scholarly journals Direct observation and analysis of TET-mediated oxidation processes in a DNA origami nanochip

2020 ◽  
Vol 48 (8) ◽  
pp. 4041-4051 ◽  
Author(s):  
Xiwen Xing ◽  
Shinsuke Sato ◽  
Nai-Kei Wong ◽  
Kumi Hidaka ◽  
Hiroshi Sugiyama ◽  
...  
Keyword(s):  
Single Molecule ◽  
High Speed ◽  
Excision Repair ◽  
Regulation Of Gene Expression ◽  
Dna Demethylation ◽  
Dna Origami ◽  
Enzymatic Reactions ◽  
Oxidation Reactions ◽  
Methyl Cytosine ◽  
Tet Enzymes

Abstract DNA methylation and demethylation play a key role in the epigenetic regulation of gene expression; however, a series of oxidation reactions of 5-methyl cytosine (5mC) mediated by ten-eleven translocation (TET) enzymes driving demethylation process are yet to be uncovered. To elucidate the relationship between the oxidative processes and structural factors of DNA, we analysed the behavior of TET-mediated 5mC-oxidation by incorporating structural stress onto a substrate double-stranded DNA (dsDNA) using a DNA origami nanochip. The reactions and behaviors of TET enzymes were systematically monitored by biochemical analysis and single-molecule observation using atomic force microscopy (AFM). A reformative frame-like DNA origami was established to allow the incorporation of dsDNAs as 5mC-containing substrates in parallel orientations. We tested the potential effect of dsDNAs present in the tense and relaxed states within a DNA nanochip on TET oxidation. Based on enzyme binding and the detection of oxidation reactions within the DNA nanochip, it was revealed that TET preferred a relaxed substrate regardless of the modification types of 5-oxidated-methyl cytosine. Strikingly, when a multi-5mCG sites model was deployed to further characterize substrate preferences of TET, TET preferred the fully methylated site over the hemi-methylated site. This analytical modality also permits the direct observations of dynamic movements of TET such as sliding and interstrand transfer by high-speed AFM. In addition, the thymine DNA glycosylase-mediated base excision repair process was characterized in the DNA nanochip. Thus, we have convincingly established the system's ability to physically regulate enzymatic reactions, which could prove useful for the observation and characterization of coordinated DNA demethylation processes at the nanoscale.

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

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Atsushi Onodera ◽  
Edahí González-Avalos ◽  
Chan-Wang Jerry Lio ◽  
Romain O. Georges ◽  
Alfonso Bellacosa ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Cell Activation ◽  
Immune Cell ◽  
Excision Repair ◽  
Dna Demethylation ◽  
T Cell Differentiation ◽  
Tet Enzymes

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.

scholarly journals TET Family of Dioxygenases: Crucial Roles and Underlying Mechanisms

2015 ◽  
Vol 146 (3) ◽  
pp. 171-180 ◽  
Author(s):  
Duo Li ◽  
Bin Guo ◽  
Haijing Wu ◽  
Lina Tan ◽  
Qianjin Lu
Keyword(s):  
Gene Expression ◽  
Therapeutic Potential ◽  
Dna Demethylation ◽  
Oxidation Reactions ◽  
Exciting Field ◽  
Tet Proteins ◽  
Active Dna Demethylation ◽  
Health And Disease ◽  
Underlying Mechanisms ◽  
Tet Enzymes

DNA methylation plays an important role in the epigenetic regulation of mammalian gene expression. TET (ten-eleven translocation) proteins, newly discovered demethylases, have sparked great interest since their discovery. TET proteins catalyze 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine in 3 consecutive Fe(II)- and 2-oxoglutarate (2-OG)-dependent oxidation reactions. TET proteins dynamically regulate global or locus-specific 5-methylcytosine and/or 5-hydroxymethylcytosine levels by facilitating active DNA demethylation. In fact, in addition to their role as methylcytosine dioxygenases, TET proteins are closely related to histone modification, interact with metabolic enzymes as well as other proteins, and cooperate in transcriptional regulation. In this review, we summarize the recent progress in this exciting field, highlighting the molecular mechanism by which TET enzymes regulate gene expression and their functions in health and disease. We also discuss the therapeutic potential of targeting TET proteins and aberrant DNA modifications.

scholarly journals Epigenetic Regulation of Genomic Stability by Vitamin C

2021 ◽  
Vol 12 ◽  
Author(s):  
John P. Brabson ◽  
Tiffany Leesang ◽  
Sofia Mohammad ◽  
Luisa Cimmino
Keyword(s):  
Dna Methylation ◽  
Vitamin C ◽  
Excision Repair ◽  
Antioxidant Properties ◽  
Genomic Stability ◽  
Dna Demethylation ◽  
Dna Hypermethylation ◽  
Active Dna Demethylation ◽  
Base Excision ◽  
Tet Enzymes

DNA methylation plays an important role in the maintenance of genomic stability. Ten-eleven translocation proteins (TETs) are a family of iron (Fe2+) and α-KG -dependent dioxygenases that regulate DNA methylation levels by oxidizing 5-methylcystosine (5mC) to generate 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). These oxidized methylcytosines promote passive demethylation upon DNA replication, or active DNA demethylation, by triggering base excision repair and replacement of 5fC and 5caC with an unmethylated cytosine. Several studies over the last decade have shown that loss of TET function leads to DNA hypermethylation and increased genomic instability. Vitamin C, a cofactor of TET enzymes, increases 5hmC formation and promotes DNA demethylation, suggesting that this essential vitamin, in addition to its antioxidant properties, can also directly influence genomic stability. This review will highlight the functional role of DNA methylation, TET activity and vitamin C, in the crosstalk between DNA methylation and DNA repair.

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