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Onodera A , González-Avalos E , Lio CJ , Georges RO , Bellacosa A , Nakayama T , Rao A
Roles of TET and TDG in DNA demethylation in proliferating and non-proliferating immune cells
Genome Biol. 2021 Jun 22;22(1) :186
PMID: 34158086    PMCID: PMC8218415    URL: https://www.ncbi.nlm.nih.gov/pubmed/34158086
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BACKGROUND: TET enzymes mediate DNA demethylation by oxidizing 5-methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Since these oxidized methylcytosines (oxi-mCs) are not recognized by the maintenance methyltransferase DNMT1, DNA demethylation can occur through "passive," replication-dependent dilution when cells divide. A distinct, replication-independent ("active") mechanism of DNA demethylation involves excision of 5fC and 5caC by the DNA repair enzyme thymine DNA glycosylase (TDG), followed by base excision repair. RESULTS: Here by analyzing inducible gene-disrupted mice, we show that DNA demethylation during primary T cell differentiation occurs mainly through passive replication-dependent dilution of all three oxi-mCs, with only a negligible contribution from TDG. In addition, by pyridine borane sequencing (PB-seq), a simple recently developed method that directly maps 5fC/5caC at single-base resolution, we detect the accumulation of 5fC/5caC in TDG-deleted T cells. We also quantify the occurrence of concordant demethylation within and near enhancer regions in the Il4 locus. In an independent system that does not involve cell division, macrophages treated with liposaccharide accumulate 5hmC at enhancers and show altered gene expression without DNA demethylation; loss of TET enzymes disrupts gene expression, but loss of TDG has no effect. We also observe that mice with long-term (1 year) deletion of Tdg are healthy and show normal survival and hematopoiesis. CONCLUSIONS: We have quantified the relative contributions of TET and TDG to cell differentiation and DNA demethylation at representative loci in proliferating T cells. We find that TET enzymes regulate T cell differentiation and DNA demethylation primarily through passive dilution of oxi-mCs. In contrast, while we observe a low level of active, replication-independent DNA demethylation mediated by TDG, this process does not appear to be essential for immune cell activation or differentiation.
1474-760x Onodera, Atsushi Orcid: 0000-0002-3715-9408 González-Avalos, Edahí Orcid: 0000-0002-6817-4854 Lio, Chan-Wang Jerry Orcid: 0000-0003-3876-6741 Georges, Romain O Orcid: 0000-0001-8207-609x Bellacosa, Alfonso Orcid: 0000-0001-6278-6801 Nakayama, Toshinori Orcid: 0000-0002-1434-2007 Rao, Anjana Orcid: 0000-0002-1870-1775 W81XWH-17-1-0136/U.S. Department of Defense/ R01 AI128589/NH/NIH HHS/United States R35 CA210043/CA/NCI NIH HHS/United States JP19H05650/Japan Society for the Promotion of Science/ 26221305/Japan Society for the Promotion of Science/ R35 CA210043/NH/NIH HHS/United States JP20gm1210003/Japan Agency for Medical Research and Development/ CA191956/NH/NIH HHS/United States R01 HG010646/NH/NIH HHS/United States 18K07164/Japan Society for the Promotion of Science/ R01 AI40127/NH/NIH HHS/United States JP20ek0410060/Japan Agency for Medical Research and Development/ Journal Article Genome Biol. 2021 Jun 22;22(1):186. doi: 10.1186/s13059-021-02384-1.