Interleukin-2 (IL-2) is a cytokine that is critical in controlling 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 NFATp and cJun. The broad goal of this research is to understand the mechanism of transcriptional activation at the human IL-2 promoter. Completion of these studies will provide insight into how a critical step during the immune response is regulated, and contribute to understanding how mRNA transcription in general is regulated, both of which are vitally important in understanding normal cell growth as well as aberrations associated with many diseases. Central to understanding IL-2 transcriptional regulation is deciphering how the promoter DNA, regulatory proteins, and chromatin contribute to setting the proper level of IL-2 transcription in T cells.
The specific aims of the proposal are the following: 1) To determine how the IL-2 core promoter elements influence the rate and extent of specific steps in the RNA polymerase II transcription reaction. 2) To characterize nucleoprotein complexes at the IL-2 enhancer in response to T cell stimulation by determining which proteins are present at the enhancer, when they bind, and how the architecture of the enhancer DNA affects its function. 3) To understand how the network of interactions between cJun, NFATp and components of TFlID function to activate transcription. 4) To reveal the contributions of individual protein-protein interactions to IL-2 transcriptional regulation in T cells by using molecular genetics and molecular inhibitors. 5) To determine what effect chromatin structure has on the mechanism of transcription at the IL-2 promoter.
Each specific aim utilizes both in vitro experiments and cell-based assays. The in vitro experiments employ a purified human RNA polymerase II transcription system. The complementary cell based experiments utilize chromatin immunoprecipitations, transient transfections, and nuclear run-on assays. The proposed experiments will reveal novel mechanisms of transcriptional regulation at the IL-2 promoter, and will provide general insight into how promoter sequences influence transcription, how protein-protein interactions contribute to high levels of synergy, and how chromatin affects the mechanism of transcription.
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