Trans-regulatory mechanisms of glucocorticoid-mediated transcriptional repression

Duke University, Durham NC

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Abstract

PROJECT ABSTRACT Glucocorticoids (GCs) are one of the most widely prescribed drugs for treating a variety of inflammatory and autoimmune diseases. Although they are potent anti-inflammatories, long-term use of GCs often results in severe metabolic side effects such as diabetes and osteoporosis. Understanding how glucocorticoids elicit transcriptional responses will help us achieve the long-term goal of developing GC therapies with reduced side effects. Treatment with GCs results in both transcriptional induction and repression. Classically, it's been proposed that the GC-inducible transcription factor, the glucocorticoid receptor (GR), represses inflammation by directly inhibiting gene transcription. Alternatively, I hypothesize that GCs primarily repress transcription by directly inducing the transcription of two transcriptional repressors: dual-specificity phosphatase 1 (DUSP1) and glucocorticoid-induced leucine zipper (GILZ). I will test this hypothesis using genomic approaches and CRISPR/Cas9-based epigenome-modifying technologies. First, I will target CRISPR/Cas9-based activators to GR binding sites that regulate expression of DUSP1 and GILZ to test the sufficiency of these binding sites for downstream gene repression. DUSP1 and GILZ have been shown to inhibit the MAPK pathway, indirectly inhibiting AP-1 transcription factor activity. I will therefore assay AP-1 genome occupancy and activity using ChIP-seq and reporter assays, respectively. I will assay genome-wide expression changes using RNA-seq. I will also test the hypothesis that loss of AP-1 binding represses GC-repressed genes. I will inhibit AP-1 DNA-binding activity using a small molecule inhibitor and assay expression changes using RNA-seq. I will assay AP-1 genome occupancy and binding-site activity using ChIP-seq and reporter assays. Second, I will test the alternative hypothesis that the GR directly represses transcription by assaying enhancer-promoter interactions before and after GC treatment using in-situ Hi-C. I will overlay this interaction data with GR and AP-1 ChIP-seq data to determine whether the GR represses transcription directly via association with enhancer-promoter interactions, or indirectly via inhibition of AP-1 bound enhancer-promoter interactions. These experiments will elucidate the extent to which DUSP1 and GILZ repress gene expression during the GC response, and will determine which transcription factors interact with the promoters of GC repressed genes. Understanding how GCs repress transcription will inform the design of more specific anti-inflammatory therapies that repress inflammatory gene expression without eliciting a metabolic response.