5-hydroxymethylcytosine (5-hmC) is an epigenetic modification (methylated cytosine) that is thought to be a derivative of 5-methylcytosine (5-mC). T4 β-glucosyltransferase catalyzes the transfer of glucose from uridine diphosphoglucose (UDP-Glucose) to the hydroxymethyl group 5-hmC, but does not modify 5-mC. This modifying enzyme can be used to differentiate these types of cytosine methylation. Specifically, after T4 β-glucosyltransferase treatment, restriction enzyme (e.g., MspI/HpaII) digestion or immunodetection can be used identify β-glucosyl-5-hydroxymethylcytosine (β-glu-5hmC) at particular loci, or genome-wide analyses can be accomplished by selective pull-down of β-glu-5hmC.

Applications

• Addition of glucose to 5-hydroxymethylcytosine (to create β-glucosyl-5-hydroxymethylcytosine) in mammalian or plant genomic DNA
• Detection of 5-hydroxymethylcytosine when coupled with digestion with a restriction enzyme that is sensitive to β-glucosyl-5-hydroxymethylcytosine