A Sensitive Approach to Map Genome-wide 5-Hydroxymethylcytosine and 5-Formylcytosine at Single-Base Resolution

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. [Display omitted] •Enzymatic approach to map genome-wide 5hmC and 5fC at single-base resolution•Identifies low-abundance 5hmC and 5fC in CpG and non-CpG contexts•Low amount of starting material can be used for 5hmC mapping•Independent of bisulfite or other chemical conversion Sun et al. established a sensitive method to map genome-wide 5-hydroxymethlycytosine and 5-formylcytosine sites at single-base resolution. The method enables detection of low-abundance 5-hydroxymethlycytosine sites, providing a more complete 5-hydroxymethlycytosine landscape, and also generated a genome-wide 5-formylcytosine map.