Summary of Study ST003182
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 PR001981. The data can be accessed directly via it's Project DOI: 10.21228/M8NQ82 This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
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.
| Study ID | ST003182 |
| Study Title | METTL3-mediated chromatin contacts promote stress granule phase separation through metabolic reprogramming during senescence |
| Study Summary | METTL3 is the catalytic subunit of the methyltransferase complex, which mediates m6A modification to regulate gene expression. In addition, METTL3 regulates transcription in an enzymatic activity-independent manner by driving changes in high-order chromatin structure. However, how these functions of MTC are coordinated remains unknown. Here we show that the methyltransferase complex coordinates its enzymatic activity-dependent and independent functions to regulate cellular senescence, a state of stable cell growth arrest. Specifically, METTL3-mediated chromatin loops induce Hexokinase 2 expression through the three-dimensional chromatin organization during senescence. Elevated Hexokinase 2 expression subsequently promotes liquid-liquid phase separation, manifesting as stress granule phase separation, by driving metabolic reprogramming. This correlates with an impairment of translation of cell-cycle related mRNAs harboring polymethylated m6A sites. In summary, our results report a coordination of m6A-dependent and -independent function of the methyltransferase complex in regulating senescence through phase separation driven by metabolic reprogramming. |
| Institute | University of Texas MD Anderson Cancer Center |
| Last Name | Zhang |
| First Name | Rugang |
| Address | 3SCR3.4121, 1901 East RD, Houston, TX, 77054 |
| rzhang11@mdanderson.org | |
| Phone | 832-748-6422 |
| Submit Date | 2024-04-29 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-05-03 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001981 |
| Project DOI: | doi: 10.21228/M8NQ82 |
| Project Title: | METTL3-mediated chromatin contacts promote stress granule phase separation through metabolic reprogramming during senescence |
| Project Summary: | METTL3 is the catalytic subunit of the methyltransferase complex, which mediates m6A modification to regulate gene expression. In addition, METTL3 regulates transcription in an enzymatic activity-independent manner by driving changes in high-order chromatin structure. However, how these functions of MTC are coordinated remains unknown. Here we show that the methyltransferase complex coordinates its enzymatic activity-dependent and independent functions to regulate cellular senescence, a state of stable cell growth arrest. Specifically, METTL3-mediated chromatin loops induce Hexokinase 2 expression through the three-dimensional chromatin organization during senescence. Elevated Hexokinase 2 expression subsequently promotes liquid-liquid phase separation, manifesting as stress granule phase separation, by driving metabolic reprogramming. This correlates with an impairment of translation of cell-cycle related mRNAs harboring polymethylated m6A sites. In summary, our results report a coordination of m6A-dependent and -independent function of the methyltransferase complex in regulating senescence through phase separation driven by metabolic reprogramming. |
| Institute: | University of Texas MD Anderson Cancer Center |
| Last Name: | Zhang |
| First Name: | Rugang |
| Address: | 3SCR3.4121, 1901 East RD, Houston, TX, 77054 |
| Email: | rzhang11@mdanderson.org |
| Phone: | 832-748-6422 |
Subject:
| Subject ID: | SU003301 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Treatment | Sample source |
|---|---|---|---|
| SA346713 | Pro_2 | Pro | lung embryonic fibroblasts |
| SA346714 | Pro_1 | Pro | lung embryonic fibroblasts |
| SA346715 | Pro_3 | Pro | lung embryonic fibroblasts |
| SA346725 | Sen_shctrl_1 | Sen_shctrl | lung embryonic fibroblasts |
| SA346726 | Sen_shctrl_3 | Sen_shctrl | lung embryonic fibroblasts |
| SA346727 | Sen_shctrl_2 | Sen_shctrl | lung embryonic fibroblasts |
| SA346716 | Sen_shHK2_MutHK2_3 | Sen_shHK2_MutHK2 | lung embryonic fibroblasts |
| SA346717 | Sen_shHK2_MutHK2_2 | Sen_shHK2_MutHK2 | lung embryonic fibroblasts |
| SA346718 | Sen_shHK2_MutHK2_1 | Sen_shHK2_MutHK2 | lung embryonic fibroblasts |
| SA346722 | Sen_shHK2_2 | Sen_shHK2 | lung embryonic fibroblasts |
| SA346723 | Sen_shHK2_3 | Sen_shHK2 | lung embryonic fibroblasts |
| SA346724 | Sen_shHK2_1 | Sen_shHK2 | lung embryonic fibroblasts |
| SA346719 | Sen_shHK2_WTHK2_3 | Sen_shHK2_WTHK2 | lung embryonic fibroblasts |
| SA346720 | Sen_shHK2_WTHK2_2 | Sen_shHK2_WTHK2 | lung embryonic fibroblasts |
| SA346721 | Sen_shHK2_WTHK2_1 | Sen_shHK2_WTHK2 | lung embryonic fibroblasts |
| Showing results 1 to 15 of 15 |
Collection:
| Collection ID: | CO003294 |
| Collection Summary: | IMR90 primary human diploid lung embryonic fibroblasts were used and cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), L-glutamine, sodium pyruvate, non-essential amino acids, sodium bicarbonate and 1% penicillin-streptomycin under low oxygen tension (2%). |
| Sample Type: | Cultured cells |
Treatment:
| Treatment ID: | TR003310 |
| Treatment Summary: | Stable isotope tracer analysis using 13C6-glucose was conducted in three biologically independent experiments for the following experimental groups: control proliferating cells (Pro), RAS-induced senescent cells via 4-OHT with control shRNA (Sen_shctrl), senescent cells with shRNA targeting HK2 (Sen_shHK2), senescent cells with shRNA targeting HK2 and rescued with wildtype Flag-HK2 (Sen_shHK2_WTHK2), and senescent cells with shRNA targeting HK2 and rescued with mutant Flag-HK2 (Sen_shHK2_MutHK2). The experiments were performed by seeding cells at a density of 3 × 10^5 cells per 6 cm dish and incubating them with 25 mM [13C6]-glucose tracer in glucose-free medium for 30 min. |
Sample Preparation:
| Sampleprep ID: | SP003308 |
| Sampleprep Summary: | Following incubation, the cells were washed with chilled PBS and incubated with 500 µl of extraction solution (80:20 v/v methanol/water) at 4 °C for 5 minutes. Next, cells were scraped with a polypropylene cell scraper and the extraction solution from each sample was collected, vortexed, and incubated on dry ice for at least 30 min. Then, each sample was centrifuged at maximum speed at 4 °C for 10 minutes, and the resulting supernatant was used for metabolic measurements. |
Combined analysis:
| Analysis ID | AN005226 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Vanquish Horizon UHPLC |
| Column | Merck SeQuant ZIC-pHILIC (150 x 2.1mm,5um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive HF-X Orbitrap |
| Ion Mode | UNSPECIFIED |
| Units | Peak Area |
Chromatography:
| Chromatography ID: | CH003953 |
| Chromatography Summary: | Hydrophilic interaction liquid chromatography (HILIC) was performed at 0.2 ml/min on a ZIC-pHILIC column (2.1 mm × 150 mm, 5 µm particle size, EMD Millipore) at 45 °C. Solvent A was 20 mM ammonium carbonate, 0.1% ammonium hydroxide, pH 9.2, and solvent B was acetonitrile. The gradient was 85% B for 2 min, 85% B to 20% B over 15 min, 20% B to 85% B over 0.1 min, and 85% B for 8.9 min. The autosampler was held at 4 °C. For each analysis, 4 µl of sample was injected. |
| Instrument Name: | Thermo Vanquish Horizon UHPLC |
| Column Name: | Merck SeQuant ZIC-pHILIC (150 x 2.1mm,5um) |
| Column Temperature: | 45 |
| Flow Gradient: | 85% B for 2 min, 85% B to 20% B over 15 min, 20% B to 85% B over 0.1 min, and 85% B for 8.9 min |
| Flow Rate: | 0.2 ml/min |
| Solvent A: | 100% water; 20 mM ammonium carbonate; 5 µM medronic acid; 0.1% ammonium hydroxide |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS004959 |
| Analysis ID: | AN005226 |
| Instrument Name: | Thermo Q Exactive HF-X Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The following parameters were used for the MS analysis: sheath gas flow rate, 40; auxiliary gas flow rate, 10; sweep gas flow rate, 2; auxiliary gas heater temperature, 350 °C; spray voltage, 3.5 kV for positive mode and 3.2 kV for negative mode; capillary temperature, 325 °C; and funnel RF level, 40. All samples were analyzed by full MS with polarity switching. Technical injections of an unlabeled sample pool were analyzed throughout the sample sequence. The unlabeled sample pool was also analyzed by data-dependent MS/MS with separate runs for positive and negative ion modes. Full MS scans were acquired at 120,000 resolution with a scan range of 65-975 m/z. Data-dependent MS/MS scans were acquired for the top 10 highest intensity ions at 15,000 resolution with an isolation width of 1.0 m/z and stepped normalized collision energy of 20-40-60. Annotation and quantitation of metabolites and carbon isotopologues with natural isotope abundance correction were performed using Compound Discoverer 3.3 software (Thermo Scientific). Metabolite measurements were normalized based on the protein concentration in the protein pellets. |
| Ion Mode: | UNSPECIFIED |
