Transcriptional programmes downstream of the activated glucocorticoid receptor

背景技术背景：本研究旨在通过培养高素质人才（HQP）的方式，探索和促进对糖皮质激素受体（GR/NR 3C 1）诱导的基因表达网络的理解。在压力下，皮质醇（一种糖皮质激素）由肾上腺皮质产生，并通过血液运送到目标组织。在那里，它作用于GR，一种促进大分子分解的转录因子，以帮助在禁食和饥饿期间维持血糖。在感染和损伤中，糖皮质激素通过减少介质的表达来抑制炎症和促进愈合，从而发挥关键的生存作用。在我的实验室里，研究的重点是糖皮质激素如何通过GR来诱导负责抑制炎症的基因的表达。由于肺上皮细胞是环境损伤和感染的重要部位，因此它们被用于模拟糖皮质激素调节的基因表达。 假设：GR依赖性基因网络涉及快速诱导的转录因子，其引起下游基因表达的不同模式并负责生理反应。 目标：先前对人肺和原代气道上皮细胞中基因表达的总体变化的表征提供了糖皮质激素最上调的转录因子的列表。四种主要高度诱导的转录因子中的三种：1）KLF 9，2）PER 1和3）ZBTB 16被选择用于独立的项目，每个项目的目的是研究GR诱导这些因子的机制，并确定它们对下游基因表达的影响。 实验方法：在每个项目的第一部分，GR拮抗剂和基因沉默将测试肺上皮细胞中每个诱导因子的GR依赖性。将通过包括基因过表达和沉默的分子方法测试每个因子（KLF 9、PER 1、ZBTB 15）对下游基因表达的影响，随后通过基因表达的微阵列分析来鉴定靶基因。将评估GR结合KLF 9、PER 1和ZBTB 16基因中控制区的能力（通过染色质免疫沉淀（ChIP））。将评估鉴定的DNA结合位点驱动转录的功能能力。同样，将评价GR和KLF 9、PER 1和ZBTB 16中的每一个结合下游靶基因中的调节位点的能力。 结果：在这个研究计划中，每个独立的项目都为HQP的逐步发展提供了理想的机会。通过描述每个转录调节因子的作用和下游基因，我们绘制了一个新的糖皮质激素在气道中的转录调节框架。这推进了对基本人类过程的核心理解，特别是糖皮质激素抗炎和其他生理作用的机制。

BACKGROUND: This mechanism-based study is designed, by way of training high quality personnel (HQP), to characterise and promote understanding of the gene expression networks induced by the glucocorticoid receptor (GR/NR3C1). During stress, cortisol (a glucocorticoid) is produced by the adrenal cortex and is carried in the blood to target tissues. There, it acts on GR, a transcription factor that promotes breakdown of macromolecules to help maintain blood glucose during fasting and starvation. In infection and injury, glucocorticoids play a key survival role by reducing expression of mediators to dampen inflammation and promoting healing. Research, in my laboratory, focuses on how glucocorticoids act via GR to induce the expression of genes that are responsible for repressing inflammation. As lung epithelial cells are an important site of environmental insult and infection, they are used to model glucocorticoid-modulated gene expression. HYPOTHESIS: GR-dependent gene networks involve rapidly induced transcription factors that elicit distinct patterns of downstream gene expression and are responsible for physiological responses. AIMS: Prior characterisation of the global changes in gene expression in the human lung and in primary airway epithelial cells provides a list of the transcription factors most up-regulated by glucocorticoids. Three of the four mostly highly induced transcription factors; 1) KLF9, 2) PER1, and 3) ZBTB16 are selected for independent projects, each aiming to characterise mechanisms by which GR induces these factors and to establish their effect on downstream gene expression. EXPERIMENTAL APPROACHES: In the first part of each project, GR antagonists and gene silencing will test the GR-dependence of each induced factor in lung epithelial cells. The effects of each factor (KLF9, PER1, ZBTB15) on downstream gene expression will be tested by molecular approaches including gene over-expression and silencing followed by microarray analysis of gene expression to identify target genes. The ability of GR to bind control regions in the KLF9, PER1 and ZBTB16 genes will be assessed (by chromatin immunoprecipitation (ChIP)). Identified DNA binding sites will be assessed for functional ability to drive transcription. Likewise, the ability of GR and each of KLF9, PER1 and ZBTB16 to bind regulatory sites in downstream target genes will be evaluated. OUTCOMES: Within this programme of research, each independent project provides an ideal opportunity for the stepwise development of HQP. By characterising, the roles and downstream genes regulated by each transcriptional regulator, we map out a novel framework of transcriptional regulation by glucocorticoids in the airways. This advances core understanding of a fundamental human process, in particular the mechanisms underlying anti-inflammatory and other physiological effects of glucocorticoids.