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

描述（由申请人提供）：激活剂和抑制剂的NF-κ B和IRF 3/7在先天免疫摘要：很多是已知的途径，激活NF-κ B，先天性和适应性免疫反应的中央协调员，但仍然有很多要了解的NF-κ B是如何保持在检查无应激细胞。我们将使用插入突变来鉴定NF-kB激活的新型抑制剂。我们还将与Xiaoxia Li和加内斯森合作，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，IRF 7和IRF 3响应Toll样受体8（TLR 8）和TLR 3的途径的新的正和负调节因子。我们已经开发了一种新的病毒载体，将新的启动子引入基因组中的许多位点。在产生的显性突变体中，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。我们已经使用这种方法来确定赖氨酸脱甲基酶FBXL 11作为一种新的NF-κ B的负调节因子，其表达受NF-κ B的调节。具体目标1A：我们将确定FBXL 11的作用机制。具体目标1B：我们将研究额外的突变体，其中不同的负调控因子的表达已被插入的启动子驱动。具体目标2：我们将研究响应TLR 8的信号通路，TLR 8通常由单链RNA激活。具体目标2A：我们将分离显性突变体，其中TLR 8依赖性启动子的激活被负调控因子抑制。具体目标2B：我们将分离显性突变体，其中IRF 7依赖性启动子的组成型激活被正调节子刺激。具体目标2C：我们将分离隐性突变体，其中信号传导所需的蛋白质的表达已被消除，使用基因捕获载体结合促进靶基因的剩余未突变等位基因的丢失或失活的策略。具体目标2D：我们将分离NF-kB已被组成性激活的隐性突变体。特定目标2 E：我们将研究我们新发现的TLR 8通路的新成分或负调节因子如何在信号传导中发挥作用。具体目标3：我们将研究响应于双链RNA（dsRNA）的信号通路，双链RNA通过TLR 3以及涉及RIG-I和同源蛋白的内部通路激活NF-κ B和IRF 3。具体目标3A：我们将鉴定作为TLR 3应答启动子对dsRNA的应答的显性负调节物的细胞蛋白质。具体目标3B：我们将分离显性突变体，其中IRF 3应答启动子已被组成型激活，以鉴定正作用蛋白。具体目标3C：我们将研究如何新发现的新的组件或负调节器的途径，响应dsRNA功能的信号。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目前对驱动和调节先天免疫重要介质的途径的理解。不受控制的炎症是一个主要的健康问题，影响许多不同的疾病，包括癌症。我们使用新的遗传学方法来研究正常的阳性和阴性对照，这些对照在不需要时保持炎症通路的控制，并在需要时激活它们。我们的研究结果有可能导致新的治疗方法在几种疾病的设置。

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.