Glucocorticoids (GCs, generically known as “steroids”) are exceptionally effective drugs in the control of a wide variety of immune-mediated diseases, including asthma. They are also used to treat viral-induced inflammation in the context of asthma exacerbations. Through reducing inflammation in tissues such as airway smooth muscle (ASM), inhaled GCs are very effective in the majority of asthma patients, whereas systemic therapy, which causes side effects, is used to treat asthma exacerbations. Despite using best therapies, over one million Americans continue to be afflicted with difficult-to-control asthma and frequent exacerbations. Thus, new pharmacologic approaches aimed at steroid signaling mechanisms in the lung, including ASM, are urgently needed to improve treatment for asthma. GCs primary biochemical function is to bind to the GC receptor (GR), leading to regulation of transcription directly by GR, which is a transcription factor. ASM is a key target tissue of GCs in asthma, and ASM exhibits hyper-contractility, remodeling and exuberant cytokine expression in asthma. Previous efforts to exploit steroid- controlled pathways as a method to improve asthma therapies directed at ASM have been predicated on the so called “transrepression” model of GR activity, in which GR is thought to reduce inflammation by antagonistic tethering to pro-inflammatory transcription factors such as NFkB. Entirely contrary to dogma, this grant application is focused instead on transcriptional induction by GR and cooperation between GR and NFkB. Our preliminary data implicate GR-NFkB cooperation as central to controlling inflammation in ASM, thus likely contributing significantly to efficacy of steroids. Mechanistically, our data suggest this pathway involves transcription of enhancer RNAs and formation of chromatin loops, and our data also suggest this mechanism is druggable. Based on our promising findings on GR-induced pathways, we have also created a novel method to interrogate GR-induced enhancer RNA signatures for genetic links to asthma. Our overarching hypothesis is that GR-mediated transcriptional induction and GR-NFkB cooperation in ASM is crucial for steroid efficacy in asthma and these pathways are linked to risk of developing asthma. We will test this hypothesis with three specific aims, which will also address crucial molecular knowledge gaps. In Aim 1, we will determine the comprehensive set of enhancer RNAs that are induced cooperatively by GR/NFkB and determine if these enhancer RNAs loop to the promoters of anti-inflammatory genes. In Aim 2, we will determine the DNA sequence requirements and additional factors involved in cooperative regulation by GR and NFkB, and we will test the therapeutic relevance of this pathway in translational models. In Aim 3, we will identify novel genetic associations between SNPs, GR-induced enhancers and asthma, and we will determine the relevance of these SNPs to GR signaling and ASM function.