Fig 2: SIRT4 deacetylates MTHFD2 at K50. (A) Overexpression of SIRT4, but not other mitochondrial sirtuins, decreases the protein level of MTHFD2. HA-tagged SIRT3–SIRT5 were individually coexpressed with Flag-tagged MTHFD2 into HEK293T cells and protein levels of MTHFD2 were detected by western blotting. (B) WT SIRT4 but not its catalytic-dead H161Y mutant decreases the endogenous MTHFD2 protein level. WT SIRT4 and H161Y mutants were transfected into HEK293T cells. The endogenous MTHFD2 protein level was detected by western blotting. (C) MTHFD2 interacts with SIRT4. The interaction between endogenous MTHFD2 and SIRT4 in HEK293T cells was determined by co-IP and western blotting. (D) Verification of direct interaction between SIRT4 and MTHFD2 using GST pull-down assay. GST-tagged SIRT4 protein was purified with GST beads and incubated with HEK293T cell lysates. The beads were collected and subjected to western blotting. (E) SIRT4 knockdown increases both MTHFD2 acetylation and protein levels. MCF7 and ZR-75-30 cells stably expressing scramble shRNA (scr) or two independent shRNAs (2# and 3#) against SIRT4, respectively, were harvested for western blotting analysis. (F) WT SIRT4, but not the H161Y mutant, decreases K50 acetylation. SIRT4 or its H161Y mutant was re-expressed into stable SIRT4-knockdown MCF7 cells. Cell extracts were analyzed by western blotting. (G) WT SIRT4 but not its H161Y mutant deacetylates MTHFD2 in vitro. Recombinant SIRT4 (with GST tag cleaved off) and MTHFD2 immunoprecipitated from HEK293T cells were incubated with or without NAD+. After in vitro deacetylation, the reaction mixture was subjected to K50 acetylation detection by western blotting.

Fig 3: The EWS‐FLI1 oncoprotein regulates the expression of genes required for the mitochondrial one‐carbon cycle. (A) A schematic illustrating the incorporation of serine and glycine into the one‐carbon cycle and production of reducing equivalents for cells. (B) The levels of formate in TC32 and SKNMC cells 48 h post‐transfection of either siNeg or siFLI1; treatment with CBR5884 40 μM for 24 h; or treatment with NCT503 10 μM for 24 h (mean ± SEM of three independent experiments). (C) Exon level expression and fold‐change (Log2 TPM) from RNA‐seq of the expression of MTHFD2 and MTHFD1L in control (siNeg) and EWS‐FLI1‐silenced (siFLI1) TC32 cells. (D) Violin plots (including median and quartiles) of the expression of MTHFD2 and MTHFD1L in EWS‐FLI1 positive EWS primary tumors (n = 58), EWS‐FLI1 positive EWS cell lines (n = 33) and normal tissues (n = 42). (E) qPCR analysis of the expression of the indicated genes (relative to siNeg‐transfected cells, mean ± s.e.m., n = 3) in EWS‐FLI1‐silenced TC32 cells (48 h). (F) Immunoblots of the indicated proteins in lysates prepared from TC32 cells, siRNA‐transfected as indicated. (G) Fold‐enrichment of EWS‐FLI1‐co‐precipitating DNA determined by qPCR of genomic DNA spanning putative EWS‐FLI1 binding sites. Data shown were calculated as a percent of input DNA and the fold‐enrichment of the anti‐FLI1 antibody compared to IgG; triplicate samples of each treatment in each experiment. (B, E, and G) Two‐way ANOVA (adjusted P‐value) ***P < 0.001. (D) Unpaired t‐test, adjusted for multiple comparisons; **q < 0.01, ***q < 0.001

Fig 4: Metabolic function of MTHFD2 promotes protein PD-L1 transcription through uridine-related-metabolites.a–b, MTHFD2-KO SW1990 cells and HPDE cells transfected with indicated expressing vectors, PD-L1 mRNA levels were analyzed by real-time PCR (a) and immunoblotting analyses were performed using the indicated antibodies (b). c The metabolomics analysis in SW1990 cells with siMTHFD2 (n = 6 independent samples). d–g SW1990 cells were treated with indicated concentration of indicated metabolites for 24 h, immunoblotting analyses were performed using the indicated antibodies (d, e) and PD-L1 mRNA levels were analyzed by real-time PCR (f, g). h, i SW1990 cells with siMTHFD2 were treated with 0.3 mM UDP or 0.6 mM UTP for 24 h, PD-L1 mRNA levels were analyzed by real-time PCR (h) and the transcriptional activity of PD-L1 promoter (from 2000 bp to 0 bp) is examined by a luciferase-based reporter assay (i). In (a) and (f–i), the values are presented as mean ± s.e.m (n = 3 independent experiments); p values (Student’s t test, two-sided) with control or the indicated groups are presented (also see Supplementary Fig. 4).

Fig 5: MTHFD2 is induced by IFN-γ and involved in IFN-γ-mediated PD-L1 regulation.a, b Indicated cancer cells transfected with control or MTHFD2 siRNAs for 24 h and then treated with 0 or 20 ng/ml IFN-γ for another 24 h, PD-L1 mRNA levels were analyzed by real-time PCR (a) and immunoblotting analyses were performed using the indicated antibodies (b). c, d Indicated cancer cells pretreated with LY294002 (c) and Rapamycin (d) with indicated concentrations and then cultured with 20 ng/ml IFN-γ, immunoblotting analyses were performed using the indicated antibodies. e Indicated cancer cells treated with 20 ng/ml IFN-γ for indicated times, immunoblotting analyses were performed using the indicated antibodies. In (a), the values are presented as mean ± s.e.m (n = 3 independent experiments); p values (Student’s t test, two-sided) with control or the indicated groups are presented (also see Supplementary Fig. 3).