Quantitative Sequencing of 5-Methylcytosine and 5-Hydroxymethylcytosine at Single-Base Resolution

TLDR

5‑Methylcytosine is converted to 5‑hydroxymethylcytosine (5hmC) in mammalian DNA by TET enzymes. The study introduces oxBS‑Seq, the first method for quantitative, single‑base resolution mapping of 5hmC in genomic DNA. oxBS‑Seq oxidizes 5hmC to 5‑formylcytosine, which is then converted to uracil by bisulfite treatment, enabling discrimination of 5hmC from 5mC at single‑base resolution. Using oxBS‑Seq, the authors mapped 800 CGIs with ~3.3% 5hmC in mouse embryonic stem cells, finding high 5hmC enrichment in CGIs associated with transcriptional regulators and LINE elements, suggesting epigenetic reprogramming and prompting questions about 5hmC dynamics and TET targeting.

Abstract

5-Methylcytosine can be converted to 5-hydroxymethylcytosine (5hmC) in mammalian DNA by the ten-eleven translocation (TET) enzymes. We introduce oxidative bisulfite sequencing (oxBS-Seq), the first method for quantitative mapping of 5hmC in genomic DNA at single-nucleotide resolution. Selective chemical oxidation of 5hmC to 5-formylcytosine (5fC) enables bisulfite conversion of 5fC to uracil. We demonstrate the utility of oxBS-Seq to map and quantify 5hmC at CpG islands (CGIs) in mouse embryonic stem (ES) cells and identify 800 5hmC-containing CGIs that have on average 3.3% hydroxymethylation. High levels of 5hmC were found in CGIs associated with transcriptional regulators and in long interspersed nuclear elements, suggesting that these regions might undergo epigenetic reprogramming in ES cells. Our results open new questions on 5hmC dynamics and sequence-specific targeting by TETs.