Article Figures & Data
Figures
- Figure 1. The Gldc-mediated GCS is activated in PSCs and during somatic cell reprogramming.
(A) The heat map from the RNA-seq analysis showed alterations in the expression of metabolic genes in mESCs and iPSCs relative to the expression of these genes in MEFs. The colours indicate the ln-transformed FPKM values. (B) Gene ontology term enrichment was assessed using the algorithm on the DAVID website. (C) RNA-seq analysis results comparing mESCs with MEFs and comparing iPSCs with MEFs. Each point represents the log2-transformed fold change for a given transcript. Genes significantly up-regulated in mESCs and iPSCs are annotated. (D) Schematic diagram of the glycine, serine, and threonine metabolism pathway. (E, F) qRT-PCR (E) and Western blot (F) analyses of the expression of the enzymes in glycine, serine, and threonine metabolic pathway in MEFs, mESCs, and iPSCs. ACTIN served as the loading control. The data were presented as the mean ± SD of three independent experiments. *P < 0.05 compared with MEFs; t test. (G) qRT-PCR analysis of the expression of Gldc, endogenous Pou5f1, Sox2, and Nanog during reprogramming at the indicated times. The mRNA levels were normalized to the expression levels on day 0. *P < 0.05 compared with the expression levels on day 0; t test. (H) The enzymatic activity of the GCS was measured in MEFs, mESCs, and iPSCs. The data were presented as the mean ± SD of three independent experiments. *P < 0.05 compared with MEFs; t test. (I) The enzymatic activity of the GCS was measured during reprogramming for the indicated times. The data were presented as the mean ± SD. *P < 0.05 compared with the activity on day 0; t test. (J) MEFs, mESCs, and iPSCs were first cultured in glycine starvation medium for 12 h, and the medium was then refreshed with medium containing 13C-labelled glycine. The amount of 13C-labelled glycine in the culture medium from MEFs, iPSCs, and mESCs was measured by GC/MS at the indicated time. The data were presented as the mean ± SD of three independent experiments. *P < 0.05 compared with MEFs; t test. (K) The enzymatic activity of the GCS was measured in V6.5 cells stably expressing shGldc or nontargeting control (NTC) shRNAs. The data were presented as the mean ± SD. *P < 0.05 compared with the NTC; t test.
Source data are available for this figure.
Source Data for Figure 1[LSA-2019-00413_SdataF1.pdf]
- Figure S1. Gldc-mediated GCS activation occurs in PSCs or during somatic cell reprogramming.
(A) List of the top 20 significantly differentially expressed genes between MEFs and mESCs or iPSCs. (B) Western blot analysis of the expression of GLDC during four factor–induced reprogramming at the indicated time points. ACTIN served as the loading control. (C) Western blot analysis of GLDC expression during three factor (Oct4, Sox2, and Klf4)–induced reprogramming at the indicated time points. ACTIN served as the loading control. qRT-PCR analysis of the expression of endogenous Pou5f1, Sox2, and Nanog during three factor (Oct4, Sox2, and Klf4)–induced reprogramming at the indicated time points. The data were presented as the mean ± SD of three independent experiments. (D) Western blot analysis of GLDC expression during Oct4, Sox2, Nanog, and Lin28A–induced reprogramming at the indicated time points. ACTIN served as the loading control. qRT-PCR analysis of the expression of endogenous Pou5f1, Sox2, and Nanog during Oct4, Sox2, Nanog, and Lin28A–induced reprogramming at the indicated time points. The data were presented as the mean ± SD of three independent experiments. (E) Western blot analysis of the expression of GLDC, OCT4, SOX2, and NANOG in V6.5 cells cultured in RA differentiation medium at the indicated time points. ACTIN served as the loading control.
Source data are available for this figure.
Source Data for Figure S1[LSA-2019-00413_SdataFS1.pdf]
- Figure 2. Sox2 and Lin28A cooperatively control Gldc expression.
(A) qRT-PCR and Western blot analysis of the expression of Gldc in MEF cells overexpressing Oct4, Sox2, Klf4, c-Myc, Nanog, and Lin28A individually. ACTIN served as the loading control. The data were presented as the mean ± SD of three independent experiments. *P < 0.05 compared with the EV control; t test. (B) qRT-PCR and Western blot analysis of the expression of Gldc in V6.5 cells stably expressing shSox2 or the NTC. The data were presented as the mean ± SD of three independent experiments. *P < 0.05 compared with the NTC; t test. ACTIN served as the loading control. (C) ChIP-qPCR analysis of potential binding site occupancy by Sox2 in the Gldc promoter in V6.5 cells using IgG or anti-Sox2 antibody. The data were presented as the mean ± SD. *P < 0.05 compared with the IgG control; t test. (D) Schematic diagram showing the Sox2-binding fragment in the Gldc promoter. (E) HEK293 cells were cotransfected with pSIN-3×flag-Sox2 and pGL2-P-Gldc wild-type, pGL2-P-Gldc mutant, or pGL2-P-EV luciferase vectors. A dual luciferase assay was performed 48 h after transfection. The data were presented as the mean ± SD. *P < 0.05 compared between the indicated groups; t test. (F) Western blot analysed the expression of GLDC in V6.5 cells stably expressing shLin28A or NTC. ACTIN served as loading control. (G) RIP assay followed by qPCR analysed the interaction between Gldc mRNA and Lin28A. Pkm2 served as positive control, Acsl6 served as negative control. Data were presented as the mean ± SD. *P < 0.05 compared with IgG; t test. (H) Western blot analysis of GLDC expression in Dgcr8−/− V6.5 cells stably expressing Lin28A wild-type, Lin28A-mutant, or empty vector (EV). ACTIN served as the loading control. (I) RIP assay was performed in V6.5 cells transfected with pSIN-3×flag-Lin28A wild-type, pSIN-3×flag-Lin28A mutant, or pSIN-3×flag-EV using anti-Flag antibody, followed by qPCR to investigate the interaction between Gldc mRNA and Lin28A. Pkm2 served as the positive control and Acsl6 served as the negative control. The data were presented as the mean ± SD. *P < 0.05 compared with empty vector; #P < 0.05 compared with Lin28A wild-type; t test. (J) Mutants of Gldc used in the dual luciferase assay. (K) HEK293 cells were cotransfected with pSIN-3×flag-Lin28A and either pSI-check2-Gldc region 1/2/3, full-length Gldc, or pSI-check2-EV luciferase vectors. A dual luciferase assay was performed 48 h after transfection. The data were presented as the mean ± SD. *P < 0.05 compared between CTR; t test. (L) HEK293 cells were cotransfected with pSIN-3×flag-Lin28A and either pSI-check2-Gldc full-length, pSI-check2-Gldc full-length with region 1/2 deleted, or pSI-check2-EV luciferase vectors. A dual luciferase assay was performed 48 h after transfection. The data were presented as the mean ± SD. *P < 0.05 compared between the indicated groups, #P < 0.05 compared with pSI-check2-Gldc full-length; t test. (M) V6.5 cells were infected with viruses expressing shSox2 and shLin28A, followed by analysis of GLDC expression by Western blotting (left) and analysis of GCS enzymatic activity (right). ACTIN served as the loading control. The data were presented as the mean ± SD. *P < 0.05 compared with the NTC; t test.
Source data are available for this figure.
Source Data for Figure 2[LSA-2019-00413_SdataF2.pdf]
- Figure S2. Sox2 and Lin28A cooperatively control Gldc expression.
(A) ChIP-seq results showing the Sox2-binding regions in the Gldc promoter. (B) qRT-PCR analysis of the expression of Gldc in V6.5 cells stably expressing shLin28A or the NTC. The data were presented as the mean ± SD of three independent experiments. (C) Western blot analysis of the expression of GLDC in Dgcr8−/− V6.5 cells stably expressing Lin28A or EV. ACTIN served as the loading control. (D) The CSD and CCHC RNA-binding domains in Lin28A were mutated as indicated. (E) CLIP-seq results showing Lin28A-binding regions in Gldc mRNA. NS, not significant.
Source data are available for this figure.
Source Data for Figure S2[LSA-2019-00413_SdataFS2.pdf]
- Figure 3. GCS promotes the production of one-carbon units and prevents the accumulation of MG.
(A) Schematic diagram of metabolic flux related to the GCS. (B) Metabolites related to the GCS were measured by LC/GC-MS in V6.5 cells stably expressing shGldc or the NTC. The data were presented as the mean ± SD. *P < 0.05 compared with the NTC; t test. (C) MG measurement with biological kit in V6.5 cells stably expressing shGldc or the NTC. The data were presented as the mean ± SD. *P < 0.05 compared with the NTC; t test. (D) LC-MS analysis of the production of 13C-labelled SAM from 13C-labelled glycine in V6.5 cells stably expressing shGldc or the NTC. The data were presented as the mean ± SD. *P < 0.05 compared with the NTC; t test. (E) GC-MS analysis of the production of 13C-labelled formate from 13C-labelled glycine in V6.5 cells stably expressing shGldc or the NTC. The data were presented as the mean ± SD. *P < 0.05 compared with the NTC; t test. (F) GC-MS analysis of the formate content in reprogramming cells expressing shGldc or the NTC. The data were presented as the mean ± SD. *P < 0.05 compared with the NTC; t test. (G) Cellular MG levels were measured in reprogramming cells expressing shGldc or the NTC. The data were presented as the mean ± SD. *P < 0.05 compared with the NTC; t test. (H, I) MEF cells with Gldc knockdown (H) or overexpression (I) were infected with virus expressing the four factors to induce iPSC formation. AP staining (upper panel) showed the iPSC colonies formed. The AP- and Ssea1-positive iPSC colonies were counted (lower panel). The data were presented as the mean ± SD. *P < 0.05 compared with the NTC/EV; t test. (J) Immunofluorescence analysis was performed in iPSCs generated from the experiment shown in (I) using anti-Oct-4/-Sox2/-Ssea1/-Nanog antibodies. Nuclei were visualized by DAPI staining. (K) Haematoxylin and eosin staining of teratoma sections derived from the iPSCs generated from the experiment shown in (I) showing the differentiation of iPSCs into cell types of the three germ layers: keratinous epithelium (a, a′), cartilage (b, b′), and glandular tissue (c, c′). Scale bars, 50 μm. (L) Formate was added to the medium of MEF cells with Gldc knockdown starting from 2 d after infection with virus expressing the four factors. AP staining (upper panel) showed the iPSC colonies formed. The AP- and Ssea1-positive iPSC colonies were counted (lower panel). The data were presented as the mean ± SD. *P < 0.05 compared with the NTC. #P < 0.05 compared with the Gldc knockdown group; t test. (M) MG was added to the medium of Gldc-overexpressing MEF cells starting from 2 d after infection with virus expressing the four factors. AP staining (upper panel) showed the iPSC colonies formed. The AP- and Ssea1-positive iPSC colonies were counted (lower panel). The data were presented as the mean ± SD. *P < 0.05 compared with the indicated groups; t test. NS, not significant.
- Figure S3. GCS promotes the production of one-carbon units and prevents the accumulation of MG.
(A) Western blot analysis of GLDC expression in V6.5 cells stably expressing shGldc and the NTC. ACTIN served as the loading control. (B) Gldc-overexpressing MEF cells were infected with virus expressing three factors to induce iPSC formation. AP staining (upper panel) showed the iPSC colonies formed. The AP- and Ssea1-positive iPSC colonies were counted (lower panel). The data were presented as the mean ± SD. *P < 0.05 compared with the EV control; t test.
Source data are available for this figure.
Source Data for Figure S3[LSA-2019-00413_SdataFS3.pdf]
- Figure 4. The GCS is required for the maintenance of PSC pluripotency by facilitating H3K4me3.
(A) GSEA of a gene set comprising stem cell pluripotency genes in shGldc- versus NTC-transfected V6.5 cells. (B, C) qRT–PCR (B) and Western blot (C) analysis of the regulation of pluripotency-related genes by Gldc in V6.5 cells. ACTIN served as the loading control. The data were presented as the mean ± SD of three independent experiments. *P < 0.05 compared with the NTC; t test. (D) GSEA of a gene set comprising genes related to one-carbon metabolism in shGldc- versus NTC-transfected V6.5 cells. (E) Western blot analysis of various H3 lysine methylation in V6.5 cells expressing shGldc or the NTC. Total H3 was used as the loading control. (F) ChIP-qPCR analysis of H3K4me3 enrichment in the promoters of Pou5f1, Sox2, Klf4, and Nanog in V6.5 cells expressing shGldc or the NTC. Actb served as a negative control. The data were presented as the mean ± SD of three independent experiments relative to the input. *P < 0.05 compared with the NTC; t test. (G) Western blot analysis of H3K4me3 modifications in V6.5 cells stably expressing shGldc or the NTC with the supplementation of 1 mM formate for 48 h. Total H3 was used as the loading control. (H) ChIP-qPCR analysis of H3K4me3 enrichment in the promoters of Pou5f1, Sox2, Klf4, and Nanog in V6.5 cells expressing shGldc or NTC with the supplementation of 1 mM formate for 48 h. The data were presented as the mean ± SD of three independent experiments relative to the input. *P < 0.05 compared with the NTC; t test. (I) qRT-PCR analysis of the expression of Pou5f1, Sox2, Klf4, and Nanog in V6.5 cells stably expressing shGldc or the NTC with the supplementation of 1 mM formate for 48 h. The data were presented as the mean ± SD of three independent experiments. *P < 0.05 compared with the NTC, #P < 0.05 compared with the Gldc knockdown group; t test. (J) qRT-PCR analysis of the expression of endogenous Pou5f1, Sox2, Klf4, and Nanog in reprogramming cells expressing shGldc or the NTC with the supplementation of 1 mM formate for 48 h. The data were presented as the mean ± SD of three independent experiments. *P < 0.05 compared with the indicated group, #P < 0.05 compared with the Gldc knockdown group; t test. (K) OICR-9429, Win6mer or vehicle (DMSO) was added to the medium of Gldc-overexpressing MEF cells starting from 2 d after infection with virus expressing the four factors. AP staining (upper panel) showed the iPSC colonies formed. The AP- and Ssea1-positive iPSC colonies were counted (lower panel). The data were presented as the mean ± SD. *P < 0.05 compared with the indicated group; t test. (L) SAM iodide was added to the medium of Gldc knockdown MEF cells starting from 2 d after infection with virus expressing the four factors. AP staining (upper panel) showed the iPSC colonies formed. The AP- and Ssea1-positive iPSC colonies were counted (lower panel). The data were presented as the mean ± SD. *P < 0.05 compared with the NTC, #P < 0.05 compared with the Gldc knockdown group; t test. NS, not significant.
Source data are available for this figure.
Source Data for Figure 4[LSA-2019-00413_SdataF4.pdf]
- Figure S4. The GCS is required for the maintenance of PSC pluripotency by facilitating H3K4me3 methylation.
(A) GSEA of a gene set comprising genes related to housekeeping in shGldc- versus NTC-transfected V6.5 cells. (B) qRT–PCR analysis of the regulation of pluripotency-related genes by Gldc in reprogramming cells. The data were presented as the mean ± SD of three independent experiments. *P < 0.05 compared with the NTC; t test. (C) Western blot analysis of various H3 lysine methylation modifications in reprogramming cells expressing shGldc or the NTC. Total H3 was used as the loading control. (D) ChIP-qPCR analysis of H3K4me3 enrichment in the promoters of Pou5f1, Sox2, Klf4, and Nanog in reprogramming cells expressing shGldc or the NTC. The data were presented as the mean ± SD of three independent experiments relative to the input. *P < 0.05 compared with the NTC; t test. (E) Western blot analysis of H3K4me3 modification in reprogramming cells expressing shGldc or the NTC with supplementation of 1 mM formate for 48 h. Total H3 was used as the loading control.
Source data are available for this figure.
Source Data for Figure S4[LSA-2019-00413_SdataFS4.pdf]
- Figure 5. The GCS protects PSCs from MG-induced senescence.
(A) Western blot analysis of the expression of senescence-related genes in V6.5 cells treated with various concentration gradients of MG. ACTIN served as the loading control. (B) Western blot analysis of the expression of senescence-related genes in reprogramming cells treated with MG. ACTIN served as the loading control. (C) Reprogramming cells were treated with MG or control, and the SA-β-gal-positive cells were counted. The data were presented as the mean ± SD. *P < 0.05 compared with CTR; t test. Scale bars, 100 μm. (D) MG was added to the medium of MEF cells starting from 2 d after infection with virus expressing the four factors. AP staining (upper panel) showed the iPSC colonies formed. The AP- and Ssea1-positive iPSC colonies were counted (lower panel). The data were presented as the mean ± SD. *P < 0.05 compared with CTR; t test. (E) Western blot analysis of the expression of senescence-related genes and argpyrimidine in V6.5 cells stably expressing shGldc or the NTC. ACTIN served as the loading control. (F) Western blot analysis of the expression of senescence-related genes and argpyrimidine in reprogramming cells expressing shGldc the NTC at various time points. ACTIN served as the loading control. (G) Reprogramming cells were infected with viruses expressing shGldc or the NTC, and SA-β-gal–positive cells were counted. The data were presented as the mean ± SD. *P < 0.05 compared with the indicated group; t test. Scale bars, 100 μm. (H) V6.5 cells stably expressing shGldc or the NTC were further infected with viruses expressing pSIN-3×flag-Glo1 or were supplemented with 1 mM carnosine, followed by qRT-PCR analysis of senescence-related genes. The data were presented as the mean ± SD of three independent experiments. *P < 0.05 compared with the NTC, #P < 0.05 compared with the Gldc knockdown group; t test. (I) V6.5 cells stably expressing shGldc or the NTC were further infected with viruses expressing pSIN-3×flag-Glo1 or EV, followed by Western blot analysis of senescence-related genes and argpyrimidine modification. ACTIN served as the loading control. (J) Reprogramming cells expressing shGldc or the NTC were further infected with viruses expressing pSIN-3×flag-Glo1 or EV, followed by Western blot analysis of senescence-related genes and argpyrimidine modification. ACTIN served as the loading control. (K) Reprogramming cells expressing shGldc or the NTC were further infected with viruses expressing pSIN-3×flag-Glo1 or EV, and SA-β-gal–positive cells were counted. The data were presented as the mean ± SD. *P < 0.05 compared with the NTC, #P < 0.05 compared with the Gldc knockdown group; t test. Scale bars, 100 μm. (L) Reprogramming cells expressing shGldc or the NTC were further infected with viruses expressing pSIN-3×flag-Glo1. AP staining (upper panel) showed the iPSC colonies formed. The AP- and Ssea1-positive iPSC colonies were counted (lower panel). The data were presented as the mean ± SD. *P < 0.05 compared with the NTC, #P < 0.05 compared with the Gldc knockdown group; t test. (M) MEFs were induced with the indicated factors. AP staining (upper panel) showed the iPSC colonies formed. The AP- and Ssea1-positive iPSC colonies were counted (lower panel). The data were presented as the mean ± SD. *P < 0.05 compared with the NTC, #P < 0.05 compared with the Gldc knockdown group; t test. NS, not significant.
Source data are available for this figure.
Source Data for Figure 5[LSA-2019-00413_SdataF5.pdf]
- Figure S5. The GCS protects pluripotent stem cells from MG-induced senescence.
(A) V6.5 cells stably expressing shGldc (targeting the 3′UTR) were further infected with viruses expressing pSIN-3×flag-Gldc, and argpyrimidine-modified proteins were analysed by Western blotting. ACTIN served as the loading control. (B) qRT-PCR analysis of the expression of senescence-related genes in reprogramming cells expressing shGldc or the NTC at various time points. The data were presented as the mean ± SD of three independent experiments. *P < 0.05 compared between the indicated groups; t test. (C) Carnosine was added to the medium of Gldc knockdown MEF cells starting from 2 d after infection with virus expressing the four factors. The AP- and Ssea1-positive iPSC colonies were counted. The data were presented as the mean ± SD. *P < 0.05 compared with the NTC, #P < 0.05 compared with the Gldc knockdown group; t test.
Source data are available for this figure.
Source Data for Figure S5[LSA-2019-00413_SdataFS5.pdf]
- Figure 6. The GCS promotes stem cell stemness and prevents cellular senescence in human stem cells.
(A, B) qRT-PCR (A) and Western blot (B) analyse the expression of the enzymes in the glycine, serine, and threonine metabolic pathway in IMR90 cells and hESCs. ACTIN served as the loading control. The data were presented as the mean ± SD of three independent experiments. (C) The enzymatic activity of the GCS was measured in IMR90 cells and hESCs. The data were presented as the mean ± SD of three independent experiments. *P < 0.05 compared with IMR90 cells; t test. (D) Western blot analysis of GLDC expression during reprogramming at the indicated time points. ACTIN served as the loading control. (E, F) Western blot analysis of GLDC expression in H9 cells stably expressing shSOX2 (E) or shLIN28A (F). ACTIN served as the loading control. (G) Western blot analysis of various H3 lysine methylation modifications in H9 cells stably expressing shGLDC or the NTC. Total H3 was used as the loading control. (H) Western blot analysis of argpyrimidine modifications in H9 cells stably expressing shGLDC or the NTC. ACTIN served as the loading control. (I) Western blot analysis of the expression of senescence-related genes in human-reprogrammed cells treated with MG. ACTIN served as the loading control. (J) Western blot analysis of the expression of senescence- or pluripotency-related genes in H9 cells stably expressing shGLDC or the NTC. ACTIN served as the loading control. (K) IMR90 cells with Gldc knockdown were infected with virus expressing the four factors to induce iPSC formation. AP staining (upper panel) showed the iPSC colonies formed. The AP-positive iPSC colonies were counted (lower panel). The data were presented as the mean ± SD. *P < 0.05 compared with the NTC; t test. (L) IMR90 cells were reprogrammed with the indicated factors. AP staining (upper panel) showed the iPSC colonies formed. The AP-positive iPSC colonies were counted (lower panel). The data were presented as the mean ± SD. *P < 0.05 compared with the NTC, #P < 0.05 compared with the Gldc knockdown group; t test.
Source data are available for this figure.
Source Data for Figure 6[LSA-2019-00413_SdataF6.pdf]
- Figure 7. Working model: The GCS determines the fate of PSCs via regulation of senescence and epigenetic modifications.
Metabolic remodelling has emerged as critical for stem cell pluripotency; here, we provide novel evidence to demonstrate that via regulation of senescence and epigenetic modifications, the GCS determines the fate of PSCs and the outcome of the cellular reprogramming.
Supplementary Materials
