Innate Immunity: Signal Transduction and Interferon Gene Regulation

描述(由申请人提供)：拟议研究的总体目标是了解I型干扰素(干扰素)基因调控对病毒感染的反应的分子机制。人和鼠型干扰素基因在感染前被抑制(诱导前抑制)，诱导后被诱导，并在诱导后被关闭(诱导后抑制)。建议的拨款旨在阐明干扰素转录控制的特定生化机制和更广泛的协调干扰素信号事件的水平。这将使用各种分子遗传和生化方法来实现，包括对小鼠的研究，在这些研究中，阻遏基因通过同源重组而失活，以及在培养细胞的研究中，这些基因将被siRNA击倒。人类和小鼠的I型干扰素基因以大簇的形式存在，携带单个干扰素-β基因和多个干扰素-α基因。建议进行实验，以确定基因间调控序列，并表征病毒感染期间转录因子结合、染色质结构和组蛋白修饰的簇范围变化。将使用染色质沉淀(CHIP)和DNA微阵列方法(CHIP)检测整个集群范围内染色质结构的变化。调控序列将通过染色质修饰的模式、使用核酸酶敏感性分析以及通过研究假定的调控序列与启动子之间的簇内相互作用来鉴定。假定的调控序列的功能作用将使用分子遗传学方法确定，该方法涉及细菌人工染色体的“重组工程”和通过同源重组插入到小鼠胚胎干细胞中。由干扰素转录调控产生的抗病毒信号事件也由不同的信号转导途径介导。非正则I？B激酶TBK1和IKK？在调解这一反应方面发挥关键作用。我们建议通过对缺乏其中一种或两种蛋白的小鼠的研究来确定这些蛋白激酶的不同功能。拟议的研究将为我们理解I型干扰素基因簇的协调和顺序激活机制以及启动抗病毒反应所需的信号通路提供重要进展。这一结果可能会导致传染病、炎症性疾病和自身免疫性疾病的治疗方法的开发，同时还可以确定抗病毒药物的治疗靶点。

DESCRIPTION (provided by applicant): The overall objective of the proposed research is to understand the molecular mechanisms of type I Interferon (IFN) gene regulation in response to virus infection. Human and mouse type IIFN genes are repressed prior to infection (pre-induction repression), induced, and switched off after induction (post- induction repression). The proposed grant aims at elucidating both specific biochemical mechanisms of IFN transcriptional control and a broader level of coordinating IFN signaling events. This will be accomplished using a variety of molecular genetic and biochemical approaches, including studies of mice in which the represser genes have been inactivated by homologous recombination and studies of cultured cells where the genes will be knocked down by siRNA. Human and mouse type I IFN genes are present in large clusters bearing a single IFN-beta and multiple IFN-alpha genes. Experiments are proposed to identify intergenic regulatory sequences and characterize cluster-wide changes in transcription factor binding, chromatin structure, and histone modification during virus infection. Cluster-wide changes in chromatin structure will be detected using chromatin precipitation (ChIP) and DNA microarray methods (chip). Regulatory sequences will be identified by patterns of chromatin modification, the use of nuclease sensitivity assays, and by studying intra-cluster interactions between putative regulatory sequences and promoters. The functional role of putative regulatory sequences will be determined using a molecular genetic approach involving "recombineering" of bacterial artificial chromosomes and insertion into mouse embryonic stem cells by homologous recombination. The antiviral signaling events that result from the transcriptional control of IFN are also mediated by distinct signal transduction pathways. The non-canonical I?B kinases TBK1 and IKK? play key roles in mediating this response. We propose to identify the distinct functions of these kinases through studies of mice lacking either or both of them. The proposed studies should provide significant advances in our understanding of the mechanisms involved in the coordinate and sequential activation of the type I IFN gene cluster and in the signaling pathways required to mount an anti-viral response. The results could lead to the development of treatments for infectious, inflammatory, and autoimmune diseases, while additionally identifying therapeutic targets for antiviral agents.