High-Resolution Analysis of 5-Hydroxymethylcytosine by TET-Assisted Bisulfite Sequencing

Abstract

DNA cytosine modification is an important epigenetic mechanism that serves critical functions in a variety of biological processes in development and disease. 5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the two most common epigenetic marks found in the mammalian genome. 5hmC is generated from 5mC by the ten-eleven translocation (TET) family of dioxygenase enzymes. This modification can reach substantial levels in certain cell types such as embryonic stem cells and neurons. Standard bisulfite sequencing techniques cannot distinguish between 5mC and 5hmC. Therefore, the method of TET-assisted bisulfite sequencing has been developed for detecting 5hmC specifically. The method is based on protection of 5hmC by glycosylation followed by complete oxidation of both 5mC and 5fC to 5caC, which converts to uracil after bisulfite treatment leaving only 5hmC remaining as a cytosine signal after PCR and sequencing. The method requires a highly active TET protein for the conversion steps. Here, we present an efficient TET protein purification method and a streamlined TAB-sequencing protocol for 5hmC analysis at single base resolution.