Genome-wide hydroxymethylation tested using the HELP-GT assay shows redistribution in cancer

Sanchari Bhattacharyya, Yiting Yu, Masako Suzuki, Nathaniel Campbell, Jozef Mazdo, Aparna Vasanthakumar, Tushar D. Bhagat, Sangeeta Nischal, Maximilian Christopeit, Samir Parekh, Ulrich Steidl, Lucy Godley, Anirban Maitra, John M. Greally, Amit Verma

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54 Scopus citations


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 bisulfate sequencing cannot distinguish between 5-mC and 5-hmC. Glycosylation of 5-hmC residues by betaglucosyl transferase (b-GT) can make CCGG residues insensitive to digestion by MspI. Restriction digestion by HpaII, MspI or MspI after b-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 b-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 pulldown 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.

Original languageEnglish (US)
JournalNucleic Acids Research
Issue number16
StatePublished - Sep 2013

ASJC Scopus subject areas

  • Genetics


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