Control of cardiac jelly dynamics by NOTCH1 and NRG1 defines the building plan for trabeculation.

In vertebrate hearts, the ventricular trabecular myocardium develops as a sponge-like network of cardiomyocytes that is critical for contraction and conduction, ventricular septation, papillary muscle formation and wall thickening through the process of compaction 1 . Defective trabeculation leads to embryonic lethality2-4 or non-compaction cardiomyopathy (NCC) 5 . There are divergent views on when and how trabeculation is initiated in different species. In zebrafish, trabecular cardiomyocytes extrude from compact myocardium 6 , whereas in chicks, chamber wall thickening occurs before overt trabeculation 7 . In mice, the onset of trabeculation has not been described, but is proposed to begin at embryonic day 9.0, when cardiomyocytes form radially oriented ribs 2 . Endocardium-myocardium communication is essential for trabeculation, and numerous signalling pathways have been identified, including Notch2,8 and Neuregulin (NRG) 4 . Late disruption of the Notch pathway causes NCC 5 . Whereas it has been shown that mutations in the extracellular matrix (ECM) genes Has2 and Vcan prevent the formation of trabeculae in mice9,10 and the matrix metalloprotease ADAMTS1 promotes trabecular termination 3 , the pathways involved in ECM dynamics and the molecular regulation of trabeculation during its early phases remain unexplored. Here we present a model of trabeculation in mice that integrates dynamic endocardial and myocardial cell behaviours and ECM remodelling, and reveal new epistatic relationships between the involved signalling pathways. NOTCH1 signalling promotes ECM degradation during the formation of endocardial projections that are critical for individualization of trabecular units, whereas NRG1 promotes myocardial ECM synthesis, which is necessary for trabecular rearrangement and growth. These systems interconnect through NRG1 control of Vegfa, but act antagonistically to establish trabecular architecture. These insights enabled the prediction of persistent ECM and cardiomyocyte growth in a mouse NCC model, providing new insights into the pathophysiology of congenital heart disease.

Enhanced dependency of KRAS-mutant colorectal cancer cells on RAD51-dependent homologous recombination repair identified from genetic interactions in Saccharomyces cerevisiae.

Activating KRAS mutations drive colorectal cancer tumorigenesis and influence response to anti-EGFR-targeted therapy. Despite recent advances in understanding Ras signaling biology and the revolution in therapies for melanoma using BRAF inhibitors, no targeted agents have been effective in KRAS-mutant cancers, mainly due to activation of compensatory pathways. Here, by leveraging the largest synthetic lethal genetic interactome in yeast, we identify that KRAS-mutated colorectal cancer cells have augmented homologous recombination repair (HRR) signaling. We found that KRAS mutation resulted in slowing and stalling of the replication fork and accumulation of DNA damage. Moreover, we found that KRAS-mutant HCT116 cells have an increase in MYC-mediated RAD51 expression with a corresponding increase in RAD51 recruitment to irradiation-induced DNA double-strand breaks (DSBs) compared to genetically complemented isogenic cells. MYC depletion using RNA interference significantly reduced IR-induced RAD51 foci formation and HRR. On the contrary, overexpression of either HA-tagged wild-type (WT) MYC or phospho-mutant S62A increased RAD51 protein levels and hence IR-induced RAD51 foci. Likewise, depletion of RAD51 selectively induced apoptosis in HCT116-mutant cells by increasing DSBs. Pharmacological inhibition targeting HRR signaling combined with PARP inhibition selectivity killed KRAS-mutant cells. Interestingly, these differences were not seen in a second isogenic pair of KRAS WT and mutant cells (DLD-1), likely due to their nondependency on the KRAS mutation for survival. Our data thus highlight a possible mechanism by which KRAS-mutant-dependent cells drive HRR in vitro by upregulating MYC-RAD51 expression. These data may offer a promising therapeutic vulnerability in colorectal cancer cells harboring otherwise nondruggable KRAS mutations, which warrants further investigation in vivo.

The metastasis suppressor RARRES3 as an endogenous inhibitor of the immunoproteasome expression in breast cancer cells.

In breast cancer metastasis, the dynamic continuum involving pro- and anti-inflammatory regulators can become compromised. Over 600 genes have been implicated in metastasis to bone, lung or brain but how these genes might contribute to perturbation of immune function is poorly understood. To gain insight, we adopted a gene co-expression network approach that draws on the functional parallels between naturally occurring bone marrow-derived mesenchymal stem cells (BM-MSCs) and cancer stem cells (CSCs). Our network analyses indicate a key role for metastasis suppressor RARRES3, including potential to regulate the immunoproteasome (IP), a specialized proteasome induced under inflammatory conditions. Knockdown of RARRES3 in near-normal mammary epithelial and breast cancer cell lines increases overall transcript and protein levels of the IP subunits, but not of their constitutively expressed counterparts. RARRES3 mRNA expression is controlled by interferon regulatory factor IRF1, an inducer of the IP, and is sensitive to depletion of the retinoid-related receptor RORA that regulates various physiological processes including immunity through modulation of gene expression. Collectively, these findings identify a novel regulatory role for RARRES3 as an endogenous inhibitor of IP expression, and contribute to our evolving understanding of potential pathways underlying breast cancer driven immune modulation.

The recently identified modifier of murine metastable epialleles, Rearranged L-Myc Fusion, is involved in maintaining epigenetic marks at CpG island shores and enhancers.

BACKGROUND: We recently identified a novel protein, Rearranged L-myc fusion (Rlf), that is required for DNA hypomethylation and transcriptional activity at two specific regions of the genome known to be sensitive to epigenetic gene silencing. To identify other loci affected by the absence of Rlf, we have now analysed 12 whole genome bisulphite sequencing datasets across three different embryonic tissues/stages from mice wild-type or null for Rlf. RESULTS: Here we show that the absence of Rlf results in an increase in DNA methylation at thousands of elements involved in transcriptional regulation and many of the changes occur at enhancers and CpG island shores. ChIP-seq for H3K4me1, a mark generally found at regulatory elements, revealed associated changes at many of the regions that are differentially methylated in the Rlf mutants. RNA-seq showed that the numerous effects of the absence of Rlf on the epigenome are associated with relatively subtle effects on the mRNA population. In vitro studies suggest that Rlf's zinc fingers have the capacity to bind DNA and that the protein interacts with other known epigenetic modifiers. CONCLUSION: This study provides the first evidence that the epigenetic modifier Rlf is involved in the maintenance of DNA methylation at enhancers and CGI shores across the genome.

Identification of novel hypomorphic and null mutations in Klf1 derived from a genetic screen for modifiers of α-globin transgene variegation.

Position-effect variegation of transgene expression is sensitive to the chromatin state. We previously reported a forward genetic screen in mice carrying a variegated α-globin GFP transgene to find novel genes encoding epigenetic regulators. We named the phenovariant strains "Mommes" for modifiers of murine metastable epialleles. Here we report positional cloning of mutations in two Momme strains which result in suppression of variegation. Both strains harbour point mutations in the erythroid transcription factor, Klf1. One (D11) generates a stop codon in the zinc finger domain and a homozygous null phenotype. The other (D45) generates an amino acid transversion (H350R) within a conserved linker between zinc fingers two and three. Homozygous MommeD45 mice have chronic microcytic anaemia which models the phenotype in a recently described family. This is the first genetic evidence that the linkers between the zinc fingers of transcription factors have a function beyond that of a simple spacer.

The first mouse mutants of D14Abb1e (Fam208a) show that it is critical for early development.

An ENU mutagenesis screen to identify novel epigenetic modifiers was established in mice carrying a multi-copy GFP transgene, which is expressed in a variegated manner in erythrocytes and is highly sensitive to epigenetic silencing. The screen has produced mouse mutants of both known modifiers of epigenetic state, such as Dnmt1 and Smarca5, and novel modifiers, such as Smchd1 and Rlf. Here we report two mouse lines generated from the screen, MommeD6 and MommeD20, with point mutations in D14Abb1e. These are the first mouse mutants of D14Abb1e (also known as Fam208a), a gene about which little is known. Heterozygous intercrosses show that homozygous mutants from both the MommeD6 and MommeD20 lines are not viable beyond gastrulation, demonstrating an important role for D14Abb1e in development. We demonstrate that haploinsufficiency for D14Abb1e effects transgene expression at the RNA level. Analysis of the predicted D14Abb1e protein sequence reveals that it contains putative nuclear localisation signals and a domain of unknown function, DUF3715. Our studies reveal that D14Abb1e is localised to the nucleus and is expressed in skin and testes.

An ENU mutagenesis screen identifies novel and known genes involved in epigenetic processes in the mouse.

BACKGROUND: We have used a sensitized ENU mutagenesis screen to produce mouse lines that carry mutations in genes required for epigenetic regulation. We call these lines Modifiers of murine metastable epialleles (Mommes). RESULTS: We report a basic molecular and phenotypic characterization for twenty of the Momme mouse lines, and in each case we also identify the causative mutation. Three of the lines carry a mutation in a novel epigenetic modifier, Rearranged L-myc fusion (Rlf), and one gene, Rap-interacting factor 1 (Rif1), has not previously been reported to be involved in transcriptional regulation in mammals. Many of the other lines are novel alleles of known epigenetic regulators. For two genes, Rlf and Widely-interspaced zinc finger (Wiz), we describe the first mouse mutants. All of the Momme mutants show some degree of homozygous embryonic lethality, emphasizing the importance of epigenetic processes. The penetrance of lethality is incomplete in a number of cases. Similarly ,abnormalities in phenotype seen in the heterozygous individuals of some lines occur with incomplete penetrance. CONCLUSIONS: Recent advances in sequencing enhance the power of sensitized mutagenesis screens to identify the function of previously uncharacterized factors and to discover additional functions for previously characterized proteins. The observation of incomplete penetrance of phenotypes in these inbred mutant mice, at various stages of development, is of interest. Overall, the Momme collection of mouse mutants provides a valuable resource for researchers across many disciplines.

No evidence for cumulative effects in a Dnmt3b hypomorph across multiple generations.

Observations of inherited phenotypes that cannot be explained solely through genetic inheritance are increasing. Evidence points to transmission of non-DNA molecules in the gamete as mediators of the phenotypes. However, in most cases it is unclear what the molecules are, with DNA methylation, chromatin proteins, and small RNAs being the most prominent candidates. From a screen to generate novel mouse mutants of genes involved in epigenetic reprogramming, we produced a DNA methyltransferase 3b allele that is missing exon 13. Mice that are homozygous for the mutant allele have smaller stature and reduced viability, with particularly high levels of female post-natal death. Reduced DNA methylation was also detected at telocentric repeats and the X-linked Hprt gene. However, none of the abnormal phenotypes or DNA methylation changes worsened with multiple generations of homozygous mutant inbreeding. This suggests that in our model the abnormalities are reset each generation and the processes of transgenerational epigenetic reprogramming are effective in preventing their inheritance.

Cezanne regulates inflammatory responses to hypoxia in endothelial cells by targeting TRAF6 for deubiquitination.

RATIONALE: Hypoxia followed by reoxygenation promotes inflammation by activating nuclear factor κB transcription factors in endothelial cells (ECs). This process involves modification of the signaling intermediary tumor necrosis factor receptor-associated factor 6 with polyubiquitin chains. Thus, cellular mechanisms that suppress tumor necrosis factor receptor-associated factor 6 ubiquitination are potential therapeutic targets to reduce inflammation in hypoxic tissues. OBJECTIVE: In this study, we tested the hypothesis that endothelial activation in response to hypoxia-reoxygenation can be influenced by Cezanne, a deubiquitinating enzyme that cleaves ubiquitin from specific modified proteins. METHODS AND RESULTS: Studies of cultured ECs demonstrated that hypoxia (1% oxygen) induced Cezanne via p38 mitogen-activated protein kinase-dependent transcriptional and post-transcriptional mechanisms. Hypoxia-reoxygenation had minimal effects on proinflammatory signaling in unmanipulated ECs but significantly enhanced Lys63 polyubiquitination of tumor necrosis factor receptor-associated factor 6, activation of nuclear factor κB, and expression of inflammatory genes after silencing of Cezanne. Thus, although hypoxia primed cells for inflammatory activation, it simultaneously induced Cezanne, which impeded signaling to nuclear factor κB by suppressing tumor necrosis factor receptor-associated factor 6 ubiquitination. Similarly, ischemia induced Cezanne in the murine kidney in vascular ECs, glomerular ECs, podocytes, and epithelial cells, and genetic deletion of Cezanne enhanced renal inflammation and injury in murine kidneys exposed to ischemia followed by reperfusion. CONCLUSIONS: We conclude that inflammatory responses to ischemia are controlled by a balance between ubiquitination and deubiquitination, and that Cezanne is a key regulator of this process. Our observations have important implications for therapeutic targeting of inflammation and injury during ischemia-reperfusion.

Dimethyloxalyglycine stimulates the early stages of gastrointestinal repair processes through VEGF-dependent mechanisms.

Dimethyloxalylglycine (DMOG) is an inhibitor of prolyl-4-hydroxylase domain enzymes. Its potential value and mechanism of actions in preventing/treating gastrointestinal injury are, however, poorly understood. We, therefore, examined the effect of DMOG on influencing gut injury and repair using a variety of in vitro and in vivo models. We performed in vitro studies utilising pro-migratory (wounded monolayer) and proliferation (using DNA quantitation) assays of human stomach (AGS) and colonic (HT29) carcinoma cells. Time course studies examined changes in hypoxia-inducible factor (HIF) and vascular endothelial growth factor (VEGF) levels, a growth factor known to be regulated via HIF. In vivo studies utilised a rat gastric (indomethacin, 20 mg/kg and 3 h restraint) damage model. DMOG stimulated migration in a dose-dependent manner, increasing migration twofold when added at 25µM (P<0.01). Additive effects were seen when DMOG was added to cells in hypoxic conditions. DMOG stimulated proliferation dose dependently, increasing proliferation threefold when added at 70 µM (P<0.01). DMOG caused upregulation of both HIF and VEGF within 4 h of administration. Addition of VEGF neutralising antibody truncated migratory and proliferative activity of DMOG by about 70%. Both oral and subcutaneous administration of DMOG decreased gastric injury without influencing intragastric pH (50% reduction in injury when 1 ml gavaged at 0.57 mM, P < 0.01). Indomethacin reduced tissue HIF and VEGF levels but this was prevented if DMOG was present. In conclusion, DMOG stimulates the early phases of gut repair and VEGF-dependent processes appear relevant. Non-peptide factors such as this may be useful to stabilise or repair gut mucosa.

Inactivation of the von Hippel-Lindau tumour suppressor gene induces Neuromedin U expression in renal cancer cells.

BACKGROUND: 209 000 new cases of renal carcinoma are diagnosed each year worldwide and new therapeutic targets are urgently required. The great majority of clear cell renal cancer involves inactivation of VHL, which acts as a gatekeeper tumour suppressor gene in renal epithelial cells. However how VHL exerts its tumour suppressor function remains unclear. A gene expression microarray comparing RCC10 renal cancer cells expressing either VHL or an empty vector was used to identify novel VHL regulated genes. FINDINGS: NMU (Neuromedin U) is a neuropeptide that has been implicated in energy homeostasis and tumour progression. Here we show for the first time that VHL loss-of-function results in dramatic upregulation of NMU expression in renal cancer cells. The effect of VHL inactivation was found to be mediated via activation of Hypoxia Inducible Factor (HIF). Exposure of VHL expressing RCC cells to either hypoxia or dimethyloxalylglycine resulted in HIF activation and increased NMU expression. Conversely, suppression of HIF in VHL defective RCC cells via siRNA of HIF-α subunits or expression of Type 2C mutant VHLs reduced NMU expression levels. We also show that renal cancer cells express a functional NMU receptor (NMUR1), and that NMU stimulates migration of renal cancer cells. CONCLUSIONS: These findings suggest that NMU may act in an autocrine fashion, promoting progression of kidney cancer. Hypoxia and HIF expression are frequently observed in many non-renal cancers and are associated with a poor prognosis. Our study raises the possibility that HIF may also drive NMU expression in non-renal tumours.

VHL inactivation induces HEF1 and Aurora kinase A.

The ciliary hypothesis for cystic renal diseases postulates that most of these conditions result from abnormalities in the primary cilium, a microtubule-based structure that acts as a sensor for extracellular cues. Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene predisposes to renal cysts and clear cell renal cell carcinoma. VHL plays a critical role in the formation of primary cilia in kidney epithelium, but the underlying mechanisms are poorly understood. Here, we demonstrate that VHL inactivation induces HEF1/Cas-L/NEDD9 and Aurora kinase A via the stabilization of hypoxia-inducible factors 1 and 2. Aurora kinase A is a mitotic kinase commonly upregulated in cancer that causes regression of the primary cilium by promoting histone deacetylase-dependent tubulin depolymerization of the ciliary axoneme. HEF1/Cas-L/NEDD9 is a component of focal adhesions that has a prominent role in inducing metastasis and that colocalizes with Aurora kinase A at the centrosome, thereby enhancing the harmful effect of Aurora kinase A on the cilium. Suppression of this pathway improved the formation of primary cilia and reduced cell motility in VHL-defective renal cancer cells. Our results highlight the gatekeeper role of VHL in the kidney epithelium.

HIF-1alpha and HIF-2alpha are differentially activated in distinct cell populations in retinal ischaemia.

BACKGROUND: Hypoxia plays a key role in ischaemic and neovascular disorders of the retina. Cellular responses to oxygen are mediated by hypoxia-inducible transcription factors (HIFs) that are stabilised in hypoxia and induce the expression of a diverse range of genes. The purpose of this study was to define the cellular specificities of HIF-1alpha and HIF-2alpha in retinal ischaemia, and to determine their correlation with the pattern of retinal hypoxia and the expression profiles of induced molecular mediators. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the tissue distribution of retinal hypoxia during oxygen-induced retinopathy (OIR) in mice using the bio-reductive drug pimonidazole. We measured the levels of HIF-1alpha and HIF-2alpha proteins by Western blotting and determined their cellular distribution by immunohistochemistry during the development of OIR. We measured the temporal expression profiles of two downstream mediators, vascular endothelial growth factor (VEGF) and erythropoietin (Epo) by ELISA. Pimonidazole labelling was evident specifically in the inner retina. Labelling peaked at 2 hours after the onset of hypoxia and gradually declined thereafter. Marked binding to Müller glia was evident during the early hypoxic stages of OIR. Both HIF-1alpha and HIF-2alpha protein levels were significantly increased during retinal hypoxia but were evident in distinct cellular distributions; HIF-1alpha stabilisation was evident in neuronal cells throughout the inner retinal layers whereas HIF-2alpha was restricted to Müller glia and astrocytes. Hypoxia and HIF-alpha stabilisation in the retina were closely followed by upregulated expression of the downstream mediators VEGF and EPO. CONCLUSIONS/SIGNIFICANCE: Both HIF-1alpha and HIF-2alpha are activated in close correlation with retinal hypoxia but have contrasting cell specificities, consistent with differential roles in retinal ischaemia. Our findings suggest that HIF-2alpha activation plays a key role in regulating the response of Müller glia to hypoxia.

Prolyl hydroxylase domain inhibitors: a route to HIF activation and neuroprotection.

Abstract Ischemic stroke is a major cause of death worldwide, and current therapeutic options are very limited. Preconditioning with an ischemic or hypoxic insult is beneficial in experimental models of ischemic stroke. Ischemia/hypoxia results in activation of numerous transcription factors, including hypoxia inducible factor (HIF), which is a master regulator of oxygen homeostasis. HIF activation induces a diverse range of target genes, encompassing a wide variety of cellular processes; including angiogenesis, energy metabolism, cell survival, radical production/scavenging, iron metabolism, stem cell homing, and differentiation. Inhibition of HIF prolyl hydroxylase domain (PHD) enzymes results in activation of HIF and is likely to mimic, at least in part, the effects of hypoxia preconditioning. A caveat is that not all consequences of HIF activation will be beneficial and some could even be deleterious. Nevertheless, PHD inhibitors may be therapeutically useful in the treatment of stroke. Prototype PHD inhibitors have shown promising results in preclinical models.

Regulation of renal epithelial tight junctions by the von Hippel-Lindau tumor suppressor gene involves occludin and claudin 1 and is independent of E-cadherin.

Epithelial-to-mesenchymal transitions (EMT) are important in renal development, fibrosis, and cancer. Loss of function of the tumor suppressor VHL leads to many features of EMT, and it has been hypothesized that the pivotal mediator is down-regulation of the adherens junction (AJ) protein E-cadherin. Here we show that VHL loss-of-function also has striking effects on the expression of the tight junction (TJ) components occludin and claudin 1 in vitro in VHL-defective clear cell renal cell carcinoma (CCRCC) cells and in vivo in VHL-defective sporadic CCRCCs (compared with normal kidney). Occludin is also down-regulated in premalignant foci in kidneys from patients with germline VHL mutations, consistent with a contribution to CCRCC initiation. Reexpression of E-cadherin was sufficient to restore AJ but not TJ assembly, indicating that the TJ defect is independent of E-cadherin down-regulation. Additional experiments show that activation of hypoxia inducible factor (HIF) contributes to both TJ and AJ abnormalities, thus the VHL/HIF pathway contributes to multiple aspects of the EMT phenotype that are not interdependent. Despite the independent nature of the defects, we show that treatment with the histone deacetylase inhibitor sodium butyrate, which suppresses HIF activation, provides a method for reversing EMT in the context of VHL inactivation.

Deletion of the von Hippel-Lindau gene in pancreatic beta cells impairs glucose homeostasis in mice.

Defective insulin secretion in response to glucose is an important component of the beta cell dysfunction seen in type 2 diabetes. As mitochondrial oxidative phosphorylation plays a key role in glucose-stimulated insulin secretion (GSIS), oxygen-sensing pathways may modulate insulin release. The von Hippel-Lindau (VHL) protein controls the degradation of hypoxia-inducible factor (HIF) to coordinate cellular and organismal responses to altered oxygenation. To determine the role of this pathway in controlling glucose-stimulated insulin release from pancreatic beta cells, we generated mice lacking Vhl in pancreatic beta cells (betaVhlKO mice) and mice lacking Vhl in the pancreas (PVhlKO mice). Both mouse strains developed glucose intolerance with impaired insulin secretion. Furthermore, deletion of Vhl in beta cells or the pancreas altered expression of genes involved in beta cell function, including those involved in glucose transport and glycolysis, and isolated betaVhlKO and PVhlKO islets displayed impaired glucose uptake and defective glucose metabolism. The abnormal glucose homeostasis was dependent on upregulation of Hif-1alpha expression, and deletion of Hif1a in Vhl-deficient beta cells restored GSIS. Consistent with this, expression of activated Hif-1alpha in a mouse beta cell line impaired GSIS. These data suggest that VHL/HIF oxygen-sensing mechanisms play a critical role in glucose homeostasis and that activation of this pathway in response to decreased islet oxygenation may contribute to beta cell dysfunction.

The A20 gene protects kidneys from ischaemia/reperfusion injury by suppressing pro-inflammatory activation.

Ischaemia followed by reperfusion (I/R) can induce inflammation and injury and is a risk factor for delayed graft function and rejection of transplanted kidneys. Inflammation is regulated by NF-kappaB transcription factors which induce pro-inflammatory molecules in endothelial cells (EC). We examined whether A20, a negative regulator of NF-kappaB, can protect kidneys from I/R injury. To mimic the fluctuations in endothelial oxygenation that occur during I/R we exposed cultured human umbilical vein EC (HUVEC) to hypoxia (1% O(2) for 4 h) followed by re-oxygenation (21% O(2) for 1 h-24 h). We observed transient expression of pro-inflammatory molecules (E-selectin, VCAM-1 and IL-8) and sustained expression of A20 in HUVEC exposed to hypoxia/re-oxygenation. The effect of A20 on endothelial responses to hypoxia/re-oxygenation was assessed. We observed that pre-treatment of HUVEC with an adenovirus containing A20 (Ad-A20) suppressed activation of NF-kappaB and induction of pro-inflammatory molecules by hypoxia/re-oxygenation, whereas a control adenovirus had little or no effect. Thus the induction of A20 may form a negative feedback loop in pro-inflammatory signalling in cells exposed to hypoxia/re-oxygenation. To validate our cell culture experiments we examined the role of A20 in renal responses to I/R. We observed that A20 was induced in rat kidneys exposed to I/R. Moreover, pre-treatment of animals with Ad-A20 significantly reduced acute tubular necrosis, renal expression of VCAM-1 and NF-kappaB activation in response to I/R, whereas pre-treatment with control adenovirus did not. Our observations suggest that A20 maintains physiological homeostasis in kidneys exposed to I/R by protecting them from inflammation and injury.

Deficiency or inhibition of oxygen sensor Phd1 induces hypoxia tolerance by reprogramming basal metabolism.

HIF prolyl hydroxylases (PHD1-3) are oxygen sensors that regulate the stability of the hypoxia-inducible factors (HIFs) in an oxygen-dependent manner. Here, we show that loss of Phd1 lowers oxygen consumption in skeletal muscle by reprogramming glucose metabolism from oxidative to more anaerobic ATP production through activation of a Pparalpha pathway. This metabolic adaptation to oxygen conservation impairs oxidative muscle performance in healthy conditions, but it provides acute protection of myofibers against lethal ischemia. Hypoxia tolerance is not due to HIF-dependent angiogenesis, erythropoiesis or vasodilation, but rather to reduced generation of oxidative stress, which allows Phd1-deficient myofibers to preserve mitochondrial respiration. Hypoxia tolerance relies primarily on Hif-2alpha and was not observed in heterozygous Phd2-deficient or homozygous Phd3-deficient mice. Of medical importance, conditional knockdown of Phd1 also rapidly induces hypoxia tolerance. These findings delineate a new role of Phd1 in hypoxia tolerance and offer new treatment perspectives for disorders characterized by oxidative stress.