Regulation of Transcription at the Human IL-2 Promoter

超出提供的空间。白介素2(IL-2)是一种细胞因子，在控制哺乳动物对感染的免疫反应中起关键作用。在免疫反应中，IL-2的产生被NFATp和cJun等激活剂在转录水平上上调。这项研究的主要目标是了解人IL-2启动子转录激活的机制。这些研究的完成将有助于深入了解免疫反应中的关键步骤是如何调控的，并有助于理解mRNA转录是如何调控的，这两者对于理解正常的细胞生长以及与许多疾病相关的异常都是至关重要的。了解IL-2转录调控的核心是破译启动子DNA、调节蛋白和染色质如何在T细胞中设定适当的IL-2转录水平。该提案的具体目的如下：1)确定IL-2核心启动子元件如何影响RNA聚合酶II转录反应中特定步骤的速度和程度。2)通过确定增强子上存在哪些蛋白质，它们何时结合，以及增强子DNA的结构如何影响其功能来表征IL-2增强子上的核蛋白复合体对T细胞刺激的响应。3)了解cJun、NFATp和TFliD组分之间的相互作用网络如何激活转录。4)利用分子遗传学和分子抑制剂，揭示单个蛋白质相互作用在T细胞IL-2转录调控中的作用。5)确定染色质结构对IL-2启动子转录机制的影响。每个特定的目标都利用了体外实验和基于细胞的分析。体外实验使用了一个纯化的人类RNA聚合酶II转录系统。基于互补细胞的实验利用染色质免疫沉淀、瞬时转基因和核连续分析。拟议的实验将揭示IL-2启动子转录调控的新机制，并将提供关于启动子序列如何影响转录、蛋白质-蛋白质相互作用如何促进高水平的协同作用以及染色质如何影响转录机制的一般见解。表演网站========================================Section End===========================================

EXCEED THE SPACE PROVIDED. 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 foUowing: 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. PERFORMANCE SITE ========================================Section End===========================================