Tuning the transcriptional response to hypoxia through HIF prolyl- and asparaginyl-hydroxylase inhibition:Sequencing Data
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PROVIDER: PRJEB14955 | ENA |
REPOSITORIES: ENA
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Project description:The hypoxia inducible factor (HIF) system orchestrates cellular responses to hypoxia in animals. HIF is an /-heterodimeric transcription factor that regulates the expression of hundreds of genes in a context dependent manner. A hypoxia-sensing component of the HIF system involves oxygen-dependent catalysis by the HIF hydroxylases; in humans there are three HIF prolyl hydroxylases (PHD1-3) and an asparaginyl hydroxylase (FIH). PHD catalysis regulates HIF levels and FIH catalysis regulates HIF activity. How differences in HIF hydroxylation status relate to variations in the induction of HIF target gene transcription is unknown. We report studies using small molecule inhibitors of the HIF hydroxylases to investigate the extent to which HIF target gene upregulation is induced by reduced PHD catalysis. The results reveal substantial differences in the role of prolyl- and asparaginyl-hydroxylation in regulating hypoxia responsive genes in cells. Selective PHD inhibitors with different structural scaffolds behave similarly. However, under the tested conditions, a broad-spectrum 2OG dioxygenase inhibitor is a better mimic of the transcriptional response to hypoxia than the selective PHD inhibitors, consistent with an important role for FIH in the hypoxic transcriptional response. Indeed, combined application of selective PHD and FIH inhibitors resulted in transcriptional induction of a subset of genes that were not fully responsive to PHD inhibition alone. Thus, for the therapeutic regulation of HIF target genes, it is important to consider both PHD and FIH activity, and in the case of some sets of target genes, simultaneous inhibition of the PHDs and FIH catalysis may be preferable.
2016-10-25 | E-MTAB-4264 | biostudies-arrayexpress
Project description:The hypoxia inducible factor (HIF) system orchestrates cellular responses to hypoxia in animals. HIF is an α/β-heterodimeric transcription factor that regulates the expression of hundreds of genes in a context dependent manner. A hypoxia-sensing component of the HIF system involves oxygen-dependent catalysis by the HIF hydroxylases; in humans there are three HIF prolyl hydroxylases (PHD1-3) and an asparaginyl hydroxylase (FIH). PHD catalysis regulates HIFα levels and FIH catalysis regulates HIF activity. How differences in HIFα hydroxylation status relate to variations in the induction of HIF target gene transcription is unknown. We report studies using small molecule inhibitors of the HIF hydroxylases to investigate the extent to which HIF target gene upregulation is induced by reduced PHD catalysis. The results reveal substantial differences in the role of prolyl- and asparaginyl-hydroxylation in regulating hypoxia responsive genes in cells. Selective PHD inhibitors with different structural scaffolds behave similarly. However, under the tested conditions, a broad-spectrum 2OG dioxygenase inhibitor is a better mimic of the transcriptional response to hypoxia than the selective PHD inhibitors, consistent with an important role for FIH in the hypoxic transcriptional response. Indeed, combined application of selective PHD and FIH inhibitors resulted in transcriptional induction of a subset of genes that were not fully responsive to PHD inhibition alone. Thus, for the therapeutic regulation of HIF target genes, it is important to consider both PHD and FIH activity, and in the case of some sets of target genes, simultaneous inhibition of the PHDs and FIH catalysis may be preferable.
2016-08-31 | E-MTAB-4300 | biostudies-arrayexpress
Project description:Skeletal muscle has an impressive ability to repair itself after a damaging insult and this response is essential to the process of muscle adaptation. In conditions such as muscular dystrophy and the sarcopenia of old age, repair is compromised leading to fibrosis and fatty tissue accumulation. Hypoxia-inducible factors (HIFs) are highly conserved regulators of gene transcription under conditions of low oxygen tension and HIF target genes such as EPO and VEGF have been associated with muscle protection and repair. We sought to interrogate the importance of HIF activation to skeletal muscle repair through the use of prolyl hydroxylase inhibitors (PHI) that stabilize HIF and activate target gene transcription in a mouse eccentric exercise limb damage model. We used microarrays to detail the global effects of prolyl hydroxylase inhibitors (PHI) in a mouse eccentric exercise limb damage model.
2017-08-23 | GSE95244 | GEO
Project description:Hypoxia Inducible Factor (HIF) prolyl hydroxylase domain (PHD) enzymes catalyse the posttranslational hydroxylation of conserved prolyl residues in the alpha-subunit of the HIF transcription factor. These modifications, which promote the degradation of HIF-alpha subunits by the pVHL E3 ligase complex and impart oxygen-dependent regulation of the HIF transcriptional response, have been extensively characterised at the molecular, cellular and organismal level. Since the discovery of the PHDs, a range of less well-characterised non-HIF substrates have been reported with the potential to confer oxygen-sensitivity to a diverse range of cellular processes. We sought to systematically compare all of the reported non-HIF substrates for their ability to support PHD-catalysed hydroxylation. We performed a comprehensive series of in vitro hydroxylation assays reacting synthetic peptides and full-length protein substrates generated by in vitro transcription and translation with purified recombinant enzyme preparations. Prolyl hydroxylation was assayed directly by mass spectrometry and radiochemical assay for hydroxyproline. Both methods enabled quantitative appraisal of enzyme-catalysed hydroxylation, with liquid chromatography mass spectrometry methods employing NMR-quantified peptide standards to calibrate retention time signatures and determine ionisation efficiencies for each target peptide. Using these approaches we assayed hydroxylation on 23 different proteins. Surprisingly, we did not detect measurable hydroxylation on any of the reported non-HIF substrates using either method. In contrast, control assays with HIF1-alpha substrate supported high stoichiometry (typically >90%) hydroxylation. Our findings suggest that PHD substrates are more restricted than has been reported.
2019-08-30 | PXD013112 | Pride
Project description:Loss of Endothelial HIF-Prolyl hydroxylase 2 (PHD2) Induces Cardiac Hypertrophy and Fibrosis
| PRJNA758078 | ENA
Project description:Acute myeloid leukaemia (AML) is a largely incurable haematological disease, for which new efficient therapeutic strategies are urgently needed. While leukaemogenesis occurs under hypoxic conditions of the bone marrow, the therapeutic tractability of the hypoxia inducible factor (HIF) system in AML is elusive. Given that inactivation of HIF-1α and HIF-2α promotes leukaemic transformation, a possible therapeutic strategy for AML treatment is to constitutively stabilise HIF- α proteins. This can be achieved by targeting the HIF-prolyl hydroxylases (PHDs), the catalysis of which promotes HIF-1α and HIF-2α degradation. Here, we demonstrate that genetic inactivation of Phd1 or Phd2 compromises initiation and progression of AML, without impacting normal haematopoiesis, thus implying the immense therapeutic potential of targeting PHDs in AML. To pharmacologically inactivate PHDs, we employed clinical grade PHD inhibitors as well as a new selective PHD inhibitor (IOX5), which stabilises HIF-α through a novel mechanism of action. Pharmacological PHD inhibition compromises AML cells in a HIF-α dependent manner to disable pro-leukaemogenic pathways in AML cells, re-program their metabolism, and induce cell death, at least in part via pro-apoptotic BNIP3. Importantly, concurrent inhibition of anti-apoptotic BCL-2 by clinically used Venetoclax potentiates the anti-leukaemic effect of PHD inhibition. Thus PHD inhibition with consequent HIF-α stabilisation is a promising new non-toxic strategy for AML treatment.
2024-02-14 | GSE232644 | GEO
Project description:On molecular level cells sense changes in oxygen availability through the prolyl-4-hydroxylase domain enzymes (PHDs), which regulate the protein stability of the α-subunit of the transcription factor hypoxia inducible factor (HIF). Especially PHD3 has been additionally associated with apoptotic cell death. We hypothesized that PHD3 plays a role in cell-fate decisions in macrophages. Therefore myeloid-specific PHD3 knock out mice (PHD3-/-) were created and analysed. PHD3-deficient bone marrow-derived macrophages (BMDM) showed no altered HIF-1alpha or HIF-2alpha stabilization or increased HIF target genes expression in normoxia or hypoxia. Macrophage M1 and M2-polarization was unchanged likewise. Compared to macrophages from wild type littermates PHD3-/- BMDM exhibited a significant reduction in TUNEL positive cells after serum withdrawal or treatment with staurosporine and s-nitroso-N-acetylpenicillamine (SNAP). Under the same conditions PHD3-/- BMDM also showed less Annexin V staining which is representative for membrane disruption and indicated a reduced early apoptosis. In an unbiased transcriptome screen we found that angiopoietin like protein 2 (Angptl2) expression was reduced in PHD3-/- BMDM under stress conditions. Addition of recombinant Angptl2 rescued the anti-apoptotic phenotype demonstrating that it is involved in the PHD3-mediated response towards apoptotic stimuli in macrophages.
2014-12-31 | GSE55693 | GEO
Project description:Hypoxia results in the changes in expression of many genes, the majority of which are mediated via the transcriptional activity of the hypoxia inducible factor (HIF) complex. However, other mechanisms of gene regulation by hypoxia are likely and include control of mRNA stability, regulation of mRNA translation and regulation mediated by micrornas. The aim of this study is to identify microRNAs which expression is regulated by hypoxia. We chose the breast cancer line MCF7 for study as we had previously characterised the expression of the components of the HIF system in that cell line and undertaken an extensive study of the gene expression profile in response to hypoxia, a prolyl hydroxylase inhibitor dimethyloxalylglycine and HIF-1a isoform manipulations (Eldvidge. G.P. et al. (2006) JBC, vol. 281, 22, 15215-15226).
2008-03-01 | E-MEXP-1111 | biostudies-arrayexpress
Project description:Collagen from the skin of wild type and prolyl 4-hydroxylase mutant mice was extracted and proline hydroxylation of the collagen-derived peptides were analyzed by LC-MS/MS.
2018-04-10 | PXD008802 | Pride
Project description:Alternative RNA splicing analysis in Hep3B cell cultured under 21% (N1,3,5) or 1.2% (H2,4,6) oxygen Hypoxia is a common characteristic of many solid tumors. The hypoxic microenvironment stabilizes hypoxia-inducible transcription factor 1? (HIF1?) and 2? (HIF2?) to activate gene transcription, which promotes tumor cell survival. 95% of human genes are alternatively spliced, producing RNA isoforms that code functionally distinct proteins. Thus, effective hypoxia response requires increased HIF target gene transcription as well as proper RNA splicing of these HIF target genes. However, it is unclear if and how hypoxia regulates RNA splicing of HIF target genes. This study determined the effects of hypoxia on alternative splicing (AS) of HIF and non-HIF target genes in Hep3B cells and characterized the role of HIF in regulating AS of HIF induced genes. The results indicated that hypoxia generally promotes exon inclusion for hypoxia-induced, but reduces exon inclusion for hypoxia reduced genes. Mechanistically, HIF activity, but not hypoxia per se is found to be necessary and sufficient to increase exon inclusion of several HIF target genes including pyruvate dehydrogenase kinase 1 (PDK1). PDK1 splicing reporters confirmed that transcriptional activation by HIF is sufficient to increase exon inclusion of PDK1 splicing reporter. In contrast, transcriptional activation of the PDK1 minigene by other transcription factor in the absence of endogenous HIF target gene activation fails to alter PDK1 RNA splicing, demonstrating a novel role of HIF target gene(s) in regulating RNA splicing of HIF target genes. Implications:This study demonstrates a novel function of HIF in regulating RNA splicing of HIF target genes. We analyzed total RNA from Hep3B cells cultured under 21% (N1,3,5) or 1.2% (H2,4,6) oxygen using the Affymetrix Human Exon 1.0 ST platform. Array data was processed by Altanalyze software version 2.0.7. Techinical replicates were performed for Nx and Hx treated Hep3B cells
2014-05-14 | E-GEOD-57613 | biostudies-arrayexpress