Mechanisms of activation of the hypoxia-inducible transcription factor 1a

Abstract: Hypoxia Inducible Factor-1 is a heterodimeric transcription factor composed of the bHLH/PAS factors HIF-1 alpha and HIF 1 beta/ARNT. Under hypoxic conditions HIF- 1 becomes activated and transcriptionally upregulates a set of target genes involved in angiogenesis, erythropoesis and glycolysis. HIF- 1 is primarily regulated through its ct subunit, which is stabilised against degradation by hypoxia. At normal oxygen levels HIF-1 alpha has a half-life of a few minutes, making it one of the most unstable proteins known. In contrast, ARNT is a constitutively stable protein. The aims of this thesis were to investigate a potential mechanism of HIF-1 alpha protein stability by hypoxia-regulated ubiquitination of the protein. Treatment of nonhypoxic cells with proteasome-specific inhibitors led to HIF-1 alpha accumulation in a similar way to hypoxia. By deletion analysis we showed that the N-terminal transactivation domain of HIF-1 alpha harbours an intrinsic destabilising sequence, removal of which lead to oxygen-independent protein stabilisation. We also demonstrated that HIF-1 alpha is directly ubiquitinated in non-hypoxic cells and that ubiquitination is dramatically reduced under hypoxic conditions, consistent with the elevated protein levels observed using proteasome inhibitors. In order to understand events downstream of HIF-1 alpha stabilisation, we studied the subcellular distribution of HIF-1 alpha in cells. We found that under hypoxic conditions wild-type HIF-1 alpha accumulates in the nucleus and that this activity was dependent on an SV40-like NLS motif in the C-terminus. Removal or point mutation of the C-terminal NLS completely abolished this effect, leading to an exclusively cytoplasmic distribution. In a series of experiments, we addressed the involvement of HIF-1 alpha with transcriptional cofactors in regulating gene expression. We have shown that HIF-1 alpha conditionally interacts via two distinct transactivation domains with the coactivators CBP1/p300, SRC-1 or TIF2 in addition to the nuclear redox regulator Ref-1 and that these interactions are important for full the transcriptional activation function of HIF-1 alpha. While the steps involved in HIF-1 alpha activation are understood in some detail the process of down-regulation post-hypoxia is less clear. We show that HIF-1 alpha is exported from the nucleus upon reoxygenation indicating a conditional export mechanism. However, HIF-1 alpha export is not dependent on the Crm1-mediated export pathway, which targets leucine-rich NES sequences, but mediated by an as yet unclear export mechanism. In conclusion, HIF-1 alpha is regulated at multiple levels from protein stability and nuclear entry to cofactor recruitment and nuclear export. The growing number of target genes under the control of HIF-1 alpha underscores the pivotal role played by this master gene regulator in cellular and systemic adaptation to hypoxia.