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.
Highlights
The discovery of 5-hydroxymethylcytosine (5-hmC), an epigenetic modification of DNA, has led to studies that have shown that this chemical modification is prevalent in ES cells and tissues such as brain and kidney [1,2,3]
TET-assisted bisulfite sequencing [10] and oxidative reduced representation bisulfite sequencing [11] have been described as assays that can analyze the hydroxymethylome at single base resolution, these assays are dependent on great depth of sequencing restricting their utility in large-scale studies
Earlier reports based on immunohistochemistry revealed decreased 5-hmC in lung and prostate cancers [20] and our data demonstrated decreased numbers of 5-hmC loci in pancreatic cancers
Summary
The discovery of 5-hydroxymethylcytosine (5-hmC), an epigenetic modification of DNA, has led to studies that have shown that this chemical modification is prevalent in ES cells and tissues such as brain and kidney [1,2,3]. PAGE 2 OF 10 involving pull down of glycosylated 5-hmC residues has been described, but requires large amounts of input DNA [12]. To overcome these limitations we developed a high-throughput single base-pair resolution assay to identify 5-hmC and 5-mC in the genome by modifying our HELP-tagging assay. By adding an extra glycosylation step before MspI digestion, we were able to interrogate both methylated and hydroxymethylated sites in the genome This assay provides a genome-wide survey of both 5-hmC and 5-mC sites of the genome and can be used to analyze large sample cohorts with modest sequencing resources. We used this assay on pancreatic cancer samples and show for the first time that 5-hmC sites are widely redistributed in cancer
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
