Publications by authors named "Peter J Ratcliffe"

164 Publications

The HIF complex recruits the histone methyltransferase SET1B to activate specific hypoxia-inducible genes.

Nat Genet 2021 07 21;53(7):1022-1035. Epub 2021 Jun 21.

Cambridge Institute of Therapeutic Immunology and Infectious Disease (CITIID), Jeffrey Cheah Biomedical Centre, Department of Medicine, University of Cambridge, Cambridge, UK.

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.
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http://dx.doi.org/10.1038/s41588-021-00887-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611696PMC
July 2021

Hypoxic and pharmacological activation of HIF inhibits SARS-CoV-2 infection of lung epithelial cells.

Cell Rep 2021 04 5;35(3):109020. Epub 2021 Apr 5.

Nuffield Department of Medicine, University of Oxford, Oxford, UK; Chinese Academy of Medical Sciences (CAMS) Oxford Institute (COI), University of Oxford, Oxford, UK. Electronic address:

COVID-19, caused by the novel coronavirus SARS-CoV-2, is a global health issue with more than 2 million fatalities to date. Viral replication is shaped by the cellular microenvironment, and one important factor to consider is oxygen tension, in which hypoxia inducible factor (HIF) regulates transcriptional responses to hypoxia. SARS-CoV-2 primarily infects cells of the respiratory tract, entering via its spike glycoprotein binding to angiotensin-converting enzyme 2 (ACE2). We demonstrate that hypoxia and the HIF prolyl hydroxylase inhibitor Roxadustat reduce ACE2 expression and inhibit SARS-CoV-2 entry and replication in lung epithelial cells via an HIF-1α-dependent pathway. Hypoxia and Roxadustat inhibit SARS-CoV-2 RNA replication, showing that post-entry steps in the viral life cycle are oxygen sensitive. This study highlights the importance of HIF signaling in regulating multiple aspects of SARS-CoV-2 infection and raises the potential use of HIF prolyl hydroxylase inhibitors in the prevention or treatment of COVID-19.
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http://dx.doi.org/10.1016/j.celrep.2021.109020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8020087PMC
April 2021

Precisely Tuned Inhibition of HIF Prolyl Hydroxylases Is Key for Cardioprotection After Ischemia.

Circ Res 2021 Apr 25;128(8):1208-1210. Epub 2021 Feb 25.

Institute of Cardiovascular Physiology, University Medical Center Göttingen, Göttingen, Germany (A.J., A.Z., K.B.-C., A.M.V., D.M.K.).

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http://dx.doi.org/10.1161/CIRCRESAHA.120.318216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8048377PMC
April 2021

Marked and rapid effects of pharmacological HIF-2α antagonism on hypoxic ventilatory control.

J Clin Invest 2020 05;130(5):2237-2251

Target Discovery Institute and.

Hypoxia-inducible factor (HIF) is strikingly upregulated in many types of cancer, and there is great interest in applying inhibitors of HIF as anticancer therapeutics. The most advanced of these are small molecules that target the HIF-2 isoform through binding the PAS-B domain of HIF-2α. These molecules are undergoing clinical trials with promising results in renal and other cancers where HIF-2 is considered to be driving growth. Nevertheless, a central question remains as to whether such inhibitors affect physiological responses to hypoxia at relevant doses. Here, we show that pharmacological HIF-2α inhibition with PT2385, at doses similar to those reported to inhibit tumor growth, rapidly impaired ventilatory responses to hypoxia, abrogating both ventilatory acclimatization and carotid body cell proliferative responses to sustained hypoxia. Mice carrying a HIF-2α PAS-B S305M mutation that disrupts PT2385 binding, but not dimerization with HIF-1β, did not respond to PT2385, indicating that these effects are on-target. Furthermore, the finding of a hypomorphic ventilatory phenotype in untreated HIF-2α S305M mutant mice suggests a function for the HIF-2α PAS-B domain beyond heterodimerization with HIF-1β. Although PT2385 was well tolerated, the findings indicate the need for caution in patients who are dependent on hypoxic ventilatory drive.
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http://dx.doi.org/10.1172/JCI133194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7190921PMC
May 2020

Genetic basis of oxygen sensing in the carotid body: HIF2α and an isoform switch in cytochrome c oxidase subunit 4.

Sci Signal 2020 01 21;13(615). Epub 2020 Jan 21.

Target Discovery Institute, University of Oxford, Oxford OX3 7FZ, UK.

The mechanistic basis of the marked oxygen sensitivity of glomus cells in the carotid body has long puzzled physiologists. In this issue of , Moreno-Domínguez show the critical importance of high levels of hypoxia-inducible factor, HIF2α/EPAS1, and the nuclear-encoded mitochondrial cytochrome c oxidase subunit, COX4I2, in glomus cell sensitivity to hypoxia.
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http://dx.doi.org/10.1126/scisignal.aba1302DOI Listing
January 2020

Co-incidence of RCC-susceptibility polymorphisms with HIF cis-acting sequences supports a pathway tuning model of cancer.

Sci Rep 2019 12 10;9(1):18768. Epub 2019 Dec 10.

NDM Research Building, University of Oxford, Old Road Campus, Headington, Oxford, OX3 7FZ, United Kingdom.

Emerging evidence suggests that dysregulation of oncogenic pathways requires precise tuning in order for cancer to develop. To test this, we examined the overlap between cis-acting elements of the hypoxia-inducible factor (HIF) pathway and cancer-susceptibility polymorphisms as defined in genome-wide association studies (GWAS). In renal cancer, where HIF is constitutively and un-physiologically activated by mutation of the von Hippel-Lindau tumour suppressor, we observed marked excess overlap, which extended to potential susceptibility polymorphisms that are below the conventional threshold applied in GWAS. In contrast, in other cancers where HIF is upregulated by different mechanisms, including micro-environmental hypoxia, we observed no excess in overlap. Our findings support a 'pathway tuning' model of cancer, whereby precise modulation of multiple outputs of specific, activated pathways is important in oncogenesis. This implies that selective pressures to modulate such pathways operate during cancer development and should focus attempts to identify their nature and consequences.
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http://dx.doi.org/10.1038/s41598-019-55098-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6904466PMC
December 2019

Structure-Activity Relationship and Crystallographic Studies on 4-Hydroxypyrimidine HIF Prolyl Hydroxylase Domain Inhibitors.

ChemMedChem 2020 02 3;15(3):270-273. Epub 2019 Dec 3.

Department of Chemistry, University of Oxford Chemistry Research Laboratory, 12 Mansfield Road, Oxford, OX1 3TA, UK.

The 2-oxoglutarate-dependent hypoxia inducible factor prolyl hydroxylases (PHDs) are targets for treatment of a variety of diseases including anaemia. One PHD inhibitor is approved for use for the treatment of renal anaemia and others are in late stage clinical trials. The number of reported templates for PHD inhibition is limited. We report structure-activity relationship and crystallographic studies on a promising class of 4-hydroxypyrimidine-containing PHD inhibitors.
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http://dx.doi.org/10.1002/cmdc.201900557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496690PMC
February 2020

Hypoxia drives glucose transporter 3 expression through hypoxia-inducible transcription factor (HIF)-mediated induction of the long noncoding RNA NICI.

J Biol Chem 2020 03 5;295(13):4065-4078. Epub 2019 Nov 5.

Department of Nephrology and Hypertension, Universitätsklinikum Erlangen and Friedrich-Alexander-Universität Erlangen-Nürnberg, Ulmenweg 18, 91054 Erlangen, Germany. Electronic address:

Hypoxia-inducible transcription factors (HIFs) directly dictate the expression of multiple RNA species including novel and as yet uncharacterized long noncoding transcripts with unknown function. We used pan-genomic HIF-binding and transcriptomic data to identify a novel long noncoding RNA oncoding ntergenic o-nduced transcript (NICI) 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 () which encodes the high-affinity glucose transporter 3 (GLUT3). Knockdown or knockout 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 is mediated through transcriptional activation rather than posttranscriptional mechanisms because knockout of NICI leads to reduced recruitment of RNA polymerase 2 to the promoter. Consistent with this we observe NICI-dependent regulation of glucose consumption and cell proliferation. Furthermore, NICI expression is regulated by the von Hippel-Lindau (VHL) tumor suppressor and is highly expressed in clear cell renal cell carcinoma (ccRCC), where SLC2A3 expression is associated with patient prognosis, implying an important role for the HIF/NICI/SLC2A3 axis in this malignancy.
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http://dx.doi.org/10.1074/jbc.RA119.009827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7105321PMC
March 2020

Lack of activity of recombinant HIF prolyl hydroxylases (PHDs) on reported non-HIF substrates.

Elife 2019 09 10;8. Epub 2019 Sep 10.

The Francis Crick Institute, London, United Kingdom.

Human and other animal cells deploy three closely related dioxygenases (PHD 1, 2 and 3) to signal oxygen levels by catalysing oxygen regulated prolyl hydroxylation of the transcription factor HIF. The discovery of the HIF prolyl-hydroxylase (PHD) enzymes as oxygen sensors raises a key question as to the existence and nature of non-HIF substrates, potentially transducing other biological responses to hypoxia. Over 20 such substrates are reported. We therefore sought to characterise their reactivity with recombinant PHD enzymes. Unexpectedly, we did not detect prolyl-hydroxylase activity on any reported non-HIF protein or peptide, using conditions supporting robust HIF-α hydroxylation. We cannot exclude PHD-catalysed prolyl hydroxylation occurring under conditions other than those we have examined. However, our findings using recombinant enzymes provide no support for the wide range of non-HIF PHD substrates that have been reported.
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http://dx.doi.org/10.7554/eLife.46490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6739866PMC
September 2019

Mechanisms of hypoxia signalling: new implications for nephrology.

Nat Rev Nephrol 2019 10 5;15(10):641-659. Epub 2019 Sep 5.

Ludwig Instute for Cancer Research and Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK.

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.
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http://dx.doi.org/10.1038/s41581-019-0182-zDOI Listing
October 2019

Conserved N-terminal cysteine dioxygenases transduce responses to hypoxia in animals and plants.

Science 2019 07;365(6448):65-69

Ludwig Institute for Cancer Research, Nuffield Department of Medicine, University of Oxford, Oxford OX3 7FZ, UK.

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- O) 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.
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http://dx.doi.org/10.1126/science.aaw0112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715447PMC
July 2019

Systemic silencing of PHD2 causes reversible immune regulatory dysfunction.

J Clin Invest 2019 06 4;129(9):3640-3656. Epub 2019 Jun 4.

Nuffield Department of Medicine Research Building, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom.

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.
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http://dx.doi.org/10.1172/JCI124099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715380PMC
June 2019

Inherent DNA-binding specificities of the HIF-1α and HIF-2α transcription factors in chromatin.

EMBO Rep 2019 01 14;20(1). Epub 2018 Nov 14.

NDM Research Building, University of Oxford, Oxford, UK

Hypoxia-inducible factor (HIF) is the major transcriptional regulator of cellular responses to hypoxia. The two principal HIF-α isoforms, HIF-1α and HIF-2α, are progressively stabilized in response to hypoxia and form heterodimers with HIF-1β to activate a broad range of transcriptional responses. Here, we report on the pan-genomic distribution of isoform-specific HIF binding in response to hypoxia of varying severity and duration, and in response to genetic ablation of each HIF-α isoform. Our findings reveal that, despite an identical consensus recognition sequence in DNA, each HIF heterodimer loads progressively at a distinct repertoire of cell-type-specific sites across the genome, with little evidence of redistribution under any of the conditions examined. Marked biases towards promoter-proximal binding of HIF-1 and promoter-distant binding of HIF-2 were observed under all conditions and were consistent in multiple cell type. The findings imply that each HIF isoform has an inherent property that determines its binding distribution across the genome, which might be exploited to therapeutically target the specific transcriptional output of each isoform independently.
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http://dx.doi.org/10.15252/embr.201846401DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322389PMC
January 2019

Nuclear entry and export of FIH are mediated by HIF1α and exportin1, respectively.

J Cell Sci 2018 11 19;131(22). Epub 2018 Nov 19.

Ludwig Institute for Cancer Research Ltd., Nuffield Department of Clinical Medicine, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, United Kingdom

Hypoxia plays a crucial role at cellular and physiological levels in all animals. The responses to chronic hypoxia are, at least substantially, orchestrated by activation of the hypoxia inducible transcription factors (HIFs), whose stability and subsequent transcriptional activation are regulated by HIF hydroxylases. Factor inhibiting HIF (FIH), initially isolated as a HIFα interacting protein following a yeast two-hybrid screen, is an asparaginyl hydroxylase that negatively regulates transcriptional activation by HIF. This study aimed to define the mechanisms that govern transitions of FIH between the nucleus and cytoplasm. We report that FIH accumulates in the nucleus within a short time window during hypoxia treatment. We provide evidence, based on the application of genetic interventions and small molecule inhibition of the HIF hydroxylases, that the nuclear localization of FIH is governed by two opposing processes: nuclear entry by 'coupling' with HIF1α for importin β1-mediated nuclear import and active export via a Leptomycin B-sensitive exportin1-dependent pathway.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.219782DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6250434PMC
November 2018

Publisher Correction: The Jumonji-C oxygenase JMJD7 catalyzes (3S)-lysyl hydroxylation of TRAFAC GTPases.

Nat Chem Biol 2018 Oct;14(10):988

Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, UK.

In the version of this article initially published, authors Sarah E. Wilkins, Charlotte D. Eaton, Martine I. Abboud and Maximiliano J. Katz were incorrectly included in the equal contributions footnote in the affiliations list. Footnote number seven linking to the equal contributions statement should be present only for Suzana Markolovic and Qinqin Zhuang, and the statement should read "These authors contributed equally: Suzana Markolovic, Qinqin Zhuang." The error has been corrected in the HTML and PDF versions of the article.
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http://dx.doi.org/10.1038/s41589-018-0104-6DOI Listing
October 2018

PHD2 inactivation in Type I cells drives HIF-2α-dependent multilineage hyperplasia and the formation of paraganglioma-like carotid bodies.

J Physiol 2018 Jun 19. Epub 2018 Jun 19.

Target Discovery Institute, University of Oxford, Oxford, UK.

Key Points: The carotid body is a peripheral arterial chemoreceptor that regulates ventilation in response to both acute and sustained hypoxia. Type I cells in this organ respond to low oxygen both acutely by depolarization and dense core vesicle secretion and, over the longer term, via cellular proliferation and enhanced ventilatory responses. Using lineage analysis, the present study shows that the Type I cell lineage itself proliferates and expands in response to sustained hypoxia. Inactivation of HIF-2α in Type I cells impairs the ventilatory, proliferative and cell intrinsic (dense core vesicle) responses to hypoxia. Inactivation of PHD2 in Type I cells induces multilineage hyperplasia and ultrastructural changes in dense core vesicles to form paraganglioma-like carotid bodies. These changes, similar to those observed in hypoxia, are dependent on HIF-2α. Taken together, these findings demonstrate a key role for the PHD2-HIF-2α couple in Type I cells with respect to the oxygen sensing functions of the carotid body.

Abstract: The carotid body is a peripheral chemoreceptor that plays a central role in mammalian oxygen homeostasis. In response to sustained hypoxia, it manifests a rapid cellular proliferation and an associated increase in responsiveness to hypoxia. Understanding the cellular and molecular mechanisms underlying these processes is of interest both to specialized chemoreceptive functions of that organ and, potentially, to the general physiology and pathophysiology of cellular hypoxia. We have combined cell lineage tracing technology and conditionally inactivated alleles in recombinant mice to examine the role of components of the HIF hydroxylase pathway in specific cell types within the carotid body. We show that exposure to sustained hypoxia (10% oxygen) drives rapid expansion of the Type I, tyrosine hydroxylase expressing cell lineage, with little transdifferentiation to (or from) that lineage. Inactivation of a specific HIF isoform, HIF-2α, in the Type I cells was associated with a greatly reduced proliferation of Type I cells and hypoxic ventilatory responses, with ultrastructural evidence of an abnormality in the action of hypoxia on dense core secretory vesicles. We also show that inactivation of the principal HIF prolyl hydroxylase PHD2 within the Type I cell lineage is sufficient to cause multilineage expansion of the carotid body, with characteristics resembling paragangliomas. These morphological changes were dependent on the integrity of HIF-2α. These findings implicate specific components of the HIF hydroxylase pathway (PHD2 and HIF-2α) within Type I cells of the carotid body with respect to the oxygen sensing and adaptive functions of that organ.
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http://dx.doi.org/10.1113/JP275996DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6138294PMC
June 2018

The Jumonji-C oxygenase JMJD7 catalyzes (3S)-lysyl hydroxylation of TRAFAC GTPases.

Nat Chem Biol 2018 07 18;14(7):688-695. Epub 2018 Jun 18.

Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, UK.

Biochemical, structural and cellular studies reveal Jumonji-C (JmjC) domain-containing 7 (JMJD7) to be a 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes (3S)-lysyl hydroxylation. Crystallographic analyses reveal JMJD7 to be more closely related to the JmjC hydroxylases than to the JmjC demethylases. Biophysical and mutation studies show that JMJD7 has a unique dimerization mode, with interactions between monomers involving both N- and C-terminal regions and disulfide bond formation. A proteomic approach identifies two related members of the translation factor (TRAFAC) family of GTPases, developmentally regulated GTP-binding proteins 1 and 2 (DRG1/2), as activity-dependent JMJD7 interactors. Mass spectrometric analyses demonstrate that JMJD7 catalyzes Fe(II)- and 2OG-dependent hydroxylation of a highly conserved lysine residue in DRG1/2; amino-acid analyses reveal that JMJD7 catalyzes (3S)-lysyl hydroxylation. The functional assignment of JMJD7 will enable future studies to define the role of DRG hydroxylation in cell growth and disease.
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http://dx.doi.org/10.1038/s41589-018-0071-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6027965PMC
July 2018

Molecular and cellular mechanisms of HIF prolyl hydroxylase inhibitors in clinical trials.

Chem Sci 2017 Nov 11;8(11):7651-7668. Epub 2017 Sep 11.

Chemistry Research Laboratory , Department of Chemistry , University of Oxford , Oxford OX1 3TA , UK . Email:

Inhibition of the human 2-oxoglutarate (2OG) dependent hypoxia inducible factor (HIF) prolyl hydroxylases (human PHD1-3) causes upregulation of HIF, thus promoting erythropoiesis and is therefore of therapeutic interest. We describe cellular, biophysical, and biochemical studies comparing four PHD inhibitors currently in clinical trials for anaemia treatment, that describe their mechanisms of action, potency against isolated enzymes and in cells, and selectivities representatives of other human 2OG oxygenase subfamilies. The 'clinical' PHD inhibitors are potent inhibitors of PHD catalyzed hydroxylation of the HIF-α oxygen dependent degradation domains (ODDs), and selective against most, but not all, representatives of other human 2OG dependent dioxygenase subfamilies. Crystallographic and NMR studies provide insights into the different active site binding modes of the inhibitors. Cell-based results reveal the inhibitors have similar effects on the upregulation of HIF target genes, but differ in the kinetics of their effects and in extent of inhibition of hydroxylation of the N- and C-terminal ODDs; the latter differences correlate with the biophysical observations.
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http://dx.doi.org/10.1039/c7sc02103hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5802278PMC
November 2017

Prolyl hydroxylase 2 inactivation enhances glycogen storage and promotes excessive neutrophilic responses.

J Clin Invest 2017 Sep 14;127(9):3407-3420. Epub 2017 Aug 14.

MRC/University of Edinburgh Centre for Inflammation Research, Queen's Medical Research Institute, University of Edinburgh, Edinburgh, United Kingdom.

Fully activated innate immune cells are required for effective responses to infection, but their prompt deactivation and removal are essential for limiting tissue damage. Here, we have identified a critical role for the prolyl hydroxylase enzyme Phd2 in maintaining the balance between appropriate, predominantly neutrophil-mediated pathogen clearance and resolution of the innate immune response. We demonstrate that myeloid-specific loss of Phd2 resulted in an exaggerated inflammatory response to Streptococcus pneumonia, with increases in neutrophil motility, functional capacity, and survival. These enhanced neutrophil responses were dependent upon increases in glycolytic flux and glycogen stores. Systemic administration of a HIF-prolyl hydroxylase inhibitor replicated the Phd2-deficient phenotype of delayed inflammation resolution. Together, these data identify Phd2 as the dominant HIF-hydroxylase in neutrophils under normoxic conditions and link intrinsic regulation of glycolysis and glycogen stores to the resolution of neutrophil-mediated inflammatory responses. These results demonstrate the therapeutic potential of targeting metabolic pathways in the treatment of inflammatory disease.
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http://dx.doi.org/10.1172/JCI90848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5669581PMC
September 2017

Multiple renal cancer susceptibility polymorphisms modulate the HIF pathway.

PLoS Genet 2017 Jul 17;13(7):e1006872. Epub 2017 Jul 17.

Department of Nephrology and Hypertension, Universitätsklinikum Erlangen und Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany.

Un-physiological activation of hypoxia inducible factor (HIF) is an early event in most renal cell cancers (RCC) following inactivation of the von Hippel-Lindau tumor suppressor. Despite intense study, how this impinges on cancer development is incompletely understood. To test for the impact of genetic signals on this pathway, we aligned human RCC-susceptibility polymorphisms with genome-wide assays of HIF-binding and observed highly significant overlap. Allele-specific assays of HIF binding, chromatin conformation and gene expression together with eQTL analyses in human tumors were applied to mechanistic analysis of one such overlapping site at chromosome 12p12.1. This defined a novel stage-specific mechanism in which the risk polymorphism, rs12814794, directly creates a new HIF-binding site that mediates HIF-1α isoform specific upregulation of its target BHLHE41. The alignment of multiple sites in the HIF cis-acting apparatus with RCC-susceptibility polymorphisms strongly supports a causal model in which minor variation in this pathway exerts significant effects on RCC development.
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http://dx.doi.org/10.1371/journal.pgen.1006872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5536434PMC
July 2017

New horizons in hypoxia signaling pathways.

Exp Cell Res 2017 07 15;356(2):116-121. Epub 2017 Mar 15.

Target Discovery Institute, University of Oxford, OX3 7FZ, UK; Francis Crick Institute, Midland Road, London NW1 1AT, UK. Electronic address:

Investigation into the regulation of the erythropoietin gene by oxygen led to the discovery of a process of direct oxygen sensing that transduces many cellular and systemic responses to hypoxia. The oxygen-sensitive signal is generated through the catalytic action of a series of 2-oxoglutarate-dependent oxygenases that regulate the transcription factor hypoxia-inducible factor (HIF) by the post-translational hydroxylation of specific amino acid residues. Here we review the implications of the unforeseen complexity of the HIF transcriptional cascade for the physiology and pathophysiology of hypoxia, and consider the origins of post-translational hydroxylation as a signaling process.
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http://dx.doi.org/10.1016/j.yexcr.2017.03.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5653532PMC
July 2017

A Ribosomopathy Reveals Decoding Defective Ribosomes Driving Human Dysmorphism.

Am J Hum Genet 2017 Mar;100(3):506-522

Department of Hematopoiesis, Sanquin, and Landsteiner Laboratory, AMC/UvA, 1066 CX Amsterdam, the Netherlands; Laboratory Genetic Metabolic Diseases, Academic Medical Center, 1105 AZ Amsterdam, the Netherlands. Electronic address:

Ribosomal protein (RP) gene mutations, mostly associated with inherited or acquired bone marrow failure, are believed to drive disease by slowing the rate of protein synthesis. Here de novo missense mutations in the RPS23 gene, which codes for uS12, are reported in two unrelated individuals with microcephaly, hearing loss, and overlapping dysmorphic features. One individual additionally presents with intellectual disability and autism spectrum disorder. The amino acid substitutions lie in two highly conserved loop regions of uS12 with known roles in maintaining the accuracy of mRNA codon translation. Primary cells revealed one substitution severely impaired OGFOD1-dependent hydroxylation of a neighboring proline residue resulting in 40S ribosomal subunits that were blocked from polysome formation. The other disrupted a predicted pi-pi stacking interaction between two phenylalanine residues leading to a destabilized uS12 that was poorly tolerated in 40S subunit biogenesis. Despite no evidence of a reduction in the rate of mRNA translation, these uS12 variants impaired the accuracy of mRNA translation and rendered cells highly sensitive to oxidative stress. These discoveries describe a ribosomopathy linked to uS12 and reveal mechanistic distinctions between RP gene mutations driving hematopoietic disease and those resulting in developmental disorders.
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http://dx.doi.org/10.1016/j.ajhg.2017.01.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5339345PMC
March 2017

Hyperplasia and hypertrophy of pulmonary neuroepithelial bodies, presumed airway hypoxia sensors, in hypoxia-inducible factor prolyl hydroxylase-deficient mice.

Hypoxia (Auckl) 2016 12;4:69-80. Epub 2016 Apr 12.

Division of Pathology, Department of Pediatric Laboratory Medicine, The Research Institute, The Hospital for Sick Children; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.

Pulmonary neuroepithelial bodies (NEBs), presumed polymodal airway sensors, consist of innervated clusters of amine (serotonin) and peptide-producing cells. While NEB responses to acute hypoxia are mediated by a membrane-bound O sensor complex, responses to sustained and/or chronic hypoxia involve a prolyl hydroxylase (PHD)-hypoxia-inducible factor-dependent mechanism. We have previously reported hyperplasia of NEBs in the lungs of -/- mice associated with enhanced serotonin secretion. Here we use a novel multilabel immunofluorescence method to assess NEB distribution, frequency, and size, together with the number and size of NEB cell nuclei, and to colocalize multiple cytoplasmic and nuclear epitopes in the lungs of -/-, +/-, and -/- mice and compare them with wild-type controls. To define the mechanisms of NEB cell hyperplasia, we used antibodies against Mash1 and Prox1 (neurogenic genes involved in NEB cell differentiation/maturation), hypoxia-inducible factor-1alpha, and the cell proliferation marker Ki67. Morphometric analysis of (% total lung area) immunostaining for synaptophysin (% synaptophysin), a cytoplasmic marker of NEB cells, was significantly increased in -/- and -/- mice compared to wild-type mice. In addition, NEB size and the number and size of NEB nuclei were also significantly increased, indicating that deficiency of s is associated with striking hyperplasia and hypertrophy of NEBs. In +/- mice, while mean % synaptophysin was comparable to wild-type controls, the NEB size was moderately increased, suggesting an effect even in heterozygotes. NEBs in all -deficient mice showed increased expression of Mash1, Prox1, Ki67, and hypoxia-inducible factor-1alpha, in keeping with enhanced differentiation from precursor cells and a minor component of cell proliferation. Since the loss of PHD activity mimics chronic hypoxia, our data provide critical information on the potential role of PHDs in the pathobiology and mechanisms of NEB cell hyperplasia that is relevant to a number of pediatric lung disorders.
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http://dx.doi.org/10.2147/HP.S103957DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5085281PMC
April 2016

Gene panel sequencing improves the diagnostic work-up of patients with idiopathic erythrocytosis and identifies new mutations.

Haematologica 2016 11 20;101(11):1306-1318. Epub 2016 Sep 20.

National Institute for Health Research (NIHR) Comprehensive Biomedical Research Centre, Oxford, UK.

Erythrocytosis is a rare disorder characterized by increased red cell mass and elevated hemoglobin concentration and hematocrit. Several genetic variants have been identified as causes for erythrocytosis in genes belonging to different pathways including oxygen sensing, erythropoiesis and oxygen transport. However, despite clinical investigation and screening for these mutations, the cause of disease cannot be found in a considerable number of patients, who are classified as having idiopathic erythrocytosis. In this study, we developed a targeted next-generation sequencing panel encompassing the exonic regions of 21 genes from relevant pathways (~79 Kb) and sequenced 125 patients with idiopathic erythrocytosis. The panel effectively screened 97% of coding regions of these genes, with an average coverage of 450×. It identified 51 different rare variants, all leading to alterations of protein sequence, with 57 out of 125 cases (45.6%) having at least one of these variants. Ten of these were known erythrocytosis-causing variants, which had been missed following existing diagnostic algorithms. Twenty-two were novel variants in erythrocytosis-associated genes (EGLN1, EPAS1, VHL, BPGM, JAK2, SH2B3) and in novel genes included in the panel (e.g. EPO, EGLN2, HIF3A, OS9), some with a high likelihood of functionality, for which future segregation, functional and replication studies will be useful to provide further evidence for causality. The rest were classified as polymorphisms. Overall, these results demonstrate the benefits of using a gene panel rather than existing methods in which focused genetic screening is performed depending on biochemical measurements: the gene panel improves diagnostic accuracy and provides the opportunity for discovery of novel variants.
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http://dx.doi.org/10.3324/haematol.2016.144063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5394871PMC
November 2016

Genetic variation at the 8q24.21 renal cancer susceptibility locus affects HIF binding to a MYC enhancer.

Nat Commun 2016 10 24;7:13183. Epub 2016 Oct 24.

Department of Nephrology and Hypertension, Universitätsklinikum Erlangen und Friedrich-Alexander-Universität (FAU) Erlangen-Nürnberg, Ulmenweg 18, 91054 Erlangen, Germany.

Clear cell renal cell carcinoma (ccRCC) is characterized by loss of function of the von Hippel-Lindau tumour suppressor (VHL) and unrestrained activation of hypoxia-inducible transcription factors (HIFs). Genetic and epigenetic determinants have an impact on HIF pathways. A recent genome-wide association study on renal cancer susceptibility identified single-nucleotide polymorphisms (SNPs) in an intergenic region located between the oncogenes MYC and PVT1. Here using assays of chromatin conformation, allele-specific chromatin immunoprecipitation and genome editing, we show that HIF binding to this regulatory element is necessary to trans-activate MYC and PVT1 expression specifically in cells of renal tubular origins. Moreover, we demonstrate that the risk-associated polymorphisms increase chromatin accessibility and activity as well as HIF binding to the enhancer. These findings provide further evidence that genetic variation at HIF-binding sites modulates the oncogenic transcriptional output of the VHL-HIF axis and provide a functional explanation for the disease-associated effects of SNPs in ccRCC.
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http://dx.doi.org/10.1038/ncomms13183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5079059PMC
October 2016

Expression of Idh1 in the Murine Subventricular Zone Stem Cell Niche Recapitulates Features of Early Gliomagenesis.

Cancer Cell 2016 10 29;30(4):578-594. Epub 2016 Sep 29.

Molecular & Population Genetics Laboratory, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK. Electronic address:

Isocitrate dehydrogenase 1 mutations drive human gliomagenesis, probably through neomorphic enzyme activity that produces D-2-hydroxyglutarate. To model this disease, we conditionally expressed Idh1 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 α-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 overexpressed Wnt, cell-cycle and stem cell genes, and shared an expression signature with human gliomas. Idh1 mutation in the major adult neurogenic stem cell niche causes a phenotype resembling gliomagenesis.
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http://dx.doi.org/10.1016/j.ccell.2016.08.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5064912PMC
October 2016

Pathways for Oxygen Regulation and Homeostasis: The 2016 Albert Lasker Basic Medical Research Award.

JAMA 2016 Sep;316(12):1252-3

McKusick-Nathans Institute of Genetic Medicine and Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland.

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http://dx.doi.org/10.1001/jama.2016.12386DOI Listing
September 2016

Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases.

Nat Commun 2016 08 26;7:12673. Epub 2016 Aug 26.

Chemistry Research Laboratory, Department of Chemistry, Oxford Centre for Integrative Systems Biology, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK.

The response to hypoxia in animals involves the expression of multiple genes regulated by the αβ-hypoxia-inducible transcription factors (HIFs). The hypoxia-sensing mechanism involves oxygen limited hydroxylation of prolyl residues in the N- and C-terminal oxygen-dependent degradation domains (NODD and CODD) of HIFα isoforms, as catalysed by prolyl hydroxylases (PHD 1-3). Prolyl hydroxylation promotes binding of HIFα to the von Hippel-Lindau protein (VHL)-elongin B/C complex, thus signalling for proteosomal degradation of HIFα. We reveal that certain PHD2 variants linked to familial erythrocytosis and cancer are highly selective for CODD or NODD. Crystalline and solution state studies coupled to kinetic and cellular analyses reveal how wild-type and variant PHDs achieve ODD selectivity via different dynamic interactions involving loop and C-terminal regions. The results inform on how HIF target gene selectivity is achieved and will be of use in developing selective PHD inhibitors.
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http://dx.doi.org/10.1038/ncomms12673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007464PMC
August 2016
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