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Studies of the regulation of erythropoietin (EPO) production by the liver and kidneys, one of the classical physiological responses to hypoxia, led to the discovery of human oxygen-sensing mechanisms, which are now being targeted therapeutically. The oxygen-sensitive signal is generated by 2-oxoglutarate-dependent dioxygenases that deploy molecular oxygen as a co-substrate to catalyse the post-translational hydroxylation of specific prolyl and asparaginyl residues in hypoxia-inducible factor (HIF), a key transcription factor that regulates transcriptional responses to hypoxia. Hydroxylation of HIF at different sites promotes both its degradation and inactivation. Under hypoxic conditions, these processes are suppressed, enabling HIF to escape destruction and form active transcriptional complexes at thousands of loci across the human genome. Accordingly, HIF prolyl hydroxylase inhibitors stabilize HIF and stimulate expression of HIF target genes, including the EPO gene. These molecules activate endogenous EPO gene expression in diseased kidneys and are being developed, or are already in clinical use, for the treatment of renal anaemia. In this Review, we summarize information on the molecular circuitry of hypoxia signalling pathways underlying these new treatments and highlight some of the outstanding questions relevant to their clinical use.

Hypoxia inducible factor (HIF) and mammalian target of rapamycin (mTOR) pathways orchestrate responses to oxygen and nutrient availability. These pathways are frequently dysregulated in cancer, but their interplay is poorly understood, in part because of difficulties in simultaneous measurement of global and mRNA-specific translation. Here, we describe a workflow for measurement of ribosome load of mRNAs resolved by their transcription start sites (TSSs). Its application to kidney cancer cells reveals extensive translational reprogramming by mTOR, strongly affecting many metabolic enzymes and pathways. By contrast, global effects of HIF on translation are limited, and we do not observe reported translational activation by HIF2A. In contrast, HIF-dependent alterations in TSS usage are associated with robust changes in translational efficiency in a subset of genes. Analyses of the interplay of HIF and mTOR reveal that specific classes of HIF1A and HIF2A transcriptional target gene manifest different sensitivity to mTOR, in a manner that supports combined use of HIF2A and mTOR inhibitors in treatment of kidney cancer. A new method for mRNA profiling of ribosome load defines the pan-genomic interplay of transcriptional and translational regulation mediated by environment-sensing HIF and mTOR pathways in kidney cancer cells, offering insights into rational therapy.

Erythropoietin (EPO) is the hormonal regulator of red cell production and provided the paradigm for oxygen-regulated gene expression that led to the discovery of hypoxia-inducible factor (HIF). In this issue of the JCI, Rankin and colleagues show, using targeted gene inactivation, that induction of Epo expression in murine liver is dependent on the integrity of HIF-2alpha, and not HIF-1alpha (see the related article beginning on page 1068). These results demonstrate distinct functions for different HIF-alpha isoforms that could potentially be exploited in therapeutic approaches to anemia.

Organisms must respond to hypoxia to preserve oxygen homeostasis. We identify a thiol oxidase, previously assigned as cysteamine (2-aminoethanethiol) dioxygenase (ADO), as a low oxygen affinity (high-K mO₂) amino-terminal cysteine dioxygenase that transduces the oxygen-regulated stability of proteins by the N-degron pathway in human cells. ADO catalyzes the conversion of amino-terminal cysteine to cysteine sulfinic acid and is related to the plant cysteine oxidases that mediate responses to hypoxia by an identical posttranslational modification. We show in human cells that ADO regulates RGS4/5 (regulator of G protein signaling) N-degron substrates, modulates G protein–coupled calcium ion signals and mitogen-activated protein kinase activity, and that its activity extends to other N-cysteine proteins including the angiogenic cytokine interleukin-32. Identification of a conserved enzymatic oxygen sensor in multicellular eukaryotes opens routes to better understanding and therapeutic targeting of adaptive responses to hypoxia.

Hypoxia-inducible transcription factors (HIFs) are fundamental to cellular adaptation to low oxygen levels, but it is unclear how they interact with chromatin and activate their target genes. Here, we use genome-wide mutagenesis to identify genes involved in HIF transcriptional activity, and define a requirement for the histone H3 lysine 4 (H3K4) methyltransferase SET1B. SET1B loss leads to a selective reduction in transcriptional activation of HIF target genes, resulting in impaired cell growth, angiogenesis and tumor establishment in SET1B-deficient xenografts. Mechanistically, we show that SET1B accumulates on chromatin in hypoxia, and is recruited to HIF target genes by the HIF complex. The selective induction of H3K4 trimethylation at HIF target loci is both HIF- and SET1B-dependent and, when impaired, correlates with decreased promoter acetylation and gene expression. Together, these findings show SET1B as a determinant of site-specific histone methylation and provide insight into how HIF target genes are differentially regulated. The histone H3K4 methyltransferase SET1B is recruited to a subset of hypoxia-inducible genes by the HIF complex. Loss of SET1B reduces HIF transcriptional activity in hypoxia and impairs tumor formation in xenograft models.

Oxygen homeostasis is maintained in plants and animals by O 2 -sensing enzymes initiating adaptive responses to low O 2 (hypoxia). Recently, the O 2 -sensitive enzyme ADO was shown to initiate degradation of target proteins RGS4/5 and IL32 via the Cysteine/Arginine N-degron pathway. ADO functions by catalysing oxidation of N-terminal cysteine residues, but despite multiple proteins in the human proteome having an N-terminal cysteine, other endogenous ADO substrates have not yet been identified. This could be because alternative modifications of N-terminal cysteine residues, including acetylation, prevent ADO-catalysed oxidation. Here we investigate the relationship between ADO-catalysed oxidation and NatA-catalysed acetylation of a broad range of protein sequences with N-terminal cysteines. We present evidence that human NatA catalyses N-terminal cysteine acetylation in vitro and in vivo. We then show that sequences downstream of the N-terminal cysteine dictate whether this residue is oxidised or acetylated, with ADO preferring basic and aromatic amino acids and NatA preferring acidic or polar residues. In vitro, the two modifications appear to be mutually exclusive, suggesting that distinct pools of N-terminal cysteine proteins may be acetylated or oxidised. These results reveal the sequence determinants that contribute to N-terminal cysteine protein modifications, with implications for O 2 -dependent protein stability and the hypoxic response. Heathcote et al. show that enzymes catalysing N-terminal cysteine oxidation and acetylation have distinct substrate preferences. The modifications are mutually exclusive in vitro, with implications for protein stability and the hypoxic response.

Key Points Hypoxia-inducible factor (HIF) is an α/β heterodimeric DNA-binding complex that directs an extensive transcriptional response to hypoxia. The activity of HIF is induced in hypoxic cells through the stabilization and activation of its α-subunit. HIFα subunits are regulated by a newly discovered signalling mechanism — that is, the oxygen-dependent enzymatic hydroxylation of specific amino-acid residues. The hydroxylation of conserved prolyl residues in two independent degradation domains in the central region of HIFα promotes interactions with the von Hippel–Lindau ubiquitylation complex, which targets HIFα for degradation by the ubiquitin–proteasome pathway. Hydroxylation at a conserved asparaginyl residue in the HIFα carboxy-terminal activation domain blocks interaction with the p300 transcriptional co-activator. To date, three HIF prolyl hydroxylases, known as prolyl hydroxylase domain (PHD)1–3, and one asparaginyl hydroxylase, known as factor inhibiting HIF (FIH), have been defined. These enzymes all belong to the non-haem, Fe 2+ -dependent, 2-oxoglutarate-dependent-oxygenase superfamily. These enzymes possess a common 'jelly-roll' (double-stranded β-helix) core and coordinate the catalytic Fe 2+ using a two-histidine, one-carboxylate 'facial triad'. During catalysis, the splitting of molecular oxygen occurs with one oxygen atom being incorporated into the HIF prolyl or asparaginyl residue and the other being incorporated into succinate during the oxidative decarboxylation of 2-oxoglutarate. The absolute requirement of the HIF hydroxylases for molecular oxygen conveys oxygen sensitivity. Additional cofactor and co-substrate requirements for Fe 2+ , the citric-acid-cycle intermediate 2-oxoglutarate and ascorbate might help these enzymes generate the flexibility that is required for an oxygen-sensing function in complex multicellular animals. The transcription factor HIF (hypoxia-inducible factor) has a central role in oxygen homeostasis in animals ranging from nematode worms to man. Recent studies have shown that this factor is regulated by an unprecedented signalling mechanism that involves post-translational hydroxylation. This hydroxylation is catalysed by a set of non-haem, Fe 2+ -dependent enzymes that belong to the 2-oxoglutarate-dependent-oxygenase superfamily. The absolute requirement of these enzymes for molecular oxygen has provided new insights into the way cells sense oxygen.

Mutations in isocitrate dehydrogenases (IDHs) have a gain‐of‐function effect leading to R (−)‐2‐hydroxyglutarate ( R‐ 2HG) accumulation. By using biochemical, structural and cellular assays, we show that either or both R ‐ and S ‐2HG inhibit 2‐oxoglutarate (2OG)‐dependent oxygenases with varying potencies. Half‐maximal inhibitory concentration (IC 50 ) values for the R ‐form of 2HG varied from approximately 25 μM for the histone N ε ‐lysine demethylase JMJD2A to more than 5 mM for the hypoxia‐inducible factor (HIF) prolyl hydroxylase. The results indicate that candidate oncogenic pathways in IDH‐associated malignancy should include those that are regulated by other 2OG oxygenases than HIF hydroxylases, in particular those involving the regulation of histone methylation. The oncometabolite 2‐hydroxyglutarate (2‐HG) inhibits chromatin‐modifying oxygenases (as histone lysine demethylases) with greater potency than HIF hydroxylases. This suggests that 2‐HG‐associated oncogenic pathways involve the regulation of histone methylation, rather than an elevated HIF response.

The hypoxic response in humans is mediated by the hypoxia‐inducible transcription factor (HIF), for which prolyl hydroxylases (PHDs) act as oxygen‐sensing components. The evolutionary origins of the HIF system have been previously unclear. We demonstrate a functional HIF system in the simplest animal, Trichoplax adhaerens : HIF targets in T. adhaerens include glycolytic and metabolic enzymes, suggesting a role for HIF in the adaptation of basal multicellular animals to fluctuating oxygen levels. Characterization of the T. adhaerens PHDs and cross‐species complementation assays reveal a conserved oxygen‐sensing mechanism. Cross‐genomic analyses rationalize the relative importance of HIF system components, and imply that the HIF system is likely to be present in all animals, but is unique to this kingdom. Schofield and colleagues demonstrate that a functional HIF system is present in the simplest animal, Trichoplax adhaerens. Their results imply that the HIF system is conserved in all animals, and reveal conservation of biochemical properties in the oxygen‐sensing machinery