Division of Pharmacology & Pharmaceutical Sciences, University of Missouri-Kansas City, Kansas City, Missouri, USA.
Cell Biochem Funct. 2023 Aug;41(6):704-712. doi: 10.1002/cbf.3825. Epub 2023 Jun 22.
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.
十-十一易位（TET）异构体（TET1-3）在表观转录调控中发挥关键作用。此外，TET2 基因突变在胶质瘤和髓系恶性肿瘤患者中经常被检测到。TET 异构体可通过迭代氧化将 5-甲基胞嘧啶氧化为 5-羟甲基胞嘧啶、5-甲酰胞嘧啶和 5-羧基胞嘧啶。TET 异构体的体内 DNA 去甲基化活性可能取决于许多因素，包括酶的结构特征、与 DNA 结合蛋白的相互作用、染色质环境、DNA 序列、DNA 长度和构象。本研究的原理是确定 TET 异构体底物中首选的 DNA 长度和构象。我们使用一种高度敏感的基于 LC-MS/MS 的方法来比较 TET 异构体的底物偏好。为此，选择了四个不同序列的 DNA 底物集（S1、S2、S3、S4）。此外，在每个集合中，合成了四种不同长度的 DNA 底物，包括 7-、13-、19-和 25-mer 核苷酸。每个 DNA 底物进一步用于三种不同的构象，即双链对称甲基化、双链半甲基化和单链单甲基化，以评估它们对 TET 介导的 5mC 氧化的影响。我们证明，小鼠 TET1（mTET1）和人 TET2（hTET2）对 13-mer dsDNA 底物具有最高的偏好性。增加或减少 dsDNA 底物的长度会降低产物形成。与它们的 dsDNA 对应物相反，ssDNA 底物的长度对 5mC 氧化没有可预测的影响。最后，我们表明 TET 异构体的底物特异性与其 DNA 结合效率相关。我们的结果表明，mTET1 和 hTET2 更喜欢 13-mer dsDNA 作为底物而不是 ssDNA。这些结果可能有助于阐明 TET 介导的 5mC 氧化的新特性，并有助于开发用于检测患者 TET2 功能的新型诊断工具。
