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Background: 5-Hydroxymethyluracil (5hmU) is a thymine base modification found in the genomes of a diverse range of organisms. To explore the functional importance of 5hmU, we develop a method for the genome-wide mapping of 5hmU-modified loci based on a chemical tagging strategy for the hydroxymethyl group., Results: We apply the method to generate genome-wide maps of 5hmU in the parasitic protozoan Leishmania sp. In this genus, another thymine modification, 5-(β-glucopyranosyl) hydroxymethyluracil (base J), plays a key role during transcription. To elucidate the relationship between 5hmU and base J, we also map base J loci by introducing a chemical tagging strategy for the glucopyranoside residue. Observed 5hmU peaks are highly consistent among technical replicates, confirming the robustness of the method. 5hmU is enriched in strand switch regions, telomeric regions, and intergenic regions. Over 90% of 5hmU-enriched loci overlapped with base J-enriched loci, which occurs mostly within strand switch regions. We also identify loci comprising 5hmU but not base J, which are enriched with motifs consisting of a stretch of thymine bases., Conclusions: By chemically detecting 5hmU we present a method to provide a genome-wide map of this modification, which will help address the emerging interest in the role of 5hmU. This method will also be applicable to other organisms bearing 5hmU.

Background: 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., Results: 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., Conclusions: 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.