomarkers of prenatal Pb exposure.     

Age-related epigenome-wide DNA methylation and hydroxymethylation in longitudinal mouse blood

DNA methylation at cytosine-phosphate-guanine (CpG) dinucleotides changes as a function of age in humans and animal models, a process that may contribute to chronic disease development. Recent studies have investigated the role of an oxidized form of DNA methylation – 5-hydroxymethylcytosine (5hmC) – in the epigenome, but its contribution to age-related DNA methylation remains unclear. We tested the hypothesis that 5hmC changes with age, but in a direction opposite to 5-methylcytosine (5mC), potentially playing a distinct role in aging. To characterize epigenetic aging, genome-wide 5mC and 5hmC were measured in longitudinal blood samples (2, 4, and 10 months of age) from isogenic mice using two sequencing methods – enhanced reduced representation bisulfite sequencing and hydroxymethylated DNA immunoprecipitation sequencing. Examining the epigenome by age, we identified 28,196 unique differentially methylated CpGs (DMCs) and 8,613 differentially hydroxymethylated regions (DHMRs). Mouse blood showed a general pattern of epigenome-wide hypermethylation and hypo-hydroxymethylation with age. Comparing age-related DMCs and DHMRs, 1,854 annotated genes showed both differential 5mC and 5hmC, including one gene – Nfic – at five CpGs in the same 250 bp chromosomal region. At this region, 5mC and 5hmC levels both decreased with age. Reflecting these age-related epigenetic changes, Nfic RNA expression in blood decreased with age, suggesting that age-related regulation of this gene may be driven by 5hmC, not canonical DNA methylation. Combined, our genome-wide results show age-related differential 5mC and 5hmC, as well as some evidence that changes in 5hmC may drive age-related DNA methylation and gene expression.     

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.     

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.     

The effects of TETs on DNA methylation and hydroxymethylation of mouse oocytes after vitrification and warming

Oocyte vitrification has extensively been applied in the field of embryo engineering and in the preservation of genetic resources of fine livestock. Following our previous work in oocyte vitrification and the level change of DNA methylation, here we further explored the dynamic change of three active demethylation proteins: Ten-Eleven-Translocation 1/2/3(TET1/2/3), 5-methylcytosine (5 mC) and 5-hydroxymethycytosine (5hmC) after vitrification and warming. In order to observe the active demethylation in vitrified oocytes, two small molecular regulators, i.e. Vitamin C (VC) and dimethyloxaloylglycine (DMOG) were used to adjust activity and level of the TET 3 protein. The results showed that the levels of 5 mC and 5hmC were significantly decreased after 2 h of vitrification (P < 0.01). Moreover, the level of TET3 protein was significantly increased after 2 h warming (P < 0.01). And the relative gene expression of TET2/3 did not change in the first 2 h, but significantly increased after 2 h (P < 0.01). When VC was added to vitrification and recovery medium, it could not significantly improve the level of TET3 gene expression, and affect 5 mC and 5hmC expression (P > 0.05). When the DMOG was added to the solutions of vitrification, the level of 5hmC showed significantly increase (P < 0.01). In conclusion, the oocyte vitrification procedure reduced DNA methylation and hydroxymethylation in MII oocytes, but adding VC and DMOG to vitrification medium can prevent the reduction of DNA hydroxymethylation by increasing activity of TET3 methylation protein after vitrification and warming.     

Altering TET dioxygenase levels within physiological range affects DNA methylation dynamics of HEK293 cells

The TET family of dioxygenases (TET1/2/3) can convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) and has been shown to be involved in active and passive DNA demethylation. Here, we demonstrate that altering TET dioxygenase levels within physiological range can affect DNA methylation dynamics of HEK293 cells. Overexpression of TET1 increased global 5hmC levels and was accompanied by mild DNA demethylation of promoters, gene bodies and CpG islands. Conversely, the simultaneous knockdown of TET1, TET2, and TET3 led to decreased global 5hmC levels and mild DNA hypermethylation of above-mentioned regions. The methylation changes observed in the overexpression and knockdown studies were mostly non-reciprocal and occurred with different preference depending on endogenous methylation and gene expression levels. Single-nucleotide 5hmC profiling performed on a genome-wide scale revealed that TET1 overexpression induced 5mC oxidation without a distribution bias among genetic elements and structures. Detailed analysis showed that this oxidation was related to endogenous 5hmC levels. In addition, our results support the notion that the effects of TET1 overexpression on gene expression are generally unrelated to its catalytic activity.     

Recognition and potential mechanisms for replication and erasure of cytosine hydroxymethylation

Cytosine residues in mammalian DNA occur in at least three forms, cytosine (C), 5-methylcytosine (M; 5mC) and 5-hydroxymethylcytosine (H; 5hmC). During semi-conservative DNA replication, hemi-methylated (M/C) and hemi-hydroxymethylated (H/C) CpG dinucleotides are transiently generated, where only the parental strand is modified and the daughter strand contains native cytosine. Here, we explore the role of DNA methyltransferases (DNMT) and ten eleven translocation (Tet) proteins in perpetuating these states after replication, and the molecular basis of their recognition by methyl-CpG-binding domain (MBD) proteins. Using recombinant proteins and modified double-stranded deoxyoligonucleotides, we show that DNMT1 prefers a hemi-methylated (M/C) substrate (by a factor of >60) over hemi-hydroxymethylated (H/C) and unmodified (C/C) sites, whereas both DNMT3A and DNMT3B have approximately equal activity on all three substrates (C/C, M/C and H/C). Binding of MBD proteins to methylated DNA inhibited Tet1 activity, suggesting that MBD binding may also play a role in regulating the levels of 5hmC. All five MBD proteins generally have reduced binding affinity for 5hmC relative to 5mC in the fully modified context (H/M versus M/M), though their relative abilities to distinguish the two varied considerably. We further show that the deamination product of 5hmC could be excised by thymine DNA glycosylase and MBD4 glycosylases regardless of context.     

Examination of the specificity of DNA methylation profiling techniques towards 5-methylcytosine and 5-hydroxymethylcytosine

DNA cytosine-5 methylation is a well-studied epigenetic pathway implicated in gene expression control and disease pathogenesis. Different technologies have been developed to examine the distribution of 5-methylcytosine (5mC) in specific sequences of the genome. Recently, substantial amounts of 5-hydroxymethylcytosine (5hmC), most likely derived from enzymatic oxidation of 5mC by TET1, have been detected in certain mammalian tissues. Here, we have examined the ability of several commonly used DNA methylation profiling methods to distinguish between 5mC and 5hmC. We show that techniques based on sodium bisulfite treatment of DNA are incapable of distinguishing between the two modified bases. In contrast, techniques based on immunoprecipitation with anti-5mC antibody (methylated DNA immunoprecipitation, MeDIP) or those based on proteins that bind to methylated CpG sequences (e.g. methylated-CpG island recovery assay, MIRA) do not detect 5hmC and are specific for 5mC unless both modified bases occur in the same DNA fragment. We also report that several methyl-CpG binding proteins including MBD1, MBD2 and MBD4 do not bind to sequences containing 5hmC. Selective mapping of 5hmC will require the development of unique tools for the detection of this modified base.