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Myc-mediated SDHA acetylation triggers epigenetic regulation of gene expression and tumorigenesis

Nature Metabolism volume 2, pages 256–269 (2020)Cite this article

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Abstract

The transcriptional role of cMyc (or Myc) in tumorigenesis is well appreciated; however, it remains to be fully established how extensively Myc is involved in the epigenetic regulation of gene expression. Here, we show that by deactivating succinate dehydrogenase complex subunit A (SDHA) via acetylation, Myc triggers a regulatory cascade in cancer cells that leads to H3K4me3 activation and gene expression. We find that Myc facilitates the acetylation-dependent deactivation of SDHA by activating the SKP2-mediated degradation of SIRT3 deacetylase. We further demonstrate that Myc inhibition of SDH-complex activity leads to cellular succinate accumulation, which triggers H3K4me3 activation and tumour-specific gene expression. We demonstrate that acetylated SDHA at Lys 335 contributes to tumour growth in vitro and in vivo, and we confirm increased tumorigenesis in clinical samples. This study illustrates a link between acetylation-dependent SDHA deactivation and Myc-driven epigenetic regulation of gene expression, which is critical for cancer progression.

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Fig. 1: Myc inhibits SDH-complex activity and causes cellular succinate accumulation by inducing SDHA acetylation.
Fig. 2: Myc increases SDHA acetylation by accelerating SKP2-mediated proteasomal degradation of SIRT3.
Fig. 3: Myc enhances H3K4me3 via SDHA acetylation-mediated increase in cellular succinate.
Fig. 4: Myc-mediated succinate accumulation and H3K4me3 activation promote its target gene expression.
Fig. 5: SDHA acetylation at Lys 335 contributes to Myc-promoted tumour progression.
Fig. 6: Myc-mediated SDHA acetylation and H3K4me3 activation trigger epigenetic regulation of gene expression and tumorigenesis.

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Data availability

Data from this study have been deposited in the GEO. Original data from ChIP-seq and RNA-seq in P493 or CA46 cells are available in the NCBI GEO (accession numbers GSE124255 and GSE141227, respectively). The datasets generated and/or analysed during the current study are available from the corresponding author upon reasonable request. Source data for Figs. 1–5 and Extended Data Figs. 1–5 are presented within the paper.

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Acknowledgements

We thank C. V. Dang for critical reading of this manuscript and valuable suggestions. This work is supported in part by National Natural Science Foundation of China (91957203, 81525022, 81930083, 31571472, 81530076, 81821001), National Key R&D Program of China (2018YFA0800300, 2018YFA0107103, 2017YFA0205600), the Chinese Academy of Sciences (XDPB10), the Program for Guangdong Introducing Innovative and Entrepreneurial Teams (2017ZT07S054) and the Program of Development Foundation of Hefei Center for Physical Science and Technology (2017FXZY004). Please address all the correspondence and requests for materials to P.G. (pgao2@ustc.edu.cn).

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Author notes

  1. These authors contributed equally: Shi-Ting Li, De Huang.

Authors and Affiliations

  1. Division of Molecular Medicine, Hefei National Laboratory for Physical Sciences at Microscale, The CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, China

    Shi-Ting Li, De Huang, Shengqi Shen, Songge Xing, Gongwei Wu, Zetan Jiang, Yijie Hao, Mengqiu Yuan, Lianbang Zhu, Ronghui Yan, Dongdong Yang, Lin Wang, Zhaoji Liu, Xin Hu, Rongbin Zhou, Kun Qu, Huafeng Zhang & Ping Gao

  2. Department of Pathology, School of Medicine, Anhui Medical University, Hefei, China

    Yongping Cai & Nana Wang

  3. Guangzhou First People’s Hospital, School of Medicine, Institutes for Life Sciences, South China University of Technology, Guangzhou, China

    Mengqiu Yuan, Zhaoji Liu & Ping Gao

  4. School of Biomedical Sciences and Engineering, Guangzhou International Campus, South China University of Technology, Guangzhou, China

    Mengqiu Yuan, Zhaoji Liu & Ping Gao

  5. Institute of Basic Medical Sciences, National Center of Biomedical Analysis, Beijing, China

    Ailing Li

  6. State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology and Toxicology, Beijing, China

    Xiaotao Duan

  7. Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China

    Ping Gao

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Contributions

P.G. and H.Z. conceived the study and supervised the experiments. S.-T.L., D.H., P.G., X.D. and H.Z. designed the experiments. X.D. performed and analysed MS mitochondrial protein acetylation. S.-T.L., D.H., S.X., G.W., Z.J., Y.H., M.Y., N.W., R.Y., D.Y., L.W., Z.L. and X.H. performed experiments. S.S., L.Z. and K.Q. analysed ChIP-seq data and RNA-seq data. Y.C. provided clinical specimens. R.Z. and A.L. provided constructive guidance and advice. P.G., H.Z., D.H. and S.-T.L. wrote the paper. All the authors read and approved the manuscript.

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Correspondence to Xiaotao Duan, Huafeng Zhang or Ping Gao.

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Extended data

Extended Data Fig. 1 Myc inhibits SDH complex activity and causes cellular succinate accumulation by inducing SDHA acetylation.

a, Western blot analysis of the nuclear marker lamin B1, cytoplasmic marker tubulin, endoplasmic reticulum marker CALR (Calreticulin) and mitochondrial marker COX4 (cytochrome c oxidase subunit IV) using the whole cell lysates or purified subcellular fractions from P493 cells. b, Diagram showing extraction of the mitochondrial fraction after P493 cells were treated with or without tetracycline (tet) for 48 hours. Mitochondrial proteins were digested, and lysine-acetylated peptides were immunoprecipitated using acetylated-lysine antibody. Enriched acetylated peptides were analyzed by nano LC-MS/MS (see Materials and Methods). c, HT1080 cells stably expressing flag-SDHA were further infected with viruses expressing non-targeting control (NTC) or Myc shRNA, followed by immunoprecipitation using anti-flag antibody, IgG (left panel) or using anti-lysine-acetylation antibody (anti-K-Ac) or IgG (right panel). Western blot analysis of Myc, flag-SDHA and SDHA acetylation. d, SDH activity was measured in HT1080 and HeLa cells infected with viruses expressing NTC or Myc shRNA for 48 hours (left panel), or in HT1080 and HeLa cells infected with pMX-GFP empty vector (EV) or pMX-GFP-Myc vector (right panel). Myc and SDHA expression were analyzed by western blotting. Data are presented as the mean ± SD of three independent experiments (n = 3). Group differences are analyzed by the two-tailed Student’s t-test. P < 0.05 compared with the NTC or EV group. e, SDH activity was measured in HT1080 cells infected with viruses expressing NTC or Myc shRNA (right panel) and further exposed to 10 mM NAM and 1 μM TSA. Immunoprecipitation assay using anti-SDHA antibody was performed using the above conditions. SDH activity was normalized to protein content. Data are presented as the mean ± SD of three independent experiments (n = 3). Group differences are analyzed by the two-tailed Student’s t-test. P < 0.05 compared with the corresponding control group. f, K335, K547 and K550 acetylated sites of SDHA were determined by LC-MS/MS. g, LC-MS/MS spectrum of the K335 acetylated peptide of SDHA. h, Primary sequence alignment of acetylated peptides from the indicated species. * denotes the acetylated K335 residues detected by nano LC-MS/MS. i, Validation of the acetyl-SDHA (K335) antibody by dot blot using a nitrocellulose membrane spotted with acetylated K335 peptide or unmodified peptide at the indicated concentration. j, Western blot analysis of Myc, SDHA K335 acetylation and endogenous SDHA levels in Raji cells expressing NTC or Myc shRNAs (sh1 and sh2), or in HT1080 cells expressing pMX-GFP empty vector (EV) or pMX-GFP-Myc vector. k, Western blot analysis of SDHA expression in HT1080 cells infected with viruses expressing NTC or SDHA shRNAs (sh1 and sh2). Cellular succinate and fumarate levels were determined using assay kits. Data are presented as the mean ± SD of three independent experiments (n = 3). Group differences are analyzed by the two-tailed Student’s t-test. P < 0.05 compared with the NTC group. l, HT1080 cells with stable knockdown of endogenous SDHA were subsequently infected with viruses expressing flag-wild-type-SDHA (WT), flag-SDHAK335R (K335R) or flag-SDHAK335Q (K335Q), followed by western blot analysis of SDHA K335 acetylation and measurement of succinate and fumarate. Relative succinate or fumarate levels of triplicate experiments mean ± SD are presented (n = 3). Group differences are analyzed by the two-tailed Student’s t-test. P < 0.05 compared with the EV group. m, Cellular succinate, fumarate or α-KG levels were determined using the LC-MS in the HT1080 cells used in (Fig. 1i, right panel). The metabolic measurements of LC-MS were normalized to cellular protein. Metabolites level of triplicate experiments mean ± SD are presented (n = 3). Group differences are analyzed by the two-tailed Student’s t-test. P < 0.05 compared with the indicated group. n and o, Cellular succinate, fumarate or α-KG levels were determined by assay kits in the HT1080 cells used in (Fig. 1i, right panel) (o), then succinate/α-KG ratio or fumarate/α-KG ratio was calculated (n). The metabolic measurements of LC-MS were normalized to cellular protein. Data are presented as the mean ± SD of three independent experiments (n = 3). Group differences are analyzed by the two-tailed Student’s t-test. P < 0.05 compared with the indicated group. n = 3 independent experiments were repeated with similar results (a, c, i, and j). Two independent LC-MS/MS experiments in P493 cells were repeated with similar results (g).

Source data

Extended Data Fig. 2 Myc increases SDHA acetylation by accelerating SKP2-mediated proteasomal degradation of SIRT3.

a, Mitochondrial proteins from P493 cells treated with or without tetracycline (tet) for 48 hours were cultured in the presence of vehicle or 1 μM TSA for 18 hours, followed by western blot analysis of lysine-acetylation (K-Ac). COX4 served as a loading control. b, Quantitative real-time PCR analysis of SIRT3 mRNA levels in P493 cells treated with or without tetracycline (tet) for 24 hours. Data are presented as the mean ± SD of three independent experiments (n = 3). Group differences are analyzed by the two-tailed Student’s t-test. P < 0.05 compared with the indicated group. c, Western blot analysis of SIRT3 in P493 cells treated with or without tetracycline (tet) for 48 hours and further cultured in the presence of vehicle or 10 μM lactacystin for 8 hours. Tubulin served as a loading control. d, Immunoprecipitation was performed using anti-flag antibody or IgG in HEK293T cells transfected with flag-SKP2 and SIRT3, followed by western blot analysis of SIRT3 and SKP2 (left panel). Immunoprecipitation was performed using anti-SIRT3 antibody or IgG in HT1080 cells, followed by western blot of SIRT3 and SKP2 (right panel). Tubulin served as a loading control. e, Quantitative real-time PCR and western blot analysis of SKP2 expression in P493 cells treated with or without tetracycline (tet) for 24 and 48 hours. Data are presented as the mean ± SD of three independent experiments (n = 3). Group differences are analyzed by the two-tailed Student’s t-test. P < 0.05 compared with the indicated group. f, Western blot analysis of SKP2 in HT1080 cells infected with viruses expressing NTC or Myc shRNA (left panel) or in HT1080 cells infected with viruses expressing pMX-GFP empty vector (EV) or pMX-GFP-Myc vector (right panel). Tubulin served as a loading control. g, Western blot analysis of SKP2 and SIRT3 protein in P493 cells (upper left panel), HT1080 cells (bottom left panel) or CA46 cells (right panel) infected with viruses expressing NTC or SKP2 shRNAs (sh1 and sh2), or infected with EV or pSIN-3×flag-SKP2. Tubulin served as a loading control. h, HT1080 cells stably expressing NTC or Myc shRNA were subsequently infected with viruses expressing empty vector (EV) or flag-SKP2 vector, followed by western blot analysis of Myc, flag-SKP2 and SIRT3 expression. i, HT1080 cells stably expressing NTC or SKP2 shRNAs were subsequently infected with viruses of EV or Myc, followed by western blot analysis of Myc, SKP2 and SIRT3 expression. Tubulin served as a loading control. j, Immunoprecipitation was performed using anti-flag antibody or IgG in HEK293T cells transfected with flag-SDHA and SIRT3, followed by western blot analysis of SIRT3 and flag-SDHA. k, CA46 cells stably expressing empty vector (EV) or SIRT3 were subsequently infected with viruses expressing NTC or Myc shRNA (left panel), or CA46 cells stably expressing NTC or SIRT3 shRNA were further infected with viruses expressing NTC or Myc shRNA (right panel), followed by western blot analysis of Myc, SIRT3, SDHA and SDHA K335 acetylation. l, P493 cells stably expressing EV or 3×flag-SIRT3 were treated with or without tetracycline (tet) for 48 hours, (left panel). P493 cells stably expressing NTC or SIRT3 shRNA were treated with or without tetracycline (tet) for 48 hours (right panel), followed by western blot analysis of SDHA K335 acetylation, SDHA, SIRT3 and Myc. Tubulin served as a loading control. m-p, Cellular succinate or α-KG levels were determined by LC-MS in the HT1080 cells used in Fig. 2i, then succinate/α-KG ratio was calculated. The succinate or α-KG levels were normalized to cellular protein. Succinate or α-KG level of triplicate experiments mean ± SD are presented (n = 3). Group differences are analyzed by the two-tailed Student’s t-test. P < 0.05 compared with the indicated group. q, Measurement of SDH activity in the CA46 cells used in (k, right panel). Relative enzyme activities of triplicate experiments mean ± SD are presented (n = 3). Group differences are analyzed by the two-tailed Student’s t-test. P < 0.05 compared with the indicated group. r-t, Cellular succinate or α-KG levels were determined with LC-MS in the CA46 cells used in (k, right panel), then succinate/α-KG ratio was calculated (r). The succinate or α-KG levels were normalized to cellular protein (s, t). Succinate or α-KG level of triplicate experiments mean ± SD are presented (n = 3). Group differences are analyzed by the two-tailed Student’s t-test. P < 0.05 compared with the indicated group. u, Measurement of SDH activity in the P493 cells used in (l, right panel). Relative enzyme activities of triplicate experiments ± SD are presented (n = 3). Group differences are analyzed by the two-tailed Student’s t-test. P < 0.05 compared with the indicated group. v-x, Cellular succinate or α-KG levels were determined with LC-MS in the P493 cells used in (l, right panel), then succinate/α-KG ratio was calculated (v). The succinate or α-KG levels were normalized to cellular protein (w, x). Succinate or α-KG level of triplicate experiments mean ± SD are presented (n = 3). Group differences are analyzed by the two-tailed Student’s t-test. P < 0.05 compared with the indicated group. n = 3 independent experiments were repeated with similar results (a, c, d and f-l).

Source data

Extended Data Fig. 3 Myc enhances H3K4me3 via SDHA acetylation-mediated increase in cellular succinate.

a, Western blot analysis of the indicated histone methylation markers and Myc in Raji cells infected with viruses expressing NTC or Myc shRNAs (sh1, sh2). b, P493 cells treated with or without tetracycline (tet) were exposed to 5 mM dimethyl α-KG (DM-αKG) for 8 hours, followed by western blot analysis of H3K4me3 and Myc from nuclear lysates and whole cell lysates, respectively. c, P493 cells treated with or without tetracycline (tet) were exposed to 10 mM dimethyl succinate (DMS) or/and 5 mM dimethyl α-KG (DM-αKG), followed by western blot analysis of H3K4me3 and Myc from nuclear lysates and whole cell lysates, respectively. H3 and tubulin served as loading controls. d, Western blot analysis of the indicated histone methylation markers from nuclear lysates and whole cell lysates in HT1080 cells infected with viruses expressing NTC or SIRT3 shRNAs (sh1 and sh2). Tubulin and H3 served as loading controls. WCL, whole-cell lysate; NL, nuclear lysate. e, HT1080 cells infected with viruses of NTC or Myc shRNA were cultured in the presence of vehicle or 10 mM NAM, followed by western blot analysis of H3K4me3, Myc and SIRT3 from nuclear lysates and whole cell lysates. Tubulin and H3 served as loading controls. WCL, whole-cell lysates; NL, nuclear lysates. f, HT1080 cells stably expressing pSIN-EV empty vector (EV) or pSIN-SIRT3 vector were subsequently infected with viruses expressing pMX-GFP empty vector (EV) or pMX-GFP-Myc vector (left panel), or HT1080 cells stably expressing NTC or SIRT3 shRNA were subsequently infected with viruses expressing NTC or Myc shRNA (right panel), followed by western blot analysis of H3K4me3, Myc and SIRT3 levels. g, HT1080 cells with stable knockdown of endogenous SDHA were infected with viruses expressing flag-wild-type-SDHA (WT) or flag-SDHAK335Q (K335Q), followed by further infection with viruses of NTC or SIRT3 shRNA and NTC or Myc shRNA. Western blot analysis of H3K4me3, Myc and SIRT3 levels. h, Western blot analysis of the in vitro demethylation reactions using H3K4me3 peptide as substrates. Antibodies used are indicated. i, HT1080 cells stably expressing NTC or sh-Myc were subsequently infected with viruses expressing NTC or sh-KDM5A, followed by western blot analysis of Myc, H3K4me3, KDM5A, H3 and tubulin expression. WCL, whole-cell lysates; NL, nuclear lysates. j, P493 cells treated with or without tetracycline (tet) for 48 h were cultured in the presence of vehicle or 10 mM dimethyl succinate (DMS) for 24 h, followed by western blot analysis of H3K4me3 (up right panel) and ChIP-seq analysis of H3K4me3 around gene TSS regions. Heat map showing H3K4me3 levels in TSS regions for the top 2500 gene promoters (left panel). Distribution analysis of H3K4me3 tags around the TSS regions of the top 2500 genes (bottom right panel). n = 3 independent experiments for western blot (up right panel) were repeated with similar results. One replicate (n = 1, each group) was used for ChIP-seq assay of P493 cells. k, H3K4me3 markers of ChIP-sequencing traces in P493 cells for the indicated genes were determined by IGV software analysis. For IGV analysis, one replicate (n = 1, each group) was used for H3K4me3 analysis in ChIP-seq assay of P493 cells. l, ChIP assay analysis of the occupancy of H3K4me3 on the indicated gene promoters in the CA46 cells used in Fig. 3g (left panel) or P493 cells used in Extended Data Fig. 3j (right panel). Data are presented as the mean ± SD of three independent experiments (n = 3). Group differences are analyzed by the two-tailed Student’s t-test. P < 0.05 compared with the NTC–DMS group or shMyc–DMS group (left panel); P < 0.05 compared with the –tet–DMS group or +tet–DMS group (right panel). n = 3 independent experiments were repeated with similar results (a-i).

Source data

Extended Data Fig. 4 Myc-mediated succinate accumulation and H3K4me3 activation promote its target gene expression.

a, CA46 cells treated with viruses expressing NTC or Myc shRNAs were cultured in the presence of vehicle or 10 mM dimethyl-succinate (DMS) for 24 hours, followed by the extraction of total RNAs for the RNA-Seq experiment. Heat map showing all the regulated genes. b, P493 cells treated with or without tetracycline (tet) were cultured in the presence of vehicle or 10 mM dimethyl-succinate (DMS) for 24 hours, followed by the extraction of total RNAs for the RNA-Seq experiment. Heat map showing all the regulated genes. c, Heat map of the RNA-seq results showing that the genes with down-regulated expression following Myc depletion were subsequently rescued by DMS treatment in P493 cells (fold-change >1.5). d, Venn diagram showing overlapping genes co-regulated by Myc and succinate based on the RNA-Seq data and ChIP-Seq data in P493 cells. Correlation significance between genes with mRNA levels and genes with H3K4me3 that were rescued by DMS based on RNA-seq and ChIP-seq, respectively, were determined using a two-tailed Spearman test, P < 0.001. e, Analysis of 585 overlapping genes by Gene Ontology (GO) term enrichment. P values were analysed with two-tailed Student’s t-test. The sufficient RNA-seq samples (n = 1, each group) were analyzed in Gene Ontology (GO) term enrichment. f, ChIP assay analysis of the occupancy of H3K4me3 on the indicated gene promoters in the CA46 cells used in (a). Data are presented as the mean ± SD of three independent experiments (n = 3). Group differences are analyzed by the two-tailed Student’s t-test. P < 0.05 compared with the NTC –DMS group; P < 0.05 compared with the shMyc–DMS group. g, ChIP assay analysis of the occupancy of H3K4me3 on the indicated gene promoters in the P493 cells used in (b). Data are presented as the mean ± SD of three independent experiments. Group differences are analyzed by the two-tailed Student’s t-test. P < 0.05 compared with the –tet –DMS group; P < 0.05 compared with the +tet –DMS group. h, P493 cells treated with or without tetracycline (tet) for 48 hours were cultured in the presence of vehicle or 500 μM 3-nitropropionic acid (3-NPA) for 24 hours, followed by western blot analysis of H3K4me3. i, Heat map of the RNA-seq data showing that the genes with down-regulated expression following Myc depletion were markedly rescued by 3-NPA treatment in P493 cells (fold-change >1.5). The statistical significance between Myc- and succinate-regulated genes was determined using Fisher’s exact probability, P < 0.001. The RNA-seq samples (n = 1, each group) used to analysed for Heatmap. j, ChIP assay studying the occupancy of H3K4me3 on the indicated gene promoters in the P493 cells used in i. Data are presented as the mean ± SD of three independent experiments (n = 3). Group differences are analyzed by the two-tailed Student’s t-test. P < 0.05 compared to the –tet +PBS group; P < 0.05 compared to the +tet +PBS group. k, The mRNA levels of the indicated genes were determined by RT-qPCR in the P493 cells used in b. Data are presented as the mean ± SD of three independent experiments (n = 3). Group differences are analyzed by the two-tailed Student’s t-test. P < 0.05 compared with the –tet –DMS group; P < 0.05 compared with the +tet –DMS group. l, The indicated proteins were determined by western blotting in the P493 cells used in k. m, P493 cells with endogenous SDHA knockdown were further constructed to stably express flag-wild-type-SDHA (WT) or flag-SDHAK335R (K335R), followed by treatment with tetracycline (tet) for 48 h or washing to remove tet for 48 hours. The mRNA levels of the indicated genes were determined by RT-qPCR. Data are presented as the mean ± SD of three independent experiments (n = 3). Group differences are analyzed by the two-tailed Student’s t-test. P < 0.05 compared with the +tet +WT group; P < 0.05 compared with the wash +WT group. n, The indicated proteins were determined by western blotting in the P493 cells used in m. The statistical significance between Myc- and succinate-regulated genes was determined using the two-sided Fisher’s exact probability, P < 0.001. The sufficient RNA-seq samples (n = 1, each group) used to analysed for Heatmap (a-c). n = 3 independent experiments were repeated with similar results (h, l and n). Tubulin and H3 served as loading controls. WCL, whole-cell lysates; NL, nuclear lysates.

Source data

Extended Data Fig. 5 SDHA acetylation at lysine 335 contributes to Myc-promoted tumour progression.

a, P493 cells with stable knockdown of endogenous SDHA were further infected with viruses expressing flag-wild-type-SDHA (WT), flag-SDHAK335R (K335R) or flag-SDHAK335Q (K335Q). Cell growth curves were determined by trypan blue counting. Data are presented as the mean ± SD (n = 3, each group). Group differences are analyzed by the two-tailed Student’s t-test. P < 0.05 compared with the corresponding indicated group. b, Endogenous SDHA was knocked down in empty vector (EV) or Myc overexpressing HT1080 cells, followed by further infection with viruses expressing flag-wild-type-SDHA (WT) or flag-SDHA-K335Q (K335Q). The cells were treated with or without 5 mM dimethyl α-KG (DM-αKG) (fresh medium containing fresh DM-αKG was replaced daily), and the cell growth of HT1080 cells was determined by trypan blue staining. Data are presented as the mean ± SD (n = 5, each group). Group differences are analyzed by the two-tailed Student’s t-test. P < 0.05 compared between indicated groups. c, Endogenous SDHA was knocked down in empty vector (EV) or Myc overexpressing HT1080 cells, followed by further infection with viruses expressing flag-wild-type-SDHA (WT) or flag-SDHA-K335R (K335R). The cells were treated with or without 5 μM CPI-455, followed by cell growth analysis with trypan blue staining. Data are presented as the mean ± SD (n = 3, each group). Group differences are analyzed by the two-tailed Student’s t-test. P < 0.05 compared with the indicated group. d, The tumor mass of the extracted tumors in Fig. 5d was measured. Data are presented as mean ± SEM (n = 5, each group). Group differences are analyzed by the two-tailed Student’s t-test. P < 0.05 compared with the corresponding indicated group. e and f, The succinate and α-KG levels were measured using detection kits with cell lysates from xenograft tumor tissues. The succinate and α-KG level were normalized to cellular protein. Data are presented as the mean ± SD (n = 5, each group). Group differences are analyzed by the two-tailed Student’s t-test. P < 0.05 compared with the indicated group. g and h, HT1080 cells used in b were injected subcutaneously into BALB/c nude mice (n = 5, each group). Mice were treated with daily i.p injection of Dimethyl α-KG (0.6 g/kg body weight) or PBS, starting one day before tumor cell implantation. Tumor growth curves were measured starting from 5 days after inoculation (g). Tumors were extracted and compared at the end of the experiment (h). Data are presented as the mean ± SD (n = 5, each group). Group differences are analyzed by the two-tailed Student’s t-test. P < 0.05 compared between indicated groups. i, Levels of Myc, SIRT3, SDHA, and K335 acetylation of SDHA and H3K4me3 were determined by western blot using the nuclear lysates or whole cell lysates of tumor tissues from each group as in h. n = 5 independent xenograft tumor tissues lysates in each group were repeated by western blot with similar results. Tubulin and H3 served as loading controls. WCL, whole cell lysates; NL, nuclear lysates. j and k, HT1080 cells used in c were injected subcutaneously into BALB/c nude mice (n = 5 for each group). Mice were treated with daily i.p injection of CPI- 455 (20 mg/kg body weight) or vehicle (4%DMSO + 35%PEG 300 + 2%Tween 80+PBS), starting one day before tumor cell implantation. Tumor growth curves were measured starting from 5 days after inoculation (j). Tumors were extracted and compared at the end of the experiment (k). Data are presented as the mean ± SD (n = 5, each group). Group differences are analyzed by the two-tailed Student’s t-test. P < 0.05 compared between indicated groups. l, Levels of Myc, SIRT3, SDHA, and K335 acetylation of SDHA and H3K4me3 were determined by western blot using the nuclear lysates or whole cell lysates of tumor tissues from each group as in k. Tubulin and H3 served as loading controls. WCL, whole-cell lysates; NL, nuclear lysates. n = 5 independent xenograft tumor tissues lysates in each group were repeated by western blot with similar results.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 3–6

Reporting Summary

Supplementary Tables

Supplementary Tables 1 and 2

Source data

Source Data Fig. 1

Unprocessed western blots. Please note that the slight difference between WB images of Fig. 1c (SDHA), 1e (SDHA), 1h (Myc) and the corresponding uncropped images in source data were caused by different exposure time.

Source Data Fig. 2

Unprocessed western blots. Please note that the slight difference between WB images of Fig. 2b (Tubulin), Fig. 2c (SIRT3), Fig. 2e (Myc), Fig. 2g (SDHA and Tubulin) and the corresponding uncropped images in source data were caused by different exposure time.

Source Data Fig. 3

Unprocessed western blots. Please note that the slight difference between WB images of Fig. 3a (Myc) and the corresponding uncropped images in source data were caused by different exposure time.

Source Data Fig. 4

Unprocessed western blots.

Source Data Fig. 5

Unprocessed western blots. Please note that the slight difference between WB images of Fig. 5e (H3K4me3) and the corresponding uncropped images in source data were caused by different exposure time.

Source Data Extended Data Fig. 1

Unprocessed western blots. Please note that the slight difference between WB images of Extended Data Fig. 1c (Tubulin) and the corresponding uncropped images in source data were caused by different exposure time.

Source Data Extended Data Fig. 2

Unprocessed western blots. Please note that the slight difference between WB images of Extended Data Fig. 2g (SKP2) and the corresponding uncropped images in source data were caused by different exposure time.

Source Data Extended Data Fig. 3

Unprocessed western blots. Please note that the slight difference between WB images of Extended Data Fig. 3g (SIRT3) and the corresponding uncropped images in source data were caused by different exposure time.

Source Data Extended Data Fig. 4

Unprocessed western blots.

Source Data Extended Data Fig. 5

Unprocessed western blots.

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Li, ST., Huang, D., Shen, S. et al. Myc-mediated SDHA acetylation triggers epigenetic regulation of gene expression and tumorigenesis. Nat Metab 2, 256–269 (2020). https://doi.org/10.1038/s42255-020-0179-8

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