Abstract
Conventional techniques for single-base resolution mapping of epigenetic modifications of DNA such as 5-hydroxymethylcytosine (5hmC) rely on the sequencing of bisulfite-modified DNA. Here we present an alternative approach called SCL-exo which combines selective chemical labeling (SCL) of 5hmC in genomic DNA with exonuclease (exo) digestion of the bead-trapped modified DNA molecules. Associated with a straightforward bioinformatic analysis, this new procedure provides an unbiased and fast method for mapping this epigenetic mark at high resolution. Implemented on mouse genomic DNA from in vitro-differentiated neural precursor cells, SCL-exo sheds light on an intrinsic lack of conservation of hydroxymethylated CpGs across vertebrates.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-0919-y) contains supplementary material, which is available to authorized users.
Highlights
The recently discovered epigenetic mark 5hydroxymethylcytosine (5hmC) results from an active DNA demethylation process which involves iterative oxidation of 5-methylcytosine (5mC) driven by Ten-eleven translocation (TET) enzymes and leads to the replacement of 5mC by an unmodified base [1,2,3,4]
Mouse epiblast-like P19 embryonal carcinoma cells were treated with retinoic acid (RA) for 48 h to induce their differentiation into neural progenitor-like cells (NPLCs) [5]
It is of note that not all DNA strands were digested by the exonuclease up to the first 5hmC since unmodified Cs were found within reads (Fig. 1e and f)
Summary
The recently discovered epigenetic mark 5hydroxymethylcytosine (5hmC) results from an active DNA demethylation process which involves iterative oxidation of 5-methylcytosine (5mC) driven by Ten-eleven translocation (TET) enzymes and leads to the replacement of 5mC by an unmodified base [1,2,3,4]. 5hmC can be mapped at single-base resolution through two rounds of MspI digestion of DNA separated by a 5hmC glucosylation step, before size selection and sequencing (RRHP) [15] Highly reproducible, this procedure does not cover all CpGs in the genome since MspI requires a CCGG context for DNA cleavage (i.e. 15 % of all CpGs). In an effort to develop an alternative approach for single-CpG resolution mapping of 5hmC genome-wide, we adapted a strategy first employed to increase the resolution of chromatin immunoprecipitation (ChIP) through the use of an exonuclease (exo) to trim DNA cross-linked to proteins up to close vicinity of intermolecular bounds (ChIP-exo [18, 19]) This new procedure, called SCL-exo, is shown here to be suited to obtain single-CpG resolution data. We uncovered that, being included in highly conserved regulatory regions of the mouse genome, a majority of hydroxymethylated cytosines are not conserved in other vertebrate species, suggesting that they might affect chromatin structure rather than directly regulate transcription factor binding
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