Activators and repressors of NFkappaB and IRF3/7 in innate immunity

摘要：NF-kB是先天免疫和适应性免疫反应的中心协调者，我们对NF-kB的激活途径了解很多，但关于NF-kB在非应激细胞中是如何被控制的，我们还需要了解很多。我们将使用插入诱变技术来鉴定NF-kB激活的新型抑制剂。我们还将与Xiaoxia Li和games Sen合作，利用他们实验室已经建立的遗传选择，发现和研究激活NF-kB， IRF7和IRF3以响应toll样受体8 （TLR8）和TLR3的途径的新型正调控因子和负调控因子。我们已经开发出一种新的病毒载体，它将新的启动子引入基因组中的许多位点。在由此产生的显性突变中，驱动下游DNA编码的蛋白质过度表达的启动子可以随意移除，证明突变是由插入引起的。我们用这种方法鉴定了赖氨酸去甲基化酶FBXL11是NF-kB的一种新的负调节因子，其表达受NF-kB调节。特异性目的1A：我们将确定FBXL11的作用机制。特异性Aim 1B：我们将研究插入启动子驱动不同负调控因子表达的其他突变体。具体目标2：我们将研究TLR8的信号通路，TLR8通常由单链RNA激活。特异性目标2A：我们将分离tlr8依赖性启动子的激活被负调节因子抑制的显性突变体。特异性目的2B：我们将分离出显性突变体，其中irf7依赖性启动子的组成激活是由阳性调节因子刺激的。具体目标2C：我们将分离隐性突变体，其中信号所需的蛋白质表达已被消除，使用基因陷阱载体结合策略，促进目标基因中剩余未突变等位基因的丢失或失活。特异性Aim 2D：我们将分离NF-kB被组成性激活的隐性突变体。具体目标2E：我们将研究我们新发现的TLR8通路的新成分或负调节因子如何在信号传导中起作用。具体目标3：我们将研究响应双链RNA （dsRNA）的信号通路，dsRNA通过TLR3激活NF-kB和IRF3，也通过涉及RIG-I和同源蛋白的内部通路。特异性目标3A：我们将鉴定作为tlr3应答启动子对dsRNA应答的显性负调节因子的细胞蛋白。特异性Aim 3B：我们将分离具有irf3响应启动子的显性突变体，以鉴定积极作用的蛋白。具体目标3C：我们将研究新发现的dsRNA信号通路的新成分或负调节因子如何在信号传导中起作用。我们强大的选择系统和强大的新遗传方法的结合，可以大大增加当前对驱动和调节先天免疫重要介质的途径的理解。不受控制的炎症是影响许多不同疾病的主要健康问题，包括癌症。我们使用新的遗传方法来研究正常的阳性和阴性控制，它们在不需要时控制炎症途径，在需要时激活它们。我们的发现有可能在几种疾病环境中导致新的治疗方法。

DESCRIPTION (provided by applicant): Activators and repressors of NFkappaB and IRF3/7 in innate immunity Abstract: Much is known about pathways that activate NF-kB, a central coordinator of innate and adaptive immune responses, but there is still much to learn about how NF-kB is kept in check in unstressed cells. We will use insertional mutagenesis to identify novel inhibitors of NF-kB activation. We will also collaborate with Xiaoxia Li and Ganes Sen to uncover and study novel positive and negative regulators of pathways that activate NF-kB, IRF7 and IRF3 in response to Toll-like receptor 8 (TLR8) and TLR3, using genetic selections that their labs have already established. We have developed a novel viral vector that introduces new promoters into many loci in the genome. In the resulting dominant mutants, the promoter, which drives the over-expression of a protein encoded by downstream DNA, can be removed at will, proving that the mutation was caused by the insertion. We have used this method to identify the lysine demethylase FBXL11 as a novel negative regulator of NF-kB whose expression is regulated by NF-kB. Specific Aim 1A: we will determine the mechanism of action of FBXL11. Specific Aim 1B: we will study additional mutants in which the expression of different negative regulators has been driven by inserted promoters. Specific Aim 2: we will study signaling pathways that respond to TLR8, which is normally activated by single-stranded RNA. Specific Aim 2A: we will isolate dominant mutants in which the activation of TLR8-dependent promoters is suppressed by negative regulators. Specific Aim 2B: we will isolate dominant mutants in which the constitutive activation of an IRF7-dependent promoter is stimulated by positive regulators. Specific Aim 2C: we will isolate recessive mutants in which the expression of a protein required for signaling has been ablated, using a gene-trap vector in combination with a strategy to promote loss or inactivation of the remaining unmutated allele of the targeted gene. Specific Aim 2D: we will isolate recessive mutants in which NF-kB has been activated constitutively. Specific Aim 2E: we will investigate how the novel components or negative regulators of the TLR8 pathway that we have newly identified function in signaling. Specific Aim 3: we will study signaling pathways that respond to double-stranded RNA (dsRNA), which activates NF-kB and IRF3 through TLR3 and also through an internal pathway involving RIG-I and homologous proteins. Specific Aim 3A: we will identify cellular proteins that function as dominant negative regulators of the response of TLR3-responsive promoters to dsRNA. Specific Aim 3B: we will isolate dominant mutants in which an IRF3-responsive promoter has been activated constitutively, to identify positively acting proteins. Specific Aim 3C: we will investigate how newly identified novel components or negative regulators of pathways that respond to dsRNA function in signaling. Our combination of robust selective systems and powerful new genetic methods can add much to the current understanding of pathways that drive and regulate important mediators of innate immunity. Uncontrolled inflammation is a major health problem that impacts many different diseases, including cancer. We use novel genetic methods to study the normal positive and negative controls that keep inflammatory pathways in check when they are not needed and activate them when they are. Our findings have the potential to lead to new therapeutic approaches in several disease settings.