Research Project
8453553
DESCRIPTION: 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.
