Demethylation of Non-CpG Sites in DNA Is Initiated by TET2 5-Methylcytosine Dioxygenase

In the mammalian genome, cytosine methylation predominantly occurs at CpG sites. In addition, a number of recent studies have uncovered extensive C5 cytosine methylation (5mC) at non-CpG (5mCpH, where H = A/C/T) sites. Little is known about the enzyme responsible for active demethylation of 5mCpH sites. Using a very sensitive and quantitative LC–MS/MS method, we demonstrate that the human TET2, an iron (II)- and 2OG-dependent dioxygenase, which is a frequently mutated gene in several myeloid malignancies, as well as in a number of other types of cancers, can oxidize 5mCpH sites in double-stranded DNA in vitro. Similar to oxidation of 5mCpG, oxidation of 5mC at CpH sites produces 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC) bases in DNA. After 5mCpG, which is the most preferred substrate, TET2 prefers 5mCpC as a substrate, followed by 5mCpA and then 5mCpT. Since the TDG/BER pathway and deformylation or decarboxylation of 5fC or 5caC, respectively, can convert 5fCpH and 5caCpH to an unmodified cytosine base in DNA, our results suggest a novel demethylation pathway of 5mCpH sites initiated by TET2 dioxygenase.

Genomic Uracil and Aberrant Profile of Demethylation Intermediates in Epigenetics and Hematologic Malignancies

DNA of all living cells undergoes continuous structural and chemical alterations resulting from fundamental cellular metabolic processes and reactivity of normal cellular metabolites and constituents. Examples include enzymatically oxidized bases, aberrantly methylated bases, and deaminated bases, the latter largely uracil from deaminated cytosine. In addition, the non-canonical DNA base uracil may result from misincorporated dUMP. Furthermore, uracil generated by deamination of cytosine in DNA is not always damage as it is also an intermediate in normal somatic hypermutation (SHM) and class shift recombination (CSR) at the Ig locus of B-cells in adaptive immunity. Many of the modifications alter base-pairing properties and may thus cause replicative and transcriptional mutagenesis. The best known and most studied epigenetic mark in DNA is 5-methylcytosine (5mC), generated by a methyltransferase that uses SAM as methyl donor, usually in CpG contexts. Oxidation products of 5mC are now thought to be intermediates in active demethylation as well as epigenetic marks in their own rights. The aim of this review is to describe the endogenous processes that surround the generation and removal of the most common types of DNA nucleobase modifications, namely, uracil and certain epigenetic modifications, together with their role in the development of hematological malignances. We also discuss what dictates whether the presence of an altered nucleobase is defined as damage or a natural modification.

L-Ascorbic Acid in the Epigenetic Regulation of Cancer Development and Stem Cell Reprogramming

Recent studies have significantly expanded our understanding of the mechanisms of L-ascorbic acid (ASC, vitamin C) action, leading to the emergence of several hypotheses that validate the possibility of using ASC in clinical practice. ASC may be considered an epigenetic drug capable of reducing aberrant DNA and histone hypermethylation, which could be helpful in the treatment of some cancers and neurodegenerative diseases. The clinical potency of ASC is also associated with regenerative medicine; in particular with the production of iPSCs. The effect of ASC on somatic cell reprogramming is most convincingly explained by a combined enhancement of the activity of the enzymes involved in the active demethylation of DNA and histones. This review describes how ASC can affect the epigenetic status of a cell and how it can be used in anticancer therapy and stem cell reprogramming.

Recent evolution of a TET-controlled and DPPA3/STELLA-driven pathway of passive DNA demethylation in mammals

Genome-wide DNA demethylation is a unique feature of mammalian development and naïve pluripotent stem cells. Here, we describe a recently evolved pathway in which global hypomethylation is achieved by the coupling of active and passive demethylation. TET activity is required, albeit indirectly, for global demethylation, which mostly occurs at sites devoid of TET binding. Instead, TET-mediated active demethylation is locus-specific and necessary for activating a subset of genes, including the naïve pluripotency and germline marker Dppa3 (Stella, Pgc7). DPPA3 in turn drives large-scale passive demethylation by directly binding and displacing UHRF1 from chromatin, thereby inhibiting maintenance DNA methylation. Although unique to mammals, we show that DPPA3 alone is capable of inducing global DNA demethylation in non-mammalian species (Xenopus and medaka) despite their evolutionary divergence from mammals more than 300 million years ago. Our findings suggest that the evolution of Dppa3 facilitated the emergence of global DNA demethylation in mammals.

Alterations in 5hmC level and genomic distribution in aging-related epigenetic drift in human adipose stem cells

Aim: To clarify mechanisms affecting the level and distribution of 5-hydroxymethylcytosine (5hmC) during aging. Materials & methods: We examined levels and genomic distribution of 5hmC along with the expression of ten–eleven translocation methylcytosine dioxygenases (TETs) in adipose stem cells in young and age-advanced individuals. Results: 5hmC levels were higher in adipose stem cells of age-advanced than young individuals (p = 0.0003), but were not associated with age-related changes in expression of TETs. 5hmC levels correlated with population doubling time (r = 0.62; p = 0.01). We identified 58 differentially hydroxymethylated regions. Hypo-hydroxymethylated differentially hydroxymethylated regions were approximately twofold enriched in CCCTC-binding factor binding sites. Conclusion: Accumulation of 5hmC in aged cells can result from inefficient active demethylation due to altered TETs activity and reduced passive demethylation due to slower proliferation.

Positive/negative ion-switching-based LC–MS/MS method for quantification of cytosine derivatives produced by the TET-family 5-methylcytosine dioxygenases

Cytosine methylation at carbon-5 (5mC) in DNA plays crucial roles in epigenetic transcriptional regulation during metazoan development. The iron (II), 2-oxoglutarate-dependent Ten-Eleven Translocation (TET)-family dioxygenases initiate active demethylation of 5mC. TET2 oxidizes 5mC in nucleic acids into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine by iterative oxidation. Mutations in the TET2 gene are frequently detected in myeloid malignancies. Despite the established and emerging roles of TET oxygenases in health and diseases, in vitro characterization of these enzymes and their mutants is still in rudimentary stages. Here, we describe an improved positive/negative ion-switching-based liquid chromatography-tandem mass spectrometry (LC–MS/MS) method that can separate and quantify modified cytosine bases produced by TET-family 5-methylcytosine dioxygenases. This method will help in further elucidate the function of epigenetically important cytosine modifications. To the best of our knowledge, this is the first study reporting ion-switching-based LC–MS/MS method to analyse cytosine variants produced in TET catalysed reactions.

Active Demethylation of Non-CpG Moieties in Animals: A Neglected Research Area

The functional role of cytosine methylation in the CpG moieties of DNA, is well established in several biological functions. The interplay between CpG methylation and hypomethylation is a well-known mechanism of modulation of gene expression. However, the role of non-CpG methylation and active dynamics of demethylation is not clearly recognized. Although some evidence exists of a role of active non-CpG demethylation in the fast dynamics of transcriptional activation in animals, few studies deal with this topic. At present, active demethylation of non-CpG moieties is a neglected research area, in spite of the promise of significant novelties.