Novel 5-methylcytosine Oxidases

DESCRIPTION With the recent discovery of 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 methylcytosine mC 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 mC 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 D 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 hmC PUBLIC HEALTH RELEVANCE 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 methylcytosine oxidases this proposal aims at providing novel reagents for better characterization of the DNA epigenomes and further understanding of their dynamic transitions