The control of gene expression in mammals relies in part on modifications to cytosine residues in DNA, which exist in at least five forms: cytosine (C), 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5- formylcytosine (5fC) and 5-carboxylcytosine (5caC). DNA methyltransferases methylate cytosine at the 5- position in the context of CpG dinucleotides, generating 5mC in the genome. Ten-eleven translocation (Tet) dioxygenases convert 5mC to 5hmC, 5fC, and 5caC in three consecutive oxidation reactions. These modifications are dynamically regulated during embryonic development and enriched in brain. To understand the function of these modifications and the regulatory mechanisms that control the levels and genomic distribution of the five forms of the cytosine, we propose to study the enzymes/proteins that generate, read, and remove these modifications. Specifically, the three aims of this proposal are to determine central aspects, enzymatically and structurally, of (1) 5mC oxidation by Tet proteins, (2) modification-specific recognition by C2H2 zinc-finger and SRA-domain proteins, and (3) 5mC and 5hmC base excision by DNA glycosylases.
During the development, mammalian germ line cells and brains undergo a series of cellular and molecular events that lead to the erasure and re-establishment of epigenetic programs and the mechanism by which erasure of 5-methylcytosine (5mC) marks takes place is not well understood. It is possible that the active erasure and the re-establishment of 5mC marks during germ line differentiation and brain development from fetus to young adult involve dynamic changes of 5mC into oxidative marks, and that these modified cytosine residues in DNA are recognized by specific protein readers with distinct roles in the maintenance of epigenetic memory. A detailed biochemical and structural analyses of generation, recognition and erasure of these marks should shed light on this issue.
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