Oxygen-dependent regulation of transcription by the hypoxia-inducible factor-1

Under limited oxygen availability (hypoxia) cells undergo rapid reprogramming in order to survive in the new environment until normoxic conditions are re-established. The observation that hypoxia can induce the expression of genes involved in erythropoiesis, angiogenesis and glucose metabolism among others, led to the identification of the hypoxia-inducible factor-1 (HIF-1) as a master regulator of the hypoxia-response pathway. HIF-1 is a heterodimeric transcriptional activator composed of two subunits (HIF-1alpha and ARNT) that belong to the bHLH/PAS family of transcription factors. At normoxia, HIF-1 activity is inhibited by rapid degradation of the alpha subunit, targeted to the 26S proteasome by interaction with the E3 ubiquitin-ligase pVHL. Under hypoxia, HIF-1alpha is stabilized and translocates to the nucleus where it dimerizes with ARNT and activates target gene expression by recruiting coactivator proteins.

The aim of this study was to investigate the structure/function relationship of HIF-1alpha N- and Cterminal activation domains (N- and C-TAD) and to analyze the mechanisms of conditional recruitment of coregulator proteins. Furthermore, we were interested in analyzing the coordinated subcellular and intranuclear redistribution of proteins involved in the hypoxic response. The information and reagents obtained in initial studies were used in the identification of new HIF-1- interacting proteins.

In the study of HIF-1alpha N-TAD, we identified several amino acid residues critical for the transactivation and degradation functions of this bifunctional domain. Although we observed a significant structural overlap between both functions, we demonstrated by mutation analysis that the transactivation activity is mediated by a longer peptide when compared to the minimal degradation box, which is circumscribed to the core of the domain. We concluded that HIF-1alpha NTAD functions as an independent transactivation domain in a CBP-dependent manner.

In silico analysis of the secondary structure of HIF-1alpha C-TAD revealed two putative alpha-helices in the initial and terminal regions of this domain. The function of these putative structures was analyzed by alanine-scanning mutagenesis and revealed two subdomains with disparate contributions to the overall activity of the C-TAD. The C-terminal helix (helix-2) proved to be critical for transactivation and for interaction with the CH1 domain of CBP. In this study we observed that colocalization of CFP-HIF-1alpha and YFP-CBP in intranuclear colocalization foci was dependent of the integrity of both HIF-1alpha transactivation domains.

We have shown that, although SRC-1 is an important coactivator of HIF-1 function, the interaction between the two proteins needs to be mediated by CBP. In vivo colocalization studies showed that CBP plays a dominant role in the intranuclear redistribution of the HIF- 1alpha/ARNT/CBP/SRC-1 complex into foci of colocalization that partially overlapped the distribution of RNA Pol IIo.

We have characterized a novel interaction interface between HIF-1alpha and CBP, that is mediated by direct binding of the N-TAD to the CH3 domain of CBP. In this study we provide evidence that, under hypoxic conditions, the high affinity of HIF-1á for limiting amounts of CBP, may interfere with other CBP-dependent pathways.

Finally, we have identified the enzyme C1-tetrahydrofolate synthase as a novel protein that participates in the regulation of HIF-1alpha activity. Overexpression of C1-tetrahydrofolate synthase dramatically increased HIF-1-mediated transactivation due to stabilization of the alpha subunit. We show that C1-tetrahydrofolate synthase stabilizes HIF-1alpha by interfering with PHD-mediated hydroxylation of Pro563 of the N-TAD and inhibiting pVHL-recruitment and protein degradation. The results from these studies have contributed to a better understanding of the mechanisms that control HIF-1 activation and provide evidence for novel levels of regulation of HIF activity.

List of scientific papers

I. Pereira T, Zheng X, Ruas JL, Tanimoto K, Poellinger L (2003). Identification of residues critical for regulation of protein stability and the transactivation function of the hypoxia-inducible factor-1alpha by the von Hippel-Lindau tumor suppressor gene product. J Biol Chem. 278(9): 6816-23. Epub 2002 Dec 4
https://pubmed.ncbi.nlm.nih.gov/12468553

II. Ruas JL, Poellinger L, Pereira T (2002). Functional analysis of hypoxia-inducible factor-1 alpha-mediated transactivation. Identification of amino acid residues critical for transcriptional activation and/or interaction with CREB-binding protein. J Biol Chem. 277(41): 38723-30. Epub 2002 Jul 19
https://pubmed.ncbi.nlm.nih.gov/12133832

III. Ruas JL, Poellinger L, Pereira T (2005). Role of CBP in regulating HIF-1-mediated activation of transcription. J Cell Sci. 118(Pt 2): 301-11. Epub 2004 Dec 22
https://pubmed.ncbi.nlm.nih.gov/15615775

IV. Ruas JL, Berchner-Pfannschmidt U, Malik S, Roeder RG, Poellinger L, Pereira T (2005). The N-terminal activation domain of the hypoxia-inducible factor-1alpha recruits CBP through the CH3 region. [Manuscript]

V. Ruas JL, Pereira T, Bakali AH, Malik S, Nordlund P, Roeder RG, Poellinger L (2005). C1-tetrahydrofolate synthase stabilizes HIF-1alpha by disrupting the interaction with the von Hippel-Lindau tumor supressor gene product. [Manuscript]