Abstract
Ten-eleven translocation (TET) enzymes catalyze stepwise oxidation of 5-methylcytosine (mC) to yield 5-hydroxymethylcytosine (hmC) and the rarer bases 5-formylcytosine (fC) and 5-carboxylcytosine (caC). Stepwise oxidation obscures how each individual base forms and functions in epigenetic regulation, and prompts the question of whether TET enzymes primarily serve to generate hmC or are adapted to produce fC and caC as well. By mutating a single, conserved active site residue in human TET2, Thr1372, we uncovered enzyme variants that permit oxidation to hmC but largely eliminate fC and caC. Biochemical analyses, combined with molecular dynamics simulations, elucidated an active site scaffold that is required for wild-type (WT) stepwise oxidation and that, when perturbed, explains the mutants' hmC-stalling phenotype. Our results suggest that the TET2 active site is shaped to enable higher-order oxidation and provide the first TET variants that could be used to probe the biological functions of hmC separately from fC and caC.
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Acknowledgements
We thank B. Niedziolka and the Wistar Institute Protein Expression Facility for help with protein expression in Sf9 cells and all members of our labs for insightful discussions. Computing time from Wayne State C&IT and additional mass spectrometry resources from I. Blair's lab are gratefully acknowledged. This work was supported by the Rita Allen Foundation Scholar Award to R.M.K. and by NIH grants (R01 GM110174 to B.A.G., R01 GM108583 to G.A.C., and F30 CA196097 to M.Y.L.).
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R.M.K., G.A.C., M.Y.L., and H.T. conceived the experiments. R.M.K., M.Y.L., D.J.C., J.E.D., X.-J.C., and B.A.G. were involved in the design and optimization of biochemical and cellular experiments, which were performed and analyzed by M.Y.L., J.E.D., and R.M.K. For MD simulations, G.A.C., H.T., M.Y.L., and R.M.K. were involved in the design of experiments, which were performed and analyzed by H.T. and G.A.C. The manuscript was written by M.Y.L., R.M.K., H.T., and G.A.C. and edited by all authors.
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Supplementary Results, Supplementary Figures 1–10 and Supplementary Tables 1–9. (PDF 2502 kb)
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Force field parameters. (TAR 81 kb)
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Liu, M., Torabifard, H., Crawford, D. et al. Mutations along a TET2 active site scaffold stall oxidation at 5-hydroxymethylcytosine. Nat Chem Biol 13, 181–187 (2017). https://doi.org/10.1038/nchembio.2250
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DOI: https://doi.org/10.1038/nchembio.2250
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