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Epigenetics Volume 16, 2021 - Issue 1
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Research Paper

Hypoxia and hypoxia mimetics differentially modulate histone post-translational modifications

Kuo-Feng Hsua Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, UK;b Tri-Service General Hospital, National Defense Medical Center, Taipei, TaiwanORCID Iconhttps://orcid.org/0000-0001-9556-9221View further author information
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Sarah E. Wilkinsa Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, UKORCID Iconhttps://orcid.org/0000-0003-1347-3328View further author information
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Richard J. Hopkinsona Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, UK;c Leicester Institute of Structural and Chemical Biology and School of Chemistry, University of Leicester, Leicester, UKORCID Iconhttps://orcid.org/0000-0001-7610-8163View further author information
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Rok Sekirnika Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, UKView further author information
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Emily Flashmana Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, UKORCID Iconhttps://orcid.org/0000-0002-4169-4278View further author information
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Akane Kawamuraa Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, UK;d Radcliffe Department of Medicine, Division of Cardiovascular Medicine, BHF Centre of Research Excellence, Wellcome Trust Centre for Human Genetics, Oxford, UK;e Chemistry - School of Natural and Environmental Sciences, Newcastle University, UKORCID Iconhttps://orcid.org/0000-0003-1169-5082View further author information
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James S.O. McCullagha Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, UKORCID Iconhttps://orcid.org/0000-0003-4733-1205View further author information
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Louise J. Walporta Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, UK;f Protein-Protein Interaction Laboratory, The Francis Crick Institute, London, UK;g Department of Chemistry, Molecular Sciences Research Hub, Imperial College London, London, UKCorrespondence[email protected]
ORCID Iconhttps://orcid.org/0000-0002-3795-8365View further author information
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Christopher J. Schofielda Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, UKCorrespondence[email protected]
ORCID Iconhttps://orcid.org/0000-0002-0290-6565View further author information
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Pages 14-27 | Received 06 Mar 2020, Accepted 22 May 2020, Published online: 01 Jul 2020

Figures & data

Figure 1. Deconvoluted MS profiles from LC-MS analysis of intact core histones under normoxia and hypoxia. Intact histone profiles from (a) HEK 293T, (b) HeLa and (c) RKO cells cultured for 24 hours under normoxia (21% O2) or severe hypoxia (0.1% O2). Coloured traces indicate the sample from control cells grown in normoxia (black) or cells treated under severe hypoxia for 24 hours (red)

Figure 1. Deconvoluted MS profiles from LC-MS analysis of intact core histones under normoxia and hypoxia. Intact histone profiles from (a) HEK 293T, (b) HeLa and (c) RKO cells cultured for 24 hours under normoxia (21% O2) or severe hypoxia (0.1% O2). Coloured traces indicate the sample from control cells grown in normoxia (black) or cells treated under severe hypoxia for 24 hours (red)

Figure 2. Histone H3.1 PTM profiles following treatment of cells with hypoxia or hypoxia mimetics. Histones were extracted from HEK 293T cells treated for 24 hours with: (a,b) normoxia (21% O2), hypoxia (1% O2), or severe hypoxia (0.1% O2); (c,d) 50, 75, 100, 125 and 150 μM DFO; (e,f) 25, 50, 75, 100 and 125 μM CP20; (g,h) 50, 100, 150 and 200 μM CoCl2. (a,c,e,g) The ion count for each mass spectral peak is expressed as a percentage of the total ion count. (b,d,f,h) The ion count for each peak as a percentage of the total ion count is expressed as a change relative to the peak intensity in control cells in each experiment. Data are expressed as mean ion counts ± SEM (n = 3). m.e.: number of methylation equivalents

Figure 2. Histone H3.1 PTM profiles following treatment of cells with hypoxia or hypoxia mimetics. Histones were extracted from HEK 293T cells treated for 24 hours with: (a,b) normoxia (21% O2), hypoxia (1% O2), or severe hypoxia (0.1% O2); (c,d) 50, 75, 100, 125 and 150 μM DFO; (e,f) 25, 50, 75, 100 and 125 μM CP20; (g,h) 50, 100, 150 and 200 μM CoCl2. (a,c,e,g) The ion count for each mass spectral peak is expressed as a percentage of the total ion count. (b,d,f,h) The ion count for each peak as a percentage of the total ion count is expressed as a change relative to the peak intensity in control cells in each experiment. Data are expressed as mean ion counts ± SEM (n = 3). m.e.: number of methylation equivalents

Figure 3. Histone H3.1 PTM profiles following treatment of cells with 2OG oxygenase inhibitors. Histones were extracted from HEK 293T cells treated for 24 hours with various concentrations of: (a) DMOG; (b) IOX2; (c) FG4592. The ion count for each peak as a percentage of the total ion count is expressed as a change relative to the control cells in each experiment. Data are means ± SEM (n = 3). m.e.: number of methylation equivalents

Figure 3. Histone H3.1 PTM profiles following treatment of cells with 2OG oxygenase inhibitors. Histones were extracted from HEK 293T cells treated for 24 hours with various concentrations of: (a) DMOG; (b) IOX2; (c) FG4592. The ion count for each peak as a percentage of the total ion count is expressed as a change relative to the control cells in each experiment. Data are means ± SEM (n = 3). m.e.: number of methylation equivalents

Figure 4. Analysis of histone H4 following treatment of cells with hypoxia, hypoxia mimetics or 2OG oxygenase inhibitors. Histones were extracted from HEK 293T cells treated for 24 hours with various concentrations of (a) hypoxia, (b) DFO, (c) CP20, (d) CoCl2, (e) DMOG, (f) IOX2 or (g) FG4592. The ion count for each peak as a percentage of the total ion count is expressed as a change relative to the control cells in each experiment. Data are means ± SEM (n = 3). No. of m.e.: number of methylation equivalents

Figure 4. Analysis of histone H4 following treatment of cells with hypoxia, hypoxia mimetics or 2OG oxygenase inhibitors. Histones were extracted from HEK 293T cells treated for 24 hours with various concentrations of (a) hypoxia, (b) DFO, (c) CP20, (d) CoCl2, (e) DMOG, (f) IOX2 or (g) FG4592. The ion count for each peak as a percentage of the total ion count is expressed as a change relative to the control cells in each experiment. Data are means ± SEM (n = 3). No. of m.e.: number of methylation equivalents

Figure 5. Changes in H3.1 PTM profiles over time following a range of hypoxic stresses. Histones were extracted from HEK 293T cells treated with (a) severe hypoxia (0.1% O2), (b) DFO (150 µM), (c) DMOG (1.25 mM), (d) CoCl2 (200 µM) for various times. The ion count for each peak as a percentage of the total ion count is expressed as a change relative to the control cells in each experiment. Data are means ± SEM (n = 3). No. of m.e: number of methylation equivalents

Figure 5. Changes in H3.1 PTM profiles over time following a range of hypoxic stresses. Histones were extracted from HEK 293T cells treated with (a) severe hypoxia (0.1% O2), (b) DFO (150 µM), (c) DMOG (1.25 mM), (d) CoCl2 (200 µM) for various times. The ion count for each peak as a percentage of the total ion count is expressed as a change relative to the control cells in each experiment. Data are means ± SEM (n = 3). No. of m.e: number of methylation equivalents
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