Abstract 183: A Genomewide Screen to Identify Novel NFAT Regulators

NFAT (Nuclear Factor of Activated T cells) transcriptions factors are directly activated by the Ca2+ activated serine-threonine protein phosphatase calcineurin. Following dephosphorylation, NFAT factors translocate to the nucleus. They share a Rel/NFkB homology DNA binding domain that functions in concert with other transcription factors such as AP-1, GATA4, NFkB, MEF2, CREB, Myc, and IRF-1 to alter the expression of genes involved in tissue and cellular differentiation as well as adaptation to extracellular stimuli. Although the fundamental aspects of how NFAT factors are regulated by phosphorylation and dephosphorylation, as well as some interacting factors, have been described; much remains to be understood about the function of these factors in cardiac myocytes and other cell types. To identify potential new NFAT regulators that could influence nuclear shuttling or its trans-activating properties, we performed a genome wide screen with a NFAT-dependent luciferase reporter and a library of cDNA expression plasmids. Roughly 20,000 cDNA expression plasmids from the Mammalian Genome Collection were individually co-transfected with a NFAT-luciferase reporter in a human immortalized cell line in 384-well plates. Approximately 2% of the genes of the library gave a positive result, including well-known NFAT partners, calcium sensitive genes and transcriptions factors. Among them, twenty potential NFAT activators were selected and NFAT-Luciferase activation was validated in a secondary screen which normalized for transfection efficiency and cell number. The results of the screen will be presented along with functional characterization of the more interesting interacting NFAT co-activators.