title: Simple and accurate single base resolution analysis of 5-hydroxymethylcytosine by catalytic oxidative bisulfite sequencing using micelle incarcerated oxidants.
abstract: Oxidation of 5-methylcytosine (5mC) is catalyzed by ten-eleven translocation (TET) enzymes to produce 5-hydroxymethylcytosine (5hmC) and following oxidative products. The oxidized nucleotides were shown to be the intermediates for DNA demethylation, as the nucleotides are removed by base excision repair system initiated by thymine DNA glycosylase. A simple and accurate method to determine initial oxidation product 5hmC at single base resolution in genomic DNA is necessary to understand demethylation mechanism. Recently, we have developed a new catalytic oxidation reaction using micelle-incarcerated oxidants to oxidize 5hmC to form 5-formylcytosine (5fC), and subsequent bisulfite sequencing can determine the positions of 5hmC in DNA. In the present study, we described the optimization of the catalytic oxidative bisulfite sequencing (coBS-seq), and its application to the analysis of 5hmC in genomic DNA at single base resolution in a quantitative manner. As the oxidation step showed quite low damage on genomic DNA, the method allows us to down scale the sample to be analyzed.
authors.affiliation: Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; CREST, Japan Agency for Medical Research and Development (AMED), 1-7-1 Otemachi, Chiyoda-ku, Tokyo 100-0004, Japan. Electronic address: seketsuf@chem.s.u-tokyo.ac.jp.
authors.forename: Seketsu
authors.initials: S
authors.lastname: Fukuzawa
authors.affiliation: Laboratory of Epigenetics, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Saori
authors.initials: S
authors.lastname: Takahashi
authors.affiliation: Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Tachibana
authors.affiliation: Laboratory of Epigenetics, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Shoji
authors.initials: S
authors.lastname: Tajima
authors.affiliation: CREST, Japan Agency for Medical Research and Development (AMED), 1-7-1 Otemachi, Chiyoda-ku, Tokyo 100-0004, Japan; Laboratory of Epigenetics, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address: suetake@protein.osaka-u.ac.jp.
authors.forename: Isao
authors.initials: I
authors.lastname: Suetake
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives* / Q000737:chemistry; D000218:Adamantane / Q000031:analogs & derivatives / Q000737:chemistry; D000818:Animals; D003497:Cyclic N-Oxides / Q000737:chemistry; D003596:Cytosine / Q000031:analogs & derivatives / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry; D053595:Embryonic Stem Cells; D007462:Iodobenzenes / Q000737:chemistry; D051379:Mice; D008823:Micelles; D009861:Onium Compounds / Q000737:chemistry; D016877:Oxidants / Q000737:chemistry*; D010084:Oxidation-Reduction; D017422:Sequence Analysis, DNA / Q000379:methods*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013447:Sulfites / Q000737:chemistry; D013696:Temperature
chemical_list: C522607:2-azaadamantane N-oxyl; C560973:5-formylcytosine; D003497:Cyclic N-Oxides; D004277:DNA, Single-Stranded; D007462:Iodobenzenes; D008823:Micelles; D009861:Onium Compounds; D016877:Oxidants; D013447:Sulfites; C000621441:bis(acetoxy)iodobenzene; C011865:5-hydroxymethylcytosine; D012967:Sodium Dodecyl Sulfate; D044503:5-Methylcytosine; D003596:Cytosine; C042345:hydrogen sulfite; D000218:Adamantane
keywords: 5-Hydroxymethylcytosine; Bisulfite sequencing; DNA demethylation; Oxidation; Single base resolution