5-hydroxymethylcytosine (5hmC) is one of the hottest topics in epigenetics in the last 5 years. 5hmC has the potential to greatly deepen our understanding of epigenetics of the brain and development. 5hmC is the first oxidative product in the active demethylation of 5-methylcytosine (5mC). The three Ten-eleven translocation (TET) enzymes oxidize each step in the demethylation of 5mC. 5mC is first converted to 5hmC, then 5-formylcytosine (5fC), then 5-carboxylcytosine (5caC), each by TET1-3 (Ito et al., 2011).
Sodium bisulfite sequencing, the gold standard for detecting 5mC, cannot discriminate between 5mC and 5hmC. Thus previous studies looking at 5mC with bisulfite have been simultaneously examining 5mC and 5hmC. Researchers have developed new methods such as oxidative bisulfite sequencing (oxBS-seq) to allow discrimination between the two (Booth et al., 2013). In oxBS-seq, 5hmC is oxidized to 5fC, then 5fC is converted to uracil by bisulfite. By comparing to a normal bisulfite run, the identity of 5hmC can be discriminated from 5mC in the original sequence.
5hmC is prevalent in embryonic stems cells and in the brain. Reduced levels of TET1 and subsequently 5hmC cause impaired self-renewal of stem cells (Freudenberg et al., 2012). In the brain and embryonic stem cells, 5hmC in enriched in promoters, gene bodies, and intergenic areas near genes and positively correlates with gene expression at these loci (Pastor et al., 2011; Song et al., 2011; Xu et al., 2011). The production of 5hmC seems to serve a functional role to promote gene expression during active demethylation. It is hypothesized that conversion of 5mC to 5hmC by TETs blocks the repressive MBD-domain containing and DNMT proteins that would typically be recruited to 5mC (Branco et al., 2011).
This process of activation by active demethylation appears to be particularly important for genes involved in brain function. 5hmC is enriched in genes involved with synaptic function in mouse and human brain (Khare et al., 2012). The reasons for this remain unclear. The plethora of such data emerging regarding 5hmC requires higher level integration and organization to gain an accurate view of the importance of this 6th base.
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 looks at the dynamics of 5hmC production in the context of DNA oxidative demethylation via TET proteins as well as mechanisms of potential regulatory roles for 5hmC. The authors also address some emerging methods for 5hmC detection.
Tan, L., and Shi, Y.G. (2012). Tet family proteins and 5-hydroxymethylcytosine in development and disease. Development 139, 1895-1902.
This review presents what is known about the roles of the TET family proteins and 5hmC in development. This review also has an interesting open questions section that poses some important questions about what remains to be studied regarding 5hmC.
- Booth, M.J., Ost, T.W., Beraldi, D., Bell, N.M., Branco, M.R., Reik, W., and Balasubramanian, S. (2013). Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine. Nat. Protoc. 8, 1841-1851.
- Branco, M.R., Ficz, G., and Reik, W. (2011). Uncovering the role of 5-hydroxymethylcytosine in the epigenome. Nat. Rev. Genet. 13, 7-13.
- 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.
- Ito, S., Shen, L., Dai, Q., Wu, S.C., Collins, L.B., Swenberg, J.A., He, C., and Zhang, Y. (2011). Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333, 1300-1303.
- 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.
- 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.
- Song, C.X., Szulwach, K.E., Fu, Y., Dai, Q., Yi, C., Li, X., Li, Y., Chen, C.H., Zhang, W., Jian, X., et al. (2011). Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine. Nat. Biotechnol. 29, 68-72.
- Xu, Y., Wu, F., Tan, L., Kong, L., Xiong, L., Deng, J., Barbera, A.J., Zheng, L., Zhang, H., Huang, S., et al. (2011). Genome-wide regulation of 5hmC, 5mC, and gene expression by Tet1 hydroxylase in mouse embryonic stem cells. Mol. Cell 42, 451-464.