Move over 5mC, there’s a new kid on the block. 5hmC is the first oxidative product in the active demethylation of 5mC, and it’s the latest, hottest topic in epigenetics. There’s a lot more to 5hmC than just some intermediate of demethylation. Research is on-going, but 5hmC appears to be vital for early differentiation (Freudenberg et al., 2012) and neuron function (Khare et al., 2012).
The recent surge of interest in 5hmC has necessitated new techniques to analyze it. Cytosines are protected from bisulfite deamination by both methylation and hydroxymethylation, meaning bisulfite cannot distinguish between 5mC and 5hmC (Huang et al., 2010). Methods such as oxidative bisulfite sequencing (OxBS-seq) and TET-assisted bisulfite sequencing (TAB-seq) use some clever chemistry to get around the similarity of 5mC and 5hmC and allow the discrimination of the two.
OxBS-seq (Oxidative bisulfite sequencing): Adds an additional oxidative step to bisulfite to discriminate between 5mC and 5hmC.
TAB-seq (Tet-assisted bisulfite sequencing): Employs a clever use of the Tet enzyme distinguish between 5mC and 5hmC using bisulfite.
5hmC Additional Reading
Branco, M.R., Ficz, G., and Reik, W. (2011). Uncovering the role of 5-hydroxymethylcytosine in the epigenome. Nat. Rev. Genet. 13, 7-13.
This review covers most of the basics on 5hmC. It examines the dynamics of 5hmC production via TET proteins as well as mechanisms of potential regulatory roles for 5hmC. The authors focus on emerging methods for 5hmC detection.
Pastor, W.A., Pape, U.J., Huang, Y., Henderson, H.R., Lister, R., Ko, M., McLoughlin, E.M., Brudno, Y., Mahapatra, S., Kapranov, P., et al. (2011). Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells. Nature 473, 394-397.
This influential paper that was one of the first to characterize 5hmC on a genome-wide scale. It also describes several novel approaches for studying 5hmC.
- Freudenberg, J.M., Ghosh, S., Lackford, B.L., Yellaboina, S., Zheng, X., Li, R., Cuddapah, S., Wade, P.A., Hu, G., and Jothi, R. (2012). Acute depletion of Tet1-dependent 5-hydroxymethylcytosine levels impairs LIF/Stat3 signaling and results in loss of embryonic stem cell identity. Nucleic Acids Res. 40, 3364-3377.
- Huang, Y., Pastor, W.A., Shen, Y., Tahiliani, M., Liu, D.R., and Rao, A. (2010). The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing. PLoS One 5, e8888.
- Khare, T., Pai, S., Koncevicius, K., Pal, M., Kriukiene, E., Liutkeviciute, Z., Irimia, M., Jia, P., Ptak, C., Xia, M., et al. (2012). 5-hmC in the brain is abundant in synaptic genes and shows differences at the exon-intron boundary. Nat. Struct. Mol. Biol. 19, 1037-1043.