Browsing by Subject "Glucocorticoid"

Quantifying the function of mammalian enhancers at the genome or population scale has been longstanding challenge in the field of gene regulation. Studies of individual enhancers have provided anecdotal evidence on which many foundational assumptions in the field are based. Genome-scale studies have revealed that the number of sites bound by a given transcription factor far outnumber the genes that the factor regulates. In this dissertation we describe a new method, chromatin immune-enriched reporter assays (ChIP-reporters), and use that approach to comprehensively test the enhancer activity of genomic loci bound by the glucocorticoid receptor (GR). Integrative genomics analyses of our ChIP-reporter data revealed an unexpected mechanism of glucocorticoid (GC)-induced gene regulation. In that mechanism, only the minority of GR bound sites acts as GC-inducible enhancers. Many non-GC-inducible GR binding sites interact with GC-induced sites via chromatin looping. These interactions can increase the activity of GC-induced enhancers. Finally, we describe a method that enables the detection and characterization of the functional effects of non-coding genetic variation on enhancer activity at the population scale. Taken together, these studies yield both mechanistic and genetic evidence that provides context that informs the understanding of the effects of multiple enhancer variants on gene expression.

Human cells are perpetually receiving and responding to a variety of intrinsic and extrinsic signals. A primary mechanism by which cells carry out these responses is via changes in the regulation of gene expression. Many studies have examined gene regulation in steady state systems, but few have investigated the genomic response to stimuli. Therefore, it is less well understood how cellular stimuli elicit dynamic gene expression responses. Here, we investigate how extracellular stimuli mediate gene expression responses via: 1) Changes in transcription factor configurations at enhancer elements; and 2) Changes in chromatin looping between putative enhancers and their target gene promoters. To study these phenomena, we used glucocorticoid (GC) treatment as a model transcriptional stimulus. This hormone steroid is known to bind to and activate the GC receptor (GR), a ligand-induced transcription factor (TF), and is therefore a highly tractable system for studying stimulus responsive gene regulation. Using this model system, we first used high-resolution TF-binding site mapping approaches to elucidate the genomic binding locations of GR and its associated cofactors. Using these approaches, we found evidence that: 1) The GR binds to the genome as both a monomer and dimer; and 2) The GR binds to the genome with AP-1 in a more relaxed configuration, while it binds FOXA1 in a more constrained configuration. We next interrogated the role of chromatin looping in mediating dynamic transcriptional responses. For this work we used high-throughput genomics methods to assay chromatin conformation across a time course of GC treatment. These studies resulted in several main findings: 1) Chromatin loops do not form in response to stimulus, but are instead pre-formed before GC treatment; 2) Chromatin looping interactions increase between distal GR binding sites and GC-responsive genes; 3) The insulator protein CTCF is depleted at stimulus responsive looping interactions; and 4) GC treatment mediates changes in higher-order chromosome compartmentalization that are concordant with gene expression responses. Together these results provide evidence for a genome topology that is pre-wired to respond to stimulus, and that subsequent transcriptional responses are mediated through GR binding to putative enhancer elements with other TFs, in a variety of genomic binding configurations.