Summary of Study ST002242

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001431. The data can be accessed directly via it's Project DOI: 10.21228/M8RD8W This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST002242
Study TitleHypoxia promotes osteogenesis via regulating the acetyl-CoA-mediated mito-nuclear communication.
Study SummaryBone-mesenchymal stem cells (MSCs) reside in a hypoxic niche that maintains their differentiation potential. Although the role of hypoxia (low oxygen concentration) in the regulation of stem cell function has been previously reported, with normoxia (high oxygen concentration) leading to impaired osteogenesis, the molecular events triggering changes in stem cell fate decisions in response to high oxygen remain elusive. Here, we study the impact of normoxia on the mito-nuclear communication with regards to stem cell differentiation. We show that normoxia-cultured MSCs undergo profound transcriptional alterations which cause irreversible osteogenesis defects. Mechanistically, high oxygen promotes chromatin compaction and histone hypo-acetylation, particularly on promoters and enhancers of osteogenic genes. Although normoxia induces metabolic rewiring resulting in high acetyl-CoA levels, histone hypo-acetylation occurs due to trapping of acetyl-CoA inside mitochondria, owing to lower CiC activity. Strikingly, restoring the cytosolic acetyl-CoA pool remodels the chromatin landscape and rescues the osteogenic defects. Collectively, our results demonstrate that the metabolism-chromatin-osteogenesis axis is heavily perturbed in response to high oxygen and identify CiC as a novel, oxygen-sensitive regulator of the MSC function.
Institute
CECAD Research Center
Last NameYang
First NameMing
AddressJoseph-Stelzmann-Straße 26, Köln, Koeln, 50931, Germany
Emailming.yang@uni-koeln.de
Phone4922147884306
Submit Date2022-08-01
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailLC-MS
Release Date2022-08-17
Release Version1
Ming Yang Ming Yang
https://dx.doi.org/10.21228/M8RD8W
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001431
Project DOI:doi: 10.21228/M8RD8W
Project Title:Hypoxia promotes osteogenesis via regulating the acetyl-CoA-mediated mito-nuclear communication.
Project Summary:Bone-mesenchymal stem cells (MSCs) reside in a hypoxic niche that maintains their differentiation potential. Although the role of hypoxia (low oxygen concentration) in the regulation of stem cell function has been previously reported, with normoxia (high oxygen concentration) leading to impaired osteogenesis, the molecular events triggering changes in stem cell fate decisions in response to high oxygen remain elusive. Here, we study the impact of normoxia on the mito-nuclear communication with regards to stem cell differentiation. We show that normoxia-cultured MSCs undergo profound transcriptional alterations which cause irreversible osteogenesis defects. Mechanistically, high oxygen promotes chromatin compaction and histone hypo-acetylation, particularly on promoters and enhancers of osteogenic genes. Although normoxia induces metabolic rewiring resulting in high acetyl-CoA levels, histone hypo-acetylation occurs due to trapping of acetyl-CoA inside mitochondria, owing to lower CiC activity. Strikingly, restoring the cytosolic acetyl-CoA pool remodels the chromatin landscape and rescues the osteogenic defects. Collectively, our results demonstrate that the metabolism-chromatin-osteogenesis axis is heavily perturbed in response to high oxygen and identify CiC as a novel, oxygen-sensitive regulator of the MSC function.
Institute:CECAD Research Center
Last Name:Yang
First Name:Ming
Address:Joseph-Stelzmann-Straße 26, Köln, Koeln, 50931, Germany
Email:ming.yang@uni-koeln.de
Phone:4922147884306

Subject:

Subject ID:SU002328
Subject Type:Cultured cells
Subject Species:Mus musculus
Taxonomy ID:10090

Factors:

Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Treatment Cellular fraction
SA214140AP02_41hypoxia cytosolic
SA214141AP02_42hypoxia cytosolic
SA214142AP02_43hypoxia cytosolic
SA214143AP02_44hypoxia cytosolic
SA214144AP02_27hypoxia mitochondrial
SA214145AP02_28hypoxia mitochondrial
SA214146AP02_26hypoxia mitochondrial
SA214147AP02_25hypoxia mitochondrial
SA214148AP02_11hypoxia whole cell
SA214149AP02_10hypoxia whole cell
SA214150AP02_09hypoxia whole cell
SA214151AP02_12hypoxia whole cell
SA214152AP02_36normoxia cytosolic
SA214153AP02_34normoxia cytosolic
SA214154AP02_35normoxia cytosolic
SA214155AP02_33normoxia cytosolic
SA214156AP02_19normoxia mitochondrial
SA214157AP02_18normoxia mitochondrial
SA214158AP02_20normoxia mitochondrial
SA214159AP02_17normoxia mitochondrial
SA214160AP02_02normoxia whole cell
SA214161AP02_03normoxia whole cell
SA214162AP02_04normoxia whole cell
SA214163AP02_01normoxia whole cell
Showing results 1 to 24 of 24

Collection:

Collection ID:CO002321
Collection Summary:To isolate mitochondrial and cytosolic cellular fractions we followed a previously published rapid subcellular fractionation method (Lee et al., 2019). Briefly, on the day of the experiment, cells were washed twice with ice-cold PBS and scraped from the flasks with 2 ml PBS. 1:4 of cells was transferred to a tube and served as the whole cell lysate (WCL). After a brief centrifugation step (10,000 g, 3 minutes, 4 °C), supernatant was removed and 300 ul of metabolite extraction solution (50%MetOH:30%acetonitrile:20% ultrapure water) was added to samples. Samples were incubated with the extraction buffer for 20 minutes in a dry-ice MetOH water-bath and were then subjected to metabolite extraction. The rest 3:4 of cells was subjected to digitonin-based cellular fractionation. More precisely, after a brief centrifugation step (10,000 g, 10 sec, 4 °C), supernatant was removed and the pellet was resuspended in 1 ml of ice-cold digitonin-PBS buffer (1mg/ml). Following quick centrifugation (10,000 g, 10 sec, 4 °C), supernatant and pellet were collected as the cytosolic and mitochondrial fractions, respectively. 4 ml of 50%MetOH:30%acetonitrile was added in the cytosolic fractions to extract metabolites. Samples were incubated for 20 minutes in a dry-ice MetOH water-bath and were then subjected to metabolite extraction. The mitochondrial fraction was resuspended in 100 ul metabolite extraction solution and incubated for 20 minutes in a dry-ice MetOH water-bath and were then subjected to metabolite extraction.
Sample Type:Cultured cells

Treatment:

Treatment ID:TR002340
Treatment Summary:Commercially available MSCs were cultured in T175 flasks under normoxic or hypoxic conditions for seven days, in cell culture medium (MEM-Alpha supplemented with 10% FBS and 1% penicillin/streptomycin).

Sample Preparation:

Sampleprep ID:SP002334
Sampleprep Summary:The cell extract suspension was incubated for 15 minutes, at 4 °C, shaking in a thermomixer at maximum speed. Samples were then centrifugated for 20 minutes at 4 °C, at 13,000 rpm. The top 80% of the supernatant was collected and subjected to LC/MS analysis.

Combined analysis:

Analysis ID AN003660
Analysis type MS
Chromatography type HILIC
Chromatography system Thermo Vanquish
Column SeQuant ZIC-pHILIC
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Orbitrap Exploris 240
Ion Mode UNSPECIFIED
Units peak area

Chromatography:

Chromatography ID:CH002712
Chromatography Summary:Chromatographic separation of metabolites was achieved using a Millipore Sequant ZIC-pHILIC analytical column (5 µm, 2.1 × 150 mm) equipped with a 2.1 × 20 mm guard column (both 5 mm particle size) with a binary solvent system. Solvent A was 20 mM ammonium carbonate, 0.05% ammonium hydroxide; Solvent B was acetonitrile. The column oven and autosampler tray were held at 40 °C and 4 °C, respectively. The chromatographic gradient was run at a flow rate of 0.200 mL/min as follows: 0–2 min: 80% B; 2-17 min: linear gradient from 80% B to 20% B; 17-17.1 min: linear gradient from 20% B to 80% B; 17.1-23 min: hold at 80% B. Samples were randomized and the injection volume was 5 µl. A pooled quality control (QC) sample was generated from an equal mixture of all individual samples and analysed interspersed at regular intervals.
Instrument Name:Thermo Vanquish
Column Name:SeQuant ZIC-pHILIC
Column Temperature:40
Flow Gradient:0-2 min: 80% B; 2-17 min: linear gradient from 80% B to 20% B; 17-17.1 min: linear gradient from 20% B to 80% B; 17.1-23 min: hold at 80% B.
Flow Rate:0.200 mL/min
Solvent A:100% water; 20 mM ammonium carbonate; 0.05% ammonium hydroxide
Solvent B:100% acetonitrile
Chromatography Type:HILIC

MS:

MS ID:MS003411
Analysis ID:AN003660
Instrument Name:Thermo Orbitrap Exploris 240
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Metabolites were measured with Vanquish Horizon UHPLC coupled to an Orbitrap Exploris 240 mass spectrometer (both Thermo Fisher Scientific) via a heated electrospray ionization source. The spray voltages were set to +3.5kV/-2.8 kV, RF lens value at 70, the heated capillary held at 320 °C, and the auxiliary gas heater held at 280 °C. The flow rate for sheath gas, aux gas and sweep gas were set to 40, 15 and 0, respectively. For MS1 scans, mass range was set to m/z=70-900, AGC target set to standard and maximum injection time (IT) set to auto. Data acquisition for experimental samples used full scan mode with polarity switching at an Orbitrap resolution of 120000. Data acquisition for untargeted metabolite identification was performed using the AcquireX Deep Scan workflow, an iterative data-dependent acquisition (DDA) strategy using multiple injections of the pooled sample. DDA full scan-ddMS2 method for AcquireX workflow used the following parameters: full scan resolution was set to 60000, fragmentation resolution to 30000, fragmentation intensity threshold to 5.0e3. Dynamic exclusion was enabled after 1 time and exclusion duration was 10s. Mass tolerance was set to 5ppm. Isolation window was set to 1.2 m/z. Normalized HCD collision energies were set to stepped mode with values at 30, 50, 150. Fragmentation scan range was set to auto, AGC target at standard and max IT at auto. Mild trapping was enabled. Metabolite identification was performed in the Compound Discoverer software (v 3.2, Thermo Fisher Scientific). Metabolite identities were confirmed using the following parameters: (1) precursor ion m/z was matched within 5 ppm of theoretical mass predicted by the chemical formula; (2) fragment ions were matched within 5 pm to an in-house spectral library of authentic compound standards analysed with the same ddMS2 method with a best match score of over 70; (3) the retention time of metabolites was within 5% of the retention time of a purified standard run with the same chromatographic method. Chromatogram review and peak area integration were performed using the Tracefinder software (v 5.0, Thermo Fisher Scientific) and the peak area for each detected metabolite was normalized against the total ion count (TIC) of that sample to correct any variations introduced from sample handling to instrument analysis. The normalized areas were used as variables for further statistical data analysis.
Ion Mode:UNSPECIFIED
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