title: A novel method for the efficient and selective identification of 5-hydroxymethylcytosine in genomic DNA.
abstract: Recently, 5-hydroxymethylcytosine (5hmC) was identified in mammalian genomic DNA. The biological role of this modification remains unclear; however, identifying the genomic location of this modified base will assist in elucidating its function. We describe a method for the rapid and inexpensive identification of genomic regions containing 5hmC. This method involves the selective glucosylation of 5hmC residues by the β-glucosyltransferase from T4 bacteriophage creating β-glucosyl-5-hydroxymethylcytosine (β-glu-5hmC). The β-glu-5hmC modification provides a target that can be efficiently and selectively pulled down by J-binding protein 1 coupled to magnetic beads. DNA that is precipitated is suitable for analysis by quantitative PCR, microarray or sequencing. Furthermore, we demonstrate that the J-binding protein 1 pull down assay identifies 5hmC at the promoters of developmentally regulated genes in human embryonic stem cells. The method described here will allow for a greater understanding of the temporal and spatial effects that 5hmC may have on epigenetic regulation at the single gene level.
authors.affiliation: Centre for Molecular Biology and Neuroscience and Institute of Medical Microbiology, Oslo University Hospital, Rikshospitalet, Norway.
authors.forename: Adam B
authors.initials: AB
authors.lastname: Robertson
authors.forename: John A
authors.initials: JA
authors.lastname: Dahl
authors.forename: Cathrine B
authors.initials: CB
authors.lastname: Vågbø
authors.forename: Pankaj
authors.initials: P
authors.lastname: Tripathi
authors.forename: Hans E
authors.initials: HE
authors.lastname: Krokan
authors.forename: Arne
authors.initials: A
authors.lastname: Klungland
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives; D002352:Carrier Proteins; D003596:Cytosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification; D053595:Embryonic Stem Cells / Q000378:metabolism; D005786:Gene Expression Regulation; D015894:Genome, Human; D023281:Genomics / Q000379:methods*; D005964:Glucosyltransferases / Q000378:metabolism; D006801:Humans; D011401:Promoter Regions, Genetic; D015800:Protozoan Proteins
chemical_list: D002352:Carrier Proteins; D015800:Protozoan Proteins; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA; D005964:Glucosyltransferases