Redox-active quinones induces genome-wide DNA methylation changes by an iron-mediated and Tet-dependent mechanism

DNA methylation has been proven to be a critical epigenetic mark important for various cellular processes. Here, we report that redox-active quinones, a ubiquitous class of chemicals found in natural products, cancer therapeutics and environment, stimulate the conversion of 5mC to 5hmC in vivo, and increase 5hmC in 5751 genes in cells. 5hmC increase is associated with significantly altered gene expression of 3414 genes. Interestingly, in quinone-treated cells, labile iron-sensitive protein ferritin light chain showed a significant increase at both mRNA and protein levels indicating a role of iron regulation in stimulating Tet-mediated 5mC oxidation. Consistently, the deprivation of cellular labile iron using specific chelator blocked the 5hmC increase, and a delivery of labile iron increased the 5hmC level. Moreover, both Tet1/Tet2 knockout and dimethyloxalylglycine-induced Tet inhibition diminished the 5hmC increase. These results suggest an iron-regulated Tet-dependent DNA demethylation mechanism mediated by redox-active biomolecules.     

Vitrification of murine mature metaphase II oocytes perturbs DNA methylation reprogramming during preimplantation embryo development

Accurate reprogramming of DNA methylation occurring in preimplantation embryos is critical for normal development of both fetus and placenta. Environmental stresses imposed on oocytes usually cause the abnormal DNA methylation reprogramming of early embryos. However, whether oocyte vitrification alters the reprogramming of DNA methylation (5 mC) and its derivatives in mouse preimplantation embryo development remains largely unknown. Here, we found that the rate of cleavage and blastocyst formation of embryos produced by IVF of vitrified matured oocytes was significantly lower than that in control counterparts, but the quality of blastocysts was not impaired by oocyte vitrification. Additionally, although vitrification neither altered the dynamic changes of 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5 fC) before 4-cell stage nor affected the levels of 5 mC and 5-carboxylcytosine (5caC) throughout the preimplantation development, vitrification significantly reduced the levels of 5hmC and 5 fC from 8-cell stage onwards. Correspondingly, vitrification did not alter the expression patterns of Tet3 in preimplantation embryos but apparently reduced the expression levels of Tet1 in 4-cell and 8-cell embryos and increased the expression levels of Tet2 at morula stage. Taken together, these results demonstrate that oocyte vitrification perturbs DNA methylation reprogramming in mouse preimplantation embryo development.     

Hydroxymethylcytosine and demethylation of the γ-globin gene promoter during erythroid differentiation

The mechanism responsible for developmental stage-specific regulation of γ-globin gene expression involves DNA methylation. Previous results have shown that the γ-globin promoter is nearly fully demethylated during fetal liver erythroid differentiation and partially demethylated during adult bone marrow erythroid differentiation. The hypothesis that 5-hydroxymethylcytosine (5hmC), a known intermediate in DNA demethylation pathways, is involved in demethylation of the γ-globin gene promoter during erythroid differentiation was investigated by analyzing levels of 5-methylcytosine (5mC) and 5hmC at a CCGG site within the 5′ γ-globin gene promoter region in FACS-purified cells from baboon bone marrow and fetal liver enriched for different stages of erythroid differentiation. Our results show that 5mC and 5hmC levels at the γ-globin promoter are dynamically modulated during erythroid differentiation with peak levels of 5hmC preceding and/or coinciding with demethylation. The Tet2 and Tet3 dioxygenases that catalyze formation of 5hmC are expressed during early stages of erythroid differentiation and Tet3 expression increases as differentiation proceeds. In baboon CD34+ bone marrow-derived erythroid progenitor cell cultures, γ-globin expression was positively correlated with 5hmC and negatively correlated with 5mC at the γ-globin promoter. Supplementation of culture media with Vitamin C, a cofactor of the Tet dioxygenases, reduced γ-globin promoter DNA methylation and increased γ-globin expression when added alone and in an additive manner in combination with either DNA methyltransferase or LSD1 inhibitors. These results strongly support the hypothesis that the Tet-mediated 5hmC pathway is involved in developmental stage-specific regulation of γ-globin expression by mediating demethylation of the γ-globin promoter.     

Ten-Eleven Translocation 1 (Tet1) Is Regulated by O-Linked N-Acetylglucosamine Transferase (Ogt) for Target Gene Repression in Mouse Embryonic Stem Cells

As a member of the Tet (Ten-eleven translocation) family proteins that can convert 5-methylcytosine (5mC) to 5-hydroxylmethylcytosine (5hmC), Tet1 has been implicated in regulating global DNA demethylation and gene expression. Tet1 is highly expressed in embryonic stem (ES) cells and appears primarily to repress developmental genes for maintaining pluripotency. To understand how Tet1 may regulate gene expression, we conducted large scale immunoprecipitation followed by mass spectrometry of endogenous Tet1 in mouse ES cells. We found that Tet1 could interact with multiple chromatin regulators, including Sin3A and NuRD complexes. In addition, we showed that Tet1 could also interact with the O-GlcNAc transferase (Ogt) and be O-GlcNAcylated. Depletion of Ogt led to reduced Tet1 and 5hmC levels on Tet1-target genes, whereas ectopic expression of wild-type but not enzymatically inactive Ogt increased Tet1 levels. Mutation of the putative O-GlcNAcylation site on Tet1 led to decreased O-GlcNAcylation and level of the Tet1 protein. Our results suggest that O-GlcNAcylation can positively regulate Tet1 protein concentration and indicate that Tet1-mediated 5hmC modification and target repression is controlled by Ogt.     

Genome-wide regulation of 5hmC, 5mC, and gene expression by Tet1 hydroxylase in mouse embryonic stem cells

DNA methylation at the 5 position of cytosine (5mC) in the mammalian genome is a key epigenetic event critical for various cellular processes. The ten-eleven translocation (Tet) family of 5mC-hydroxylases, which convert 5mC to 5-hydroxymethylcytosine (5hmC), offers a way for dynamic regulation of DNA methylation. Here we report that Tet1 binds to unmodified C or 5mC- or 5hmC-modified CpG-rich DNA through its CXXC domain. Genome-wide mapping of Tet1 and 5hmC reveals mechanisms by which Tet1 controls 5hmC and 5mC levels in mouse embryonic stem cells (mESCs). We also uncover a comprehensive gene network influenced by Tet1. Collectively, our data suggest that Tet1 controls DNA methylation both by binding to CpG-rich regions to prevent unwanted DNA methyltransferase activity, and by converting 5mC to 5hmC through hydroxylase activity. This Tet1-mediated antagonism of CpG methylation imparts differential maintenance of DNA methylation status at Tet1 targets, ultimately contributing to mESC differentiation and the onset of embryonic development.     

Structure of Naegleria Tet-like dioxygenase (NgTet1) in complexes with a reaction intermediate 5-hydroxymethylcytosine DNA

The family of ten-eleven translocation (Tet) dioxygenases is widely distributed across the eukaryotic tree of life, from mammals to the amoeboflagellate Naegleria gruberi. Like mammalian Tet proteins, the Naegleria Tet-like protein, NgTet1, acts on 5-methylcytosine (5mC) and generates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) in three consecutive, Fe(II)- and α-ketoglutarate-dependent oxidation reactions. The two intermediates, 5hmC and 5fC, could be considered either as the reaction product of the previous enzymatic cycle or the substrate for the next cycle. Here we present a new crystal structure of NgTet1 in complex with DNA containing a 5hmC. Along with the previously solved NgTet1–5mC structure, the two complexes offer a detailed picture of the active site at individual stages of the reaction cycle. In the crystal, the hydroxymethyl (OH-CH₂-) moiety of 5hmC points to the metal center, representing the reaction product of 5mC hydroxylation. The hydroxyl oxygen atom could be rotated away from the metal center, to a hydrophobic pocket formed by Ala212, Val293 and Phe295. Such rotation turns the hydroxyl oxygen atom away from the product conformation, and exposes the target CH₂ towards the metal-ligand water molecule, where a dioxygen O₂ molecule would occupy to initiate the next round of reaction by abstracting a hydrogen atom from the substrate. The Ala212-to-Val (A212V) mutant profoundly limits the product to 5hmC, probably because the reduced hydrophobic pocket size restricts the binding of 5hmC as a substrate.     

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.     

PGC7, H3K9me2 and Tet3: regulators of DNA methylation in zygotes

In zygotes, a global loss of DNA methylation occurs selectively in the paternal pronucleus before the first cell division, concomitantly with the appearance of modified forms of 5-methylcytosine. The adjacent maternal pronucleus and certain paternally-imprinted loci are protected from this process. Nakamura et al. recently clarified the molecular mechanism involved: PGC7/Stella/Dppa3 binds to dimethylated histone 3 lysine 9 (H3K9me2), thereby blocking the activity of the Tet3 methylcytosine oxidase in the maternal genome as well as at certain imprinted loci in the paternal genome.DNA methylation is a crucial epigenetic modification that regulates imprinting (differential silencing of maternal or paternal alleles) and repression of retrotransposons and other parasitic DNA, as well as possibly X-chromosome inactivation and cellular differentiation. DNA methylation needs to be faithfully maintained throughout the life cycle, since loss of DNA methylation can result in gene dosage problems, dysregulation of gene expression, and genomic instability due to retrotransposon reactivation¹. Nevertheless, genome-wide loss of DNA methylation has been observed during germ cell development² and in the paternal pronucleus soon after fertilization³.For almost a decade, the global decrease of DNA methylation observed in the paternal genome within a few hours of fertilization was ascribed to an “active”, replication-independent process³. The maternal pronucleus is spared and instead undergoes “passive”, replication-dependent demethylation during early embryogenesis, arising from inhibition of the DNA maintenance methyltransferase Dnmt1 (Dnmt1 is normally recruited to newly-replicated DNA because of the high affinity of its obligate partner, UHRF1, for hemi-methylated DNA strands, which are produced from symmetrically-methylated CpG dinucleotides as a result of DNA replication). The basis for active and passive demethylation of the paternal and maternal genomes remained a mystery until proteins of the TET family – TET1, TET2 and TET3 in humans – were discovered to be Fe(II)- and 2-oxoglutarate-dependent enzymes capable of oxidizing 5-methylcytosine (5mC) in DNA^4,5,6. TET enzymes serially convert 5mC into 5-hydroxymethyl-cytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC)^5,7,8.With the generation of specific antibodies to 5hmC, it became clear that the supposed “active demethylation” of the paternal pronucleus in mouse zygote after fertilization was due to the inability of anti-5mC antibodies to recognize 5hmC and other 5mC oxidation products^9,10. The enzyme responsible for 5mC oxidation was shown to be Tet3, which unlike Tet1 and Tet2 is highly expressed in mouse oocytes and zygotes. RNAi-mediated depletion of Tet3 decreased the staining of the paternal pronucleus with 5hmC, suggesting that immediately after fertilization, Tet3 in the zygote selectively oxidizes 5mC in the paternal genome to 5hmC^9,10.How is the maternal pronucleus protected from Tet3 activity? Nakamura et al.¹¹ previously showed that zygotes lacking PGC7/Stella/Dppa3 lose asymmetric regulation of DNA methylation, instead showing global loss of 5mC staining in both paternal and maternal pronuclei. This was correlated with hypomethylation at several maternally-imprinted loci (Peg1, Peg3, Peg10) in PGC7-deficient zygotes, as judged by bisulfite sequencing. Further, certain paternally-imprinted loci (H19, Rasgrf1), which are normally protected from global loss of methylation in the paternal genome, also became hypomethylated in PGC7-deficient zygotes. These data suggested that PGC7 protects the maternal genome, as well as certain paternally imprinted loci, from loss of 5mC.In their recent publication, Nakamura et al.¹² elegantly extended these findings to address the mechanism involved. Based on the fact that a major difference between maternal and paternal genomes is that the maternal genome contains histones, whereas the DNA of the entering sperm is tightly packaged with protamine, they asked whether PGC7 recognizes specific histone marks. Indeed, the maternal genome harbors considerable levels of the histone mark H3K9me2¹¹, leading them to examine whether PGC7 distinguishes maternal and paternal genomes by recognizing H3K9me2 in the maternal genome. Using wild-type (WT) ES cells and ES cells deficient in the G9a lysine methyltransferase which generates H3K9me2 mark, they showed that PGC7 associated loosely with nucleosomes and chromatin lacking H3K9me2, but tightly if H3K9me2 was present. The binding was recapitulated using recombinant bacterially-expressed PGC7 and histone tail peptides, indicating a direct interaction of PGC7 with the H3K9me2 mark. In agreement, genomic loci enriched with H3K9me2 recruited PGC7 as judged by chromatin immunoprecipitation (ChIP), but this recruitment was abrogated in G9a-deficient ES cells. These data indicated that PGC7 targets genomic regions occupied by nucleosomes containing H3K9me2 (Figure 1); an interesting extension would be to ask whether loss of maternal G9a also results in 5hmC conversion in the maternal pronucleus in zygotes.Open in a separate windowFigure 1Schematic view of paternal (left) and maternal (right) genomes soon after fertilization. Paternal and maternal pronuclei are indicated with immunostaining results in the boxes. PGC7 binds H3K9me2 in the maternal pronucleus and at certain paternally-imprinted loci (H19, Rasgrf1) in the paternal pronucleus, thereby potentially regulating chromatin organization to interfere with Tet3 accessibility.Next, Nakamura et al.¹² tested by immunocytochemistry whether PGC7 in zygotes also required H3K9me2. It is known that H3K9me2 staining is concentrated in the maternal but not the paternal pronucleus¹³. Using conventional staining methods in which the cells are first fixed and then permeabilized to allow antibodies to enter the cell, the authors observed in their earlier study that PGC7 bound to both pronuclei¹¹. Remarkably, by simply reversing the order of the fixation and permeabilization steps – permeabilizing first to allow the loss of loosely bound proteins by dissociation, then fixing and staining – they found that PGC7 associated much more tightly with the maternal pronucleus that bears H3K9me2 mark. Injection of mRNA encoding Jhdm2a (an H3K9me1/ me2-specific demethylase) into zygotes eliminated staining for H3K9me2 as well as PGC7 in the maternal pronucleus, and concomitantly caused loss of 5mC and acquisition of 5hmC. Taken together, these data strongly suggested that PGC7 was selectively recruited to the maternal pronucleus through binding H3K9me2, and that this binding protected zygotic maternal DNA from oxidation of 5mC to 5hmC and beyond (Figure 1).These findings led Nakamura et al. to investigate how PGC7 controls Tet3 activity in zygotes. They showed (in cells that were permeabilized before fixation and immunocytochemistry) that Tet3 was tightly associated only with the paternal pronucleus in WT zygotes, but was present in both pronuclei in PGC7-deficient zygotes. When PGC7 was prevented from binding to the maternal pronucleus by injection of Jhdm2a mRNA, Tet3 became tightly associated with both pronuclei. In other words, loss of PGC7 or loss of H3K9me2 that recruits PGC7 had the same effect – eliminating selective association of Tet3 with the paternal genome. The implication is that PGC7 – which preferentially binds the maternal genome – somehow promotes the selective binding of Tet3 to the paternal genome, thus permitting rapid 5mC oxidation in paternal but not maternal DNA (Figure 1).PGC7 is a small protein (150 amino acids (aa) in the mouse, 159 aa in humans) whose sequence is only moderately conserved. Nakamura et al.¹² showed that the binding of PGC7 to H3K9me2 required the N-terminal half of PGC7, whereas its ability to exclude Tet3 from the maternal pronucleus required the C-terminal half. It is unclear how Tet3 exclusion is mediated. One possibility is that the C-terminal region of PGC7 sterically excludes Tet3 from binding, either to DNA or to a chromatin mark; another is that the C-terminal region of PGC7 is capable of altering chromatin configuration to prevent the binding of Tet3 to chromatin. In support of the latter hypothesis, the rate with which micrococcal nuclease (MNase) digested high-molecular weight chromatin was significantly slower in WT ES cells in which PGC7 was present, compared to PGC7^−/− and G9a^−/− ES cells in which PGC7 was either absent or not recruited to DNA because of the loss of H3K9me2 mark. In contrast, DNA methylation did not alter the chromatin association of PGC7 or its ability to protect high-molecular weight chromatin from MNase digestion, as shown by using Dnmt1^−/−Dnmt3a^−/−Dnmt3b^−/− triple knockout ES cells that completely lack DNA methylation.How does PGC7 protect paternally-imprinted loci from Tet3-mediated 5mC oxidation? Although the haploid sperm genome is mostly packaged with protamine, a genome-wide analysis revealed that 4% of the genome of mature human sperm bears nucleosomes located at developmental and imprinted genes¹⁴. Nakamura et al.¹² found that among paternally-imprinted differentially methylated regions (DMRs), the H19 and Rasgrf1 DMRs contained H3K9me2 whereas the Meg3 DMR did not, consistent with their previous finding that in PGC7-deficient zygotes, the H19 and Rasgrf1 DMRs were hypomethylated but the Meg3 DMR was unaffected¹¹. Therefore, PGC7 may be recruited to paternally-imprinted loci through H3K9me2-containing nucleosomes that pre-exist in the sperm haploid genome upon fertilization. Alternatively, Nakamura et al. point out that protamine in the sperm is replaced soon after fertilization by the histone H3.3 variant, which in somatic cells does not bear H3K9me2 mark.In conclusion, Nakamura et al.¹² demonstrate unambiguously that PGC7 specifically binds to H3K9me2 in the maternal genome in zygotes, where its global occupancy excludes Tet3 and inhibits Tet3-mediated 5mC oxidation. This novel finding provides new insights into the global alterations of DNA methylation status that occur during early embryogenesis. Follow-up questions abound. First, can PGC7 protect other methylated loci such as transposable elements and the X-chromosome? It would be interesting to assess H3K9me2 at these loci. Second, how does the N-terminal half of PGC7 recognize H3K9me2? Structural characterization of this interaction may elucidate a novel epigenetic “reader” domain specific for H3K9me2. Third, PGC7 is a marker for cells of the inner cell mass, and is co-expressed with Tet1 and Tet2 rather than Tet3 in ESCs¹⁵. Does PGC7 also antagonize Tet1 and Tet2 and protect imprinted loci in ESCs? Fourth, how does PGC7 inhibit the access of Tet3 to chromatin? Considering that PGC7 is small and is not equipped with known enzymatic domains, it is likely that PGC-interacting proteins, rather than PGC7 itself, function to regulate chromatin status. Fifth, how is Tet3 recruited to paternal chromatin – are there specific histone or other epigenetic marks that facilitate Tet3 recruitment? Finally, while technically challenging, it seems imperative to identify the target genes of PGC7 and Tet3, by profiling the genomic location of 5hmC and other 5mC oxidation products in the paternal and maternal genomes of zygotes from WT, Tet3-deficient and PGC7-deficient mice.     

Tet1 regulates epigenetic remodeling of the pericentromeric heterochromatin and chromocenter organization in DNA hypomethylated cells

Pericentromeric heterochromatin (PCH), the constitutive heterochromatin of pericentromeric regions, plays crucial roles in various cellular events, such as cell division and DNA replication. PCH forms chromocenters in the interphase nucleus, and chromocenters cluster at the prophase of meiosis. Chromocenter clustering has been reported to be critical for the appropriate progression of meiosis. However, the molecular mechanisms underlying chromocenter clustering remain elusive. In this study, we found that global DNA hypomethylation, 5hmC enrichment in PCH, and chromocenter clustering of Dnmt1-KO ESCs were similar to those of the female meiotic germ cells. Tet1 is essential for the deposition of 5hmC and facultative histone marks of H3K27me3 and H2AK119ub at PCH, as well as chromocenter clustering. RING1B, one of the core components of PRC1, is recruited to PCH by TET1, and PRC1 plays a critical role in chromocenter clustering. In addition, the rearrangement of the chromocenter under DNA hypomethylated condition was mediated by liquid-liquid phase separation. Thus, we demonstrated a novel role of Tet1 in chromocenter rearrangement in DNA hypomethylated cells.     

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.     

MicroRNA-29b/Tet1 regulatory axis epigenetically modulates mesendoderm differentiation in mouse embryonic stem cells

Ten eleven translocation (Tet) family-mediated DNA oxidation on 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) represents a novel epigenetic modification that regulates dynamic gene expression during embryonic stem cells (ESCs) differentiation. Through the role of Tet on 5hmC regulation in stem cell development is relatively defined, how the Tet family is regulated and impacts on ESCs lineage development remains elusive. In this study, we show non-coding RNA regulation on Tet family may contribute to epigenetic regulation during ESCs differentiation, which is suggested by microRNA-29b (miR-29b) binding sites on the Tet1 3′ untranslated region (3′ UTR). We demonstrate miR-29b increases sharply after embyoid body (EB) formation, which causes Tet1 repression and reduction of cellular 5hmC level during ESCs differentiation. Importantly, we show this miR-29b/Tet1 regulatory axis promotes the mesendoderm lineage formation both in vitro and in vivo by inducing the Nodal signaling pathway and repressing the key target of the active demethylation pathway, Tdg. Taken together, our findings underscore the contribution of small non-coding RNA mediated regulation on DNA demethylation dynamics and the differential expressions of key mesendoderm regulators during ESCs lineage specification. MiR-29b could potentially be applied to enrich production of mesoderm and endoderm derivatives and be further differentiated into desired organ-specific cells.     

Placental 5-methylcytosine and 5-hydroxymethylcytosine patterns associate with size at birth

Altered placental function as a consequence of aberrant imprinted gene expression may be one mechanism mediating the association between low birth weight and increased cardiometabolic disease risk. Imprinted gene expression is regulated by epigenetic mechanisms, particularly DNA methylation (5mC) at differentially methylated regions (DMRs). While 5-hydroxymethylcytosine (5hmC) is also present at DMRs, many techniques do not distinguish between 5mC and 5hmC. Using human placental samples, we show that the expression of the imprinted gene CDKN1C associates with birth weight. Using specific techniques to map 5mC and 5hmC at DMRs controlling the expression of CDKN1C and the imprinted gene IGF2, we show that 5mC enrichment at KvDMR and DMR0, and 5hmC enrichment within the H19 gene body, associate positively with birth weight. Importantly, the presence of 5hmC at imprinted DMRs may complicate the interpretation of DNA methylation studies in placenta; future studies should consider using techniques that distinguish between, and permit quantification of, both modifications.     

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]     

Hydroxylation of methylated DNA by TET1 in chondrocyte differentiation of C3H10T1/2 cells

DNA methylation is closely involved in the regulation of cellular differentiation, including chondrogenic differentiation of mesenchymal stem cells. Recent studies showed that Ten–eleven translocation (TET) family proteins converted 5-methylcytosine (5mC) to 5-hydroxymethylcytosine, 5-formylcytosine and 5carboxylcytosine by oxidation. These reactions constitute potential mechanisms for active demethylation of methylated DNA. However, the relationship between the DNA methylation patterns and the effects of TET family proteins in chondrocyte differentiation is still unclear. In this study, we showed that DNA hydroxylation of 5mC was increased during chondrocytic differentiation of C3H10T1/2 cells and that the expression of Tet1 was particularly enhanced. Moreover, knockdown experiments revealed that the downregulation of Tet1 expression caused decreases in chondrogenesis markers such as type 2 and type 10 collagens. Furthermore, we found that TET proteins had a site preference for hydroxylation of 5mC on the Insulin-like growth factor 1 (Igf1) promoter in chondrocytes. Taken together, we showed that the expression of Tet1 was specifically facilitated in chondrocyte differentiation and Tet1 can regulate chondrocyte marker gene expression presumably through its hydroxylation activity for DNA.     

Tet family proteins and 5-hydroxymethylcytosine in development and disease

Over the past few decades, DNA methylation at the 5-position of cytosine (5-methylcytosine, 5mC) has emerged as an important epigenetic modification that plays essential roles in development, aging and disease. However, the mechanisms controlling 5mC dynamics remain elusive. Recent studies have shown that ten-eleven translocation (Tet) proteins can catalyze 5mC oxidation and generate 5mC derivatives, including 5-hydroxymethylcytosine (5hmC). The exciting discovery of these novel 5mC derivatives has begun to shed light on the dynamic nature of 5mC, and emerging evidence has shown that Tet family proteins and 5hmC are involved in normal development as well as in many diseases. In this Primer we provide an overview of the role of Tet family proteins and 5hmC in development and cancer.     

Dynamic reprogramming of 5-hydroxymethylcytosine during early porcine embryogenesis

DNA active demethylation is an important epigenetic phenomenon observed in porcine zygotes, yet its molecular origins are unknown. Our results show that 5-methylcytosine (5mC) converts into 5-hydroxymethylcytosine (5hmC) during the first cell cycle in porcine in vivo fertilization (IVV), IVF, and SCNT embryos, but not in parthenogenetically activated embryos. Expression of Ten-Eleven Translocation 1 (TET1) correlates with this conversion. Expression of 5mC gradually decreases until the morula stage; it is only expressed in the inner cell mass, but not trophectoderm regions of IVV and IVF blastocysts. Expression of 5mC in SCNT embryos is ectopically distinct from that observed in IVV and IVF embryos. In addition, 5hmC expression was similar to that of 5mC in IVV cleavage-stage embryos. Expression of 5hmC remained constant in IVF and SCNT embryos, and was evenly distributed among the inner cell mass and trophectoderm regions derived from IVV, IVF, and SCNT blastocysts. Ten-Eleven Translocation 3 was highly expressed in two-cell embryos, whereas TET1 and TET2 were highly expressed in blastocysts. These data suggest that TET1-catalyzed 5hmC may be involved in active DNA demethylation in porcine early embryos. In addition, 5mC, but not 5hmC, participates in the initial cell lineage specification in porcine IVV and IVF blastocysts. Last, SCNT embryos show aberrant 5mC and 5hmC expression during early porcine embryonic development.