Transcriptional Elongation in NF-kB Mediated Inflammation

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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.