Roles of TET and TDG in DNA demethylation in proliferating and non-proliferating immune cells

Abstract

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.

TET2 Regulates 5-Hydroxymethylcytosine Signature and CD4+ T-Cell Balance in Allergic Rhinitis

“… 37 38 Most articles concerning these oxidization products focused on 5hmC, and there was little research into the function of 5fC and 5caC in immune cells. 39 40 A recent study by Onodera et al . 39 has showed that although loss of TET enzymes disrupted gene expression, loss of TDG enzymes resulted in the accumulation of 5fC/5caC with no effects.…”

“… 39 40 A recent study by Onodera et al . 39 has showed that although loss of TET enzymes disrupted gene expression, loss of TDG enzymes resulted in the accumulation of 5fC/5caC with no effects. Changes in 5fC and 5caC made only a minor contribution to the differentiation and activation of Th2 cells and LPS-activated macrophages.…”

“…Changes in 5fC and 5caC made only a minor contribution to the differentiation and activation of Th2 cells and LPS-activated macrophages. 39 Caldwell et al . 40 identified 5fC/5caC, rather than 5hmC, which was the major driver of DNA demethylation during induced pluripotent stem cells reprogramming, and played important roles in gene regulation and chromatin reorganization.…”

Conventional and pathogenic Th2 cells in inflammation, tissue repair, and fibrosis

“…GATA3 protein stability is also important for the functions of Th2 cells ( 20 , 21 ). Through direct or indirect interaction with epigenetic modifiers, GATA3 is involved in chromatin remodeling at the Th2 cytokine gene loci: histone H3K9 acetylation ( 22 , 23 ), histone H3K4 methylation ( 20 ), and DNA demethylation ( 24 ). In addition, epigenetic modifications of the Gata3 gene control the stable expression of GATA3 proteins ( 20 , 25 , 26 ).…”

Molecular Characterization of the Clinical and Tumor Immune Microenvironment Signature of 5-methylcytosine-Related Regulators in non-small Cell Lung Cancer

“…DNA 5mC methylation is the classic epigenetic process, which is controlled by “writers” (DNA methyltransferases), “erasers” (DNA methyltransferases), and “readers” ( Ito et al, 2011 ; Du et al, 2015 ; Lio et al, 2020 ). With the discovery of 5mC regulators, recent studies suggested that DNA cytosine modifications may act as epigenetic markers in tumorigenesis ( Wu and Zhang, 2010 ; Cavalcante et al, 2020 ; Jiang, 2020 ; Mo et al, 2020 ) and can regulate tumor microenvironment (TME) infiltrating cells ( Chen et al, 2020 ; Zhao et al, 2021 ; Onodera et al, 2021 ). However, the comprehensive roles of TME cell infiltration directed by 5mC regulators in NSCLC remain unclear.…”