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.
Three forms of methylated cytosines are present in the eukaryotic genome: 3-methylcytosine, 4-methylcytosine and 5-methylcytosine. 3-methylcytosines create methyl lesions, which impair local DNA function and flexibility, resulting in replication and transcription error. On the other hand, 5-methylcytosine is usually present at the gene promoter which blocks transcription and translation. Fe(II)/2OG-dependent nucleic acid-modifying enzymes are the class of enzymes responsible for the demethylation of these modified cytosines. ALKBH2 and 3 remove 3-methylcytosine via a one-step direct demethylation process. On the other hand, active demethylation of 5mC is initiated by Ten-Eleven Translocation (TET)-family dioxygenases. Via oxidative demethylation, TET1-3 converts 5mC into 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine. Remarkably, recent findings demonstrate that ALKBH2,3 possess oxidative demethylation properties, along with direct demethylation. On the other hand, the TET family of enzymes possess direct demethylation properties along with oxidative demethylation. Here we review the importance of methylated cytosines in human DNA, their origin, function and removal. In addition, we discuss the recent findings of extraordinary flexibility of Fe(II)/2OG-dependent nucleic acid-modifying enzymes ALKBH2,3 and TET family of enzymes in cytosine demethylation, as well as their impact on epigenetics.
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