With the recent discovery of 5-hydroxymethylcytosine in mammalian DNA and the TET enzyme family responsible for its generation, it is clear that the mammalian DNA epigenome is much more complex than had been thought. A lot of questions remain to be answered about the in vivo roles and the in vitro properties of the TET enzymes. In this fast-track application, we propose studying the TET-like enzymes in the single-cell protist Naegleria gruberi. They are much smaller in size than the human TET enzymes and are much easier to produce and engineer. A panel of biochemical assays will be carried out to thoroughly characterize the in vitro properties of the active TET-like enzymes in N. gruberi.
We aim at providing these enzymes as reagents to the research market. Since 5-methylcytosine (5mC) is an important epigenetic marker in mammalian genomic DNA, these enzymes should enable many creative experiments, e.g., direct sequencing of the epigenome, because they functionalize the chemically inert 5mC. To enhance their versatility, we propose using enzyme engineering to look for mutants with altered or improved properties. For this purpose, we will determine the 3D structure of at least one active TET-like enzymes. We plan to apply the wild-type enzymes or derived mutants in several epigenomic applications and determine the methylome/hydroxymethylome changes during the differentiation process of N. gruberi, which may shed lights on the similar processes in the more complex biological systems. In summary, our studies will bring a number of novel enzymatic reagents to the research market and generate scientific insights on epigenetic regulation involving 5hmC.
Dynamic DNA epigenomes provide a basis for the phenotypic variation of a single genome. Many pathological processes, including cancer, are associated with aberrant landscapes of the epigenome. By studying a unique family of 5-methylcytosine oxidases, this proposal aims at providing novel reagents for better characterization of the DNA epigenomes and further understanding of their dynamic transitions.
|Hashimoto, Hideharu; Pais, June E; Dai, Nan et al. (2015) Structure of Naegleria Tet-like dioxygenase (NgTet1) in complexes with a reaction intermediate 5-hydroxymethylcytosine DNA. Nucleic Acids Res 43:10713-21|
|Pais, June E; Dai, Nan; Tamanaha, Esta et al. (2015) Biochemical characterization of a Naegleria TET-like oxygenase and its application in single molecule sequencing of 5-methylcytosine. Proc Natl Acad Sci U S A 112:4316-21|
|Hashimoto, Hideharu; Zhang, Xing; Vertino, Paula M et al. (2015) The Mechanisms of Generation, Recognition, and Erasure of DNA 5-Methylcytosine and Thymine Oxidations. J Biol Chem 290:20723-33|
|Hashimoto, Hideharu; Pais, June E; Zhang, Xing et al. (2014) Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA. Nature 506:391-5|
|Horton, John R; Wang, Hua; Mabuchi, Megumu Yamada et al. (2014) Modification-dependent restriction endonuclease, MspJI, flips 5-methylcytosine out of the DNA helix. Nucleic Acids Res 42:12092-101|