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 are still in rudimentary stages. Here, we describe an improved positive/negative ion-switching based 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.
The ten‐eleven translocation (TET) isoforms (TET1‐3) play critical roles in epigenetic transcription regulation. In addition, mutations in the TET2 gene are frequently detected in patients with glioma and myeloid malignancies. TET isoforms can oxidize 5‐methylcytosine to 5‐hydroxymethylcytosine, 5‐formylcytosine, and 5‐carboxylcytosine, by iterative oxidation. The in vivo DNA demethylation activity of TET isoforms may depend on many factors including enzyme's structural features, its interaction with DNA‐binding proteins, chromatin context, DNA sequence, DNA length, and configuration. The rationale for this study is to identify the preferred DNA length and configuration in the substrates of TET isoforms. We have used a highly sensitive LC‐MS/MS‐based method to compare the substrate preference of TET isoforms. To this end, four DNA substrate sets (S1, S2, S3, S4) of different sequences were chosen. In addition, in each set, four different lengths of DNA substrates comprising 7‐, 13‐, 19‐, and 25‐mer nucleotides were synthesized. Each DNA substrate was further used in three different configurations, that is, double stranded symmetrically‐methylated, double stranded hemi‐methylated, and single stranded single‐methylated to evaluate their effect on TET‐mediated 5mC oxidation. We demonstrate that mouse TET1 (mTET1) and human TET2 (hTET2) have highest preference for 13‐mer dsDNA substrates. Increasing or decreasing the length of dsDNA substrate reduces product formation. In contrast to their dsDNA counterparts, the length of ssDNA substrates did not have a predictable effect on 5mC oxidation. Finally, we show that substrate specificity of TET isoforms correlates with their DNA binding efficiency. Our results demonstrate that mTET1 and hTET2 prefer 13‐mer dsDNA as a substrate over ssDNA. These results may help elucidate novel properties of TET‐mediated 5mC oxidation and help develop novel diagnostic tools to detect TET2 function in patients.
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.
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