The Behaviour of 5-Hydroxymethylcytosine in Bisulfite Sequencing

By Yun Huang, William A. Pastor, Yinghua Shen, Mamta Tahiliani, David R. Liu, Anjana Rao. PLoS ONE, 2010

741 citations recorded by the source corpus.

Background We recently showed that enzymes of the TET family convert 5-mC to 5-hydroxymethylcytosine (5-hmC) in DNA. 5-hmC is present at high levels in embryonic stem cells and Purkinje neurons. The methylation status of cytosines is typically assessed by reaction with sodium bisulfite followed by PCR amplification. Reaction with sodium bisulfite promotes cytosine deamination, whereas 5-methylcytosine (5-mC) reacts poorly with bisulfite and is resistant to deamination. Since 5-hmC reacts with bisulfite to yield cytosine 5-methylenesulfonate (CMS), we asked how DNA containing 5-hmC behaves in bisulfite sequencing. Methodology/Principal Findings We used synthetic oligonucleotides with different distributions of cytosine as templates for generation of DNAs containing C, 5-mC and 5-hmC. The resulting DNAs were subjected in parallel to bisulfite treatment, followed by exposure to conditions promoting cytosine deamination. The extent of conversion of 5-hmC to CMS was estimated to be 99.7%. Sequencing of PCR products showed that neither 5-mC nor 5-hmC undergo C-to-T transitions after bisulfite treatment, confirming that these two modified cytosine species are indistinguishable by the bisulfite technique. DNA in which CMS constituted a large fraction of all bases (28/201) was much less efficiently amplified than DNA in which those bases were 5-mC or uracil (the latter produced by cytosine deamination). Using a series of primer extension experiments, we traced the inefficient amplification of CMS-containing DNA to stalling of Taq polymerase at sites of CMS modification, especially when two CMS bases were either adjacent to one another or separated by 1–2 nucleotides. Conclusions We have confirmed that the widely used bisulfite sequencing technique does not distinguish between 5-mC and 5-hmC. Moreover, we show that CMS, the product of bisulfite conversion of 5-hmC, tends to stall DNA polymerases during PCR, suggesting that densely hydroxymethylated regions of DNA may be underrepresented in quantitative methylation analyses.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/a0bb963f280affc8dde5aa6380369d33709cb8a4

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In Molecular Biology

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Citations in the corpus

• The mysterious presence of a 5-methylcytosine oxidase in the Drosophila genome (2013, background): “would have detected both 5hmC and 5mC due to its inability to distinguish between the 2 base modifications.(56,57) It may be possible that some highly repetitive regions of the Drosophila genome could contain 5mC/5hmC but”
• 5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming. (2011, background): “Note that both modifications, 5mC and 5hmC, are resistant to bisulphite induced deamination and thus cannot be discriminated in bisulphite sequencing reaction”
• Base-Resolution Analysis of 5-Hydroxymethylcytosine in the Mammalian Genome (2012, neutral): “TAB-Seq of Model DNA and Specific Loci Traditional bisulfite sequencing cannot discriminate 5mC from 5hmC because both resist deamination by bisulfite treatment (Huang et al., 2010; Jin et al., 2010).”
• DNA methylome analysis using short bisulfite sequencing data (2012, background): “Bisulfite treatment of 5-hydroxymethylcytosine (5hmC) yields a similar intermediate to 5mC, meaning that BS-seq can be used to detect whether a position is (hydroxy-) methylated but not to determine the exact type of modification 21,25 ( Fig.”
• Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine (2010, methodology): “Widely used methods to probe 5-mC, such as bisulfite sequencing and methylation-sensitive restriction digestion, cannot discriminate between 5-hmC and 5-m”
• Analysing and interpreting DNA methylation data (2012, background): “Disadvantages include the high cost of whole-genome resequencing and the difficulty of discriminating between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) 144,145 .”
• Acute exercise remodels promoter methylation in human skeletal muscle. (2012, methodology): “The bisulfite modification technique used in this study does not distinguish between methylated and hydroxyl methylated cytosines (Huang et al., 2010).”
• Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine (2011, background): “Sodium bisulfite sequencing, which cannot distinguish between 5mC and 5hmC (38, 43), has been used by several laboratories to demonstrate active DNA demethylation—that is, conversion of 5mC to C—of certain sequences in zygotes.”
• Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells (2011, methodology): “Since conventional bisulfite sequencing does not distinguish 5mC and 5hmC (Huang et al., 2010; Jin et al., 2010; Nestor et al., 2010), we have not formally ruled out the possibility that 5hmC is present at a subset of CpG sites at the Elf5 promoter.”
• DNA methylation and cancer. (2010, background): “hmC is structurally similar to mC and both nucleotides are not converted to uracil upon bisulfite treatment, implying that a proportion of genomic loci identified as methylated may actually be hydroxymethylated (Huang et al., 2010).”