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Oxidation of thymine by TET2 dioxygenase carrying leukemia-derived neomorphic mutations

ABSTRACT: TET enzymes oxidize 5-methylcytosine (mC) in DNA, contributing to the regulation of gene transcription and genome functions. Deficiency of TET2 promotes the development of clonal hematopoiesis and various blood cancers, yet the mechanism by which TET2 mutations facilitate tumorigenesis remains to be explored. Here, we report that a subset of TET2 mutations identified in leukemia patients alter the substrate specificity of TET2 from acting on mC to thymine. This neomorphic activity results from substitutions at key residues involved in the interactions with the mC base, including Asn1387 and His1904. Recombinant human TET2 proteins harboring the mutation of these residues can catalyze the oxidation of thymine to 5-hydroxymethyluracil (hmU) and 5-formyluracil (fU). Exogenous expression of the TET2 Asn1387Thr (N1387T) mutant in transfected cells leads to hmU accumulation, with the highest level in cells lacking SMUG1-initiated base excision repair pathway. Endogenous knock-in of N1300T, the murine equivalent of N1387T, in mouse embryonic stem cells induces hmU production, causing DNA lesions and transcriptional activation of DNA damage response genes. These findings suggest that certain patient-derived TET2 mutations acquire unexpected gain-of-functions beyond an impairment in mC oxidation, and thymine oxidation by the neomorphic mutants may impose risks to genome stability, presumably contributing to malignant transformation.

ORGANISM(S): Mus musculus

PROVIDER: GSE278366 | GEO | 2025/02/12

REPOSITORIES: GEO

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Project description:Ten-eleven translocation (TET) proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytsosine (5fC), and 5-carboxylcytosine (5caC). 5fC/5caC can be excised and repaired by the base excision repair (BER) pathway, implicating 5mC oxidation in active DNA demethylation. Genome-wide DNA methylation is erased in the transition from metastable states to ground state of embryonic stem cells (ESCs) and in migrating primordial germ cells (PGCs), while some resistant regions become demethylated only in gonadal PGCs. Understanding the mechanisms underlying global hypomethylation in naïve ESCs and developing PGCs will be useful for realizing cellular pluripotency and totipotency. In this study, we found that PRDM14, the PR-domain-containing transcriptional regulator, accelerates the TET-BER cycle, resulting in the promotion of active DNA demethylation in ESCs. Induction of PRDM14 expression rapidly removed the 5mC associated with transient elevation of 5hmC at pluripotency-associated genes, germline-specific genes, and imprinted loci but not across the entire genome, which resemble second wave of DNA demethylation in gonadal PGCs. PRDM14 physically interacts with TET1/TET2 and enhances the recruitment of TET1/TET2 at target loci. Knockdown of Tet1/Tet2 impaired transcriptional regulation and DNA demethylation by PRDM14. The repression of the BER pathway by administration of pharmacological inhibitors against APE1 and PARP1 and the knockdown of thymine DNA glycosylase (TDG) also impaired DNA demethylation by PRDM14. Furthermore, DNA demethylation induced by PRDM14 normally takes place in the presence of aphidicolin, which is an inhibitor of G1/S progression. Together, our analysis provides mechanistic insight into DNA demethylation in naive pluripotent stem cells and developing PGCs. To investigate the function of TET1/TET2 in transcriptional regulation by PRDM14 in ESCs, we exploited microarray analysis using total mRNA derived from Scramble, Scramble + PRDM14, Tet1/Tet2 KD, Tet1/Tet2 KD + PRDM14 mouse ESC.

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Oxidation of thymine by TET2 dioxygenase carrying leukemia-derived neomorphic mutations

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