| Abstrakt: |
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. Significance statement: Ten‐eleven translocation (TET) isoforms play a crucial role in epigenetic regulation of gene expression by modifying DNA methylation patterns. In addition, these isoforms are particularly important in embryonic development and stem cell maintenance, where they regulate the dynamic changes in DNA methylation patterns that are critical for proper cell fate specification and tissue differentiation. Aberrant DNA methylation patterns are also a hallmark of cancer, and TET enzymes have been implicated in cancer progression and metastasis. Therefore, understanding the significance of TET enzymes has important implications for our understanding of both normal development and disease. Although, it has been reported that TET isoforms can oxidize 5mC present in dsDNA along with ssDNA and RNA, little is known about the minimum substrate length required for the optimum activity of TET isoforms. Here, by systematically varying length and configuration of DNA substrates combined with a highly sensitive LC‐MS/MS method, we demonstrate that mTET1 and 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. Furthermore, our results demonstrate that mTET1 and hTET2 prefer dsDNA as a substrate over ssDNA. Finally, we show that substrate specificity of TET isoforms correlates with their DNA binding efficiency. It is important to note that the length of linker DNA can vary from 5 to 60 nucleotides depending on the context and specific protein−DNA interactions involved. Therefore, identifying that hTET2 and mTET1 prefer double‐stranded 13‐mer DNA as substrates is significant because it provides insight into the minimal substrate DNA length, probably within the linker region, with which TET proteins interact for optimum oxidative product formation. It is worth mentioning that TETs work in conjunction with various regulatory proteins and factors that influence their binding and activity. These factors encompass DNA‐binding proteins, chromatin structure, and specific DNA sequence motifs. The presence of these additional factors can impact the specific DNA length and context required for optimal TET activity. TETs exhibit their effects on individual DNA molecules as well as larger genomic regions like CpG islands, which consist of frequently methylated CpG sites. Additionally, the DNA sequence context, including neighboring nucleotides and specific DNA motifs, can impact TET activity. Certain DNA sequences can facilitate the binding and recognition of TET enzymes and other associated DNA‐binding proteins, thereby altering the minimum DNA length required for their activity. Thus, the minimum length of DNA required for DNA demethylase activity in vivo may vary depending on the specific TET enzyme and its associated factors. Importantly, our results demonstrate that the size of the target region can influence the 5mC oxidation states (i.e., 5hmC, 5fC, and 5caC) which can dictate fundamental epigenetic regulatory processes in health and diseases. [ABSTRACT FROM AUTHOR] |
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