Transcriptional regulation by glucocorticoids: a comparative transcriptome analysis preceding mechanistic and functional assessments of KLF9 induction

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dc.contributor.advisorNewton, Robert
dc.contributor.authorMostafa, Mahmoud
dc.contributor.committeememberGiembycz, Mark A.
dc.contributor.committeememberCobb, Jennifer A.
dc.dateFall Convocation
dc.date.accessioned2022-11-15T17:42:58Z
dc.date.embargolift2022-09-15
dc.date.issued2020-09-15
dc.description.abstractRegulation 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.
dc.identifier.citationMostafa, M. (2020). Transcriptional regulation by glucocorticoids: a comparative transcriptome analysis preceding mechanistic and functional assessments of KLF9 induction (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.
dc.identifier.urihttp://hdl.handle.net/1880/115469
dc.identifier.urihttps://dx.doi.org/10.11575/PRISM/40436
dc.language.isoenen
dc.language.isoEnglish
dc.publisher.facultyGraduate Studiesen
dc.publisher.facultyCumming School of Medicine
dc.publisher.institutionUniversity of Calgaryen
dc.rightsUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.en
dc.subjectGlucocorticoid
dc.subjectinhaled corticosteroid (ICS)
dc.subjectasthma
dc.subjectairway epithelial cells
dc.subjectair-liquid interface (ALI)
dc.subjectgene regulation
dc.subjectchromatin immunoprecipitation (ChIP)
dc.subjectenhancer RNA (eRNA)
dc.subjectKrüppel-like factor 9 (KLF9)
dc.subjectglucocorticoid receptor (GR
dc.subjectNR3C1)
dc.subject.classificationBiology--Molecular
dc.subject.classificationBiology--Cell
dc.subject.classificationBiology--Bioinformatics
dc.titleTranscriptional regulation by glucocorticoids: a comparative transcriptome analysis preceding mechanistic and functional assessments of KLF9 induction
dc.typedoctoral thesis
thesis.degree.disciplineMedicine – Cardiovascular/Respiratory Science
thesis.degree.grantorUniversity of Calgaryen
thesis.degree.grantorUniversity of Calgary
thesis.degree.nameDoctor of Philosophy (PhD)
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