Modulation of IFN Action via Novel Regulatory Factors

描述（由申请人提供）：细胞因子干扰素家族调节几种生理反应，如抗病毒、抗肿瘤和免疫功能。它们在临床上用于治疗许多癌症、病毒性疾病和神经退行性疾病。通过与其他细胞因子相互作用，IFN形成控制肿瘤细胞生长和宿主对病原体防御的细胞间信号分子的大网络。本实验室先前的研究在IRFg/p48/ISGF 3 γ基因的启动子中鉴定了一种新的反应元件，称为γ-IFN激活的转录元件（GATE）。在上一个资助期间，我们已经确定了两个GATE结合因子的特征，并确定了其中一个转录因子CCAAT/增强子结合蛋白-β（C/EBP-β）。该因子调节许多不同的过程，包括细胞分化、能量代谢、免疫应答、肿瘤生长和凋亡。小鼠C/EBP-β基因的缺失导致免疫缺陷、巨噬细胞依赖的先天抗肿瘤和抗菌防御的丧失。由于IRF 9是迄今为止已知的唯一的C/EBP-β依赖性IFN-γ调节基因，因此我们假设可能存在在IFN-γ/C/EBP-β途径控制下的其他未定义的基因。我们现在已经从野生型和C/EBP-β缺失小鼠中产生了巨噬细胞系，并鉴定了几个基因。其中之一是死亡相关蛋白激酶1（DAPK 1），它是细胞凋亡、细胞周期和转移的重要调节因子。该基因在几种人类癌症中经常失活和/或缺失。在具体的目标1中，我们建议研究C/EBP-β在调节DAPK基因中的作用，使用启动子突变分析、体内足迹法、染色质组蛋白免疫沉淀（CHIP）测定来证明C/EBP-β与DAPK基因调节的生理相关性。在具体目标2中，我们将研究MAP激酶在调节IFN诱导的DAPK转录中的作用。我们还提出了一种新的蛋白质，TRAP 220，作为一个调节IFN驱动的转录反应，通过C/EBP-β的人类转录介质机制的组成部分的参与的初步证据。我们将使用RNAi和ChIP测定研究TRAP 220在调节IFN-γ驱动的DAPK转录中的生理相关性。最后，我们将研究MAPK/CEBP/TRAP 220相互作用在不表达内源性DAPK 1的人癌细胞中的破坏。这项研究将整合通过C/EBP-β控制细胞死亡反应的信号通路和转录控制机制。

DESCRIPTION (provided by applicant): The interferon family of cytokines regulates several physiologic responses, such as antiviral, antitumor, and immune functions. They are in clinical use for the therapy of a number of cancers, viral diseases and neurodegenerative disorders. By interacting with other cytokines IFNs form a large network of intercellular signaling molecules that control neoplastic cell growth and host defenses against pathogens. Previous studies from our laboratory identified a novel response element called, the gamma-IFN activated transcriptional element (GATE), in the promoter of the IRFg/p48/ISGF3gamma gene. During the last funding period, we have characterized two GATE-binding factors and identified one of them as transcription factor CCAAT/Enhancer Binding protein-beta (C/EBP-beta). This factor regulates a number of disparate process including cell differentiation, energy metabolism, immune response, tumor growth and apoptosis. The deletion of C/EBP-beta gene in mice causes immunodeficiency, loss of macrophage dependent innate antitumor and antibacterial defenses. Since IRF9 was the only C/EBP-beta dependent IFN-gamma regulated gene known thus far we hypothesized that there might be other undefined genes under the control of IFN-gamma/C/EBP-beta pathway. We have now generated macrophage cell lines from wildtype and C/EBP-beta null mice and identified several genes. One of them is the death associated protein kinasel (DAPK1), a crucial regulator of apoptosis, cell cycle, and metastasis. This gene is frequently inactivated and/or deleted in several human cancers. In specific aim 1, we propose to investigate the role of C/EBP-beta in regulating the DAPK gene, using promoter mutational analysis, in vivo foot printing, chromatin histone immunoprecipitation (CHIP) assays to demonstrate the physiologic relevance of C/EBP-beta to DAPK gene regulation. In specific aim 2 we will investigate the role of MAPKinases in regulating IFN-induced DAPK-transcription. We also present preliminary evidence for the involvement of a novel protein, TRAP220, a component of the human transcriptional mediator machinery as a regulator of IFN-driven transcriptional response through C/EBP-beta. We will investigate the physiologic relevance of TRAP220 in regulating IFN-gamma driven transcription of DAPK using RNAi and ChIP assays. Lastly, we will investigate the disruption of MAPK/CEBP/TRAP220 interactions in human cancer cells that do not express endogenous DAPK1. This study will integrate signaling pathways and transcriptional control mechanisms that control cellular death responses through C/EBP-beta.