Reprogramming the cellular response to hypoxia

Abstract

HIF-1 is a heterodimeric transcription factor comprising HIF-1[alpha] and HIF-1[Beta] subunits. Cellular response to hypoxia relies on the dimerisation of HIF-1? and HIF-1? in the nucleus, forming HIF-1, which then initiates transcription of target genes. Both subunits are constitutively expressed, but the ? subunit is degraded in an oxygen-dependent manner, so it is only stabilised in hypoxia. HIF-1 expression is deregulated in many cancers and has been associated with resistance to chemotherapy. Therefore, understanding the molecular mechanisms of hypoxic response is key to developing cancer therapeutics.

This thesis describes the construction and validation of a novel method to study the HIF-1 pathway by reprogramming cellular behaviour. This is achieved by targeting HIF-1[alpha]/HIF-1[Beta] dimerisation with an endogenously expressed molecular inhibitor. The compound, cyclo-CLLFVY, was previously identified through screening of a library of biologically synthesised cyclic peptides. In this study, the peptide sequence was genetically-encoded, between split Nostoc punctiforme DnaE inteins, onto the chromosome of a mammalian cell line, to allow inducible expression and in situ cyclisation of the peptide. The utility of this methodology was demonstrated via identification of genes specifically transactivated by HIF-1 or HIF-2 transcription factors in hypoxia, in the integrated cell line. Furthermore, cell viability assays showed that specific inhibition of HIF-1 dimerisation in these cells increased cellular sensitivity to glucose deprivation and to inhibition of glycolysis.

In addition, the epigenetic regulation of HIF-1[alpha] was studied. HIF-1 transactivates the expression of its [alpha]-subunit resulting in positive autoregulation, which contributes to the build up of HIF-1[alpha] at the onset of hypoxia. This autoregulation is dependent on an unmethylated CpG site in the hypoxia response element (HRE) within the promoter of the HIF-1[alpha] gene. Given the key role of HIF-1 in the development of the mammalian embryo, the methylation status of the HIF-1[alpha] HRE in developing tissue was sought. The HIF-1[alpha] HRE was unmethylated in several embryonic tissues, suggesting that transactivation of HIF-1[alpha] plays a role in HIF-1-mediated gene expression during development.

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Identifiers

ORCID for Ali Tavassoli: orcid.org/0000-0002-7420-5063

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