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（IFN）基因调控对病毒感染的反应的分子机制。人和小鼠IIFN型基因在感染前被抑制（诱导前抑制），被诱导，并在诱导后关闭（诱导后抑制）。拟议的补助金旨在阐明IFN转录控制的特定生化机制和更广泛的协调IFN信号事件的水平。这将使用各种分子遗传学和生物化学方法来实现，包括对小鼠的研究，其中阻遏物基因已通过同源重组失活，以及对培养细胞的研究，其中基因将被siRNA敲低。人和小鼠I型IFN基因以携带单个IFN-β和多个IFN-α基因的大簇存在。实验提出，以确定基因间的调控序列和集群范围内的转录因子结合，染色质结构和组蛋白修饰病毒感染过程中的变化的特点。将使用染色质沉淀（ChIP）和DNA微阵列方法（芯片）检测染色质结构的全细胞变化。调控序列将通过染色质修饰的模式、使用核酸酶敏感性测定以及通过研究推定的调控序列和启动子之间的簇内相互作用来鉴定。将使用分子遗传学方法确定推定的调控序列的功能作用，所述分子遗传学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小鼠胚胎干细胞。由IFN的转录控制引起的抗病毒信号传导事件也由不同的信号转导途径介导。非典型的I？B激酶TBK 1和IKK？在调解这种反应中发挥关键作用。我们建议通过对缺乏其中一种或两种激酶的小鼠的研究来确定这些激酶的不同功能。拟议的研究应提供显着的进步，我们的理解的机制，参与协调和顺序激活的I型IFN基因簇和安装抗病毒反应所需的信号通路。这些结果可能会导致感染性，炎症和自身免疫性疾病的治疗方法的发展，同时还确定了抗病毒剂的治疗靶点。

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.