Dataset Information

In vivo genome-wide profiling reveals a tissue-specific role for 5-formylcytosine.

ABSTRACT: Genome-wide methylation of cytosine can be modulated in the presence of TET and thymine DNA glycosylase (TDG) enzymes. TET is able to oxidise 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). TDG can excise the oxidative products 5fC and 5caC, initiating base excision repair. These modified bases are stable and detectable in the genome, suggesting that they could have epigenetic functions in their own right. However, functional investigation of the genome-wide distribution of 5fC has been restricted to cell culture-based systems, while its in vivo profile remains unknown.Here, we describe the first analysis of the in vivo genome-wide profile of 5fC across a range of tissues from both wild-type and Tdg-deficient E11.5 mouse embryos. Changes in the formylation profile of cytosine upon depletion of TDG suggest TET/TDG-mediated active demethylation occurs preferentially at intron-exon boundaries and reveals a major role for TDG in shaping 5fC distribution at CpG islands. Moreover, we find that active enhancer regions specifically exhibit high levels of 5fC, resulting in characteristic tissue-diagnostic patterns, which suggest a role in embryonic development.The tissue-specific distribution of 5fC can be regulated by the collective contribution of TET-mediated oxidation and excision by TDG. The in vivo profile of 5fC during embryonic development resembles that of embryonic stem cells, sharing key features including enrichment of 5fC in enhancer and intragenic regions. Additionally, by investigating mouse embryo 5fC profiles in a tissue-specific manner, we identify targeted enrichment at active enhancers involved in tissue development.

SUBMITTER: Iurlaro M

PROVIDER: S-EPMC4928330 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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In vivo genome-wide profiling reveals a tissue-specific role for 5-formylcytosine.

Iurlaro Mario M McInroy Gordon R GR Burgess Heather E HE Dean Wendy W Raiber Eun-Ang EA Bachman Martin M Beraldi Dario D Balasubramanian Shankar S Reik Wolf W

Genome biology 20160629 1

<h4>Background</h4>Genome-wide methylation of cytosine can be modulated in the presence of TET and thymine DNA glycosylase (TDG) enzymes. TET is able to oxidise 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). TDG can excise the oxidative products 5fC and 5caC, initiating base excision repair. These modified bases are stable and detectable in the genome, suggesting that they could have epigenetic functions in their own right. However ...[more]

PMID: 27356509

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Project description:Background: Genome-wide methylation of cytosine can be modulated in the presence of TET and thymine DNA glycosylase (TDG) enzymes. TET enzymes are able to oxidise 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). TDG can excise the oxidative products 5fC and 5caC, initiating base excision repair. Furthermore, these modified bases are stable and detectable in the genome, raising the possibility that they could have epigenetic functions in their own right. To date, functional investigation of the genome-wide distribution of 5fC has been restricted to cell culture based systems, while its in vivo profile, in particular during development, remains unknown. Results: Here we describe the first analysis of the in vivo genome-wide profile of 5fC, across a range of dissected tissues from both wild type and Tdg-deficient E11.5 mouse embryos. Changes in the formylation profile of cytosine upon depletion of TDG suggest TET/TDG-mediated active demethylation occurs preferentially at intron-exon boundaries, and reveals a major role for TDG in shaping 5fC distribution at CpG islands. Moreover, we find enhancer regions exhibit high levels of 5fC, which accumulates at tissue-specific enhancers implicating a role in embryonic development. Conclusions: The tissue-specific distribution of 5fC can be regulated by the collective contribution of TET-mediated oxidation and excision by TDG. We show that the in vivo profile of 5fC during embryonic development resembles that of embryonic stem cells, sharing key features including enrichment of 5fC in enhancer and intragenic regions. Additionally, by investigating 5fC profiles in a tissue-specific manner from mouse embryos, we identified a targeted enrichment at active enhancers involved in tissue development. 5-formylcytosine has been mapped genomewide by pull-down and sequencing in mouse hindbrain, heart, carcass and liver. Each tissue was replicated in two different mice except for hindbrain which was replicated in four different mice.

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.

Dataset Information

In vivo genome-wide profiling reveals a tissue-specific role for 5-formylcytosine.

Publications

In vivo genome-wide profiling reveals a tissue-specific role for 5-formylcytosine.

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