Gene regulatory circuitry underlying the dynamic control of glucocorticoid signaling

项目总结/摘要 糖皮质激素（如皮质醇）是应激诱导和昼夜节律调节的类固醇激素，调节基因表达。 通过结合并因此激活糖皮质激素受体（GR）转录因子来表达。 糖皮质激素反应性基因调控是高度动态的，需要多层次的动态调控 对健康发育和生理至关重要的机制。糖皮质激素信号在 糖皮质激素在调节炎症中的作用，并且合成糖皮质激素被广泛用作抗炎药物。 然而，它们的长期使用有许多不健康的副作用，类似于慢性的不良健康影响。 压力，这至少部分是由于长期暴露造成的动态控制的丧失。 控制糖皮质激素反应动力学的细胞内和分子遗传机制 基因活性仍然知之甚少。这个项目将解决知识差距，重点是klf 9，一个GR 一种靶基因，编码一种普遍表达的转录因子，在神经和 免疫系统，最近被牵连在慢性应激诱导的病理学。基于我们 根据初步数据和现有文献，我们假设Klf 9的功能是GR激活的前馈， 阻遏物，以执行GR靶基因的子集的瞬时和/或昼夜节律表达，包括其自身和 GR拮抗剂fkbp 5。我们将通过评估Klf 9在斑马鱼中的动态作用来验证这一假设。 通过比较fkbp 5、klf 9和其他GR的表达动力学来控制GC反应性基因表达 野生型斑马鱼和我们最近生产的Klf 9缺陷系的幼虫中的靶基因（特异性 目标1）。此外，我们将在klf 9基因座中引入表位标签序列（特异性目标2），其将 促进未来旨在通过染色质免疫沉淀鉴定直接Klf 9靶基因的实验， 高通量测序（ChIP-seq），并与我们已经产生的菌株组合， 缺乏功能性GR并且其中GR是表位标记的，全面鉴定 由GR和Klf 9共同监管。因此，该项目将确定Klf 9是否是基因中的关键节点。 GR反应基因表达的动态调控网络，并提供基础， 未来的研究将系统地阐明更广泛的发育和神经内分泌功能 klf 9在糖皮质激素反应基因调控方面的作用。

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.