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Hypoxia inducible transcription factors (HIFs) directly dictate the expression of multiple RNA species including novel and as yet uncharacterized long non-coding transcripts with unknown function. We used pan-genomic HIF-binding and transcriptomic data to identify a novel long non-coding RNA NICI (Non-coding Intergenic Co-Induced transcript) on chromosome 12p13.31 which is regulated by hypoxia via HIF-1 promoter-binding in multiple cell types. CRISPR/Cas9-mediated deletion of the hypoxia-response element revealed co-regulation of NICI and the neighboring protein-coding gene, solute carrier family 2 member 3 (SLC2A3) which encodes the high-affinity glucose transporter 3 (GLUT3). Knock-down or knock-out of NICI attenuated hypoxic induction of SLC2A3 indicating a direct regulatory role of NICI in SLC2A3 expression, which was further evidenced by CRISPR/Cas9-VPR mediated activation of NICI expression. We also demonstrate that regulation of SLC2A3 is mediated through transcriptional activation rather than post-transcriptional mechanisms since knock-out of NICI leads to reduced recruitment of RNA polymerase 2 to the SLC2A3 promoter. Consistent with this we observe NICI-dependent regulation of glucose consumption and cell proliferation. Furthermore, NICI expression is regulated by the VHL tumour suppressor and is highly expressed in clear cell renal cancer, where SLC2A3 expression is associated with patient prognosis, implying an important role for the HIF/NICI/SLC2A3 axis in this malignancy.

Hypoxia is central to both ischaemic and neoplastic diseases. However, the non-coding transcriptional response to hypoxia is largely uncharacterized. We undertook integrated genomic analyses of both non-coding and coding transcripts using massively parallel sequencing and interfaced this data with pan-genomic analyses of hypoxia-inducible factor (HIF) and RNApol2 binding in hypoxic cells. These analyses revealed that all classes of RNA are profoundly regulated by hypoxia and implicated HIF as a major direct regulator of both the non-coding and coding transcriptome, acting predominantly through release of pre-bound promoter-paused RNApol2. These findings indicate that the transcriptional response to hypoxia is substantially more extensive than previously considered.

Physiological effects of cellular hypoxia are sensed by prolyl hydroxylase (PHD) enzymes which regulate HIFs. Genetic interventions on HIF/PHD pathways reveal multiple phenotypes that extend the known biology of hypoxia. Recent studies unexpectedly implicate HIF in aspects of multiple immune and inflammatory pathways. However such studies are often limited by systemic lethal effects and/or use tissue-specific recombination systems, which are inherently irreversible, un-physiologically restricted and difficult to time. To study these processes better we developed recombinant mice which express tetracycline-regulated shRNAs broadly targeting the main components of the HIF/PHD pathway, permitting timed bi-directional intervention. We have shown that stabilization of HIF levels in adult mice through PHD2 enzyme silencing by RNA interference, or inducible recombination of floxed alleles, results in multi-lineage leukocytosis and features of autoimmunity. This phenotype was rapidly normalized on re-establishment of the hypoxia-sensing machinery when shRNA expression was discontinued. In both situations these effects were mediated principally through the Hif2a isoform. Assessment of cells bearing regulatory T cell markers from these mice revealed defective function and pro-inflammatory effects in vivo. We believe our findings have shown a new role for the PHD2/Hif2a couple in the reversible regulation of T cell and immune activity.

Tammie Bishop, Peter J Ratcliffe Nuffield Department of Medicine, University of Oxford, Oxford, UK Abstract: By the early 1900s, the close matching of oxygen supply with demand was recognized to be a fundamental requirement for physiological function, and multiple adaptive responses to environment hypoxia had been described. Nevertheless, the widespread operation of mechanisms that directly sense and respond to levels of oxygen in animal cells was not appreciated for most of the twentieth century with investigators generally stressing the regulatory importance of metabolic products. Work over the last 25 years has overturned that paradigm. It has revealed the existence of a set of “oxygen-sensing” 2-oxoglutarate dependent dioxygenases that catalyze the hydroxylation of specific amino acid residues and thereby control the stability and activity of hypoxia-inducible factor. The hypoxia-inducible factor hydroxylase pathway regulates a massive transcriptional cascade that is operative in essentially all animal cells. It transduces a wide range of responses to hypoxia, extending well beyond the classical boundaries of hypoxia physiology. Here we review the discovery and elucidation of these pathways, and consider the opportunities and challenges that have been brought into focus by the findings, including new implications for the integrated physiology of hypoxia and therapeutic approaches to ischemic/hypoxic disease. Keywords: hypoxia, oxygen-sensing, HIF, 2-oxoglutarate oxygenase, hydroxylase, cell signaling

Summary Isocitrate dehydrogenase 1 mutations drive human gliomagenesis, probably through neomorphic enzyme activity that produces D-2-hydroxyglutarate. To model this disease, we conditionally expressed Idh1R132H in the subventricular zone (SVZ) of the adult mouse brain. The mice developed hydrocephalus and grossly dilated lateral ventricles, with accumulation of 2-hydroxyglutarate and reduced -ketoglutarate. Stem and transit amplifying/progenitor cell populations were expanded, and proliferation increased. Cells expressing SVZ markers infiltrated surrounding brain regions. SVZ cells also gave rise to proliferative subventricular nodules. DNA methylation was globally increased, while hydroxymethylation was decreased. Mutant SVZ cells over-expressed Wnt, cell cycle and stem cell genes, and shared an expression signature with human gliomas. Idh1R132H mutation in the major adult neurogenic stem cell niche causes a phenotype resembling gliomagenesis. Significance Few curative treatments are available for human brain tumors. IDH1R132H is a driver mutation in gliomas and other malignancies, probably causing tumorigenesis through D-2-hydroxyglutarate accumulation, although the downstream mechanisms remain unclear. We found that adult mice expressing Idh1R132H in the brain subventricular zone (SVZ) develop features of gliomagenesis, including increased numbers of neural stem cells and their progeny. Other abnormalities included cellular infiltration into surrounding brain regions, reminiscent of tumor invasion. The gene expression profile of the Idh1R132H SVZ closely overlaps those of human gliomas. Likely, non-exclusive tumorigenic mechanisms included promotion of a neural stem cell phenotype, Wnt pathway activation, maintenance of telomeres, and DNA hypermethylation. Our Idh1R132H mouse provides a system for assessing brain tumor therapies in vivo.

Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor that directs a broad range of cellular responses to hypoxia. Recent studies have defined a set of 2-oxoglutarate and Fe(II)-dependent dioxygenases that modify HIF-alpha subunits by prolyl and asparaginyl hydroxylation. These processes potentially provide a dual system of control, down-regulating both HIF-alpha stability and transcriptional activity. Although genetic analyses in both primitive organisms and mammalian cells have demonstrated a critical role for the prolyl hydroxylase pathway in the regulation of HIF, analogous studies have not been performed on the HIF asparaginyl hydroxylase pathway, and its role in directing the expression of endogenous HIF transcriptional targets has not yet been clearly defined. Here we demonstrate, using small interfering RNA-mediated FIH suppression and controlled overexpression by a doxycycline-inducible system, that alterations in FIH expression in both directions have reciprocal effects on the expression of a range of HIF target genes. These effects were observed in normoxic and severely hypoxic cells but not anoxic cells. Evidence for FIH activity in severely hypoxic cells contrasted with results for the prolyl hydroxylase PHD2, suggesting that these enzymes display different oxygen dependence in vivo, with PHD2 requiring higher levels of oxygen for biological activity. Our results demonstrate an important physiological role for FIH in regulating HIF-dependent target genes over a wide range of oxygen tensions and indicate that inhibition of FIH has the potential to augment HIF target gene expression even in severe hypoxia.

Oxygen-dependent prolyl hydroxylation of hypoxia-inducible factor (HIF) by a set of closely related prolyl hydroxylase domain enzymes (PHD1, 2 and 3) regulates a range of transcriptional responses to hypoxia. This raises important questions about the role of these oxygen-sensing enzymes in integrative physiology. We investigated the effect of both genetic deficiency and pharmacological inhibition on the change in ventilation in response to acute hypoxic stimulation in mice. Mice exposed to chronic hypoxia for 7 days manifest an exaggerated hypoxic ventilatory response (HVR) (10.8 ± 0.3 versus 4.1 ± 0.7 ml min(-1) g(-1) in controls; P < 0.01). HVR was similarly exaggerated in PHD2(+/-) animals compared to littermate controls (8.4 ± 0.7 versus 5.0 ± 0.8 ml min(-1) g(-1); P < 0.01). Carotid body volume increased (0.0025 ± 0.00017 in PHD2(+/-) animals versus 0.0015 ± 0.00019 mm(3) in controls; P < 0.01). In contrast, HVR in PHD1(-/-) and PHD3(-/-) mice was similar to littermate controls. Acute exposure to a small molecule PHD inhibitor (PHI) (2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetic acid) did not mimic the ventilatory response to hypoxia. Further, 7 day administration of the PHI induced only modest increases in HVR and carotid body cell proliferation, despite marked stimulation of erythropoiesis. This was in contrast with chronic hypoxia, which elicited both exaggerated HVR and cellular proliferation. The findings demonstrate that PHD enzymes modulate ventilatory sensitivity to hypoxia and identify PHD2 as the most important enzyme in this response. They also reveal differences between genetic inactivation of PHDs, responses to hypoxia and responses to a pharmacological inhibitor, demonstrating the need for caution in predicting the effects of therapeutic modulation of the HIF hydroxylase system on different physiological responses.

Interactions between biological pathways and molecular oxygen require robust mechanisms for detecting and responding to changes in cellular oxygen availability, to support oxygen homeostasis. Peptidylglycine α-amidating monooxygenase (PAM) catalyzes a two-step reaction resulting in the C-terminal amidation of peptides, a process important for their stability and biological activity. Here we show that in human, mouse, and insect cells, peptide amidation is exquisitely sensitive to hypoxia. Different amidation events on chromogranin A, and on peptides processed from proopiomelanocortin, manifest similar striking sensitivity to hypoxia in a range of neuroendocrine cells, being progressively inhibited from mild (7% O2) to severe (1% O2) hypoxia. In developing Drosophila melanogaster larvae, FMRF amidation in thoracic ventral (Tv) neurons is strikingly suppressed by hypoxia. Our findings have thus defined a novel monooxygenase-based oxygen sensing mechanism that has the capacity to signal changes in oxygen availability to peptidergic pathways.

Hypoxia-inducible expression has been demonstrated for many groups of mammalian genes, and studies of transcriptional control have revealed the existence of hypoxia-responsive elements (HREs) in the cis-acting sequences of several of these genes. These sequences generally contain one or more binding sites for a heterodimeric DNA binding complex termed hypoxia-inducible factor-1 (HIF-1). To analyze this response further, Chinese hamster ovary cells were stably transfected with plasmids bearing HREs linked to genes encoding immunoselectable cell surface markers, and clones that showed reduced or absent hypoxia-inducible marker expression were selected from a mutagenized culture of cells. Analysis of these cells revealed several clones with transacting defects in HRE activation, and in one the defect was identified as a failure to express the alpha-subunit of HIF-1. Comparison of hypoxia-inducible gene expression in wild type, HIF-1alpha-defective, and HIF-1alpha-complemented cells revealed two types of response. For some genes (e.g. glucose transporter-1), hypoxia-inducible expression was critically dependent on HIF-1alpha, whereas for other genes (e.g. heme oxygenase-1) hypoxia-inducible expression appeared largely independent of the expression of HIF-1alpha. These experiments show the utility of mutagenesis and selection of mutant cells in the analysis of mammalian transcriptional responses to hypoxia and demonstrate the operation of HIF-1alpha-dependent and HIF-1alpha-independent pathways of hypoxia-inducible gene expression in Chinese hamster ovary cells.

Studies of regulation of the haematopoietic growth factor erythropoietin led to the unexpected discovery of a widespread system of direct oxygen sensing that regulates gene expression in animals. The oxygen-sensitive signal is generated by a series of non-haem Fe(II)- and 2-oxoglutarate-dependent dioxygenases that catalyse the post-translational hydroxylation of specific residues in the transcription factor hypoxia-inducible factor (HIF). These hydroxylations promote both oxygen-dependent degradation and oxygen-dependent inactivation of HIF, but are suppressed in hypoxia, leading to the accumulation of HIF and assembly of an active transcriptional complex in hypoxic cells. Hypoxia-inducible factor activates an extensive transcriptional cascade that interfaces with other cell signalling pathways, microRNA networks and RNA-protein translational control systems. The relationship of these cellular signalling pathways to the integrated physiology of oxygen homeostasis and the implication of dysregulating these massive physiological pathways in diseases such as cancer are discussed.

Hypoxia inducible factor (HIF) directs an extensive transcripti onal cascade that transduces numerous adaptive responses to hypoxia. Pan-genomic analyses, using chromatin immunoprecipitation and transcript profiling, h ave revealed large numbers of HIF-binding sites that are generally associate d with hypoxia- inducible transcripts, even over long chromosomal distances. Ho wever, these studies do not define the specific targets of HIF-binding sites and do not reveal how induction of HIF affects chromatin conformation over distan tly connected functional elements. To address these questions we deployed a r ecently developed chromosome conformation assay that enables simultaneo us high- resolution analyses from multiple viewpoints. These assays defi ned specific long- range interactions between intergenic HIF-binding regions and o ne or more promoters of hypoxia-inducible genes, revealing the existence o f multiple enhancer-promoter, promoter-enhancer and enhancer-enhancer inte ractions. However, neither short-term activation of HIF by hypoxia, nor l ong-term stabilization of HIF in von Hippe l-Lindau (VHL) defective cells greatly altered these interactions, indicating that at least under these condit ions HIF can operate on pre-existing patterns of chromatin-chromatin interac tions that define potential transcriptional targets and permit rapid gene activat ion by hypoxic stress.

Tumor hypoxia induces the up-regulation of a gene program associated with angiogenesis, glycolysis, adaptation to pH, and apoptosis via the hypoxia-inducible transcription factors (Hifs) 1 and 2. Disruption of this pathway has been proposed as a cancer therapy. Here, we use short interfering RNAs to compare specific inactivation of Hif-1alpha or Hif-2alpha and show markedly different cell type-specific effects on gene expression and cell migration. Remarkably, among a panel of hypoxia-inducible genes, responses were critically dependent on Hif-1 alpha but not Hif-2 alpha in both endothelial and breast cancer cells but critically dependent on Hif-2 alpha in renal carcinoma cells.

Hypoxia-inducible factor (HIF) plays an important role in determining patterns of gene expression in cancer. HIF is down-regulated in oxygenated cells by a series of Fe (II) and 2-oxoglutarate dependent dioxygenases that hydroxylate specific residues in the regulatory HIF-alpha subunits. Because these enzymes require ascorbate for activity in vitro we analyzed the effects of ascorbate on HIF in human cancer cell lines. Ascorbate at physiological concentrations (25 micro M) strikingly suppressed HIF-1alpha protein levels and HIF transcriptional targets, particularly when the system was oncogenically activated in normoxic cells. Similar results were obtained with iron supplementation. These results indicate that both ascorbate and iron availability have major effects on HIF, and imply that the system is commonly regulated by limiting hydroxylase activity under normoxic tissue culture conditions.

Solid tumors contain regions of hypoxia, a physiological stress that can activate cell death pathways and, thus, result in the selection of cells resistant to death signals and anticancer therapy. Bcl2/adenovirus EIB 19kD-interacting protein 3 (BNIP3) is a cell death factor that is a member of the Bcl-2 proapoptotic family recently shown to induce necrosis rather than apoptosis. Using cDNA arrays and serial analysis of gene expression, we found that hypoxia induces up-regulation of BNIP3 and its homologue, Nip3-like protein X. Analysis of human carcinoma cell lines showed that they are hypoxically regulated in many tumor types, as well as in endothelial cells and macrophages. Regulation was hypoxia inducible factor-1-dependent, and hypoxia inducible factor-1 expression was suppressed by von Hippel-Lindau protein in normoxic cells. Northern blotting and in situ hybridization analysis has revealed that these factors are highly expressed in human tumors compared with normal tissue and that BNIP3 is up-regulated in perinecrotic regions of the tumor. This study shows that genes regulating cell death can be hypoxically induced and are overexpressed in clinical tumors.

The von Hippel-Lindau tumor suppressor protein acts as the substrate recognition component of a ubiquitin E3 ligase that targets hypoxia-inducible factor (HIF)-alpha subunits for proteolysis. Stabilization of HIF-alpha subunits has been described in VHL-defective cell lines, leading to HIF activation and up-regulation of hypoxia-inducible mRNAs. Mutations of the von Hippel-Lindau tumor suppressor protein are found in most clear cell renal cell carcinomas (CC-RCCs) but not other renal tumors, raising a question about the importance of activation of the HIF pathway in CC-RCC development. To address this question, we have examined the expression of HIF-alpha subunits in 45 primary renal tumors and related this to tumor subtype, the presence of VHL mutations, and measures of angiogenesis. We show that HIF-alpha is up-regulated in the majority of CC-RCCs, and that the pattern of expression is biased toward the HIF-2alpha isoform. Expression of HIF-alpha proteins was associated significantly with up-regulation of VEGF mRNA and protein and increased microvessel density. Up-regulation of HIF-alpha in CC-RCC was found to involve increased mRNA as well as protein expression, suggesting that both VHL-dependent and VHL-independent mechanisms are involved. These results suggest that activation of the HIF pathway is functionally important in CC-RCC development and might provide a new therapeutic target.