The ten-eleven translocation (TET) enzymes oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine. These species are implicated as intermediates to active demethylation in the mammalian cell cycle but recent reports show their involvement in regulatory roles. In this study, we present kinetic data that explains the modus operandi of the TET enzymes in vitro and relate our data to the role of TETs in the maintenance of 5mC forms as epigenetic states in vivo. We show how products of the TET reaction display inhibitory effects resulting in the reduction of the rates and conversion efficiencies of the oxidation steps. We further connect the biochemistry of TETs to their utility in single-base resolution mapping of 5mC in genomes. To date, bisulfite sequencing (BS) is considered the gold standard for detection of 5mC in genomes, however, BS does have its drawbacks. DNA is commonly damaged and degraded by the chemical bisulfite reaction resulting in libraries that demonstrate high GC-bias and are enriched for methylated regions. The TET-dependent, 5mC-sequencing enzymatic approach presented in this work is more robust than BS and exhibits insignificant levels of DNA damage, resulting in longer fragments and minimal GC bias. This allows for 5mC mapping over a wide range of DNA input amounts with superior sequencing metrics and higher CpG coverage.

In vitro biochemistry of the TET enzymes and their application in a novel bisulfite-free, enzyme-dependent methylome sequencing methodology

2019 Fall National Meeting

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