Gene regulatory circuitry underlying the dynamic control of glucocorticoid signaling

项目摘要/摘要 糖皮质激素(如皮质醇)是一种应激诱导和昼夜节律调节的类固醇激素，调节基因 通过与糖皮质激素受体(GR)转录因子结合从而激活其表达。 糖皮质激素反应基因调控是高度动态的，需要多层次的动态控制 对健康发育和生理至关重要的机制。糖皮质激素信号转导起着中枢作用 在调节炎症中起作用，合成的糖皮质激素被广泛用作抗炎药。 然而，它们的长期使用有许多不健康的副作用，类似于慢性 压力，这至少部分是由于暴露的长期性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学机制 基因活动仍然知之甚少。该项目将解决这一知识差距，重点是KLF9，a GR 编码普遍表达的转录因子的靶基因，在神经和神经系统中起重要作用 免疫系统，这是最近被牵连的慢性应激诱导的病理。基于我们的 初步数据和现有文献我们假设KLF9作为GR激活的前馈发挥作用 抑制物以强制GR靶基因子集的瞬时和/或昼夜节律表达，包括其自身和 GR拮抗剂FKBP5。我们将通过评估KLF9在动态变化中的作用，在斑马鱼中检验这一假说 通过比较FKBP5、KLF9和其他GR的表达动态来控制GC反应基因的表达 野生型斑马鱼幼体的靶基因和我们最近培育的KLF9缺陷系(特异性 目标1)。此外，我们将在KLF9基因座中引入表位标签序列(特定目标2)，这将 促进未来的实验，旨在通过染色质免疫沉淀和 高通量测序(CHIP-SEQ)，并与我们已经产生的菌株相结合 缺乏功能性GR，其中GR是表位标记的，全面鉴定这组基因 由GR和KLF9共同监管。因此，该项目将确定KLF9是否是基因中的关键节点 动态控制GR反应基因表达的调控网络，并为 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 KLF9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.