title: Base-Resolution Analysis of 5-Hydroxymethylcytosine by One-Pot Bisulfite-Free Chemical Conversion with Peroxotungstate.
abstract: 5-Hydroxymethylcytosine (hmC) is an essential intermediate in the active DNA demethylation pathway. Here we report a new base-resolution method for measuringhmC by combining peroxotungstate-mediated oxidation and sequencing analysis. We reveal that an oxidized product ofhmC, trihydroxylated thymine (thT), tolerated the incorporation of dATP as a substrate in the process of DNA polymerase elongation. By comparing the results of Sanger sequencing before and after the oxidation, we observed thathmC sites on single-stranded DNAs could be discriminated from unmethylated cytosines. We found that a thermal cycle condition during peroxotungstate treatment enhanced the oxidation reaction ofhmC in double-stranded DNA. Furthermore, Illumina sequencing analysis ofhmC-containing synthetic genome fragments enabled us to identify simultaneously the positions ofhmC in base resolution. This bisulfite-free simplehmC detection technique could facilitate the acquisition of epigenomic information.
authors.affiliation: Department of Chemistry and Biotechnology, The University of Tokyo , 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors.forename: Gosuke
authors.initials: G
authors.lastname: Hayashi
authors.affiliation: Department of Chemistry and Biotechnology, The University of Tokyo , 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors.forename: Kenta
authors.initials: K
authors.lastname: Koyama
authors.affiliation: Department of Chemistry and Biotechnology, The University of Tokyo , 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors.forename: Hidefumi
authors.initials: H
authors.lastname: Shiota
authors.affiliation: Research Center for Advanced Science and Technology, The University of Tokyo , 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
authors.forename: Asuka
authors.initials: A
authors.lastname: Kamio
authors.affiliation: Research Center for Advanced Science and Technology, The University of Tokyo , 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Umeda
authors.affiliation: Research Center for Advanced Science and Technology, The University of Tokyo , 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
authors.forename: Genta
authors.initials: G
authors.lastname: Nagae
authors.affiliation: Research Center for Advanced Science and Technology, The University of Tokyo , 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Aburatani
authors.affiliation: Department of Chemistry and Biotechnology, The University of Tokyo , 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.|Research Center for Advanced Science and Technology, The University of Tokyo , 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
authors.forename: Akimitsu
authors.initials: A
authors.lastname: Okamoto
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives* / Q000737:chemistry; D020029:Base Pairing; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics; D012644:Selenium Radioisotopes; D013447:Sulfites / Q000737:chemistry; D017973:Tungsten Compounds / Q000737:chemistry*
chemical_list: D012644:Selenium Radioisotopes; D013447:Sulfites; D017973:Tungsten Compounds; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; C055707:bis(N,N,N-trimethylamino)ethylselenide-Se iodide; D004247:DNA; C042345:hydrogen sulfite; C045951:tungstate