There is exciting news regarding a new method to detect 5-hmC quantitatively at high resolution – using any sequencing platform. The new method, termed oxidative bisulfite sequencing (oxBS-Seq) , was applied to explore the 5-hmC mark’s potential functional role in epigenetic plasticity. First author Michael J. Booth, along with collaborators from the Wolf Reik lab at the Babraham Institute, and Shankar Balasubramanian lab at Cambridge University, have reported their work in Science Magazine.
In brief, this method uses potassium perruthenate (KRuO4) oxidation to convert 5hmC to 5fC (formylcytosine), and is followed up by conventional bisulfite conversion. Through this process C and 5-hmC sites convert to Uracil, whereas 5-mC does not. OxBS-Seq data is then subtracted from BS-Seq data. The group validated their experimental results by comparing results with meDIP-seq and an enzymatic method of 5-hmC detection using beta glucosyltransferase.
What is epigenetic plasticity exactly? Plasticity has previously been applied to brain development. Just think of the mind’s adaptation ability given environmental input. (Surprise, surprise this ability is inversely correlated with age. LOL!) The term is also apt in the context of epigenetics given the dynamic, bidirectional nature of DNA methylation. Last year in Nature, the Wolf Reik group noted a link between the balance of 5hmC and 5mC in ES cells, and the balance of pluripotency and differentiation. Mice ES cells show an overall inverse pattern of high 5-hmC to low 5-mC at gene transcription start sites, perhaps helping to maintain epigenetic plasticity. What could this mean for reprogramming? What does this pattern look like in differentiated adult tissues, with much higher levels of 5-hmC? Why can hydroxymethylation losses be significantly divergent from regular methylation losses in cancers? Is 5hmC a “pause button” of sorts, rather than a full stop? Is youth wasted on the young? But I diverge…
This method is one Epigeneticists have been waiting for. Wolf Reik head of the Epigenetics Institute Strategic Programme at the Babraham Institute remarked in a press release, “It has recently become apparent that in addition to DNA methylation, there are other modifications of DNA, such as for example hydroxymethylation. This suggests that DNA modifications are more dynamic than we previously thought. With the new method we are now in a position to map these modifications at great precision, and to relate them to stem cell function, ageing, and perhaps more generally to how the environment interacts with the genome.” The bisulfite method has a limitation because it can not discriminate between 5-mC and 5-hmC. Individual genes can be quantitated for hydroxymethylation by enzymatic assays, such as NEB’s EpiMark 5-hmC and 5-mC Analysis kit, upstream of PCR and seqeuncing. Enrichment methods are used upstream of Single molecule real time sequencing (SMRT) providing high resolution, but provide non-quantitative data on 5-hmC. With oxBS-Seq, functions for hydroxymethylation can be targeted by demonstrating single nucleotide pair resolution divergent 5-hmC and 5-mC patterns, and exploring correlating cellular molecular events.
This is a brilliant method advance, which provides opportunity for scientists to discover the utilities of DNA hydroxymethylation. The paper is a must read…further intriguing details included. Cheers to these Brit researchers!
Booth, M., Branco, M., Ficz, G., Oxley, D., Krueger, F., Reik, W., & Balasubramanian, S. (2012). Quantitative Sequencing of 5-Methylcytosine and 5-Hydroxymethylcytosine at Single-Base Resolution Science DOI: 10.1126/science.1220671