Browsing by Author "Mostafa, Mahmoud"

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    Transcriptional regulation by glucocorticoids: a comparative transcriptome analysis preceding mechanistic and functional assessments of KLF9 induction
    (2020-09-15) Mostafa, Mahmoud; Newton, Robert; Giembycz, Mark A.; Cobb, Jennifer A.
    Regulation of gene expression by glucocorticoid receptor (GR) is not only central to numerous endocrine processes, but also critical for the control of inflammation by glucocorticoids administered for the treatment of inflammatory diseases, including asthma. Advancing our knowledge of glucocorticoid biology as well as developing GR ligands with improved therapeutic profile are hindered by an inadequate understanding of the mechanisms and impacts of gene regulation by glucocorticoids. Furthermore, owing to their pleiotropic effects on many different target cells, transcriptomic responses to glucocorticoids vary widely between cell types. This confounds the identification of key glucocorticoid-regulated genes. In this thesis, transcriptomic responses to glucocorticoid were compared between A549, BEAS-2B, and primary HBE cells. These are models for airway epithelial cells, which represent a key player in asthma pathogenesis and response to inhaled glucocorticoid therapies. While the variability in glucocorticoid-modulated gene expression between epithelial cell variants was surprisingly high, the genes regulated in common may represent key players in eliciting glucocorticoid effects. Among the genes that were induced in common, transcriptional regulators, such as KLF9, were highly enriched and hypothesized to contribute to later gene expression changes, including repression of inflammatory genes. Mechanistically, ligand-activated GR binds and activates multiple conserved enhancer regions upstream of the KLF9 gene. These include a promoter proximal region that was appeared to be essential for the constitutive expression and glucocorticoid-mediated induction of KLF9. In fact, GR recruitment and transcriptional activation at KLF9 upstream enhancers were highly conserved among cell lines and primary cells, demonstrating reliability of cell lines in mechanistic interrogations of conserved genes. Assessment of KLF9 functions was attempted via transcriptome analysis of CRISPR-edited KLF9 KO lines. While procedural artifacts hindered the identification of transcriptomic impacts of KLF9 induction, candidate gene investigations following overexpression or knockdown of KLF9 suggested a role for the constitutively expressed KLF9 in limiting the expression of AKAP12 and RGS2. Yet, the functional impacts of glucocorticoid-induced KLF9 in pulmonary epithelial cells remain undetermined. Collectively, these results establish a platform for identifying key glucocorticoid-regulated genes in the airways, and guiding the selection of cell line models for in vitro investigations of such genes.