Regulation of Transcription at the Human IL-2 Promoter

描述：（由申请人提供）：白细胞介素-2（IL-2）是一种细胞因子，在哺乳动物对感染的免疫应答中发挥作用。在免疫应答期间，IL-2的产生在转录水平上被激活剂如NFAT 1和cJun上调。本研究的主要目的是了解人IL-2启动子的转录激活机制。另一个目标是揭示当人类T细胞对激活和无反应性信号做出反应时，三种蛋白质-蛋白质相互作用在多大程度上控制全基因组转录。这些研究的结果将为了解mRNA转录是如何调节的提供重要的见解，这对于理解正常细胞生长以及与许多疾病相关的畸变至关重要。这些研究将解释启动子DNA元件，蛋白质-蛋白质相互作用和协同核蛋白复合物如何有助于在不同刺激条件下设定T细胞中IL-2转录水平。1）建立IL-2启动子在共刺激和无反应性过程中分子组成的动力学曲线，并确定启动子近端停顿是否参与IL-2调节。2)确定三种蛋白质-蛋白质相互作用在T细胞活化过程中IL-2启动子上发生的分子事件程序中的作用。3)了解特定蛋白质-蛋白质相互作用如何控制共刺激和无反应性T细胞中发生的两种不同转录程序。4)研究转录激活因子、不同的DNA元件和核蛋白结构对IL-2启动子协同激活的作用机制。5)通过测量含有特定激活子和辅激活子的确定区域的复合物的相互作用亲和力和亚基化学计量，了解转录因子之间相互作用的生物物理性质。这些研究利用基于细胞的和生物化学测定。这项研究的创新方面之一是使用抑制肽来研究cJun，NFAT 1和TFIID复合物的两个亚基之间的特定蛋白质-蛋白质相互作用在人类T细胞中设置转录程序的功能。拟议的实验将揭示在IL-2启动子和其他启动子在人类T细胞的转录调控的新机制，提供一般的洞察：如何启动子元件影响协同转录激活，如何蛋白质-蛋白质相互作用有助于转录调控全基因组，以及如何核蛋白复合物的形成和功能的天然人类启动子的调控区。

DESCRIPTION: (provided by applicant): 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.