Quantification of Oxidized 5-Methylcytosine Bases and TET Enzyme Activity

In eukaryotic DNA, cytosine can be enzymatically modified to yield up to four epigenetic base variants. DNA methyltransferases convert cytosine to 5-methylcytosine (mC), which plays critical roles in gene regulation during development. Ten-eleven translocation (TET) enzymes can sequentially oxidize mC to three products: 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxylcytosine (caC). These oxidized bases have been found in numerous mammalian cell types, where they potentially carry out independent epigenetic functions and aid in DNA demethylation. To gain insight into the mechanisms and functions of TET family enzymes, rigorous approaches are needed to quantify genomic cytosine modifications in cells and track TET enzyme activity in vitro. Here, we present tools developed by our lab and others to report on each of the five forms of cytosine (unmodified, mC, hmC, fC, and caC) with high specificity and sensitivity. We provide detailed protocols for qualitative and quantitative analysis of cytosine modifications in genomic DNA by dot blotting and LC-MS/MS. We then describe methods for generating synthetic oligonucleotide substrates for biochemical studies, provide optimized reaction conditions, and introduce several chemoenzymatic assays, as well as HPLC, mass spectrometry, and scintillation counting methods to quantify cytosine modifications in vitro. These approaches enable mechanistic studies of TET activity, which are key to understanding the role of these enzymes in epigenetic regulation.