Structural and Functional Versatility of NFAT

描述（由申请人提供）：拟议研究的长期目标是了解活化T细胞的核因子（NFAT）如何在不同的细胞环境中调节不同的转录程序，并探索该转录因子家族的治疗潜力。NFAT调节不同组织和细胞中的特定基因表达;其活性由钙-钙调蛋白依赖性钙调磷酸酶控制，钙调磷酸酶是免疫抑制剂CsA和FK 506的主要靶点。这些药物的副作用，以及越来越多的证据表明钙调磷酸酶/NFAT途径可能被靶向用于其他临床应用，表明选择性操纵特定的NFAT转录程序可能是开发针对多种疾病的治疗方法的策略，包括自身免疫，心脏肥大和癌症。基础研究设计基于组合基因调控模型，其中NFAT与不同的转录因子伴侣相互作用以控制特定的基因表达。NFAT与其他转录因子之间形成的各种转录复合物可能是选择性靶向特定NFAT功能的潜在靶标。该项目的最后一个资助周期表明，NFAT可以通过与不同的转录因子如Fos-Jun和FOXP 3合作来调节不同的T细胞转录程序。拟议继续开展的这一项目将侧重于NFAT和加塔之间的合作机制。这两个转录因子家族的成员已被证明在多种细胞过程中功能协同和/或物理相互作用，其中许多具有重要的临床意义，如T细胞发育，心脏和骨骼肌肥大。目的1是确定NFAT 1：GATA 3复合物的高分辨率结构，并使用结构指导突变来分析这两个转录因子之间的结合机制。目的二是通过多种生化方法和结构导向突变技术分析NFAT 1和GATA 3在溶液中的DNA桥连作用。这些研究将测试NFAT和加塔是否可以在生物化学水平上直接介导长距离DNA相互作用，以及它们是否合作将两个DNA分子桥接在一起作为其转录协同作用机制的一部分。目的3是将目的2的生化研究扩展到细胞培养模型，以探索NFAT 1和GATA 3在远程基因调控中的作用。这些研究将为深入了解NFAT和加塔之间的合作机制提供全面的见解，这将为进一步研究它们在体内的生理作用奠定基础。这些研究将显著推进NFAT通过与不同转录因子的不同伙伴关系激活特定基因表达的机制的基础知识，并有助于解决靶向特定NFAT复合物用于治疗开发的可行性。 公共卫生相关性：钙调神经磷酸酶/NFAT通路由于是免疫抑制剂CsA和FK 506的主要靶点而受到广泛关注。越来越多的研究也将钙调磷酸酶/NFAT途径的功能扩展到许多医学上重要的过程。这些研究不仅拓宽了靶向钙调磷酸酶/NFAT通路的药物的潜在临床应用，而且还提出了是否可以靶向钙调磷酸酶/NFAT通路的特定分支以获得治疗益处的问题。拟议的研究旨在了解NFAT在不同细胞环境中调节不同转录程序的机制，并利用这些知识探索钙调神经磷酸酶/NFAT途径的治疗潜力。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.