Studies In Structure And Catalytic Mechanism Of TET2:Key Enzyme In Mammalian DNA Demethylation Process

Methylation of the fifth position of cytosine (5-methylcytosine,5mC) is a highly conserved epigenetic modification of DNA found in most plant, animal and fungi models. Dynamic regulation of DNA methylation has a profound impact on genome stability, gene expression and development. It’s well known how DNA methylation is established but the mechanism how DNA modification is erased remains ambiguious. Many candidates from the known repertoire of DNA modifying enzymes have historically been proposed to function in DNA demethylation, including DNA cytosine deaminases, DNA glycosylases and other DNA repair factors. However, none of the above has been recognized as a unifying mechanism because it seems that their roles have often seemed specific to individual biological system or physiological condition.The report of TET enzymes that can oxidize 5mC into 5hmC in 2009 is a milestone in uncovering DNA demethylation mechanisms, introducing 5-hydroxylcytosine (5hmC) as a key intermediate in active demethylation pathways. Followed studies demonstrate TET is capable to consecutively oxidize 5hmC, yielding 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) which might be reverted to cytosine through thymine DNA glycosylase (TDG)-mediated base excision repair to complete the cycle. As DNA demethylation initiator, TET proteins play important roles in diverse biological processes and mutations of TET are frequently observed in cancer, especially myeloid malignance. The lack of three dimensional structure prevents us from understanding the mechanism of substrate recognition and catalysis by TET.To disclose the myth, we solved the crystal structure of TET2-5mC complex at 2.02A. The structure shows that two zinc fingers bring the Cys-rich and DSBH domains together to form a compact catalytic domain. The Cys-rich domain stabilizes the DNA above the DSBH core. TET2 specifically recognizes CpG dinucleotide and shows substrate preference for 5mC in a CpG context. A hydrophobic loop from Cys-rich domain unwind the DNA helix, making 5mC flip into the catalytic cavity with the methyl group orientated to catalytic Fe(Ⅱ) for reaction. The methyl group is not involved in TET2-DNA contacts so that the catalytic cavity allows TET2 to accommodate 5mC derivatives for further oxidation. Mutations of Fe(Ⅱ)/NOG-chelating, DNA-interacting and zinc-chelating residues frequently observed in human cancers significantly decrease activity of TET2.Our studies illustrate three dimensional packing of TET2 and provide a structural basis for understanding the mechanisms of TET-mediated 5mC oxidation. Biochemical data demonstrate mutaions in myeloid malignancy cause diseases due to loss of function. Considering TET’s close relation to tumorgenesis, our work also provide theoretical basis for targeted drug design.