A sensitive approach to map genome-wide 5-hydroxymethylcytosine and 5-formylcytosine at single-base resolution

Mol Cell. 2015 Feb 19;57(4):750-761. doi: 10.1016/j.molcel.2014.12.035. Epub 2015 Jan 29.


Mapping genome-wide 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) at single-base resolution is important to understand their biological functions. We present a cost-efficient mapping method that combines 5hmC-specific restriction enzyme PvuRts1I with a 5hmC chemical labeling enrichment method. The sensitive method enables detection of low-abundance 5hmC sites, providing a more complete 5hmC landscape than available bisulfite-based methods. This method generated a genome-wide 5fC map at single-base resolution. Parallel analyses revealed that 5hmC and 5fC in non-CpG context exhibit lower abundance, more dynamically, than those in CpG context. In the genic region, distribution of 5hmCpG and 5fCpG differed from 5hmCH and 5fCH (H = A, T, C). 5hmC and 5fC were distributed distinctly at regulatory protein-DNA binding sites, depleted in permissive transcription factor binding sites, and enriched at active and poised enhancers. This sensitive bisulfite conversion-free method can be applied to biological samples with limited starting material or low-abundance cytosine modifications.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • 5-Methylcytosine / analogs & derivatives
  • Animals
  • Base Sequence
  • Cytosine / analogs & derivatives*
  • Cytosine / chemistry
  • DNA Restriction Enzymes / chemistry
  • Embryonic Stem Cells
  • Epigenesis, Genetic
  • Gene Library
  • Histones / metabolism
  • Mice
  • Restriction Mapping / methods*


  • Histones
  • 5-hydroxymethylcytosine
  • 5-Methylcytosine
  • Cytosine
  • DNA Restriction Enzymes

Associated data

  • GEO/GSE64186