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Doctoral defence of Nicholas Downes, BSc, 29 Oct 2021: Hypoxia widely alters gene expression in endothelial cells

The doctoral dissertation in the field of Molecular Medicine and Cardiovascular Genomics will be examined at the Faculty of Health Sciences at the Kuopio campus.

What is the topic of your doctoral research?  Why is it important to study the topic?

Gene regulation allows cells to dynamically adapt and respond to a wide variety of stimuli and environmental cues. Whilst these complex gene regulatory programs are typically robust, coordinated and interconnected, perturbations and insufficiencies in their capacity can inextricably contribute to the development of various complex diseases. Hypoxia, the state of insufficient oxygen supply, appears as a pathophysiological factor in a number of these complex diseases, such as cardiovascular disease. When cells are exposed to hypoxic conditions, they implement a variety of adaptive measures to not only help them survive, but also resolve the cause of the hypoxia. However, in patients with chronic vascular disease, these responses are often impaired due to the presence of multiple complicating comorbidities, such as aging and diabetes mellitus.

The primary aim of this thesis was to elucidate the key drivers of the cellular adaptation to hypoxia, but also identify novel regulators and reveal how they integrate to redirect the transcriptome. Given the significance that hypoxia plays in disease pathogenesis and perpetuation, further understanding of the regulatory landscape that shapes the cell response to hypoxia will provide better opportunities to identify suitable, efficacious targets for next generation therapeutic approaches.

What were the key findings or observations of your doctoral research?  

Through the combined use of nascent and steady-state transcriptomic approaches, not only could we identify the changes in the hypoxic transcriptome, but also establish at which regulatory level these changes were mediated. Hypoxia was seen to alter the expression of approximately 300 genes in endothelial cells, where they affected processes such as metabolism, growth factor secretion, migration, and proliferation. Modelling of the transcriptomic dynamics in hypoxia revealed that these changes occurred as a result of increased rates of transcription, rather than through changes in rates of RNA processing or decay. Enhanced transcriptional output occurred as a result of combined increased de novo RNA polymerase II initiation events and induced promoter-proximal RNA polymerase II pause release. The majority of the differentially expressed genes in hypoxia were regulated through the canonical hypoxia inducible factor (HIF) family of transcription factors. However, despite the functional and structural similarities between the major paralogues, HIF1a and HIF2a, less than 20% of their target genes were co-regulated, with each paralogue regulating distinct biological processes in a non-compensatory manner.

Using unbiased RNA transcript identification approaches, we were also able to show a large proportion of the transcriptome that was altered under hypoxic conditions were of non-coding origin. Interestingly, a significant subset of these non-coding RNAs originated from genomic regulatory regions residing close to protein coding genes, indicative of possible regulatory roles. However, correlation analyses did not reveal a global role for these non-coding genes in hypoxia. Notably however, a novel non-coding RNA overlapping the HIF1A locus appeared to be highly induced in hypoxia. Mechanistic studies revealed that hypoxia induced the transcription of this non-coding RNA, whereby it reduced the expression of HIF1A in cis. As such, we identified a transcriptional regulatory circuit that temporally controls the expression of HIF1A in hypoxic conditions, and therefore contributes to modulating the hypoxia cellular response.

The doctoral dissertation of Nicholas Downes, BSc, entitled Transcriptional gene regulation in endothelial cells in response to hypoxia will be examined at the Faculty of Health Sciences. The Opponent in the public examination will be Assistant Professor Sven Heinz of the University of California, San Diego, and the Custos will be Professor Seppo Ylä-Herttuala of the University of Eastern Finland.

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Dissertation online

Public examination