PT - JOURNAL ARTICLE AU - Booth, Michael J. AU - Branco, Miguel R. AU - Ficz, Gabriella AU - Oxley, David AU - Krueger, Felix AU - Reik, Wolf AU - Balasubramanian, Shankar TI - Quantitative Sequencing of 5-Methylcytosine and 5-Hydroxymethylcytosine at Single-Base Resolution AID - 10.1126/science.1220671 DP - 2012 May 18 TA - Science PG - 934--937 VI - 336 IP - 6083 4099 - http://science.sciencemag.org/content/336/6083/934.short 4100 - http://science.sciencemag.org/content/336/6083/934.full SO - Science2012 May 18; 336 AB - Methylation of the cytosine base in eukaryotic DNA (5mC) is an important epigenetic mark involved in gene silencing and genome stability. Methylated cytosine can be enzymatically oxidized to 5-hydroxymethylcytosine (5hmC), which may function as a distinct epigenetic mark—possibly involved in pluripotency—and it may also be an intermediate in active DNA demethylation. To be able to detect 5hmC genome-wide and at single-base resolution, Booth et al. (p. 934, published online 26 April) developed a 5hmC sequencing chemistry that selectively oxidizes 5hmC to 5-formylcytosine and then to uracil while leaving 5mC unchanged. Using this method, mouse embryonic stem cell genomic DNA was sequenced to reveal that 5hmC is found enriched at intragenic CpG islands and long interspersed nuclear element–1 retrotransposons.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.