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:TET proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC are excised by mammalian DNA glycosylase TDG, implicating 5mC oxidation in DNA demethylation. Here we show that the genomic locations of 5fC can be determined by coupling chemical reduction with biotin tagging. Genome-wide mapping of 5fC in mouse embryonic stem cells (mESCs) reveals that 5fC preferentially occurs at poised enhancers among other gene regulatory elements. Application to Tdg null mESCs further suggests that 5fC production coordinates with p300 in remodeling epigenetic states of enhancers. This process, which is not influenced by 5hmC, appears to be associated with further oxidation of 5hmC and commitment to demethylation through 5fC. Finally, we resolved 5fC at base-resolution by hydroxylamine-based protection from bisulfite-mediated deamination, thereby confirming sites of 5fC accumulation. Our results reveal roles of active 5mC/5hmC oxidation and TDG-mediated demethylation in epigenetic tuning at regulatory elements. We report here a chemical labeling method that effectively differentiates 5fC from 5mC, 5hmC, and 5caC in genomic DNA. First, we quantitatively protect endogenous 5hmC with a regular glucose using b-glucosytransferase-catalyzed 5hmC glucosylation. Then, we selectively reduce 5fC with NaBH4 to 5hmC, and chemically label the resulting 5hmC (from 5fC) with an azide-modified glucose. Biotin can be installed subsequently for specific enrichment of 5fC. Our method thereby provides an effective tool of general utility for the genomic localization of 5fC. Here we provide genome-wide profiles of 5hmC, 5fC, and p300 in Tdg fl/fl and Tdg-/- mESCs as well as a 5fC control (Non-NaBH4) and polyA RNA-Seq expression data. Genome-wide profiles of 5hmC and 5fC in mESCs differentiated to embryoid bodies are also included. We also report the development and application of a single-base resolution method for the detection of 5fC in genomic DNA by hydroylamine mediated protection of 5fC from deamination during bisulfite treatment, or 5fC Chemical Assisted Bisulfite Sequencing (fCAB-Seq). We applied this method in parallel with conventional ChIP-Methyl-Seq to H3K4me1 ChIP enriched DNA from Tdg fl/fl and Tdg-/- mice.

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Thymine DNA glycosylase regulates cell-cycle-driven p53

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