Characterization of 5-methylcytosine dioxygenase Tet2 and rescue of mutant Tet2 activity by using turbo co-substrate
Date
2023Metadata
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Regulation of epigenetic transcription mediated by 5-methylcytosine (5mC) plays a critical role in eukaryotic development. Demethylation of these epigenetic marks is accomplished by sequential oxidation by ten-eleven translocation dioxygenases (TET1-3), followed by thymine-DNA glycosylase-dependent base excision repair. Inactivation of the TET2 gene due to genetic mutations or other epigenetic mechanisms is associated with poor prognosis in patients with diverse cancers, especially hematopoietic malignancies. Herein, we describe an efficient single-step purification of enzymatically active untagged human TET2 dioxygenase using cation-exchange chromatography. We further provide a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach that can separate and quantify the four normal DNA bases (A, T, G, and C), as well as the four modified cytosine bases (5-methyl, 5-hydroxymethyl, 5-formyl, and 5-carboxyl). This method can be used to evaluate the activities of wild-type and mutant TET2 dioxygenases.
In the mammalian genome, cytosine methylation predominantly occurs at CpG sites. In addition, several 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 the active demethylation of 5mCpH sites. Using a highly sensitive and quantitative LC–MS/MS method, we demonstrated that 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 the oxidation of 5mCpG, the 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 preferred substrate, TET2 prefers 5mCpC as a substrate, followed by 5mCpA and 5mCpT. Because the TDG/BER pathway can convert 5fCpH and 5caCpH to an unmodified cytosine base in DNA1, our results suggest a novel demethylation pathway of 5mCpH sites initiated by TET2 dioxygenase.
TET isoforms (TET1-3) play a critical role in epigenetic transcriptional regulation. In addition, mutations in TET2 are frequently detected in patients with glioma and myeloid malignancies. TET isoforms can oxidize 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine via iterative oxidation. However, little is known about the preference for DNA length and configuration as the optimum substrates for TET isoforms. We used a highly sensitive LC-MS/MS-based method to compare the substrate preferences of the TET isoforms. To this end, four DNA substrate sets (S1, S2, S3, and S4) with different sequences were chosen. In addition, four different lengths of DNA substrates comprising 7-, 13-, 19-, and 25-mer nucleotides were synthesized in each set. 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 effects on TET-mediated 5mC oxidation. We demonstrated that mouse TET1 (mTET1) and human TET2 (hTET2) had the highest preference for 13-mer dsDNA substrates. Increasing or decreasing the length of the dsDNA substrate reduced the product formation. In contrast to their dsDNA counterparts, the length of the ssDNA substrates did not exhibit a consistent pattern of 5mC oxidation. Finally, we showed that the substrate specificity of TET isoforms correlates with their DNA-binding efficiency. Our results demonstrated that mTET1 and hTET2 prefer 13-mer dsDNA as a substrate over ssDNA.
The pathogenesis of malignant evolution has been linked to the epigenetic repression of tumor suppressor genes (TSG). One such epigenetic mechanism observed in myelodysplastic syndrome (MDS) is the acquired progressive methylation of CpG islands in gene promoters, leading to transcriptional repression. The TET family of hydroxylases/dioxygenases, which includes TET1-3, has recently been identified as iron (II)- and 2OG-dependent dioxygenases (2-OGDDs). TET2 gene mutations occur frequently in myeloid malignancies, such as myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), myeloproliferative neoplasms, and secondary acute myeloid leukemia derived from these conditions. In cancer cells, the normal regulation of 2-OGDDs activity is disrupted, leading to changes in gene expression and epigenetic modifications. The 2OG analogs mimic the chemical structure of 2OG and modulate the activity of 2-OGDDs. 2OG analogs have been shown to restore the activity of clinically relevant 2-OGDDs and modulate the epigenetic landscape of cancer cells. The objective of this study was to develop effective strategies using 2OG analogs to enhance the activity of TET2 harboring point mutations at the R1896 residue. A library of 11 compounds was designed to mimic the chemical structure of the 2OG. By screening these compounds, eight 2OG analogs that could specifically rescue the mutant TET2 activity were identified. The catalytic activity of the TET2 R1896S mutant was enhanced by up to 90% compared with that of the wild-type enzyme. Furthermore, we demonstrated that TET2 clinical mutations in the R1896A and R1896F residues could be rescued using this alternate co-substrate approach.
The crystal structure of the catalytic domain of TET2 revealed that the two zinc fingers bring together the cysteine-rich domain and the double-stranded β-helix fold domain, creating a compact catalytic domain. The cysteine-rich domain stabilizes DNA above the double-stranded β-helix fold domain, and the catalytic cavity allows for insertion of 5mC, with the methyl group positioned towards the catalytic Fe (II) for oxidation. This cysteine-rich domain is conserved among all three TET family members and catalyzes substrate oxidation. Numerous TET2 mutations are documented, primarily within the dioxygenase domain. However, there is limited information regarding the structural and functional effects of these TET2 missense variants. The TET2-DNA crystal structure identified 12 amino acid residues in the active site that interact with the DNA substrate. In this study, these residues were mutated to alanine to disrupt their interaction with DNA and to investigate the resulting oxidation patterns of CpG and non-CpG substrates. Mutated residues affected the activity of hTET2, particularly on CpG substrates. Notably, three active-site mutations (Y1295A, R1302A, and H1904A) showed significantly higher oxidation efficiencies on CpG substrates than the wild-type enzyme. By performing alanine scanning on the DNA-interacting residues, it was found that the catalytic cavity of TET2 can accommodate different DNA sequences, and stability of the TET2-DNA complex relies on multiple amino acid residues. This suggests that a single-point mutation can be compensated for without rendering the enzyme catalytically inactive. However, further in vivo studies and molecular dynamics simulations are required to confirm these findings.
Table of Contents
Introduction -- Cloning, expression, purification of TET methylcytosine dioxygenase 2 and development of an in vitro assay system -- Characterization of TET methylcytosine dioxygenase 2 mediated 5mC oxidation in non-CpG context -- Substrate DNA length regulates the activity of TET 5-methylcytosine dioxygenases -- Use of 2-oxoglutarate analogs for rescuing mutations in TET methylcytosine dioxygenase 2 -- Functional characterization of active site mutations in TET methylcytosine dioxygenase 2 -- Conclusions
Degree
Ph.D. (Doctor of Philosophy)