Modulation of IFN Action via Novel Regulatory Factors

描述(申请人提供)：干扰素家族的细胞因子调节几种生理反应，如抗病毒、抗肿瘤和免疫功能。它们在临床上用于治疗一些癌症、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形成了一个巨大的细胞间信号分子网络，控制着肿瘤细胞的生长和宿主对病原体的防御。本实验室以前的研究发现，在IRFg/P48/ISGF3γ基因的启动子中有一个新的反应元件，称为γ-干扰素激活的转录元件(GATE)。在上一次资助期间，我们鉴定了两个门结合因子，并确定其中一个为转录因子CCAAT/增强子结合蛋白-β(C/EBP-β)。该因子调控一系列不同的过程，包括细胞分化、能量代谢、免疫反应、肿瘤生长和细胞凋亡。小鼠C/EBP-β基因缺失导致免疫缺陷，丧失巨噬细胞依赖的天然抗肿瘤和抗菌防御能力。由于IRF9是迄今已知的唯一依赖于C/EBP-β的干扰素-γ调节基因，我们推测可能还有其他未定义的基因受干扰素-γ/C/EBP-β途径的控制。我们现在已经从野生型和C/EBP-β基因缺失的小鼠中建立了巨噬细胞系，并鉴定了几个基因。其中之一是死亡相关蛋白激动素(DAPK1)，它是细胞凋亡、细胞周期和转移的关键调节因子。该基因在几种人类癌症中经常失活和/或缺失。在特定目的1中，我们建议通过启动子突变分析、活体足印分析、染色质组蛋白免疫沉淀(ChIP)实验，研究C/EBP-β在DAPK基因调控中的作用，以证明C/EBP-β与DAPK基因调控的生理学相关性。在特定的目标2中，我们将研究MAPKinase在调节干扰素诱导的DAPK转录中的作用。我们还提出了一种新的蛋白质TRAP220参与的初步证据，TRAP220是人类转录调节机制的一个组成部分，通过C/EBP-β调节干扰素驱动的转录反应。我们将利用RNAi和芯片分析来研究TRAP220在调节干扰素-γ驱动的DAPK转录中的生理学相关性。最后，我们将研究不表达内源性DAPK1的人癌细胞中MAPK/CEBP/TRAP220相互作用的中断。这项研究将整合信号通路和转录控制机制，通过C/EBP-β控制细胞死亡反应。

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.