Transcriptional programmes downstream of the activated glucocorticoid receptor

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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.*********