The bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) and GSK J4 inhibitors were both purchased from Selleck, USA. Working solutions of BPTES and GSK J4 inhibitors were reconstituted and diluted in dimethyl sulfoxide (DMSO). Dimethyl-α-ketoglutarate (dm-αKG) was purchased from Sigma-Aldrich, USA. The concentrations of BPTES, dm-αKG and GSK J4 in this study were 1.5 μmol/L, 4 mmol/L and 2 μmol/L respectively, based on the previous literature[18–20].
All the cells were maintained in the cell incubator at 37 °C with 5% CO2. The mouse embryonic stem cell line bearing Venus-tagged Blimp1 reporter was a kind gift from Jiahao Sha's lab (Nanjing Medical University, China). mESC culture and PGCLC induction were performed as previously described. Briefly, mESCs were cultured in N2B27 medium with 2i/Lif (PD0325901&CHIR9902: Selleck; Lif: Millipore, Germany). N2B27 medium consists of 50% DMEM/F12, 50% Neurobasal medium with 1×N2, 1×B27, 2 mmol/L Glutamax, 0.1 mmol/L NEAA, 1 mmol/L sodium pyruvate (Gibco, USA) and 0.1 mmol/L β-mercaptoethanol (Sigma-Aldrich). mESCs were maintained on a dish coated with 0.01% poly-L-ornithine solution (Sigma-Aldrich) and laminin (Invitrogen, USA) under a feeder-free condition.
To induce EpiLCs, approximately 2×105 mESCs were dissociated into single cells by TrypLE Select Enzyme (Gibco) and plated on fibronectin (Santa Cruz, USA)-coated 3.5 cm dishes in N2B27 medium containing 20 ng/mL activin A (Peprotech, USA), 12 ng/mL bFGF (Peprotech) and 1% KnockOut serum replacement (KSR) (Gibco).
To induce PGCLCs, Day 2 EpiLCs were dissociated into single cells and seeded in wells of low-cell-binding U-bottom 96-well plates (2500 cells/well) (Corning, USA). The PGCLC medium contains GMEM supplemented with 15% KSR, 1 mmol/L sodium pyruvate, 0.1 mmol/L NEAA, 2 mmol/L L-glutamine (Gibco), 0.1 mmol/L β-mercaptoethanol, and 1% penicillin/streptomycin (Hyclone, USA) in the presence of BMP4 (500 ng/mL), Lif (1000 U/mL), SCF (100 ng/mL), and EGF (50 ng/mL) (Peprotech).
To inhibit glutaminolysis, the experiment was performed as detailed below: during PGCLC induction, cells were incubated with PGCLC culture medium and 1.5 μmol/L BPTES (targeting glutaminase). The medium was changed every 24 hours and the cells were resupplemented with the indicated concentration of BPTES.
The untargeted metabolomics profiling was performed on XploreMET platform using gas chromatography-mass spectrometry (Metabo-Profile, China). The sample preparation procedures were followed as previously published. The differential metabolites were obtained using univariate statistical analysis. Student's t-test. The P value together with log1.5 fold change (FC) is introduced with a cutoff value of 0.05 and 1.5 for P value and log1.5 FC, respectively.
Cell aggregates were harvested and fixed for 15 minutes at room temperature in 4% paraformaldehyde in PBS (4% PFA) (Sigma-Aldrich). After fixation, samples were washed with PBS for 3 times (Sangon Biotech, China), 5 minutes per time, and then dehydrated through 10% and 20% sucrose each for 1 hour at 4 °C. Subsequently, samples were embedded in OCT (SAKURA, USA), and sectioned at 5 μm. Sections were permeabilized with 0.1% Triton X-100 in PBS (PBST) for 20 minutes and blocked for 1 hour with 1% bovine serum albumin (BSA) in PBS at room temperature (Sangon Biotech). Then, sections were incubated with the diluted primary antibody overnight at 4 °C. Next day, the sections were washed with PBST for 3 times, 5 minutes per time, and incubated with the diluted secondary fluorescence-conjugated antibody for 1 hour at room temperature in the dark. After being washed again, the sections were mounted with anti-fade mounting medium (Vectorlabs, USA). All images were obtained on ZEISS LSM700 confocal microscope (Carl Zeiss AG, Germany) and processed with Zeiss Zen Desk (Blue Edition). The antibodies mentioned are as follows: Rabbit polyclonal antibody-H3K27me3 (Millipore), Rabbit monoclonal antibody-Blimp1 (CST, USA), and Alexa Fluor 555 Donkey anti-Rabbit (IgG) secondary antibody (Thermo Fisher Scientific, USA).
For sample preparation, cell aggregates were harvested, washed with PBS and dissociated by TrypLE Select Enzyme for ~10 minutes at 37 °C. Dissociated cells were incubated in PE anti-c-Kit antibody/PBS (1:200) for 30 minutes in the dark at 37 °C (Biolegend, USA). Venus-tagged Blimp1 (Blimp1-Venus) positive PGCLCs can be detected with FITC channel on the BD FACSVerse (BD, USA). Data were analyzed with FlowJo software (Treestar, USA).
Total RNAs from cell aggregates were isolated with TRIzol reagent according to the instructions (Invitrogen). Total RNA (500 ng) was reverse transcribed into cDNA using the RT reagent Kit (Takara, Japan). qPCR was performed in triplicate on ABI Q5 (Thermo Fisher Scientific) using ChamQ SYBR qPCR Master Mix for quantification of the target gene expression (Vazyme, China). Relative expression was normalized to Actb for each sample. The primers for quantitative reverse transcription PCR (qRT-PCR) amplification are summarized in Table 1 .
Gene Forward primer (5′-3′) Reverse primer(5′-3′) Blimp1 AGCATGACCTGACATTGACACC CTCAACACTCTCATGTAAGAGGC Prdm14 ACAGCCAAGCAATTTGCACTAC TTACCTGGCATTTTCATTGCTC T ATCAGAGTCCTTTGCTAGGTAG GTTACAATCTTCTGGCTATGC Tfap2c GGGCTTTTCTCTCTTGGCTGGT TCCACACGTCACCCACACAA Glud1 CTACGGCCGATTGACCTTCA TGTGCGCATAATTTGCCTGG Actb CATTGCTGACAGGATGCAGAAGG TGCTGGAAGGTGGACAGTGAGG
Table 1. Primers for quantitative reverse transcription PCR analysis
Glutamate dehydrogenase (GDH) activity assay was measured by using the GDH assay kit (Biovision, USA). Cells were seeded into 96-well plates and harvested 48 hours later. Cell aggregates were washed with cold PBS and then homogenized in 200 μL ice-cold Assay Buffer. After being centrifuged (13 000 g for 10 minutes), 50 μL supernatants were transferred into a new 96-well plate and added with 100 μL of reaction mix composed of 82 μL of GDH Assay Buffer, 8 μL of GDH Developer and 10 μL of glutamate for each well. The concentration of GDH was measured at a wavelength of 450 nm in a microplate reader (Biotek, USA) after incubation for 3 minutes at 37 °C (A0), and then measured again every 10 minutes (A1-An). We chose the period of linear range to calculate the GDH activity of the tested samples.
αKG assay was measured by using the αKG assay kit (Biovision). Cells (2×105) were rapidly homogenized with 100 µL of ice cold α-KG assay buffer and deproteinized using 10 kDa molecular weight cut off spin columns (Biovision). 50 µL samples were added into wells of a 96-well plate and 50 μL of reaction mix was added for each well. The samples were incubated for 30 minutes at 37 °C and measured OD at 570 nm in a microplate reader (Biotek). We chose the period of linear range to calculate the αKG concentrations of the tested samples.
All assays were performed for at least three times. Comparisons were made using unpaired two-tailed Student's t-tests and data were presented as mean±SD. In all cases, P values smaller than 0.05 were considered to be statistically significant. Statistical significance was set at *P<0.05,**P<0.01 and***P<0.001. Data were analyzed with GraphPad Prism8.0.
α-ketoglutarate promotes the specialization of primordial germ cell-like cells through regulating epigenetic reprogramming
- Received Date: 2019-12-26
- Accepted Date: 2020-06-05
- Rev Recd Date: 2020-05-20
- Available Online: 2020-08-06
- Publish Date: 2021-01-28
Abstract: There is growing evidence that cellular metabolism can directly participate in epigenetic dynamics and consequently modulate gene expression. However, the role of metabolites in activating the key gene regulatory network for specialization of germ cell lineage remains largely unknown. Here, we identified some cellular metabolites with significant changes by untargeted metabolomics between mouse epiblast-like cells (EpiLCs) and primordial germ cell-like cells (PGCLCs). More importantly, we found that inhibition of glutaminolysis by bis-2- (5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES) impeded PGCLC specialization, but the impediment could be rescued by addition of α-ketoglutarate (αKG), the intermediate metabolite of oxidative phosphorylation and glutaminolysis. Moreover, adding αKG alone to the PGCLC medium accelerated the PGCLC specialization through promoting H3K27me3 demethylation. Thus, our study reveals the importance of metabolite αKG in the germ cell fate determination and highlights the essential role of cellular metabolism in shaping the cell identities through epigenetic events.
|Citation:||Ming Xing, Na Wang, Hanyi Zeng, Jun Zhang. α-ketoglutarate promotes the specialization of primordial germ cell-like cells through regulating epigenetic reprogramming[J]. The Journal of Biomedical Research, 2021, 35(1): 36-46. doi: 10.7555/JBR.34.20190160|