title: Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution.
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.
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, UK.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Booth
authors.forename: Miguel R
authors.initials: MR
authors.lastname: Branco
authors.forename: Gabriella
authors.initials: G
authors.lastname: Ficz
authors.forename: David
authors.initials: D
authors.lastname: Oxley
authors.forename: Felix
authors.initials: F
authors.lastname: Krueger
authors.forename: Wolf
authors.initials: W
authors.lastname: Reik
authors.forename: Shankar
authors.initials: S
authors.lastname: Balasubramanian
mesh_terms: D044503:5-Methylcytosine / Q000032:analysis* / Q000031:analogs & derivatives; D000818:Animals; D018899:CpG Islands; D003596:Cytosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D019175:DNA Methylation; D053595:Embryonic Stem Cells / Q000502:physiology; D044127:Epigenesis, Genetic; D007426:Genes, Intracisternal A-Particle; D059014:High-Throughput Nucleotide Sequencing; D020084:Long Interspersed Nucleotide Elements; D051379:Mice; D010084:Oxidation-Reduction; D012211:Rhenium / Q000737:chemistry; D017422:Sequence Analysis, DNA; D013447:Sulfites; D014158:Transcription, Genetic; D014498:Uracil / Q000737:chemistry
chemical_list: C560973:5-formylcytosine; D013447:Sulfites; C011865:5-hydroxymethylcytosine; C015428:perrhenate; D014498:Uracil; D044503:5-Methylcytosine; D012211:Rhenium; D003596:Cytosine; D004247:DNA; C042345:hydrogen sulfite