TAB-seq employs a clever use of the Tet enzyme distinguish between 5mC and 5hmC using bisulfite. The Tet enzymes are responsible for step-wise oxidative demethylation of 5mC in vivo. 5mC is first converted to 5hmC, then 5-formylcytosine (5fC), then 5-carboxylcytosine (5caC). 5caC or 5fC are then converted to unmodified C by Terminal deoxynucleotidyl transferase (Ito et al., 2011).
In TAB-seq, genomic 5hmC is first protected with protected by glucosylation. The DNA is then treated with a Tet enzyme, converting 5mC to 5caC, while leaving the glycosylated 5hmC untouched. Any C’s read in the resulting sequence are thus interpreted as 5hmC. The major advantage of TAB-seq over OxBSseq is that it actually measures 5hmC and does not infer it. This means that if 5hmC is the only mark you are interested in, TAB-seq is a cheaper and more streamlined to perform. On the downside, TAB-seq relies on the activity of the Tet enzyme which is not 100% efficient and expensive to produce (Yu et al., 2012).
TAB-seq was introduced in 2012 (Yu et al., 2012). Since, it has been used to study the role of 5hmC in cancers (Mariani et al., 2013) brain function (Chopra et al., 2014), and cognitive disorders such as autism (Zhubi et al., 2014).
TAB-seq Additional Reading
This nature protocol provides a good background on TAB-seq as well as comparison to related techniques, applications, and limitations.
This review describes the endogenous role of the TET proteins in demethylation. The review also describes methods for assaying 5hmC including OxBS-seq and TAB-seq and how they compare to each other and others.
- Chopra, P., Papale, L.A., White, A.T., Hatch, A., Brown, R.M., Garthwaite, M.A., Roseboom, P.H., Golos, T.G., Warren, S.T., and Alisch, R.S. (2014). Array-based assay detects genome-wide 5-mC and 5-hmC in the brains of humans, non-human primates, and mice. BMC Genomics 15, 131-2164-15-131.
- 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.
- Mariani, C.J., Madzo, J., Moen, E.L., Yesilkanal, A., and Godley, L.A. (2013). Alterations of 5-hydroxymethylcytosine in human cancers. Cancers (Basel) 5, 786-814.
- Yu, M., Hon, G.C., Szulwach, K.E., Song, C.X., Zhang, L., Kim, A., Li, X., Dai, Q., Shen, Y., Park, B., et al. (2012). Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome. Cell 149, 1368-1380.
- Zhubi, A., Chen, Y., Dong, E., Cook, E.H., Guidotti, A., and Grayson, D.R. (2014). Increased binding of MeCP2 to the GAD1 and RELN promoters may be mediated by an enrichment of 5-hmC in autism spectrum disorder (ASD) cerebellum. Transl. Psychiatry. 4, e349.