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

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.

List of scientific papers

I. Kallio PJ, Wilson WJ, OBrien S, Makino Y, Poellinger L (1999). Regulation of the hypoxia-inducible transcription factor 1alpha by the ubiquitin-proteasome pathway. J Biol Chem. 274(10): 6519-25.
https://pubmed.ncbi.nlm.nih.gov/10037745

II. Kallio PJ, Okamoto K, OBrien S, Carrero P, Makino Y, Tanaka H, Poellinger L (1998). Signal transduction in hypoxic cells: inducible nuclear translocation and recruitment of the CBP/p300 coactivator by the hypoxia-inducible factor-1alpha. EMBO J. 17(22): 6573-86.
https://pubmed.ncbi.nlm.nih.gov/9822602

III. Carrero P, Okamoto K, Coumailleau P, OBrien S, Tanaka H, Poellinger L (2000). Redox-regulated recruitment of the transcriptional coactivators CREB-binding protein and SRC-1 to hypoxia-inducible factor 1alpha. Mol Cell Biol. 20(1): 402-15.
https://pubmed.ncbi.nlm.nih.gov/10594042

IV. OBrien S, Poellinger L (2002). Regulation of the intracellular localisation of the hypoxia-inducible factor 1alpha. [Manuscript]