The longterm interest of this laboratory is to define the mechanisms of inducible gene transcription in activated T cells during the immune response. NFAT proteins are inducible transcription factors that play a key role in this process: a large number of genes induced by activate T cells, including IL-2, IL-3, IL-4, IL-5, IL-8, IL-10, IL-13, GM-CSF, IFN-g, CD40L and FasL are thought to be target genes for NFAT. Together, these gene products specify the differentiation, migration and function of helper and cytolytic T cells, B cells, mast cells, NK cells and eosinophils, thereby determining the overall outcome of the immune response. Many of the known NFAT sites in inducible genes are composite NFAT-AP-1 sites, to which NFAT proteins bind cooperatively with the bZIP (basic-region leucine zipper)-family transcription facto AP-1 (Fos-Jun). The overall theme of this proposal is to determine how NFAT proteins cooperate with Fos-Jun proteins and other potential partner proteins in the nucleus of activated T cells to turn on inducible genes. The focus is entirely on endogenous gene expression in primary T cells and untransformed T cell lines, and the use of transformed cell lines or transient reporter assays has been avoided as far as possible.
Aim I is to assess the extent of functional redundancy within the NFAT family, by evaluating the extent to which genes induced by individual NFAT proteins are overlapping or unique. Constitutively active versions of NFAT-proteins will be expressed in T cells under conditions where the endogenous proteins are either not activated or are selectively blocked, and expression of inducible genes will be assessed. In parallel, the technique of chromatin immunoprecipitation (CHIP) will be adapted to identify target genes for NFAT proteins.
Aim 2 is to examine in detail the activation of the gene encoding IL-10, an immunosuppressive and anti-inflammatory cytokine with potential for treating diverse clinical conditions including insulin-dependent diabetes, allergic inflammation, transplant rejection, and tumor growth. The effects of NFAT, IL-4 and IL, on the early phase of IL-10 expression in naive T cells, and on its acute transcription in differentiated T cells will be assessed.
Aim 3 is define the importance of the NFAT-AP-1 interaction in vivo, using NFAT proteins mutated so that they are unable to cooperate with Fos and Jun.
Aim 4 is to identify novel NFAT composite sites at which NFAT cooperates with partner proteins other than Fos and Jun, and to delineate the structural basis for the known interaction between NFAT and GATA proteins.
Aim 5 is to investigate the structure, function, and regulation of a new NFAT protein that does not cooperate with AP-1.
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