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Oncometabolites suppress DNA repair by disrupting local chromatin signalling

Nature volume 582, pages 586–591 (2020)Cite this article

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Abstract

Deregulation of metabolism and disruption of genome integrity are hallmarks of cancer1. Increased levels of the metabolites 2-hydroxyglutarate, succinate and fumarate occur in human malignancies owing to somatic mutations in the isocitrate dehydrogenase-1 or -2 (IDH1 or IDH2) genes, or germline mutations in the fumarate hydratase (FH) and succinate dehydrogenase genes (SDHA, SDHB, SDHC and SDHD), respectively2,3,4. Recent work has made an unexpected connection between these metabolites and DNA repair by showing that they suppress the pathway of homology-dependent repair (HDR)5,6 and confer an exquisite sensitivity to inhibitors of poly (ADP-ribose) polymerase (PARP) that are being tested in clinical trials. However, the mechanism by which these oncometabolites inhibit HDR remains poorly understood. Here we determine the pathway by which these metabolites disrupt DNA repair. We show that oncometabolite-induced inhibition of the lysine demethylase KDM4B results in aberrant hypermethylation of histone 3 lysine 9 (H3K9) at loci surrounding DNA breaks, masking a local H3K9 trimethylation signal that is essential for the proper execution of HDR. Consequently, recruitment of TIP60 and ATM, two key proximal HDR factors, is substantially impaired at DNA breaks, with reduced end resection and diminished recruitment of downstream repair factors. These findings provide a mechanistic basis for oncometabolite-induced HDR suppression and may guide effective strategies to exploit these defects for therapeutic gain.

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Fig. 1: Oncometabolites directly suppress HDR.
Fig. 2: Oncometabolites suppress the stepwise recruitment of HDR factors to DNA DSBs.
Fig. 3: Oncometabolites suppress HDR via inhibition of KDM4B.
Fig. 4: Aberrant H3K9 methylation impairs HDR and mechanistically underlies oncometabolite induced HDR deficiency.

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All data generated or analysed during this study are included in this published article (and its supplementary information files, including source data). There are no restrictions on data availability.

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Acknowledgements

We thank D. Hegan and A. Dhawan for their assistance. This work was supported by the NIH (grants R01ES005775 and R35CA197574 to P.M.G., and R01CA215453 to R.S.B.) and by the American Cancer Society (Research Scholar Grant to R.S.B.). P.L.S. was supported by the NIH National Institute of General Medical Sciences training grant T32GM007223.

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Author notes

  1. These authors jointly supervised this work: Ranjit S. Bindra, Peter M. Glazer

Authors and Affiliations

  1. Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, USA

    Parker L. Sulkowski, Sebastian Oeck, Jonathan Dow, Nicholas G. Economos, Yanfeng Liu, Katelyn Noronha, Ranjit S. Bindra & Peter M. Glazer

  2. Department of Genetics, Yale University School of Medicine, New Haven, CT, USA

    Parker L. Sulkowski, Jonathan Dow, Nicholas G. Economos & Peter M. Glazer

  3. Department of Medical Oncology, University of Duisburg-Essen, Essen, Germany

    Sebastian Oeck

  4. Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA

    Lily Mirfakhraie & Megan C. King

  5. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA

    Katelyn Noronha

  6. Karmanos Cancer Institute, Wayne State University, Detroit, MI, USA

    Xun Bao & Jing Li

  7. Department of Urology, University of California at Los Angeles, Los Angeles, CA, USA

    Brian M. Shuch

  8. Department of Pathology, Yale University School of Medicine, New Haven, CT, USA

    Ranjit S. Bindra

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Contributions

P.L.S. designed and performed experiments and contributed to all aspects of the study. S.O. designed experiments and performed and analysed imaging studies and laser micro-stripe irradiation. J.D. and N.E. performed DNA repair and cell biology assays and contributed to data analysis and compiling of the manuscript. Y.L. designed and performed the tumour growth delay assays. B.S., X.B. and K.N. provided reagents and contributed to the experiments. L.M. and M.C.K. designed and performed end-resection assays. J.L. performed LC/MS assays. P.M.G. and R.S.B. conceptualized and supervised the study and interpreted the data. P.M.G., R.S.B., M.C.K. and P.L.S. wrote the manuscript.

Corresponding authors

Correspondence to Ranjit S. Bindra or Peter M. Glazer.

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Competing interests

R.S.B., P.M.G. and P.L.S. are inventors on US patent application no. 62/344,678 submitted by and assigned to Yale University, which covers compositions and methods for targeting and treating HDR-deficient tumours. P.M.G and R.S.B are founders of and consultants for Cybrexa Therapeutics. P.M.G. is a consultant for pHLIP. Neither company has any conceptual or financial connection to this manuscript.

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Peer review information Nature thanks Brendan Price, Yue Xiong and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Elevated oncometabolites induce H3K9me3 and influence global gene expression, but do not alter expression of HDR genes.

a, Liquid chromatography with mass spectrometry (LC–MS) quantification of 2HG levels in cells with engineered IDH1 mutations and in cells with endogenous IDH1 and IDH2 mutations, as indicated. Note that engineered IDH1-mutant cells produce 2HG at levels similar to the endogenous mutant cell lines, and that the HT1080 cells with CRISPR–Cas9 knockout of the mutant IDH1R132C allele (IDH1KO/+) show reduction of 2HG production to levels similar to those seen in IDH1 wild-type cells. Levels of 2HG are also quantified in endogenous mutant IDH1 or IDH2 cells after treatment with the mutant-IDH1-specific inhibitor AGI-5198 and the mutant-IDH2-specific inhibitor AG-221. b, Western blot analysis of H3K9me3 levels in cell lines with WT IDH1 or engineered to express mutant IDH1, as indicated, compared with the HT1080 cell line with an endogenous IDH1R132C mutation. c, d, LC–MS analysis of succinate (c) and fumarate (d) in: YUNK1 cells with shRNA suppression of SDHB or FH compared with shCTRL (non-targeting) control cells, the endogenous FH−/− UOK 262 renal cell carcinoma cell line with and without FH cDNA complementation (clones 1–3), and in the FH−/− NCCFH1 renal cell carcinoma cell line. e, Western blot analysis of H3K9me3 in YUNK1 cells with shRNA suppression of SDHB or FH compared to shCTRL (non-targeting) control cells, the endogenous FH−/− UOK 262 renal cell carcinoma cell line with and without FH cDNA complementation, and in the FH−/− NCCFH1 renal cell carcinoma cell line. This experiment was repeated twice with similar results. f, Western blot analysis of H3K9me3 in HT1080 cells (IDH1R132C/+) and in HT1080 cells with CRISPR–Cas9 knockout of the mutant IDH1 allele (IDH1KO/+), and with or without treatment with 500 μM 2HG, 1 mM AGI-5198 or 1 mM AGI-5198 and 500 μM 2HG. This experiment was repeated twice with similar results. g, Western blot analysis of H3K9me3 in WT IDH1 U87 glioblastoma cells and in U87 cells with CRISPR–Cas9 knock-in of the mutant IDH1R132H allele and with or without treatment with 500 μM 2HG, 1 μM AGI-5198 or 1 μM AGI-5198 and 500 μM 2HG. This experiment was repeated three times with similar results. h, Western blot analysis of H3K9me3 in FH−/− UOK 262 cells with or without FH cDNA complementation (clones 1–3) and with or without treatment with 30 μM dimethyl fumarate. This experiment was repeated twice with similar results. i, Plot of microarray gene expression analysis of astrocytes either overexpressing IDH1WT or IDH1R132H. Genes were plotted in descending order based on the ratio of IDH1WT expression levels to that of IDH1R132H. Genes with a high value are highly expressed in IDHWT cells compared to IDH1R13H cells, and therefore represent genes that are putatively suppressed in IDH1R132H-expressing cells. j, Heat map representation of microarray expression analysis of HDR-associated genes in matched pairs of otherwise isogenic IDHWT and IDH1R132H mutant cells: IDHWT and IDH1R132H/+ HCT116 cells; human immortalized astrocytes expressing IDHWT or IDH1R132H, and IDHWT and IDH1R132H/+ HeLa cells. k, Dot plot of expression levels of HDR-associated genes from the TCGA lower-grade glioma mRNA-seq dataset. For each gene, patient samples are separated by IDH status. l, Western blot analyses of IDH1(R132H), total IDH1, FH, SDHB, RAD51, ATM, BRCA2, TIP60, RPA and MRE11 in YUNK1 cells with shRNA suppression of SDHB or FH compared with shCTRL (non-targeting) control cells and in astrocytes overexpressing IDH1(WT) or IDH1(R132H). This experiment was repeated four times with similar results. m, n, Representative images of neutral comet assays performed in immortalized astrocytes overexpressing IDH1(WT) or IDH1(R132H) or treated with 2HG, 2 mM succinate or 30 μM dimethyl fumarate (m) and in YUNK1 cells after shRNA suppression of FH or SDHB or addition of 500 μM octyl-(R)-2HG, 2 mM succinate or 30 μM dimethyl fumarate (n). Scale bars, 400 μm. o, LC–MS quantification of 2HG in astrocytes after 2 h treatment with 500 μM R-octyl-2HG. p, Western blot analysis of H3K9me3 levels in astrocytes treated with 500 μM R-octyl-2HG for 2, 4 or 12 h, compared to vehicle control (DMSO cells) or astrocytes expressing IDH1(R132H). This experiment was repeated two times with similar results. q, LC–MS quantification of succinate in YUNK1 cells after 2 h treatment with 2 mM succinate. r, Western blot analysis of H3K9me3 levels in YUNK1 cells treated with 2 mM succinate for 2, 4 or 12 h, compared to vehicle control (DMSO) cells or YUNK1 cells with shRNA suppression of SDHB. This experiment was repeated two times with similar results. s, LC–MS quantification of fumarate in YUNK1 cells after 2 h treatment with 30 μM dimethyl fumarate. t, Western blot analysis of H3K9me3 levels in YUNK1 cells treated with 2 mM succinate for 2, 4 or 12 h, compared to vehicle control (DMSO) cells or YUNK1 cells with shRNA suppression of FH. This experiment was repeated two times with similar results. In a, c, d, o, q and s, data are mean ± s.e.m. with n = 3 biological replicates; statistical analysis is by two-tailed unpaired t-test; df = 4, with P values as indicated.

Source Data

Extended Data Fig. 2 Oncometabolites directly impair formation of RAD51 and BRCA1 foci after ionizing radiation and cause radiosensitization.

a, Quantification of RAD51 nuclear foci at the indicated time points after 2 Gy ionizing radiation (IR), in IDHWT or IDH1R132H/+ HeLa cells. Statistical analysis of time course by ANOVA, F = 35.15, df = 1. Statistical analysis at specific time points by two-tailed unpaired t-test, df = 4. b, Representative images of RAD51 nuclear foci 4 h after 2 Gy ionizing radiation in IDHWT or IDH1R132H/+ HeLa cells. These experiments were performed three times as quantified in a. Scale bar, 10 μm. c, Western blot analysis of FH and SDHB in HEK293FT cells with shCTRL (non-targeting control) or shRNA suppression of SDHB or FH. These experiments were repeated six times. d, Quantification of RAD51 nuclear foci at the indicated time points following 2 Gy ionizing radiation in HEK293FT cells with shRNA suppression of SDHB or FH compared to non-targeting control (shCTRL) cells, and in shCTRL cells treated with either 2 mM succinate or 30 μM dimethyl fumarate. Statistical analysis of time course by ANOVA. F values versus control: shSDHB, F = 61.55; shFH, F = 195.3; +succinate, F = 258.2; +fumarate, F = 242.4; df = 1 for all tests. Statistical analysis at indicated time points by two-tailed unpaired, t-test; df = 4. e, Representative images of RAD51 nuclear foci at 4 h post ionizing radiation in HEK293FT cells with shRNA suppression of SDHB or FH compared to non-targeting control (shCTRL) cells. Scale bar, 10 μm. These experiments were performed three times and are quantified in d. (f) Quantification of cells with RAD51 foci-positive nuclei (>10 foci per nucleus) 4 h after 2 Gy ionizing radiation treatment of parental U87 glioma cells (IDH1WT) and in U87 cells with CRISPR–Cas9-mediated knock-in of an IDH1R132H allele at the endogenous locus (IDH1R132H/+). Cells were treated as indicated with or without 500 μM octyl-(R)-2HG (2HG) for 24 h, 1 mM AG1-5198 for 5 days or a combination thereof, before irradiation. g, Quantification of RAD51 foci-positive nuclei in SNU1079 (IDH1R132C/+) and RBE (IDH1R132S/+) cholangiocarcinoma cells, and SW1353 (IDH2R172K/+) chondrosarcoma cells 4 h after 2 Gy ionizing radiation. Mutant IDH1 cells were treated with or without 2HG, AGI-5198 or AGI-5198 + 2HG as in f and the IDH2-mutant SW1353 cells were treated with or without 2HG or AG-221 or AG-221 + 2HG. h, Representative images of BRCA1 nuclear foci at 4 h after 2 Gy ionizing radiation. Scale bar, 10 μm. This experiment was repeated three times and is quantified in i and j. i, Quantification of BRCA1 nuclear foci at the indicated time points after 2 Gy ionizing radiation, in IDHWT or IDH1R132H/+ HeLa cells. Statistical analysis of time course by ANOVA; F = 41.35, df = 1. Statistical analysis at specific time points by two-tailed unpaired t-test; df = 4. j, Quantification of BRCA1 nuclear foci at the indicated time points following 2 Gy ionizing radiation in YUNK1 cells with shRNA suppression of SDHB or FH compared to non-targeting control (shCTRL) cells, and in shCTRL cells treated with either 2 mM succinate or 30 μM dimethyl fumarate. Statistical analysis of time course by ANOVA. F values versus control: shSDHB, F = 20.92; shFH, F = 37.42; +succinate, F = 19.89; +fumarate F = 11.57, df = 1 for all tests. Statistical analysis at indicated time points by two-tailed unpaired, t-test, df = 4. k, Quantification of BRCA1 foci-positive cells (>10 nuclear BRCA1 foci) 4 h after 2 Gy ionizing radiation in SNU1079 (IDH1R132C/+), RBE (IDH1R132S/+) and HT1080 (IDH1R132C/+) cells treated with 1 μM AGI-5198 or DMSO control. l, m, Quantification (l) and representative images (m) of BRCA1 nuclear foci 4 h after 2 Gy ionizing radiation in SW1353 (IDH2R172K/+) cells treated as indicated with DMSO (control), 500 μM 2HG, 5 μM AG-221 or 5 μM AG-221 and 500 μM 2HG. Scale bar, 10 μm. Images in m are from experiments that were repeated three times and are quantified in l. n, Clonogenic survival assay in U87 glioblastoma cells with or without mutant IDH1(R132H) treated with the indicated doses of ionizing radiation. o, Tumour growth delay assay in IDH1WT and IDH1R132H/+ U87 tumour xenografts. Mice were treated with a single dose of 8 Gy ionizing radiation or mock irradiated at a tumour volume of 150 mm3. N = 8 mice per group each with a single tumour. Statistical analysis by ANOVA (IDH1WT, F = 4.25, df = 1; IDH1R132H, F = 13.76, df = 1). p, Western blot analysis of H3K9me3 in U87 IDH1WT and U87 IDH1R132H/+ tumour xenografts. This experiment was performed three times with similar results. In f, g, k, l, data are mean ± s.e.m. with n = 3 biological replicates; statistical analysis by two-tailed unpaired t-test; df = 4. In a, d, i, j, n, data are mean ± s.e.m. of 3 biological replicates; statistical analysis by ANOVA: P values are indicated.

Source Data

Extended Data Fig. 3 Supporting data for DSB–ChIP assays to analyse recruitment of DNA repair factors to an I-SceI-induced, site-specific DNA DSB.

a, Top, schematic of the qPCR analysis to assess I-SceI cleavage at the DSB–ChIP site. Bottom, qPCR analysis to assay site-specific DNA DSB induction 1 h after addition of the ligands Shield-1 and triamcinolone. Reduced amplification of the genomic DNA across the I-SceI target indicates that cleavage at that site has occurred; short bars indicate reduced PCR amplification and therefore successful cleavage. b–j, Pertaining to Fig. 2b, line graphs of percent input values for DSB–ChIP assays performed with the indicated antibodies in cells treated as indicated with either DMSO, 500 μM ocyl-R-2HG, 2 mM succinate or 30 μM dimethyl fumarate, for the following factors: γH2A.X (b; +2HG, F = 10.8; +succinate, F = 2.682; +fumarate, F = 17.8); SUV39H1 (c; +2HG, F = 0.33; +succinate, F = 3.5; +fumarate, F = 0.07); H3K9me3 (d; +2HG, F = 124.4; +succinate, F = 25.21; +fumarate, F = 517); TIP60 (e; +2HG, F = 218; +succinate, F = 340.7; +fumarate, F = 248.6); MRE11 (f; +2HG, F = 97.7; +succinate, F = 209.9; +fumarate, F = 71.2); ATM (g; +2HG, F = 46.8; +succinate, F = 31.7; +fumarate, F = 47.3); BRCA1 (h; +2HG, F = 50.0; +succinate, F = 50.7; +fumarate, F = 24.1); RPA32 (i; +2HG, F = 23.59; +succinate, F = 22.3; +fumarate, F = 16.0); and RAD51 (j; +2HG, F = 43.3; +succinate, F = 61.8; +fumarate, F = 11.75) at the indicated time points after addition of triamcinolone and Shield-1 to induce an I-SceI break in DSB–ChIP U2OS cells. k, l, Line graphs of per cent input values for DSB–ChIP assays performed with IgG controls for rabbit IgG (k) and mouse IgG (l) at the indicated time points post addition of triamcinolone and Shield-1 to induce an I-SceI break in DSB–ChIP cells treated with either DMSO, 500 μM octyl-R-2HG, 2 mM succinate or 30 μM dimethyl fumarate. m–u, Antibody validation experiments for the DSB–ChIP assays. Each panel includes a western blot analysis of target-protein knockdown by siRNA (with three biological replicates in each case and quantification of western blot band intensities normalized to β-actin loading control and presented below each lane), an accompanying bar graph quantifying the knockdown data showing mean ± s.e.m., with dots indicating individual values for each of the biological replicates, and then a DSB–ChIP assay performed with the same antibody for the respective target protein with and without siRNA knockdown, as follows: γH2A.X (m; F = 12.3, df = 1); SUV39H1 (n; F = 10.49, df = 1); H3K9me3 ChIP assay after siRNA knockdown of the H3K9 methyltransferase SUV39H1 (as shown in n) (o; F = 10.34, df = 1); TIP60 (p; F = 8.13, df = 1); MRE11 (q; F = 26.1, df = 1); ATM (r; F = 29.9, df = 1); BRCA1 (s; F = 23.17, df = 1), RPA32 (t; F = 97.5, df = 1) and RAD51 (u; F = 9.4, df = 1). In a, data are mean ± s.e.m. with n = 3 biological replicates; statistical analysis by two-tailed unpaired t-test; df = 4, P values as indicated. In b–u, lines run through the mean ± s.e.m. of three biological replicates; statistical analysis by ANOVA; P values indicated.

Source Data

Extended Data Fig. 4 Elevated metabolites have minimal effects on cell cycle distribution, growth rates and NHEJ DNA repair.

a, b, Cell cycle profile plots based on DNA content by flow cytometry for U2OS DSB–ChIP cells treated with either DMSO (control) (a) or with the indicated metabolites: 500 μM octyl-R-2HG, 2 mM succinate, or 30 μM dimethyl fumarate (b). c, Quantification of cell cycle analyses in: YUNK1 cells with shRNA suppression of SDHB or FH compared to shCTRL (non-targeting) controls; U2OS DSB–ChIP cells treated with DMSO, 500 μM octyl-R-2HG, 2 mM succinate, or 30 μM dimethyl fumarate; astrocytes expressing IDH1(WT) or IDH1(R132H); SNU1079 (IDH1R132C/+) cells treated with DMSO or 1 μM AGI-5198; and UOK 262 FH−/− renal cell carcinoma cells with and without FH cDNA complementation. d, Serial cell counts over time of the indicated cells in standard culture conditions: U87 IDH1R132H/+ glioblastoma cells compared to IDH1WT U87 cells; astrocytes expressing IDH1(WT) or IDH1(R132H); UOK 262 FH−/− renal cell carcinoma cells with and without FH cDNA complementation; and YUNK1 cells with shRNA suppression of SDHB or FH compared to shCTRL (non-targeting) control cells. Data are mean ± s.e.m. of three biological replicates; P values by ANOVA are indicated. Additional statistics: for U87, F = 24.69 (df = 1); astrocytes, F = 26.00 (df = 1); UOK262, F = 29.64 (df = 1); YUNK1 shSDHB, F = 16.11 (df = 1), and YUNK1 shFH, F = 89.47 (df = 1). e, pDNA-PKcs foci formation (using an antibody to phosphorylated DNA-PKcs) at 4 h after 2 Gy ionizing radiation quantified as percentage foci-positive nuclei (>10 foci per nucleus) in: SNU1079 cells (IDH1R132H/+) and HT1080 (IDH1R132C/+) cells treated with 1 μM AGI-5198 or DMSO control; U87 glioblastoma IDH1WT and IDH1R132H/+ cells; astrocytes overexpressing IDH1(WT) or IDH1(R132H); UOK 262 FH−/− renal cell carcinoma cells with and without FH cDNA complementation; and NCCFH1 FH−/− renal cell carcinoma cells with or without FH complementation. Representative images from SNU1079 (IDH1R132C/+) cells with and without AGI-5198 treatment are shown. Scale bar, 20 μm. f, Schematic of the luciferase based NHEJ assay. g, Quantification of relative NHEJ by the luciferase-based NHEJ assay in HeLa cells, Hela cells after siRNA suppression of KU80 (also known as XRCC5, positive control for NHEJ deficiency), IDH1R132H/+ HeLa cells, astrocytes overexpressing IDH1(WT) or IDH1(R132H), U87 glioblastoma IDH1WT and U87 IDH1R132H/+ cells, and YUNK1 cells with shRNA suppression of SDHB or FH, compared to shCTRL (non-targeting) control cells. h, Western blot analysis to confirm KU80 knockdown after siRNA suppression of KU80 in HeLa cells. This experiment was performed two times with similar results. i, Schematic of the EJ5–GFP NHEJ reporter. j, Quantification of NHEJ using the EJ5 chromosomally integrated reporter assay in U2OS-EJ5 reporter cells after treatment of cells with 500 μM octyl-R-2HG, 2 mM succinate or 30 μM dimethyl fumarate, compared to DMSO control and to cells with siRNA knockdown of KU80. k, Quantification of KU80 levels after siRNA suppression of KU80 in U2OS-EJ5 reporter cells. This experiment was performed two times with similar results. l, Schematic diagram of the EJ2–GFP reporter to assay MMEJ. m, Quantification of EJ2 MMEJ reporter activity in EJ2-HEK293FT cells after treatment with 500 μM octyl-R-2HG, 2 mM succinate or 30 μM dimethyl fumarate compared to DMSO control. Treatment with PARP inhibitor (PARPi) BMN-673 is used as a positive control for PARP-dependent MMEJ. n, Western blot levels of poly-ADP-ribose (PAR) polymers and poly-ribosylated proteins levels after treatment with 10 nM of the PARPi BMN-673. This experiment was performed 2 times with similar results. In c, e, g, j, m, data are mean ± s.e.m. with n = 3 biological replicates; statistical analysis by two-tailed unpaired t-test; df = 4.

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Extended Data Fig. 5 Elevated metabolites disrupt normal H3K9me3 deposition at sites of DNA damage caused by laser-stripe micro-irradiation.

a, Quantification of H3K9me3 intensity in the laser micro-irradiated stripe above background after laser micro-irradiation induction of DNA damage in HeLa cells treated with or without 2HG or expressing IDH1(R132H), as indicated. (F = 49.27, df = 1). b, c, Representative immunofluorescence of γH2AX and H3K9me3 in cell nuclei in parental U87 glioblastoma cells (WT IDH1) and U87 cells with CRISPR–Cas9-mediated knock-in of an IDH1R132H allele at the endogenous locus (IDH1R132H/+) without DNA damage (b) or 1 min after laser micro-irradiation induction of DNA damage at 150 μJ per pixel (c). Scale bars, 20 μm. d, Quantification of H3K9me3 intensity in the laser micro-irradiated stripe above background after the indicated time points in the U87 cell line matched pair after laser micro-irradiation. (F = 5.360, df = 1). e, f, Representative images of undamaged (e) and laser micro-stripe irradiated (f) YUNK1 cells with shRNA suppression of SDHB or FH compared to non-targeting control shRNA (shCTRL), or in YUNK1 cells treated with 2 mM succinate or 30 μM dimethyl fumarate. Cells were pretreated with exogenous metabolites 24 h before micro-stripe irradiation and then were analysed 1 min after irradiation. Scale bars, 20 μm. g, Quantification of H3K9me3 intensity in the laser micro-irradiated stripes above background at the indicated time points after laser micro-irradiation in the YUNK1 cells treated as indicated. (shSDHB, F = 31.25, df = 1; +succinate, F = 33.80, df = 1; shFH, F = 32.25, df = 1; +fumarate, F = 44.39, df = 1). h, i, Representative images of undamaged (h) and laser micro-stripe irradiated (i) HEK293FT cells with shRNA suppression of SDHB or FH compared to non-target control shRNA (shCTRL), or in HEK293FT cells treated with 2 mM succinate or 30 μM dimethyl fumarate. Cells were pretreated with exogenous metabolites 24 h before micro-stripe irradiation and were analysed at 1 min after irradiation. Scale bars, 20 μm. j, Quantification of H3K9me3 intensity in the laser micro-irradiated stripe above background at the indicated times after laser micro-irradiation in the HEK293FT cells treated as indicated. (shSDHB, F = 8.68, df = 1; +succinate, F = 13.75, df = 1; shFH, F = 10.84, df = 1; +fumarate F = 14.05, df = 1). k, l, Representative images of undamaged (k) and laser micro-stripe irradiated (l) SW1353 (IDH2R172K/+) chondrosarcoma cells treated as indicated with DMSO, 500 μM octyl-(R)-2HG, 5 μM AG-221 or 5 μM AG-221 and 500 μM octyl-(R)-2HG. Scale bars, 20 μm. m, Quantification of H3K9me3 intensity in the laser micro-irradiated stripe above background at 2 min after laser micro-irradiation in the SW1353 (IDH1R172K/+) chondrosarcoma cells treated as indicated. In a, d, g, j, line runs through the mean ± s.e.m. with n = 3 biological replicates for each time point; statistical analysis by ANOVA. In m, data are mean ± s.e.m. with n = 3 biological replicates; statistical analysis by two-tailed unpaired t-test; df = 4, P values are indicated.

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Extended Data Fig. 6 Oncometabolites impair TIP60 and ATM recruitment and activation following treatment of cells with ionizing radiation.

a, Quantification of TIP60 foci-positive nuclei 1 h after 2 Gy ionizing radiation in SW1353 (IDH2R172K/+) cells treated as indicated with DMSO (control), 500 μM 2HG, 5 μM AG-221 or 5 μM AG-221 and 500 μM 2HG. b, Quantification and representative images of TIP60 foci-positive nuclei 4 h after 2 Gy ionizing radiation in FH-deficient UOK 262 renal cell carcinoma cells (FH−/−) and in three subclones complemented with FH cDNA, with or without treatment with 30 μM dimethyl fumarate for 24 h before 2 Gy ionizing radiation. Scale bars, 100 μm. c, Quantification and representative images of phosphorylated ATM on residue S1981 (pATM S1981) foci-positive nuclei 4 h after 2 Gy ionizing radiation in HT1080 fibrosarcoma cells (IDH1R132C/+) and in HT1080 cells with CRISPR–Cas9-mediated knockout of the IDH1R132C allele (IDH1KO/+) treated as indicated with DMSO control, 2HG, AGI-5198 or AGI-5198 + 2HG. Scale bars, 100 μm. d, Quantification and representative images of pATM (S1981) foci-positive nuclei 4 h after 2 Gy ionizing radiation in FH-deficient UOK 262 renal cell carcinoma cells (FH−/−) and in a subclone complemented with FH cDNA. Scale bars, 100 μm. e, Quantification of cells with TIP60 foci-positive nuclei (>10 foci per nucleus) 1 h after 2 Gy ionizing radiation in U87 IDH1WT and U87 IDH1R132H/+ glioblastoma cells, immortalized astrocytes overexpressing IDH1(WT) or IDH1(R132H), IDH1WT and IDH1R132H/+ HeLa cells, and YUNK1 and HEK293FT cells with shRNA suppression of SDHB (shSDHB) or FH (shFH), compared to non-targeting control shRNA (shCTRL). f, Quantification of cells with pATM S1981 foci-positive nuclei (>10 foci per nucleus) 1 h after 2 Gy ionizing radiation in immortalized astrocytes overexpressing IDH1(WT) or IDH1(R132H), IDH1WT HeLa cells, IDH1R132H/+ HeLa cells, and YUNK1 and HEK293FT cells with shRNA suppression of SDHB (shSDHB) or FH (shFH) compared to non-targeting control shRNA (shCTRL). g, Representative immunofluorescent of TIP60 and pATM S1981 nuclear foci 1 h after 2 Gy ionizing radiation in HEK293FT cells with shRNA suppression of SDHB or FH compared to non-targeting control shRNA (shCTRL). Scale bar, 20 μm. h, Quantification of cells with pATM S1981 foci-positive nuclei (>10 foci per nucleus) and representative immunofluorescent images of pATM S1981 foci 1 h after 2 Gy ionizing radiation in SNU1079 (IDH1R132C/+) cholangiocarcinoma cells treated with IDH1 inhibitor (1 μM AGI-5198) or DMSO control. Scale bars, 100 μm. i, Western blot analysis of pATM S981 and total ATM 1 h after 2 Gy ionizing radiation in YUNK1 cells with shRNA suppression of SDHB (shSDHB) or FH (shFH) compared to non-targeting control shRNA (shCTRL) and in immortalized astrocytes overexpressing IDH1(WT) or IDH1(R132H). This experiment was performed three times with similar results. j, Western blot analysis of phospho-ATM S1981 (pATM) and phospho-S*Q motifs in YUNK1 cells with shRNA suppression of SDHB (shSDHB) as compared to a non-targeting control shRNA (shCTRL). This experiment was performed two times with similar results. k, Quantification and representative images of pATM and TIP60 foci colocalization in HeLa cells treated with DMSO control (CTRL), 500 μM octyl-(R)-2HG, 2 mM succinate or 30 μM dimethyl fumarate. Scale bar, 5 μm. l, Western blot analysis of pATM S1981 and total ATM, phospho-ATR and total ATR, phospho-RPA32 and total RPA32, phospho-CHK1 and total CHK1, phospho-DNA-PKcs and total DNA-PKcs, and phospho-CHK2 and total CHK2 in IDH1WT or IDH1R132H/+ U87 glioblastoma cells 1 h after 5 Gy ionizing radiation, compared to unirradiated controls. This experiment was performed two times with similar results. For a–f, h, k, data are mean ± s.e.m. with n = 3 biological replicates; statistical analysis by two-tailed unpaired t-test; df = 4, P values as indicated.

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Extended Data Fig. 7 αKG supplementation rescues oncometabolite-induced HDR deficiency.

a, b, Western blot analysis of H3K9me3 levels in the following cells with or without treatment with 2 mM αKG for 96 h: SNU1079 IDH1R132C/+ cholangiocarcinoma cells (a), U87 IDH1WT and IDH1R132H/+ glioblastoma cells (b), UOK 262 FH−/− renal cell carcinoma cells with and with FH cDNA complementation, and YUNK1 cells with shRNA suppression of SDHB (shSDHB) or FH (shFH). These experiments were repeated twice with similar results. c, Quantification of cells with TIP60 foci-positive nuclei (>10 foci per nucleus) at 1 h post 2 Gy ionizing radiation in the following cells with or without treatment with 2mM αKG for 96 h: IDH1WT and IDH1R132H/+ U87 glioblastoma cells, IDH1WT and IDH1R132H/+ HeLa cells, UOK 262 FH−/− renal cell carcinoma cells, YUNK1 cells with shRNA suppression of SDHB (shSDHB) or FH (shFH), compared to non-targeting control shRNA (shCTRL), and HEK293FT cells with shRNA suppression of SDHB (shSDHB) or FH (shFH) compared to non-targeting control shRNA (shCTRL). d–f, Quantification of cells with pATM S1918 foci-positive nuclei (>10 foci per nucleus) at 1 h after 2 Gy ionizing radiation in the following cells treated or not with 2 mM αKG for 48 h as indicated: HEK293FT cells with shRNA suppression of SDHB (shSDHB) or FH (shFH) compared to non-targeting control shRNA (shCTRL) (d), IDH1WT and IDH1R132H/+ HeLa cells (e), and YUNK1 cells with shRNA suppression of SDHB (shSDHB) or FH (shFH) compared to non-targeting control shRNA (shCTRL) (f). g, Quantification of cells with RAD51 foci-positive nuclei (>10 foci per nucleus) at 4 h post 2 Gy ionizing radiation in U87 IDH1WT and IDH1R132H/+ glioblastoma cells treated with either 2 mM αKG for 48 h or with DMSO control. h, Quantification of cells with RAD51 foci-positive nuclei (>10 foci per nucleus) at 4 h post 2 Gy ionizing radiation in SNU1079 (IDH1R132C/+) cholangiocarcinoma cells pre-treated with DMSO, 2 mM αKG or AGI-5198 for 48 h. i, Western blot analysis of H3K9me3 and total H3 levels in U2OS EJ-DR cells treated with DMSO (control), 2 mM αKG, 500 μM octyl-R-2HG, 2 mM succinate, 30 μM dimethyl fumarate, or the indicated combinations of αKG plus 2HG, succinate or fumarate. This experiment was repeated twice with similar results. j, ChIP analysis of H3K9me3 occupancy at the DSB–ChIP reporter locus in U2OS cells in the absence of a DSB after treatment with DMSO (control), 500 μM octyl-R-2HG, 2 mM succinate or 30 μM dimethyl fumarate, in all cases with or without 2 mM αKG, as indicated. k–r, Heat maps of the relative occupancy of the indicated factors at the site-directed DSB in U2OS cells as measured by ChIP and normalized to the uninduced controls. The assay was performed at the indicated time points post addition of Shield-1 and triamcinolone in DMSO-treated cells (control) (k), cells treated with 2 mM αKG (l), cells treated with 500 μM octyl-(R)-2HG (m), cells treated with 2 mM αKG and 500 μM octyl-(R)-2HG (n), cells treated with 2 mM succinate (o), cells treated with 2 mM succinate and 2 mM αKG (p), cells treated with 30 μM dimethyl fumarate (q), and cells treated with 30 μM dimethyl fumarate and 2 mM αKG (r). The heat maps for 2HG alone, succinate alone, fumarate alone and DMSO control alone are reproduced from Fig. 2b and are presented again here for comparison. s–aa, Line graphs of percent input values for DSB–ChIP assays with antibodies (corresponding to the heat maps in l, n, p, r) for γH2A.X (s; αKG + 2HG, F = 0.04, df = 1; αKG + succinate, F = 2.76, df = 1; αKG + fumarate, F = 0.18, df = 1); SUV39H1 (t; αKG + 2HG, F = 0.73; df = 1, αKG + succinate, F = 0.55, df = 1; αKG + fumarate, F = 0.09, df=1); H3K9me3 (u; αKG + 2HG, F = 0.076, df = 1; αKG + succinate, F = 4.05, df = 1; αKG + fumarate, F = 8.910, df = 1); TIP60 (v; αKG + 2HG, F = 1.32, df = 1; αKG + succinate, F = 1.98, df = 1; αKG + fumarate, F = 107.8, df = 1); MRE11 (w; αKG + 2HG, F = 0.53, df = 1; αKG + succinate, F = 1.2, df = 1; αKG + fumarate, F = 2.3, df = 1); ATM (x; αKG + 2HG, F = 14.8, df = 1, αKG + succinate, F = 0.31, df = 1; αKG + fumarate, F = 8.67, df = 1); BRCA1 (y; αKG + 2HG, F = 3.3, df = 1; αKG + succinate, F = 2.1, df = 1; αKG + fumarate F = 1.5, df = 1); RPA32 (z; αKG + 2HG, F = 0.003, df = 1; αKG + succinate, F = 1.78, df = 1, αKG + fumarate, F = 0.57, df = 1); and RAD51 (aa; αKG + 2HG, F = 1.4, df = 1; αKG + succinate, F = 2.4, df = 1; αKG + fumarate, F = 3.10, df = 1) at the indicated time points after addition of triamcinolone and Shield-1 to induce an I-SceI break in the U2OS DSB–ChIP cells. Line graphs corresponding with k, m, o, q, are presented in Extended Data Fig. 3b–j. ab, Quantification of HDR efficiency as measured by restoration of a functional GFP gene following I-SceI induction of a DSB in the DR–GFP reporter in U2OS cells following pre-treatment with DMSO, 500 μM octyl-R-2HG, 2 mM succinate or 30 μM dimethyl fumarate, and with no αKG, 1 mM αKG or 2 mM αKG as indicated. In c–h, j, and ab, data are mean ± s.e.m. with n = 3 biological replicates; statistical analysis by two-tailed unpaired t-test; df=4. In s–aa, lines run through the mean ± s.e.m. with n = 3 biological replicates for each time point; statistical analysis by ANOVA; P values are indicated.

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Extended Data Fig. 8 Overexpression of KDM4A and KDM4B suppresses elevated H3K9me3 and rescues HDR deficiency in oncometabolite-producing cells.

a–c, Western blot analysis of H3K9me3 and total H3 levels in the cholangiocarcinoma cell line SNU1079 (IDH1R132C/+) (a), and renal cell carcinoma cell lines UOK262 (FH−/−) (b) and NCCFH1 (FH−/−) (c) treated as indicated or after transfection with vectors for expression of haemagglutinin (HA)-tagged KDM4A, catalytically inactive KDM4A(H188A), KDM4B, catalytically inactive KDM4B(H189A), KDM4C and KDM6A or vectors for expression of Flag-tagged ALKBH2, ALKBH3 and JMJD4. This experiment was repeated twice with similar results. d, Quantification of RAD51-positive foci in SNU1079 cells after the indicated treatment or transfection. e–j, Quantification of TIP60 foci-positive nuclei (>10 foci per nucleus) (e, f), RAD51 foci-positive nuclei (g, h), and mean comet-tail moment in UOK 262 and NCCFH1 FH−/− renal cell carcinoma cell lines (i, j), after transfection with FH expression constructs or after with vectors for expression of HA-tagged KDM4A, catalytically inactive KDM4A(H188A), KDM4B, catalytically inactive KDM4B(H189A), KDM4C, or KDM6A or vectors for expression of Flag-tagged ALKBH2, ALKBH3 or JMJD4. In g, h, cells treated with 2 mM αKG or DMSO control are also included in the analysis. k, Western blots showing H3K9me3 and total H3 levels after transfection with constructs for expression of HA-tagged KDM4A or KDM4B in YUNK1 shSDHB or shFH cells. This experiment was repeated twice with similar results. l, Quantification of neutral comet assay in immortalized astrocytes expressing IDH1 or IDH1(R132H) and in YUNK1 shCTRL, shSDHB and shFH cells, with or without overexpression of KDM4A or KDM4B as indicated, or with 24 h treatment with 2 mM αKG. m, n, Quantification of RAD51 nuclear foci 6 h after 2 Gy ionizing radiation in IDH1WT or IDH1R132/+ HeLa cells (m) and in HEK293FT cells (n) with shRNA suppression of SDHB (shSDHB) or FH (shFH), compared with non-targeting shRNA (shCTRL). Cells were irradiated after 24 h pretreatment with αKG or 24 h after transfection with expression vectors for KDM4A or KDM4B. o, p, Quantification of BRCA1 nuclear foci 4 h after 2 Gy ionizing radiation in IDH1WT or IDH1R132H/+ HeLa cells (o) and HEK293FT cells (p) with shRNA suppression of SDHB (shSDHB) or FH (shFH), compared with non-targeting shRNA (shCTRL). Cells were irradiated after 24 h pretreatment with αKG or 24 h after transfection with expression vectors for KDM4A or KDM4B. q, Quantification of neutral comet assays performed in WT and HIF1A-knockout mouse embryonic fibroblasts (MEFs) after treatment with 500 μM octyl-(R)-2HG, 2 mM succinate or 30 μM dimethyl fumarate, compared to DMSO control. r, Western blot analysis of HIF-1α after indicated treatment of WT and HIF1A-knockout MEFs with 500 μM octyl-(R)-2HG, 2 mM succinate or 30 μM dimethyl fumarate. Hypoxia exposure at 1% O2 for 24 h is used as a positive control for HIF-1α stabilization. s, Western blot analysis of KDM4A and KDM4B expression and H3K9me3 levels in KDM4A-knockout and KDM4B-knockout YUNK1 cell lines. This experiment was repeated twice with similar results. t, u, Quantification of RAD51 foci-positive cells 4 h after 2 Gy ionizing radiation (t) and comet-tail moment (u) in parental YUNK1 cells, KDM4A-knockout YUNK1 cells, and KDM4B-knockout YUNK1 cells with overexpression constructs for KDM4A or KDM4B or mock transfection, as indicated. v, Western blot analysis of KDM4A and KDM4B expression and of H3K9me3 levels in KDM4A- and KDM4B-knockout YUNK1 cells compared to parental YUNK1 controls after transfection with overexpression constructs for HA-tagged KDM4A and KDM4B open reading frames, as indicated. This experiment was repeated twice with similar results. In d–h, j, l–q, t, u, data are mean ± s.e.m. with n = 3 biological replicates; statistical analysis by two-tailed unpaired t-test; df = 4, P values are indicated.

Source Data

Extended Data Fig. 9 Inhibition of KDM4B mediates oncometabolite-induced HDR deficiency.

a, Quantification of neutral comet assay (a) and quantification of RAD51 foci-positive cells (>10 foci per nucleus) (b) 4 h after 2 Gy ionizing radiation in parental YUNK1 cells, KDM4A-knockout YUNK1 cells, and KDM4B-knockout YUNK1 cells after siRNA suppression of either KDM4A or KDM4B, or non-targeting siRNA control (siCTRL), as indicated. c, Quantification of RAD51 foci-positive cells (c) and quantification of neutral comet assay (d) in KDM4B-knockout YUNK1 cells transfected with expression constructs for either KDM4B(WT) or the catalytically inactive KDM4B(H189A). e, Quantification by ChIP of baseline H3K9me3 levels (in the absence of a DSB) at the DSB–ChIP reporter locus in U2OS cells after siRNA suppression of either KDM4A or KDM4B, compared to non-targeting control siRNA. f, Validation of siRNA suppression of KDM4A and KDM4B and documentation of H3K9me3 levels by western blot in the U2OS DSB–ChIP cells. This experiment was repeated twice with similar results. g–o, Per cent input values for DSB–ChIP assays performed after siRNA suppression of KDM4A or KDM4B with antibodies (corresponding to Fig. 3g–i) for γH2A.X (g; siKDM4A, F = 0.0, df = 1; siKDM4B, F = 0.02, df = 1); SUV39H1 (h; siKDM4A, F = 60.85, df = 1; siKDM4B, F = 0.98, df = 1); H3K9me3 (i; siKDM4A, F = 1.4, df = 1; siKDM4B, F = 28.3, df = 1); TIP60 (j; siKDM4A, F = 15.2, df = 1; siKDM4B, F = 41.3, df = 1); MRE11 (k; siKDM4A, F = 15.5, df = 1; siKDM4B, F = 69.3, df = 1); ATM (l; siKDM4A, F = 0.1, df = 1; siKDM4B, F = 15.4, df = 1); BRCA1 (m; siKDM4A, F = 5.5, df = 1; siKDM4B, F = 19.94, df = 1); RPA32 (n; siKDM4A, F = 1.9, df = 1; siKDM4B, F = 24.5, df = 1); and RAD51(o; siKDM4A, F = 0.88, df = 1; siKDM4B, F = 7.4, df = 1) at the indicated time points after addition of triamcinolone and Shield-1 to induce an I-SceI break in DSB–ChIP U2OS cells. p, Quantification and representative images of neutral comet assays performed in DSB–ChIP U2OS cells after siRNA suppression of KDM4 or KDM4B compared to a non-targeting control siRNA (siCTRL). Scale bars, 400 μm. q, Western blot analysis of IDH1(R132H) expression in parental YUNK1, KDM4A-knockout YUNK1 cells and KDM4B-knockout YUNK1 cells treated with either doxycycline (DOX; to induce expression of IDH1(R132H)) or vehicle control, and western blot analysis of global H3K9me3 and total H3 levels. This experiment was repeated twice with similar results. r, s, Quantification of TIP60 (r) and RAD51 (s) foci-positive cells (>10 foci per nucleus) after 2 Gy ionizing radiation (at 1 h for TIP60 and 4 h for RAD51) in parental, KDM4A-knockout and KDM4B-knockout YUNK1 cells, treated with either doxycycline or vehicle control, and also treated as indicated with DMSO, 500 μM octyl-(R)-2HG, 1 mM AGI-5198 or both 500 μM octyl-(R)-2HG and 1 mM AGI-5198. t, Western blot analysis of KDM4A and KDM4B levels in HT1080 cells (IDH1R132C/+) and in HT1080 cells with CRISPR–Cas9 knockout of the mutant IDH1 allele (IDH1KO/+) transfected with scramble siRNA control (siSCR), siKDM4A or siKDM4B, and western blot analysis of global H3K9me3 and total H3 levels. This experiment was repeated twice with similar results. u, v, Quantification of TIP60 (u) and RAD51 (v) foci-positive cells (>10 foci per nucleus) after 2 Gy ionizing radiation in HT1080 cells (IDH1R132C/+) and in HT1080 cells with CRISPR–Cas9 knockout of the mutant IDH1R132C allele (IDH1KO/+) transfected with siSCR, siKDM4A or siKDM4B, and treated with or without 500 μM 2HG, 1 mM AGI-5198 or both 1 mM AGI-5198 and 500 μM 2HG. w, Quantification of mean comet-tail moment in parental, KDM4A-knockout and KDM4B-knockout YUNK1 cells treated with either 500 μM octyl-R-2HG or DMSO control. x, Quantification of cell proliferation by serial cell counts over time of parental, KDM4A-knockout (two independent clones) and KDM4B-knockout YUNK1 (two independent clones) cells. y, Western blot analysis of KDM4A and KDM4B expression levels in U87 glioma cells with KDM4A or KDM4B knockout, compared to parental U87 cells, and western blot analysis of global H3K9me3 and total H3 levels. This experiment was repeated twice with similar results. z, Quantification of cell proliferation by serial cell counts of parental, KDM4A-knockout (two independent clones) and KDM4B-knockout (two independent clones) U87 cells. aa, Western blot analysis of expression of mutant H3 constructs and analysis of H3K9me3 levels after expression of the indicated H3 mutants in SNU1079 IDH1R132C cholangiocarcinoma cells. Note that only H3K9M can reduce global H3K9me3 levels in IDH1-mutant cells. This experiment was repeated twice with similar results. ab, Western blot analysis of IDH1(R132H), H3K4me3, H3K9me3, H3K27me3, H3K36me3 and total H3 in IDHWT and IDH1R132H/+ U87 glioblastoma cells. This experiment was repeated twice with similar results. ac, Quantification of 2HG levels by fluorometric 2HG detection assay in SNU1079 (IDH1R132C/+) cholangiocarcinoma cells transfected with either H3.3(WT) or H3.3(K9M) expression constructs. In a–e, p, r, s, u–w, ac, data are mean ± s.e.m. with n = 3 biological replicates; statistical analysis by two-tailed unpaired t-test; df = 4. In f, h, i–q, data are mean ± s.e.m. with n = 3 biological replicates; statistical analysis by ANOVA; P values as indicated.

Source Data

Extended Data Fig. 10 Oncometabolites induce HDR deficiency via hypermethylation of H3K9.

a, b, Quantification of neutral comet assay performed in SNU1079 (IDH1R132C/+) cholangiocarcinoma cells (a) and UOK262 FH−/− renal cell carcinoma cells (b) after expression of the indicated H3.3 mutants. c, Quantification of neutral comet assay performed in YUNK1 shSDHB, shFH or shCTRL cells transfected with expression constructs for H3.3(WT) versus H3.3(K9M). Cells were assayed 24 h after transfection with the indicated construct. d, Western blot analysis of global H3K9me3 levels in YUNK1 shSDHB or shFH cells transfected with vector for expression of H3.3(WT) or H3.3(K9M). This experiment was repeated twice with similar results. e, Western blot analysis of pATM and total ATM levels in shCTRL, shSDHB and shFH YUNK1 cells transfected with expression constructs for H3.3(WT) or H3.3(K9M), at 1 h after ionizing radiation (5 Gy) or without ionizing radiation. This experiment was repeated twice with similar results. f, Quantification over time by ChIP of H3K9me3, H3K4me3, H3K27me3 and H3K36me3 levels after induction of the site-specific DSB in U2OS DSB–ChIP cells after DMSO treatment. The H3K9me3 data is also presented in Fig. 2 and Extended Data Fig. 3 for relevant comparisons. g, Western blot analysis of H3K9M mutant construct expression and H3K9me3 levels in U2OS DSB–ChIP cells after expression of H3K9M or WT H3, or treatment with DMSO control or 500 μM octyl-(R)-2HG. This experiment was repeated twice with similar results. h, ChIP analysis of H3K9me3 occupancy at the DSB–ChIP locus in U2OS cells (in the absence of an induced DSB), after transfection with construct for expression of H3.3(WT) or H3.3(K9M) expression construct after indicated treatment with either 500 μM 2HG or DMSO control. i–q, Per cent input values for assays performed in U2OS cells after transfection with expression construct for H3.3(WT) or H3.3(K9M) and treated with 500 μM 2HG or DMSO as indicated (corresponding to Fig. 4b–e), with antibodies for H3K9me3 (i; F = 54.63, df = 1), γH2A.X (j; F = 1.95, df = 1), SUV39H1 (k; F = 34.11, df = 1), TIP60 (F = 126.6, df = 1), MRE11 (m; F = 7.9, df=1), ATM (n; F = 0.75, df = 1), BRCA1 (o; F = 119.5, df = 1), RPA32 (p; F = 10.34, df = 1), RAD51 (q; F = 80.2, df = 1) and total H3 (r; F = 120.6, df = 1) at the indicated time points after addition of triamcinolone and Shield-1 to induce an I-SceI break. s, Line graphs of percent input values for DSB–ChIP assays performed with antibodies for KDM4A and KDM4B in U2OS cells at the indicated time points after addition of triamcinolone and Shield-1 to induce an I-SceI break. t, Quantification of neutral comet assay performed in YUNK1 cells with shSDHB, shFH or shCTRL, transfected with constructs for expression of H3.3(WT) or H3.3(K9R). Cells were assayed 24 h after transfection with the indicated construct. u, v, Quantification of TIP60 foci-positive nuclei (>10 foci per nucleus) (u) at 1 h post 2 Gy ionizing radiation and RAD51 foci-positive nuclei at 4 h post 2 Gy ionizing radiation (v) in immortalized astrocytes and YUNK1 cells transfected with constructs for expression of H3.3(WT) or H3.3(K9R). w, PARP inhibitor sensitivity in IDH1WT and IDH1R132H/+ U87 glioblastoma cells transfected with expression constructs for H3.3(WT), H3.3(K9M) or H3.3(K9R). (IDHWT: H3.3(WT) vs H3.3(K9R), F = 8.7, df = 1; IDH1R132H: H3.3(WT) vs H3.3(K9M), F = 23.0, df = 1). x, Quantification of genomic H3K9me3 peaks across chromosomes by analysis of ChIP-sequencing data for H3K9me3 in a matched pair of immortalized human astrocyte cell lines expressing IDH1(WT) or IDH1(R132H)28. Peaks were called using HOMER (v.4.10) and are defined as spanning at least 1,000 base pairs and at least 2,500 base pairs apart, filtered by P ≤ 0.01. n = 2 technical replicates. y, qPCR analysis of amplicons that span the Cas9–guide RNA cleavage target sites to asses I-SceI cleavage at the H3K9me3 differentially methylated, H3K9me3 both low, and H3K9me3 both high loci in IDH1WT and IDH1R132H/+ astrocytes. Reduced amplification of the genomic DNA across the Cas9–guide RNA target sites indicates that cleavage at the target site has occurred. z, aa, ChIP analysis of MRE11 (z) and RPA32 (aa) at H3K9me3 differentially methylated, H3K9me3 low, and H3K9me3 high loci as identified in x, in the IDH1WT and IDH1R132H astrocyte cell lines 12 h after Cas9 nucleofection. ab–ac, Quantification of TIP60 foci-positive nuclei (>10 foci per nucleus) at 1 h after 2 Gy ionizing radiation (ab) and RAD51 foci-positive nuclei at 4 h after 2 Gy ionizing radiation (ac) in immortalized astrocytes and YUNK1 cells transfected with siRNA to knock down SUV39H1 or non-targeting control (siCTRL). ad, Western blot analysis of SUV39H1 levels in YUNK1 cells and immortalized astrocytes transfected with siRNA to knock down SUV39H1 or non-targeting control siRNA (siCTRL). In f, i–s, w, data are mean ± s.e.m.; statistical analysis by ANOVA, with P values as indicated. In a–c, h, t–v, y–ac, data are mean ± s.e.m. with n = 3 biological replicates; statistical analysis by two-tailed unpaired t-test; df = 4, P values are indicated.

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Supplementary Figure 1

The original source images for all data obtained by electrophoretic separation. The full scanned images show the uncropped form with molecular weight markers. Blots are labelled according to the corresponding Figure panel within the main or Extended Data Figures. The red boxes indicate which bands were used to assemble the figures.

Supplementary Figure 2

Representative flow cytometry data with gating strategies.

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Sulkowski, P.L., Oeck, S., Dow, J. et al. Oncometabolites suppress DNA repair by disrupting local chromatin signalling. Nature 582, 586–591 (2020). https://doi.org/10.1038/s41586-020-2363-0

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