Transcriptional Elongation in NF-kB Mediated Inflammation

可诱导的活性氧(ROS)在先天免疫反应(MR)中起着重要的调节作用。 通过控制1RF3和NF-KB/ROIA的磷酸化。它需要RERA的磷酸化。 可诱导Lys上的乙酰化，并与阳性延伸因子-b(PTEF-b)复合体结合。PTEF-b 细胞周期蛋白依赖性激酶(CDK)-9和溴域-4(Brd4)蛋白复合体诱导RNAPol11Ser2 磷酸化以诱导下游基因的转录延长。P1将检验这一假设 ROS通过调节NFKappaB/活化状态调节先天炎症和抗病毒活性 RelA和IRF3在呼吸道上皮细胞中有三个特定的作用目的。目标1将研究ROS如何修改 核因子-kappaB/relA诱导Brd4-CDK9复合体募集介导炎性细胞因子 制作。RELA Lys310对ROS诱导的PTEF-b招募的需求将通过以下测试 染色质免疫沉淀(CHIP)法检测WT重组后的小鼠胚胎成纤维细胞 RelA及其非乙酰化Lys310 Arg突变体对TNFa、RSV和豚草花粉颗粒NADPH的响应 氧化物酶。ROS在诱导启动子相关的失活CDK9清除和替换中的作用 激活的CDK9将在小气道上皮细胞(SAE)中被检测。Brd4作为适配器的作用 蛋白质将使用siRNA敲除或体内竞争使用赖氨酸乙酰化组蛋白H4进行检测 多肽。目标2将研究ROS如何介导IRF3通路信号。我们将调整可感应性 ROS在SAE中检测1RF3转位的动力学，使用我们新的选择性反应监测 (SRM)测定。ROS对1RF3增强体形成的要求将通过芯片检测来测试 IFNp和干扰素刺激基因(ISG)促进抗氧化调节。我们将确定是否 IRF3通过耗尽CDK-7和CDK-9的siRNA诱导抗病毒转录起始或延长。目标3将 研究转录延长如何在RSV体内感染中介导ILR的作用。多么 抑制NF-KB-PTEF-b募集和CDK9影响细胞炎症、抗病毒免疫、呼吸道 病理和呼吸道高反应性(AHR)将在BALB/c小鼠模型中使用核因子-KB进行测试 抑制性Ikky/Nemo结合结构域(NBD)和小分子CDK9抑制剂。这些研究将 建立ROS启动的转录延伸通路在MR中的作用，从而奠定基础 用于选择性调节呼吸道粘膜炎症和呼吸道合胞病毒毛细支气管炎的新方法。

Inducible reactive oxygen species (ROS) play a critical regulatory role in the innate immune response (MR) by controlling the phosphorylation of both 1RF3 and NF-KB/ROIA. RelA phosphorylation is required for its inducible acetylation at Lys and binding to the positive elongation factor-b (PTEF-b) complex. The PTEF-b complex of cyclin-dependent kinase (CDK)-9 and bromodomain-4 (Brd4) proteins induces RNA Pol 11 Ser2 phosphorylation to induce transcriptional elongation in downstream genes. P1 will test the hypothesis that ROS modulate innate inflammatory and antiviral activities by modulating the activation states of NFKappaB/ RelA and iRF3 in the airway epithelium in three specific aims. Aim 1 will examine how ROS modify NF-KappaB/RelA to induce recruitment of the Brd4-CDK9 complex mediating inflammatory cytokine production. The requirement of RelA Lys310 for ROS-induced PTEF-b recruitment will be tested by chromatin immunoprecipitation (ChIP) assays in RelA''' mouse embryonic fibroblasts reconstituted with WT RelA or its non-acetylated Lys310 Arg mutant in response to TNFa, RSV and ragweed pollen particle NADPH oxidase. The role of ROS in inducing promoter-associated inactive CDK9 clearance and replacement with activated CDK9 will be determined in small airway epithelial cells (SAEs). The role of Brd4 as an adapter protein will be examined using siRNA knockdown or in vivo competition using Lys-acetylated histone H4 peptides. Aim 2 will examine how ROS mediate IRF3 pathway signaling. We will modulate inducible ROS to examine the kinetics of 1RF3 translocation in SAEs, using our novel selected reaction monitoring (SRM) assay. The requirement of ROS for 1RF3 enhanceosome formation will be tested by ChIP assay on IFNp and interferon-stimulated gene (ISG) promoters ¿ antioxidant modulation. We will determine whether IRF3 induces antiviral transcriptional initiation or elongation by siRNA depletion of CDK-7 and -9. Aim 3 will examine how transcriptional elongation mediates the effect of the ilR in RSV infection in vivo. How inhibiting NF-KB-PTEF-b recruitment and CDK9 affects cellular inflammation, anti-viral immunity, airway pathology and airway hyper-reactivity (AHR) will be tested in a BALB/c mouse model using an NF-KB inhibitory IKKy/NEMO binding domain (NBD) peptide and small molecule CDK9 inhibitor. These studies will establish the role of the ROS-initiated transcriptional elongation pathway in the MR and so lay the foundation for novel approaches to selectively modulate inflammation in airway mucosa and RSV bronchiolitis.