Hypoxia-inducible factors (HIFs) mediate the transcriptional adaptation of hypoxic cells. The extensive transcriptional programm regulated by HIFs involves the induction of genes controlling angiogenesis, cellular metabolism, cell growth, metastasis, apoptosis, extracellular matrix remodeling and others. HIF is a heterodimer of HIF-α and HIF-β subunits. In addition to HIF-1α, HIF-2α has evolved as an isoform that contributes differently to the hypoxic adaptation by performing non-redundant functions. Poly (ADP-ribose) polymerase-1 (PARP-1) is a nuclear protein involved in the control of DNA repair and gene transcription by modulating chromatin structure and acting as part of gene-specific enhancer/promoter-binding complexes. Previous results have shown that PARP-1 regulates HIF-1 activity. In this study, we focused on the cross-talk between HIF-2α and PARP-1. By using different approaches to suppress PARP-1, we show that HIF-2α mRNA expression, protein levels and HIF-2-dependent gene expression, such as ANGPTL4 and erythropoietin (EPO), are regulated by PARP-1. This regulation occurs at both the transcriptional and post-trancriptional level. We also show a complex formation between HIF-2α with PARP-1. This complex is sensitive to PARP inhibition and seems to protect against the von Hippel-Lindau-dependent HIF-2α degradation. Finally, we show that parp-1(-/-) mice display a significant reduction in the circulating hypoxia-induced EPO levels, number of red cells and hemoglobin concentration. Altogether, these results reveal a complex functional interaction between PARP-1 and the HIF system and suggest that PARP-1 is involved in the fine tuning of the HIF-mediated hypoxic response in vivo.

By controlling HIFa hydroxylation and stability, the prolyl hydroxylase domain (PHD)-containing proteins are essential to the maintenance of oxygen homeostasis; therefore these enzymes are tightly regulated. Small ubiquitin-like modifier (SUMO) is a 10-kDa protein readily conjugated to lysine residues of the targeted proteins in a process termed SUMOylation. In this study, we introduce SUMO conjugation as a novel regulator of PHD3 (also known as EGLN3). PHD3 SUMOylation occurs at a cluster of four lysines at the C-terminal end of the protein. Furthermore, PHD3 SUMOylation by SUMO2 or SUMO3 contributes to PHD3-mediated repression of HIF1-dependent transcriptional activity. Interestingly, PHD3-SUMO conjugation does not affect PHD3 hydroxylase activity or HIF1a stability, providing new evidence for a dual role of PHD3 in HIF1 regulation. Moreover, we show that hypoxia modulates PHD3-SUMO conjugation and that this modification inversely correlates with HIF1 activation. PHD3 SUMOylation highlights a new and additional layer of regulation that is likely required to fine-tune HIF function.

29Hypoxia Inducible Factor (HIF) is the master transcriptional regulator that orchestrates cellular 30 adaptation to low oxygen. HIF is tightly regulated via the stability of its α-subunit, which is 31 subjected to oxygen-dependent proline hydroxylation by Prolyl-Hydroxylase Domain 32 containing proteins (PHDs/EGLNs), and ultimately targeted for proteasomal degradation 33 through poly-ubiquitination by von-Hippel-Lindau protein (pVHL). However, sustained HIF-α 34 signalling is found in many tumours independently of oxygen availability pointing towards the 35 relevance of non-canonical HIF-α regulators. In this study, we establish the Ubiquitin Specific 36Protease 29 (USP29) as direct post-translational activator of HIF-α in a variety of cancer cell 37 lines. USP29 binds to HIF-α, decreases poly-ubiquitination and thus protects HIF-α from 38 proteasomal degradation. Deubiquitinating activity of USP29 is essential to stabilise not only 39 HIF-1α but also HIF-2α, via their C-termini in an oxygen/PHD/pVHL-independent manner. 40 Furthermore, in prostate cancer samples the expression of USP29 correlates with the HIF-41 target gene CA9 (carbonic anhydrase 9) as well as disease progression and severity. 42 43 44 45 46 47 48 49 50 51 52 53 54