Interleukin-2 (IL-2) is a cytokine that functions in the mammalian immune response to infection. During an immune response, production of IL-2 is upregulated at the level of transcription by activators such as NFAT1 and cJun. The main goal of this research is to understand the mechanisms of transcriptional activation at the human IL-2 promoter. An additional goal is to reveal the extent to which three protein-protein interactions control transcription genome-wide when human T cells respond to both activating and anergic signals. The results of these studies will provide significant insight into how mRNA transcription is regulated, which is vitally important in understanding normal cell growth as well as aberrations associated with many diseases. The studies will decipher how promoter DNA elements, protein-protein interactions, and a synergistic nucleoprotein complex contribute to setting the level of IL-2 transcription in T cells under different stimulatory conditions.
The specific aims of the proposal are the following: 1) To establish a kinetic profile of the molecular composition at the IL-2 promoter during co-stimulation and anergy, and determine whether promoter-proximal pausing is involved in IL-2 regulation. 2) To determine the role of three protein-protein interactions in setting the program of molecular events that occurs at the IL-2 promoter during T cell activation. 3) To obtain an understanding of how specific protein-protein interactions control the two different transcriptional programs that occur in co-stimulated and anergic T cells. 4) To investigate the mechanism by which transcriptional activators, distinct DNA elements, and nucleoprotein structure contribute to combinatorial synergistic activation at the IL-2 promoter. 5) To understand the biophysical properties of interactions between transcription factors by measuring the interaction affinities and subunit stoichiometries of complexes containing defined regions of specific activators and coactivators. These studies utilize both cell-based and biochemical assays. One of the innovative aspects of this research is the use of inhibitory peptides to study the function of specific protein-protein interactions between cJun, NFAT1, and two subunits of the TFIID complex in setting transcriptional programs in human T cells. The proposed experiments will reveal novel mechanisms of transcriptional regulation at the IL-2 promoter and other promoters in human T cells by providing general insight into: how promoter elements influence synergistic transcriptional activation, how protein-protein interactions contribute to transcriptional regulation genome-wide, and how nucleoprotein complexes form and function at the regulatory region of a natural human promoter. ? ? ?
