Enzymatic Approaches and Bisulfite Sequencing Cannot Distinguish Between 5-Methylcytosine and 5-Hydroxymethylcytosine in DNA

Abstract

DNA cytosine methylation (5mC) is highly abundant in mammalian cells and is associated with transcriptional repression. Recently, hydroxymethylcytosine (hmC) has been detected at high levels in certain human cell types; however, its roles are unknown. Due to the structural similarity between 5mC and hmC, it is unclear whether 5mC analyses can discriminate between these nucleotides. Here we show that 5mC and hmC are experimentally indistinguishable using established 5mC mapping methods, thereby implying that existing 5mC data sets will require careful re-evaluation in the context of the possible presence of hmC. Potential differential enrichment of 5mC and hmC DNA sequences may be facilitated using a 5mC monoclonal antibody.

Placental 5-methylcytosine and 5-hydroxymethylcytosine patterns associate with size at birth

“…29 Both 5mC and 5hmC are resistant to deamination by bisulfite conversion, so that the cytosine methylation quantified using bisulfite conversion represents the sum of 5mC and 5hmC levels. 31 Although the techniques used in our study do not permit us to compare the relative enrichment of 5mC and 5hmC quantitatively, the presence of 5hmC within DMRs in imprinted regions in human placenta mean that studies of DNA methylation using conventional bisulfite conversion might overestimate 5mC levels at DMRs, particularly if the results of such studies are used to infer potential effects of altered methylation on gene expression. Our findings of significant enrichment of 5hmC and a lower enrichment of 5mC at DMR2 relative to the other DMRs analyzed suggest that studies using bisulfite conversion techniques that report small changes in cytosine methylation at DMRs, including at this region, 18 could overestimate the amount of 5mC present.…”

“…28 5-hydroxymethylcytosine (5hmC) is a recently described cytosine modification that may be an intermediate in the DNA demethylation pathway 29 and is present in the placenta. 30 Since commonly used methods of assessing DNA methylation using bisulfite conversion do not discriminate between 5mC and 5hmC, 31 previous studies reporting altered DNA methylation at placental DMRs in association with fetal growth may be confounded. We used specific enrichment techniques to map 5mC and 5hmC at the DMRs controlling the expression of IGF2 and CDKN1C in human placenta, and additionally explored relationships between 5mC, 5hmC, gene expression, and fetal growth across the full range of normal birth weight.…”

DNA Methylation in Mammalian and Non-Mammalian Organisms

“…The importance of 5hmC and its cousins in epigenetics is that the hydroxymethyl group is suggested to alter the biological properties of methylated DNA (Ndlovu et al, 2011). The rediscovery of 5hmC also presents an unanticipated experimental problem, as conventional techniques were originally unable to distinguish between 5mC and 5hmC in DNA (Nestor et al, 2010). Recent technical developments can now distinguish prominent 5hmC sites in the genome (C.X.…”

Germ cell-specific DNA methylation and genome diploidization in primitive vertebrates

“…The methylation status at the CpG island in the egg and sperm were directly examined with the bisulfite sequencing technique. Because bisulfite sequencing cannot distinguish 5-methyl-cytosine and 5-hydroxymethyl-cytosine, 28 the bisulfite sequencing outputs might include both types of DNA modification. Our results showed that the promoter CpG island of ntl in sperm was unmethylated in all the examined clones, as would be expected for a CpG island.…”