title: Genome-wide hydroxymethylation tested using the HELP-GT assay shows redistribution in cancer.
abstract: 5-hydroxymethylcytosine (5-hmC) is a recently discovered epigenetic modification that is altered in cancers. Genome-wide assays for 5-hmC determination are needed as many of the techniques for 5-methylcytosine (5-mC) determination, including methyl-sensitive restriction digestion and bisulfite sequencing cannot distinguish between 5-mC and 5-hmC. Glycosylation of 5-hmC residues by beta-glucosyl transferase (β-GT) can make CCGG residues insensitive to digestion by MspI. Restriction digestion by HpaII, MspI or MspI after β-GT conversion, followed by adapter ligation, massive parallel sequencing and custom bioinformatic analysis allowed us determine distribution of 5-mC and 5-hmC at single base pair resolution at MspI restriction sites. The resulting HpaII tiny fragment Enrichment by Ligation-mediated PCR with β-GT (HELP-GT) assay identified 5-hmC loci that were validated at global level by liquid chromatography-mass spectrometry (LC-MS) and the locus-specific level by quantitative reverse transcriptase polymerase chain reaction of 5-hmC pull-down DNA. Hydroxymethylation at both promoter and intragenic locations correlated positively with gene expression. Analysis of pancreatic cancer samples revealed striking redistribution of 5-hmC sites in cancer cells and demonstrated enrichment of this modification at many oncogenic promoters such as GATA6. The HELP-GT assay allowed global determination of 5-hmC and 5-mC from low amounts of DNA and with the use of modest sequencing resources. Redistribution of 5-hmC seen in cancer highlights the importance of determination of this modification in conjugation with conventional methylome analysis.
authors.affiliation: Cancer Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA, Department of Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA, Department of Pathology, Johns Hopkins School of Medicine, 1550 Orleans Street, Baltimore MD 21231, USA, Department of Medicine, University of Chicago, 5841 S. Maryland Avenue, Chicago, IL 60637, USA and Department of Medicine, Hematology and Medical Oncology, Mount Sinai School of Medicine, 1470 Madison Avenue, New York, NY 10029, USA.
authors.forename: Sanchari
authors.initials: S
authors.lastname: Bhattacharyya
authors.forename: Yiting
authors.initials: Y
authors.lastname: Yu
authors.forename: Masako
authors.initials: M
authors.lastname: Suzuki
authors.forename: Nathaniel
authors.initials: N
authors.lastname: Campbell
authors.forename: Jozef
authors.initials: J
authors.lastname: Mazdo
authors.forename: Aparna
authors.initials: A
authors.lastname: Vasanthakumar
authors.forename: Tushar D
authors.initials: TD
authors.lastname: Bhagat
authors.forename: Sangeeta
authors.initials: S
authors.lastname: Nischal
authors.forename: Maximilian
authors.initials: M
authors.lastname: Christopeit
authors.forename: Samir
authors.initials: S
authors.lastname: Parekh
authors.forename: Ulrich
authors.initials: U
authors.lastname: Steidl
authors.forename: Lucy
authors.initials: L
authors.lastname: Godley
authors.forename: Anirban
authors.initials: A
authors.lastname: Maitra
authors.forename: John M
authors.initials: JM
authors.lastname: Greally
authors.forename: Amit
authors.initials: A
authors.lastname: Verma
mesh_terms: D044503:5-Methylcytosine / Q000032:analysis / Q000031:analogs & derivatives; D000818:Animals; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic; D003596:Cytosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D004273:DNA, Neoplasm / Q000737:chemistry*; D015870:Gene Expression; D015894:Genome, Human; D023281:Genomics / Q000379:methods; D016695:Glycosyltransferases / Q000378:metabolism; D006801:Humans; D051379:Mice; D010190:Pancreatic Neoplasms / Q000235:genetics; D016133:Polymerase Chain Reaction
chemical_list: D004273:DNA, Neoplasm; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; D016695:Glycosyltransferases