The long-term objectives of the proposed research are to understand how the nuclear factor of activated T cells (NFAT) regulates distinct transcription programs in different cellular contexts and to explore the therapeutic potential of this family of transcription factors. NFAT regulates specific gene expression in diverse tissues and cells;its activity is controlled by the calcium-calmodulin dependent calcineurin, which is the major target of the immunosuppressant drugs CsA and FK506. The side effects of these drugs, and growing evidence indicating that the calcineurin/NFAT pathway may be targeted for other clinical applications, suggest that selective manipulation of specific NFAT transcription programs may be a strategy to develop therapeutics against a variety of diseases, including autoimmunity, cardiac hypertrophy and cancer. The basic research design is based on a model of combinatorial gene regulation wherein NFAT interacts with different transcription factor partners to control specific gene expression. The various transcription complexes formed between NFAT and other transcription factors could be potential targets for selective targeting of specific NFAT functions. The last funding cycle of this project has demonstrated that NFAT can regulate distinct T cell transcription programs by cooperating with different transcription factors such as Fos-Jun and FOXP3. The proposed continuation of this project will focus on the cooperative mechanisms between NFAT and GATA. Members of these two families of transcription factors have been shown to functionally synergize and/or physically interact in a variety of cellular processes, many of which have important clinical implications, such as T cell development, and heart and skeletal muscle hypertrophy.
Aim 1 is to determine the high-resolution structure of the NFAT1:GATA3 complex and use structure-guided mutations to analyze the binding mechanism between these two transcription factors.
Aim 2 is to analyze DNA bridging by NFAT1 and GATA3 in solution by a variety of biochemical methods and structure-guided mutagenesis. These studies will test if NFAT and GATA can directly mediate long-range DNA interaction at the biochemical level and if they cooperate to bridge two DNA molecules together as part of their mechanism of transcription synergy.
Aim 3 is to extend the biochemical studies of Aim 2 to a cell culture model to explore the roles of NFAT1 and GATA3 in long-range gene regulation. The proposed studies will provide comprehensive insights into the cooperative mechanisms between NFAT and GATA, which will serve as a foundation for further studying their physiological roles in vivo. These studies will significantly advance the basic knowledge on the mechanisms by which NFAT activates specific gene expression through diverse partnerships with distinct transcription factors and help address the feasibility of targeting specific NFAT complexes for therapeutic development.
The calcineurin/NFAT pathway has attracted much attention because it is the major target of the immunosuppressants CsA and FK506. Increasing studies have also expanded the function of the calcineurin/NFAT pathway to many medically important processes. These studies not only broaden the potential clinical application of drugs targeting the calcineurin/NFAT pathway, but also raise the question if specific branches of the calcineurin/NFAT pathway could be targeted for therapeutic benefits. The proposed studies seek to understand the mechanisms by which NFAT regulates diverse transcription programs in different cellular contexts and use this knowledge to explore the therapeutic potential of the calcineurin/NFAT pathway.
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