Gene regulatory circuitry underlying the dynamic control of glucocorticoid signaling

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PROJECT SUMMARY / ABSTRACT Glucocorticoids (e.g. cortisol) are stress-induced and circadian-regulated steroid hormones that regulate gene expression by binding to and thus activating the Glucocorticoid Receptor (GR) transcription factor. Glucocorticoid-responsive gene regulation is highly dynamic, requiring multiple levels of dynamic control mechanisms that are critical for healthy development and physiology. Glucocorticoid signaling plays a central role in regulating inflammation, and synthetic glucocorticoids are widely prescribed as anti-inflammatory drugs. However, their extended use has many unhealthy side effects, similar to the adverse health effects of chronic stress, which is due at least in part to the loss of dynamic control resulting from the chronicity of the exposure. The intracellular and molecular-genetic mechanisms that control the dynamics of glucocorticoid responsive gene activity remain poorly understood. This project will address that knowledge gap, focusing on klf9, a GR target gene that encodes a ubiquitously-expressed transcription factor with important roles in the nervous and immune systems, which has recently been implicated in chronic stress-induced pathology. Based on our preliminary data and the available literature we hypothesize that Klf9 functions as a GR-activated feedforward repressor to enforce transient and/or circadian expression of a subset of GR-target genes, including itself and the GR antagonist fkbp5. We will test that hypothesis in zebrafish by assessing the role of Klf9 in the dynamic control of GC-responsive gene expression, by comparing expression dynamics of fkbp5, klf9, and other GR target genes in larvae of wild-type zebrafish and a Klf9-deficient line that we have recently produced (Specific Aim 1). In addition, we will introduce an epitope tag sequence into the klf9 locus (Specific Aim 2), which will facilitate future experiments aimed at identifying direct Klf9-target genes by chromatin immunoprecipitation and high throughput sequencing (ChIP-seq), and in combination with strains that we have already generated that lack a functional GR and in which the GR is epitope tagged, comprehensively identify the set of genes that is jointly regulated by the GR and Klf9. The project will thus determine whether Klf9 is a critical node in the gene regulatory network underlying dynamic control of GR-responsive gene expression, and provide a foundation for future research that will systematically elucidate the broader developmental and neuroendocrine functionality of klf9 with respect to glucocorticoid-responsive gene regulation.