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

ABSTRACT: 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 (beta-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. Comparison of sequencing libraries after HpaII, MspI and MspI+ beta-GT conversion resulted in locus specific 5-mC and 5-hmC determination. A custom bioinformatics pipeline was created to identify 5-hmC sites that were validated at global level by LS-MS and the locus specific level by qRT-PCR 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. 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. Redistribution of 5-hmC seen in cancer highlights the importance of examining this modification in conjugation with conventional methylome analysis.

ORGANISM(S): Homo sapiens

PROVIDER: GSE42723 | GEO | 2013/07/30

SECONDARY ACCESSION(S): PRJNA182946

REPOSITORIES: GEO

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Project description:5-methylcytosine (5-mC) can be oxidized to 5-hydroxymethylcytosine (5-hmC). Genome-wide profiling of 5-hmC thus far indicated 5-hmC may not only be an intermediate form of DNA demethylation but could also constitute an epigenetic mark per se. We describe a cost-effective and selective method to detect both the hydroxymethylation and methylation status of cytosines in more than 1.8 million MspI sites in the human genome. This method involves the selective glucosylation of 5-hmC residues, short-sequence tag generation and high-throughput sequencing. We tested this method by screening H9 human embryonic stem cells and their differentiated embroid body cells, and found that differential hydroxymethylation preferentially occur in bivalent genes during cellular differentiation. Especially, our results support hydroxymethylation can regulate key transcription regulators with bivalent marks through demethylation and affect cellular decision on choosing active or inactive state of these genes upon cellular differentiation. We developed a cost-effective and selective method to detect both the hydroxymethylation and methylation status of cytosines in more than 1.8 million MspI sites in the human genome. In this method, we took advantage of the differential enzymatic sensitivities of the isoschizomers MspI and HpaII. HpaII cleaves only a completely unmodified site, any modification at either cytosine blocks the cleavage, while MspI recognizes and cleaves both 5-mC and 5-hmC, but not the newly discovered 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). Furthermore, beta-glucosyltransferase (beta-GT) can transfer a glucose to the hydroxyl group of 5-hmC and generate beta-glucosyl-5-hydroxymethylcytosine (5-ghmC) that blocks MspI digestion. Thus, either by combining beta-GT treatment with MspI digestion or simply applying MspI/HpaII digestion, short sequence tags generated can be used for inferring hydroxymethylation or methylation status in around 1.8 million cytosine sites in the human genome. We tested this method by screening H9 human embryonic stem cells and their differentiated embroid body cells.

Project description:5-hydroxymethylcytosine (5-hmC), a derivative of 5-methylcytosine (5-mC), is abundant in the brain for unknown reasons. We mapped the genomic distribution of 5-hmC and 5-mC in human and mouse tissues using glucosylation of 5-hmC coupled with restriction enzyme digestion, and interrogation on microarrays. We detected 5-hmC enrichment in genes with synapse-related functions in the brain. We also identified significant, tissue-specific differential distributions of these DNA modifications at the exon-intron boundary, in both human and mouse. This boundary change was mainly due to 5-hmC in the brain, but due to 5-mC in non-neural contexts. This pattern was replicated in multiple independent datasets, and the brain-specific change in 5-hmC was validated using single-molecule sequencing. Moreover, in the brain, constitutive exons contained higher levels of 5-hmC, relative to alternatively-spliced exons. Our study suggests a novel role for 5-hmC in RNA splicing and synaptic function in the brain The M-NM-2-glucosyltransferase (BGT) enzyme transfers a glucose molecule specifically to the hydroxymethyl group of 5-hmC, thus rendering it resistant to digestion by the methylation insensitive MspI enzyme at the ChmCGG target site; 5-hmC is thus detected by differential resistance to MspI-digestion with and without glucosylation of genomic DNA (gDNA). HpaII (targets the same site, CCGG) cannot cut CmCGG or ChmCGG, and conceptually its difference with MspI digestion is a measure of both 5-mC and 5-hmC. Subtraction of 5-hmC from the HpaII-based estimate therefore measures 5-mC. These estimates were measured on respective Affymetrix whole-genome tiling arrays (2.0 R ) MspI - APRIL_fc1.ch02_coverage.bed Undigested DNA - APRIL_fc1.ch01_coverage.bed GluMspI - APRIL_fc1.ch04_coverage_NONTARGET.bed GluMspI - APRIL_fc1.ch04_coverage.bed MspI - APRIL_fc1.ch02_coverage_NONTARGET.bed Undigested DNA - APRIL_fc1.ch01_coverage_NONTARGET.bed MspI - MARCH_fc1.ch02_coverage_NONTARGET.bed Undigested DNA - MARCH_fc1.ch01_coverage_NONTARGET.bed GluMspI - MARCH_fc1.ch04_coverage_NONTARGET.bed GluMspI - MARCH_fc1.ch04_coverage.bed MspI - MARCH_fc1.ch02_coverage.bed Undigested DNA - MARCH_fc1.ch01_coverage.bed MspI - MAY_fc1.ch02_coverage.bed GluMspI - MAY_fc1.ch04_coverage_NONTARGET.bed GluMspI - MAY_fc1.ch04_coverage.bed Undigested DNA - MAY_fc1.ch01_coverage.bed MspI - MAY_fc1.ch02_coverage_NONTARGET.bed Undigested DNA - MAY_fc1.ch01_coverage_NONTARGET.bed

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

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