Background: 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 commonly used to assay 5-methylcytosine (5-mC), including conventional methyl-sensitive restriction digest and bisulfite sequencing, are incapable of distinguishing between 5-mC and 5-hmC.

Results: Glycosylation of 5-hmC residues by beta-Glucosyl Transferase (β-GT) can make CCGG residues insensitive to digestion by MspI. We used this premise to modify the HELP-tagging assay to identify both 5-mC and 5-hmC loci in the genome. The HELP tagging assay uses massive parallel sequencing to analyze the cytosine methylation status of 2.1 million CpGs in the genome. Libraries were generated from genomic DNA digested with HpaII, MspI and β-GT +MspI. Comparison of HpaII and MspI digested samples led to determination of 5-mC loci, while comparison of β-GT +MspI with MspI digested samples identified 5-hmC residues. This modified “HELP-GT” assay allowed multiplexing of 8 libraries per sequencing lane to generate an average of 6-10 million HpaII/MspI reads per sample with an average depth of coverage between 6-11x for each CCGG site. A custom bioinformatics pipeline was created to identify 5-hmC sites that were validated at global level by LS-MS and at the locus specific level by qRT-PCR of 5-hmC pulldown DNA. Hydroxymethylation at both promoter and intragenic locations correlated positively with gene expression. This assay was then used to analyze 5-hmC profiles of two pancreatic cancer cell lines (Pa03C and Pa04C) that were compared with pancreatic control cells (HPNE). Analysis of pancreatic cancer samples revealed striking redistribution of 5-hmC sites in cancer cells with significantly increased 5-hmC at Promoters, Gene bodies and Transcription factor binding sites. 5-hmC was also increased at many oncogenic promoters such as GATA6 in pancreatic cancer and correlated with its overexpression. 5-hmC profiles were also able to distinguish between cancer and control cells with greater discrimination (unsupervised hierarchical clustering) when compared to 5-mC patterns.

Conclusions: The HELP-GT assay allows a high resolution, simultaneous determination of 5-hmC and 5-mC loci from small amounts of DNA with the utilisation of modest sequencing resources. This assay is able to provide single base pair resolution analysis of over 1 million sites in the human genome with the use of 1μg of genomic DNA. Redistribution of 5-hmC seen in cancer highlights the importance of examining this modification in conjugation with conventional methylome analysis.

Citation Format: Sanchari Bhattacharyya, Yiting Yu, Masako Suzuki, Nathaniel Cambpell, Jozef Mazdo, Aparna Vasantkumar, Tushar D. Bhagat, Sangeeta Nischal, Ulrich Steidl, Lucy Godley, Anirban Maitra, John M. Greally, Amit Verma. Genome wide hydroxymethylation tested using the HELP-GT assay shows redistribution in cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4231. doi:10.1158/1538-7445.AM2013-4231