title: Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells.
abstract: 5-hydroxymethylcytosine (5hmC) is a modified base present at low levels in diverse cell types in mammals. 5hmC is generated by the TET family of Fe(II) and 2-oxoglutarate-dependent enzymes through oxidation of 5-methylcytosine (5mC). 5hmC and TET proteins have been implicated in stem cell biology and cancer, but information on the genome-wide distribution of 5hmC is limited. Here we describe two novel and specific approaches to profile the genomic localization of 5hmC. The first approach, termed GLIB (glucosylation, periodate oxidation, biotinylation) uses a combination of enzymatic and chemical steps to isolate DNA fragments containing as few as a single 5hmC. The second approach involves conversion of 5hmC to cytosine 5-methylenesulphonate (CMS) by treatment of genomic DNA with sodium bisulphite, followed by immunoprecipitation of CMS-containing DNA with a specific antiserum to CMS. High-throughput sequencing of 5hmC-containing DNA from mouse embryonic stem (ES) cells showed strong enrichment within exons and near transcriptional start sites. 5hmC was especially enriched at the start sites of genes whose promoters bear dual histone 3 lysine 27 trimethylation (H3K27me3) and histone 3 lysine 4 trimethylation (H3K4me3) marks. Our results indicate that 5hmC has a probable role in transcriptional regulation, and suggest a model in which 5hmC contributes to the 'poised' chromatin signature found at developmentally-regulated genes in ES cells.
authors.affiliation: Harvard Medical School, Immune Disease Institute and Program in Cellular and Molecular Medicine, Children's Hospital Boston, Boston, Massachusetts 02115, USA.
authors.forename: William A
authors.initials: WA
authors.lastname: Pastor
authors.forename: Utz J
authors.initials: UJ
authors.lastname: Pape
authors.forename: Yun
authors.initials: Y
authors.lastname: Huang
authors.forename: Hope R
authors.initials: HR
authors.lastname: Henderson
authors.forename: Ryan
authors.initials: R
authors.lastname: Lister
authors.forename: Myunggon
authors.initials: M
authors.lastname: Ko
authors.forename: Erin M
authors.initials: EM
authors.lastname: McLoughlin
authors.forename: Yevgeny
authors.initials: Y
authors.lastname: Brudno
authors.forename: Sahasransu
authors.initials: S
authors.lastname: Mahapatra
authors.forename: Philipp
authors.initials: P
authors.lastname: Kapranov
authors.forename: Mamta
authors.initials: M
authors.lastname: Tahiliani
authors.forename: George Q
authors.initials: GQ
authors.lastname: Daley
authors.forename: X Shirley
authors.initials: XS
authors.lastname: Liu
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Ecker
authors.forename: Patrice M
authors.initials: PM
authors.lastname: Milos
authors.forename: Suneet
authors.initials: S
authors.lastname: Agarwal
authors.forename: Anjana
authors.initials: A
authors.lastname: Rao
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives; D000818:Animals; D019921:Biotinylation; D002460:Cell Line; D003596:Cytosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D019175:DNA Methylation; D053595:Embryonic Stem Cells / Q000378:metabolism*; D005091:Exons / Q000235:genetics; D018507:Gene Expression Regulation, Developmental / Q000235:genetics; D016678:Genome / Q000235:genetics*; D005947:Glucose / Q000378:metabolism; D051379:Mice; D010504:Periodic Acid / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*; D024363:Transcription Initiation Site; D014158:Transcription, Genetic / Q000235:genetics
chemical_list: D010504:Periodic Acid; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; C009288:metaperiodate; D005947:Glucose