Project description:Active DNA demethylation, the enzymatic removal of methyl groups from DNA initially triggered by their successive oxidation, is a fundamental process critical to regulating cellular identity. The major enzymes involved in this process have been recently elucidated, but other important components in this pathway and the independent contributions of oxidized 5-methylcytosine derivatives to the regulation of gene expression remain unclear. A major obstacle to the elucidation of this pathway is the low abundance of these derivatives and limited power of current detection technologies. Here, we first develop a technique for their detection with APOBEC3A conversion and sequencing (APOBEC-seq) to directly discriminate deamination-insensitive oxidized cytosines from methylated or unmethylated counterparts, which now facilitates robust DNA demethylation analysis suitable to interrogate the active DNA demethylation pathway by a comprehensive set of experiments. Our results demonstrate that APOBEC-seq is a powerful tool for the detection of oxidized cytosines, revealing insights about the relationship between oxidized gene re-activation and demethylation as a function of TDG, APEX1, and the MBD family of proteins. We also report a ubiquitous binding of TDG to all active unmethylated and unoxidized promoters – regardless of RNA polymerase subtype – suggesting that, together with TET family of enzymes, the presence of TDG at these regions may safeguard active genes from DNA hypermethylation-induced silencing and may explain the failures of CRISPR/dCas9-based DNA methylation editing tools to introduce persistent DNA hypermethylation at targeted promoters. We also report an interaction between MBD3/NuRD and TDG which recruits TDG to its targets and may shed light on the critical role of MBD3 in development. Finally, we apply APOBEC-seq to profile oxidation in vivo in the mouse cortex and report a dramatic tissue-specific pattern of oxidation in genes and enhancers.

Project description:Thymine DNA Glycosylase (TDG) is an enzyme of the base excision repair mechanism and removes damaged or mispaired bases from DNA via hydrolysis of the glycosidic bond. Specificity is of high importance for such a glycosylase, so as to avoid the damage of intact DNA. Among the substrates reported for TDG are mispaired uracil and thymine but also formyl-cytosine and carboxyl-cytosine. Methyl-cytosine and hydroxylmethyl-cytosine are, in contrast, not processed by the TDG enzyme. We have in this work employed molecular dynamics simulations to explore the conformational dynamics of DNA carrying a formyl-cytosine or carboxyl-cytosine and compared those to DNA with the non-cognate bases methyl-cytosine and hydroxylmethyl-cytosine, as amino and imino tautomers. Whereas for the mispairs a wobble conformation is likely decisive for recognition, all amino tautomers of formyl-cytosine and carboxyl-cytosine exhibit the same Watson-Crick conformation, but all imino tautomers indeed form wobble pairs. The conformational dynamics of the amino tautomers in free DNA do not exhibit differences that could be exploited for recognition, and also complexation to the TDG enzyme does not induce any alteration that would indicate preferable binding to one or the other oxidised methyl-cytosine. The imino tautomers, in contrast, undergo a shift in the equilibrium between a closed and a more open, partially flipped state, towards the more open form upon complexation to the TDG enzyme. This stabilisation of the more open conformation is most pronounced for the non-cognate bases methyl-cytosine and hydroxyl-cytosine and is thus not a likely mode for recognition. Moreover, calculated binding affinities for the different forms indicate the imino forms to be less likely in the complexed DNA. These findings, together with the low probability of imino tautomers in free DNA and the indifference of the complexed amino tautomers, suggest that discrimination of the oxidised methyl-cytosines does not take place in the initial complex formation.

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Bisulfite-free base-resolution sequencing of oxidized cytosines (APOBEC-seq) reveals a ubiquitous role of thymine DNA glycosylase in active gene promoters and an interaction with MBD3/NuRD

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