The Hypoxia Inducible Factor (HIF) is a master regulator of the cellular response to hypoxia. Its levels and activity are controlled by dioxygenases called Prolyl-Hydroxylases and Factor Inhibiting HIF (FIH). In order to activate genes, HIF has to access sequences in DNA that are integrated in chromatin. It is known that the chromatin remodelling complex SWI/SNF (Switch/Sucrose non-fermenting) is essential for HIF activity. However, no additional information exists about the role of other chromatin remodelling enzymes. This thesis describes the role of ISWI (Imitation Switch) in the cellular response to hypoxia. It was found that unlike SWI/SNF, ISWI depletion enhances HIF activity without altering its levels. Further analysis revealed that ISWI acts in a specific manner on certain HIF target genes, increasing expression of CA9 and BNIP3, whilst reducing expression of Glut3, and leaving many others unaltered. Loss of ISWI also causes impairment in the transcription of FIH, a crucial regulator of the hypoxia response, and Rb a protein involved in cell cycle regulation. Mechanistically, the de-repression of CA9 is dependent on the reduction of FIH and is rescued by FIH overexpression. This is in contrast to the de-repression of BNIP3 which is dependent on the Rb/E2F1 axis and is rescued by E2F1 knockdown. Importantly, loss of ISWI by siRNA knockdown alters the cellular response to hypoxia by preventing autophagy and inducing apoptosis. These results demonstrate a novel role for ISWI as a survival factor during the cellular response to hypoxia. The JmjC family of enzymes are capable of tri-methyl histone demethylation and require molecular oxygen to function making them putative oxygen sensors. Moreover, microarray array data within this thesis confirms that 10 of the JmjC demethylases are up-regulated in U2OS cells when exposed to hypoxia. To further investigate chromatin structure in hypoxia a live cell microscopy imaging chamber was designed and tested. Using this chamber, and applying a novel technique to measure chromatin compaction in vivo, it is shown, for the first time, that hypoxia induces compaction in chromatin. This compaction is independent of HIF-1a and occurs rapidly within 30 minutes of hypoxia exposure.
|Date of Award
|Sonia Rocha (Supervisor)