Investigation of the tet family dioxygenases and their role in epigenetics
DNA methylation is an epigenetic marker that is known to be important in regulating gene expression and cellular processes. Recently a second DNA modification, 5-hydroxy methylcytosine (5-hmC), has shown in the mammalian genome. In addition, the TET (Ten-Eleven Translocation gene) family (1-3) of dioxygenases have been shown to generate 5-hmC from 5-methylcytosine (5-mC). New evidence suggests 5-hmC modifications is connected with embryonic stem (ES) cell pluripotency, helping to dictate differentiation, as well as being involved in higher brain cognitive functions. Finding a small molecule that selectively targets one or all of the TET family of dioxygenases, could provide a flexible and sensitive tool for exposing the role of the TET enzymes and of the newly discovered 5-hmC base modification. A platform for screening iron(II)/2-oxoglutarate-dependent dioxygenases (such as TETs 1-3) has been established and it is a coupled enzyme assay that detects succinate formation, the carboxylated by-product of 2-oxoglutarate, during the dioxygenase catalytic cycle. The activity of iron(II)/2-oxoglutarate-dependent dioxygenases will be monitored with the SPECTROstar Nano using the oxidation of NADH, to NAD, at 340 nm. Once the platform has been optimized on the SPECTROstar Nano, an HTS screen will be performed in 1536 well plates to find potential TET inhibitors.