Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

PARylated PDHE1α generates acetyl-CoA for local chromatin acetylation and DNA damage repair

Nature Structural & Molecular Biology volume 30, pages 1719–1734 (2023)Cite this article

Subjects

Abstract

Chromatin relaxation is a prerequisite for the DNA repair machinery to access double-strand breaks (DSBs). Local histones around the DSBs then undergo prompt changes in acetylation status, but how the large demands of acetyl-CoA are met is unclear. Here, we report that pyruvate dehydrogenase 1α (PDHE1α) catalyzes pyruvate metabolism to rapidly provide acetyl-CoA in response to DNA damage. We show that PDHE1α is quickly recruited to chromatin in a polyADP-ribosylation-dependent manner, which drives acetyl-CoA generation to support local chromatin acetylation around DSBs. This process increases the formation of relaxed chromatin to facilitate repair-factor loading, genome stability and cancer cell resistance to DNA-damaging treatments in vitro and in vivo. Indeed, we demonstrate that blocking polyADP-ribosylation-based PDHE1α chromatin recruitment attenuates chromatin relaxation and DSB repair efficiency, resulting in genome instability and restored radiosensitivity. These findings support a mechanism in which chromatin-associated PDHE1α locally generates acetyl-CoA to remodel the chromatin environment adjacent to DSBs and promote their repair.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: PDHE1α is recruited to chromatin in line with nuclear acetyl-CoA generation and chromatin acetylation in response to DNA damage.
Fig. 2: PDHE1α-catalyzed local acetyl-CoA is required for chromatin acetylation in response to DNA damage.
Fig. 3: PARP1 activity controls the recruitment of PDHE1α to sites of DNA damage.
Fig. 4: PARylation of PDHE1α by PARP1 is essential for its enrichment at DNA damage sites.
Fig. 5: PDHE1α PARylation coordinated with its enzyme activity is required for chromatin accessibility following DNA damage.
Fig. 6: PDHE1α recruitment to chromatin facilitates DNA damage repair and maintains genome integrity.
Fig. 7: PDHE1α is a critical mediator of cellular resistance to DNA-damaging treatment.
Fig. 8: Working model of the role of PDHE1α in the DDR.

Similar content being viewed by others

Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium repository via the PRIDE partner repository with the dataset identifier PXD040676. The STRING database (https://cn.string-db.org/) was used for the functional network analysis of the proteomics data. The raw sequencing data and the processed data are available from the Gene Expression Omnibus (GEO) under accession number GSE237929. The imaging datasets generated and analyzed during the current study are not publicly available, as the large amount of data could not be uploaded to a repository, but are available from the corresponding author upon request. Source data are provided with this paper.

References

  1. Jackson, S. P. & Bartek, J. The DNA-damage response in human biology and disease. Nature 461, 1071–1078 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Ceccaldi, R., Rondinelli, B. & D’Andrea, A. D. Repair pathway choices and consequences at the double-strand break. Trends Cell Biol. 26, 52–64 (2016).

    CAS  PubMed  Google Scholar 

  3. Ciccia, A. & Elledge, S. J. The DNA damage response: making it safe to play with knives. Mol. Cell 40, 179–204 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Smerdon, M. J. DNA repair and the role of chromatin structure. Curr. Opin. Cell Biol. 3, 422–428 (1991).

    CAS  PubMed  Google Scholar 

  5. Soria, G., Polo, S. E. & Almouzni, G. Prime, repair, restore: the active role of chromatin in the DNA damage response. Mol. Cell 46, 722–734 (2012).

    CAS  PubMed  Google Scholar 

  6. Price, B. D. & D’Andrea, A. D. Chromatin remodeling at DNA double-strand breaks. Cell 152, 1344–1354 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Kim, J. J., Lee, S. Y. & Miller, K. M. Preserving genome integrity and function: the DNA damage response and histone modifications. Crit. Rev. Biochem. Mol. Biol. 54, 208–241 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Zhang, J. et al. Histone lysine modifying enzymes and their critical roles in DNA double-strand break repair. DNA Repair (Amst.) 107, 103206 (2021).

    CAS  PubMed  Google Scholar 

  9. Downs, J. A. et al. Binding of chromatin-modifying activities to phosphorylated histone H2A at DNA damage sites. Mol. Cell 16, 979–990 (2004).

    CAS  PubMed  Google Scholar 

  10. Murr, R. et al. Histone acetylation by Trrap-Tip60 modulates loading of repair proteins and repair of DNA double-strand breaks. Nat. Cell Biol. 8, 91–99 (2006).

    CAS  PubMed  Google Scholar 

  11. Sivanand, S. et al. Nuclear acetyl-CoA production by ACLY promotes homologous recombination. Mol. Cell 67, 252–265 e256 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Shogren-Knaak, M. et al. Histone H4-K16 acetylation controls chromatin structure and protein interactions. Science 311, 844–847 (2006).

    CAS  PubMed  Google Scholar 

  13. Xu, Y. et al. Histone H2A.Z controls a critical chromatin remodeling step required for DNA double-strand break repair. Mol. Cell 48, 723–733 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Xu, Y. et al. The p400 ATPase regulates nucleosome stability and chromatin ubiquitination during DNA repair. J. Cell Biol. 191, 31–43 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Etchegaray, J. P. & Mostoslavsky, R. Interplay between metabolism and epigenetics: a nuclear adaptation to environmental changes. Mol. Cell 62, 695–711 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Kinnaird, A., Zhao, S., Wellen, K. E. & Michelakis, E. D. Metabolic control of epigenetics in cancer. Nat. Rev. Cancer 16, 694–707 (2016).

    CAS  PubMed  Google Scholar 

  17. Pietrocola, F., Galluzzi, L., Bravo-San Pedro, J. M., Madeo, F. & Kroemer, G. Acetyl coenzyme A: a central metabolite and second messenger. Cell Metab. 21, 805–821 (2015).

    CAS  PubMed  Google Scholar 

  18. Menzies, K. J., Zhang, H., Katsyuba, E. & Auwerx, J. Protein acetylation in metabolism – metabolites and cofactors. Nat. Rev. Endocrinol. 12, 43–60 (2016).

    CAS  PubMed  Google Scholar 

  19. Sivanand, S., Viney, I. & Wellen, K. E. Spatiotemporal control of acetyl-CoA metabolism in chromatin regulation. Trends Biochem. Sci. 43, 61–74 (2018).

    CAS  PubMed  Google Scholar 

  20. Tsuchiya, Y., Pham, U. & Gout, I. Methods for measuring CoA and CoA derivatives in biological samples. Biochem. Soc. Trans. 42, 1107–1111 (2014).

    CAS  PubMed  Google Scholar 

  21. Yi, C. H. et al. Metabolic regulation of protein N-alpha-acetylation by Bcl-xL promotes cell survival. Cell 146, 607–620 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Kim, J. W. & Dang, C. V. Multifaceted roles of glycolytic enzymes. Trends Biochem. Sci. 30, 142–150 (2005).

    CAS  PubMed  Google Scholar 

  23. Siebert, G. & Humphrey, G. B. Enzymology of the nucleus. Adv. Enzymol. Relat. Areas Mol. Biol. 27, 239–288 (1965).

    PubMed  Google Scholar 

  24. Gao, X. L., Wang, H. Z., Yang, J. J., Liu, X. W. & Liu, Z. R. Pyruvate kinase M2 regulates gene transcription by acting as a protein kinase. Mol. Cell 45, 598–609 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Monaghan, R. M. & Whitmarsh, A. J. Mitochondrial proteins moonlighting in the nucleus. Trends Biochem. Sci. 40, 728–735 (2015).

    CAS  PubMed  Google Scholar 

  26. Li, X. et al. Nucleus-translocated ACSS2 promotes gene transcription for lysosomal biogenesis and autophagy. Mol. Cell 66, 684–697.e9 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Chen, W. et al. The SESAME complex regulates cell senescence through the generation of acetyl-CoA. Nat. Metab. https://doi.org/10.1038/s42255-021-00412-9 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Sutendra, G. et al. A nuclear pyruvate dehydrogenase complex is important for the generation of acetyl-CoA and histone acetylation. Cell 158, 84–97 (2014).

    CAS  PubMed  Google Scholar 

  29. Chen, J. et al. Compartmentalized activities of the pyruvate dehydrogenase complex sustain lipogenesis in prostate cancer. Nat. Genet. 50, 219–228 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Zhang, J. et al. mTORC2-mediated PDHE1α nuclear translocation links EBV-LMP1 reprogrammed glucose metabolism to cancer metastasis in nasopharyngeal carcinoma. Oncogene 38, 4669–4684 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Nagaraj, R. et al. Nuclear localization of mitochondrial TCA cycle enzymes as a critical step in mammalian zygotic genome activation. Cell 168, 210–223 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Liu, C. & Yu, X. ADP-ribosyltransferases and poly ADP-ribosylation. Curr. Protein Pept. Sci. 16, 491–501 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Wei, H. & Yu, X. Functions of PARylation in DNA damage repair pathways. Genomics Proteomics Bioinformatics 14, 131–139 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Messner, S. & Hottiger, M. O. Histone ADP-ribosylation in DNA repair, replication and transcription. Trends Cell Biol. 21, 534–542 (2011).

    CAS  PubMed  Google Scholar 

  35. Kumbhar, R. et al. Poly(ADP-ribose) binding and macroH2A mediate recruitment and functions of KDM5A at DNA lesions. J. Cell Biol. 220, e202006149 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Chen, Q. et al. ADP-ribosylation of histone variant H2AX promotes base excision repair. EMBO J. 40, e104542 (2021).

    CAS  PubMed  Google Scholar 

  37. Li, Z. M. et al. Destabilization of linker histone H1.2 is essential for ATM activation and DNA damage repair. Cell Res. 28, 756–770 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Leidecker, O. et al. Serine is a new target residue for endogenous ADP-ribosylation on histones. Nat. Chem. Biol. 12, 998–1000 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Krishnakumar, R. & Kraus, W. L. The PARP side of the nucleus: molecular actions, physiological outcomes, and clinical targets. Mol. Cell 39, 8–24 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Robson, M. et al. Olaparib for metastatic breast cancer in patients with a germline BRCA mutation. N. Engl. J. Med. 377, 523–533 (2017).

    CAS  PubMed  Google Scholar 

  41. Ledermann, J. et al. Olaparib maintenance therapy in platinum-sensitive relapsed ovarian cancer. N. Engl. J. Med. 366, 1382–1392 (2012).

    CAS  PubMed  Google Scholar 

  42. Clouaire, T. et al. Comprehensive mapping of histone modifications at DNA double-strand breaks deciphers repair pathway chromatin signatures. Mol. Cell 72, 250–262 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Iacovoni, J. S. et al. High-resolution profiling of γH2AX around DNA double strand breaks in the mammalian genome. EMBO J. 29, 1446–1457 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Trefely, S. et al. Quantitative subcellular acyl-CoA analysis reveals distinct nuclear metabolism and isoleucine-dependent histone propionylation. Mol. Cell 82, 447–462.e6 (2022).

    CAS  PubMed  Google Scholar 

  45. Behal, R. H., Buxton, D. B., Robertson, J. G. & Olson, M. S. Regulation of the pyruvate dehydrogenase multienzyme complex. Annu. Rev. Nutr. 13, 497–520 (1993).

    CAS  PubMed  Google Scholar 

  46. Arce-Molina, R. et al. A highly responsive pyruvate sensor reveals pathway-regulatory role of the mitochondrial pyruvate carrier MPC. eLife 9, e53917 (2020).

    PubMed  PubMed Central  Google Scholar 

  47. Hansen, L. L., Brown, G. K., Kirby, D. M. & Dahl, H. H. Characterization of the mutations in three patients with pyruvate dehydrogenase E1α deficiency. J. Inherit. Metab. Dis. 14, 140–151 (1991).

    CAS  PubMed  Google Scholar 

  48. Parrini, E. et al. Diagnostic targeted resequencing in 349 patients with drug-resistant pediatric epilepsies identifies causative mutations in 30 different genes. Hum. Mutat. 38, 216–225 (2017).

    CAS  PubMed  Google Scholar 

  49. Lissens, W. et al. Mutation analysis of the pyruvate dehydrogenase E1α gene in eight patients with a pyruvate dehydrogenase complex deficiency. Hum. Mutat. 7, 46–51 (1996).

    CAS  PubMed  Google Scholar 

  50. Gong, F., Chiu, L. Y. & Miller, K. M. Acetylation reader proteins: linking acetylation signaling to genome maintenance and cancer. PLoS Genet. 12, e1006272 (2016).

    PubMed  PubMed Central  Google Scholar 

  51. Li, X., Qian, X. & Lu, Z. Local histone acetylation by ACSS2 promotes gene transcription for lysosomal biogenesis and autophagy. Autophagy 13, 1790–1791 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Carrer, A. et al. Acetyl-CoA metabolism supports multistep pancreatic tumorigenesis. Cancer Discov. 9, 416–435 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Lee, J. V. et al. Acetyl-CoA promotes glioblastoma cell adhesion and migration through Ca2+-NFAT signaling. Genes Dev. 32, 497–511 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Liu, X. et al. Acetate production from glucose and coupling to mitochondrial metabolism in mammals. Cell 175, 502–513.e13 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Sulkowski, P. L. et al. Oncometabolites suppress DNA repair by disrupting local chromatin signalling. Nature 582, 586–591 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Chen, L. L. & Xiong, Y. Tumour metabolites hinder DNA repair. Nature 582, 492–494 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Gibson, B. A. & Kraus, W. L. Small molecules, big effects: a role for chromatin-localized metabolite biosynthesis in gene regulation. Mol. Cell 41, 497–499 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Trefely, S., Lovell, C. D., Snyder, N. W. & Wellen, K. E. Compartmentalised acyl-CoA metabolism and roles in chromatin regulation. Mol. Metab. https://doi.org/10.1016/j.molmet.2020.01.005 (2020).

  59. Jiang, Y. H. et al. Local generation of fumarate promotes DNA repair through inhibition of histone H3 demethylation. Nat. Cell Biol. 17, 1158–1168 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Gustafsson, N. M. S. et al. Targeting PFKFB3 radiosensitizes cancer cells and suppresses homologous recombination. Nat. Commun. 9, 3872 (2018).

    PubMed  PubMed Central  Google Scholar 

  61. Tallis, M., Morra, R., Barkauskaite, E. & Ahel, I. Poly(ADP-ribosyl)ation in regulation of chromatin structure and the DNA damage response. Chromosoma 123, 79–90 (2014).

    CAS  PubMed  Google Scholar 

  62. Guo, Y., Fan, B. & Li, M. PARP molecular functions and applications of PARP inhibitors in cancer treatment. Genome Instab. Dis. 4, 137–153 (2023).

    CAS  Google Scholar 

  63. Gibson, B. A. et al. Chemical genetic discovery of PARP targets reveals a role for PARP-1 in transcription elongation. Science 353, 45–50 (2016).

    CAS  PubMed Central  Google Scholar 

  64. Li, N. et al. PARP inhibition suppresses growth of EGFR-mutant cancers by targeting nuclear PKM2. Cell Rep. 15, 843–856 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Gupte, R., Liu, Z. Y. & Kraus, W. L. PARPs and ADP-ribosylation: recent advances linking molecular functions to biological outcomes. Genes Dev. 31, 101–126 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. Ahel, I. et al. Poly(ADP-ribose)-binding zinc finger motifs in DNA repair/checkpoint proteins. Nature 451, 81–85 (2008).

    CAS  PubMed  Google Scholar 

  67. Zhang, L. & Li, D. Q. MORC2 regulates DNA damage response through a PARP1-dependent pathway. Nucleic Acids Res. 47, 8502–8520 (2019).

    CAS  PubMed Central  Google Scholar 

  68. Hassa, P. O. et al. Acetylation of poly(ADP-ribose) polymerase-1 by p300/CREB-binding protein regulates coactivation of NF-κB-dependent transcription. J. Biol. Chem. 280, 40450–40464 (2005).

    CAS  PubMed  Google Scholar 

  69. Hanahan, D. & Weinberg, R. A. Hallmarks of cancer: the next generation. Cell 144, 646–674 (2011).

    CAS  PubMed  Google Scholar 

  70. Vernieri, C. et al. Targeting cancer metabolism: dietary and pharmacologic interventions. Cancer Discov. 6, 1315–1333 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Sun, L., Zhang, H. & Gao, P. Metabolic reprogramming and epigenetic modifications on the path to cancer. Protein Cell https://doi.org/10.1007/s13238-021-00846-7 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  72. Vander Heiden, M. G. Targeting cancer metabolism: a therapeutic window opens. Nat. Rev. Drug Discov. 10, 671–684 (2011).

    Google Scholar 

  73. Martinez-Outschoorn, U. E., Peiris-Pages, M., Pestell, R. G., Sotgia, F. & Lisanti, M. P. Cancer metabolism: a therapeutic perspective. Nat. Rev. Clin. Oncol. 14, 113 (2017).

    PubMed  Google Scholar 

  74. Cai, Z. et al. Phosphorylation of PDHA by AMPK drives TCA cycle to promote cancer metastasis. Mol. Cell 80, 263–278.e7 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  75. Vander Heiden, M. G., Cantley, L. C. & Thompson, C. B. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324, 1029–1033 (2009).

    Google Scholar 

  76. Stacpoole, P. W. Therapeutic targeting of the pyruvate dehydrogenase complex/pyruvate dehydrogenase kinase (PDC/PDK) axis in cancer. J. Natl Cancer Inst. https://doi.org/10.1093/jnci/djx071 (2017).

  77. Aymard, F. et al. Transcriptionally active chromatin recruits homologous recombination at DNA double-strand breaks. Nat. Struct. Mol. Biol. 21, 366–U172 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  78. Sanjana, N. E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods 11, 783–784 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  79. Hou, T. et al. SIRT6 coordinates with CHD4 to promote chromatin relaxation and DNA repair. Nucleic Acids Res. 48, 2982–3000 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. Liao, W., McNutt, M. A. & Zhu, W. G. The comet assay: a sensitive method for detecting DNA damage in individual cells. Methods 48, 46–53 (2009).

    CAS  PubMed  Google Scholar 

  81. Hou, T. et al. Cytoplasmic SIRT6-mediated ACSL5 deacetylation impedes nonalcoholic fatty liver disease by facilitating hepatic fatty acid oxidation. Mol. Cell 82, 4099–4115.e9 (2022).

    CAS  PubMed  Google Scholar 

  82. Zhao, Y., Shen, Y., Wen, Y. & Campbell, R. E. High-performance intensiometric direct- and inverse-response genetically encoded biosensors for citrate. ACS Cent. Sci. 6, 1441–1450 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  83. Nelson, J. D., Denisenko, O. & Bomsztyk, K. Protocol for the fast chromatin immunoprecipitation (ChIP) method. Nat. Protoc. 1, 179–185 (2006).

    CAS  PubMed  Google Scholar 

  84. Dejardin, J. & Kingston, R. E. Purification of proteins associated with specific genomic loci. Cell 136, 175–186 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Natural Science Foundation of China (grant nos. 32090030, 81720108027 and 81530074 to W.-G.Z.; grant no. 82002986 to J.Z.); the National Key R&D Program of China (grant no. 2017YFA0503900 to W.-G.Z.); the Science and Technology Program of Guangdong Province in China (grant no. 2017B030301016 to W.-G.Z.); Guangdong Basic and Applied Basic Research Foundation (grant nos. 2019A1515110041 and 2021A1515011126 to J.Z.); the Shenzhen Municipal Commission of Science and Technology Innovation (grant nos. JCYJ20200109114214463 and JCYJ20220818100015032 to W.-G.Z.; grant no. RCYX20210706092040047 to Y.T.); and Shenzhen University 2035 Program for Excellent Research to W.-G.Z. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the paper. We thank G. Legube (Université Paul Sabatier, Toulouse, France) for providing the AsiSI-ER-U2OS-AID cells; Y. Zhao (East China University of Science and Technology, Shanghai, China) for providing the citrate sensor construct pcDNA3.1-Citron1-NLS; S. Lin (Xiamen University, Fujian, China) for providing the 13C-isotope-labeled citrate reagent; and J. Tamanini of ETediting, UK for English language editing before submission.

Author information

Authors and Affiliations

  1. International Cancer Center, Guangdong Key Laboratory of Genome Instability and Human Disease Prevention, Marshall Laboratory of Biomedical Engineering, Department of Biochemistry and Molecular Biology, Shenzhen University Medical School, Shenzhen, China

    Jun Zhang, Feng Chen, Yuan Tian, Wenchao Xu, Qian Zhu, Zhenhai Li, Lingyu Qiu, Xiaopeng Lu, Xiangyu Liu, Baohua Liu & Wei-Guo Zhu

  2. Guangdong Key Laboratory of Genome Instability and Human Disease Prevention, Marshall Laboratory of Biomedical Engineering, Department of Cell Biology and Medical Genetics, Shenzhen University Medical School, Shenzhen, China

    Bin Peng & Xingzhi Xu

  3. Shenzhen Key Laboratory of Synthetic Genomics, Guangdong Provincial Key Laboratory of Synthetic Genomics, CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China

    Haiyun Gan

  4. Shenzhen Key Laboratory for Systemic Aging and Intervention, National Engineering Research Center for Biotechnology (Shenzhen), Shenzhen University Medical School, Shenzhen, China

    Baohua Liu

Authors
  1. Jun Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Feng Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yuan Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wenchao Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Qian Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Zhenhai Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Lingyu Qiu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Xiaopeng Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Bin Peng
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Xiangyu Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Haiyun Gan
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Baohua Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Xingzhi Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Wei-Guo Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.Z. and W.-G.Z. conceptualized the project. J.Z., Y.T., Q.Z., B.P., X.Liu, X.Lu and H.G. were responsible for the study methodology. J.Z., F.C., W.X., Z.L. and L.Q. performed investigations. J.Z. and F.C. wrote the original draft of the paper. J.Z., X.X. and W.-G.Z. reviewed and edited the paper. J.Z. and W.-G.Z. were responding for funding acquisition. X.Liu, B.L. and X.X. were responsible for resources. W.-G.Z. supervised the study.

Corresponding author

Correspondence to Wei-Guo Zhu.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Carolina Perdigoto and Dimitris Typas were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Additional information

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

Extended data

Extended Data Fig. 1 PDHE1α is recruited to chromatin in response to DNA damage.

a, Western blot showing specific antibody signals for pan-Ac, γH2AX, and H3 in the chromatin and histone fractions from HeLa cells treated with either ionizing radiation (IR) or etoposide. CBB, Coomassie Brilliant Blue. b, Immunofluorescence staining showing pan-Ac and γH2AX signal in laser micro-irritation induced DNA damage sites; nuclei were counterstained with DAPI. Scale bars, 5 μm. c, ChIP-PCR analysis showing the pan-Ac signal distribution of AsiSI-induced DSBs on one side of the chromatin flanking Chr1_6 (chr1:89,231,184-89,231,191). d, Nuclear and cytosolic acetyl-CoA levels were quantified according to a recent protocol (Trefely et al., Mol Cell 82, 447, 2022). Data in c and d are representative of three independent replicates. The statistical analyses were performed using a two-tailed student’s t-test, and shows the mean ± s.d. e, The acetyl-CoA levels in washing/storage buffer fractions collected from the functional nuclei extraction procedure were quantified using a commercial kit. f, ChIP-PCR analysis showing the binding of PDHE1α and RAD51 to DSB sites. RAD51 and IgG were used as positive and negative control respectively. Data in e and f are representative of six and three independent replicates respectively. The statistical analyses were performed using a two-tailed student’s t-test, and shows the mean ± s.d. g, Western blot showing the knockdown efficiency of each siRNA. h, Western blot showing the nuclear and chromatin distribution of multiple components of the PDH complex in HeLa cells treated with IR. i, The activity of acetyl-CoA producing enzymes were confirmed by examining acetyl-CoA levels. n = 3 biological independent replicates were performed. The statistical analyses were performed using a two-tailed student’s t-test, and shows the mean ± s.d. j, Dynamics of PDHE1α and ACSS2 accumulation at DNA damage sites monitored by laser micro-irradiation-coupled live-cell imaging. GFP signal accumulation intensity at DSB stripes was quantified from three independent replicates (n = 20 for each group), the curve shows the mean ± s.e.m. Scale bars, 5 μm.

Source data

Extended Data Fig. 2 PDHE1α-catalyzed local acetyl-CoA is required for chromatin acetylation in response to DNA damage.

a, Dynamics of citrate metabolism at DNA damage sites monitored by laser micro-irradiation-coupled live-cell imaging on HeLa cells overexpressing the highly responsive citrate sensor construct, NLS-Citron1/pCDNA3.1. The fluorescence intensity at DSB stripes was quantificated from three independent replicates (n = 20 for each group), the curve shows the mean ± s.e.m. Scale bars, 5 μm. b-c, Volcano plots of the acetyl-modified proteins identified in Fig. 2B. Relative acetylation levels in the IR (b) and 4-OHT (c)-induced DNA damage groups versus the control are plotted on the x-axis as mean log2 ratios. Negative log10 PEP (the maximal posterior error probability for peptides) values are plotted on the y-axis. Significantly enriched proteins are denoted by red and blue dots, 13C isotope-labeled proteins are denoted by green dots, and all others are denoted by gray dots. d-e, Venn diagram showing all identified (d) and 13C isotope-labeled (e) that overlap between significant lysine acetylation sites (upper) and between acetylated proteins (lower) in IR- and 4-OHT-induced DNA damage versus control groups. f, Western blot showing the histone acetylation marks in acid-extracted fractions and the knockdown efficiency in whole cell lysate from HeLa cells transfected with siRNAs targeting PDHE1α, ACLY, and ACSS2 or NC. g-h, Immunofluorescence staining showing the colocalization of γH2AX with pan-Ac (g) and H3K9ac (h) signal in laser micro-irradiatiation induced DNA damage sites, nuclei were counterstained with DAPI. Pan-lysine acetylation intensity at DSB stripes was quantified from three independent replicates where data were collected from 78 cells for g and 51 cells for h group. The statistical analyses were performed using a two-tailed student’s t-test, and shows the mean ± s.d. Scale bars, 5 μm. i, Quantifition of total 13C-isotope -abelled Kac intensity around DSB sites in 13C-pyruvate, 13C-acetate and 13C-citrate cultured cells.

Source data

Extended Data Fig. 3 PARP1 activity controls the recruitment of PDHE1α to sites of DNA damage.

a, Co-Immunoprecipitation (Co-IP) analysis of the interaction of PDHE1α and PARP1 in Flag-PARP1 and HA-PDHE1α overexpressed cells. b, Immunoprecipitation analysis of the endogeneous interaction between PDHE1α and PARP1 in the whole cell lysate fraction extracted from HeLa cells. c, Western blot of the PDHE1α signal at chromatin fraction from HeLa cells treated with different doses of olaparib. PAR signaling was used as a positive control of olaparib treatment efficiency. d, Western blot showing that treatment with the PARP1 inhibitors, olaparib and AG14361, had no effect on total protein levels in whole cell lysates extracted from HeLa cells. e, Western blot showing the PDHE1α signal in the chromatin fraction from MEF-WT and MEF-PARP1−/− cells treated with etoposide (10 μM) for 1 h or IR (3 Gy). f, MEF PARP1−/− cells were rescued with Flag-vector and Flag-PARP1 constructs. Western blot showing PDHE1α recruitment in chromatin fractions from HeLa cells following DNA damage. g, Western blot showing the PDHE1α signal in the chromatin fraction from HeLa cells transfected with NC siRNA and siRNA targeting PARP1 and treated with 3 Gy IR. h-i, Dynamics of PDHE1α accumulation at DNA damage sites monitored by laser micro-irradiation-coupled live-cell imaging of HeLa cells stably expressing shNC and shPARP1 constructs (h) and PARP1 inhibitor treated cells (i). GFP signal accumulation intensity at DSB stripes was quantified from three independent replicates (n = 20 for each group), the curve shows the mean ± s.e.m. Scale bars, 5 μm.

Source data

Extended Data Fig. 4 PAR binding accelerates PDHE1α PARylation by PARP1.

a, In vitro PARylation assay performed using purified His-PDHE1α in the presence or absence of recombinant PARP1 enzyme, NAD+, and sonicated DNA as well as the PARP1 inhibitor, olaparib. b, PDHE1α was PARylated following DNA damage. c, Co-IP analysis showing that PDHE1α is PARylated by PARP1 in chromatin fractions from HeLa transfected with either control or PARP1 siRNAs. Cells were irradiated with 3 Gy IR. d, PDHE1α is PARylated by PARP1 on the chromatin. HeLa cells were pretreated with PARP inhibitor and then treated with or without IR. The cellular fractions were isolated and subjected to IP with anti-PDHE1α antibody, and the immunoprecipitates were blotted with specific antibodies. e, PDHE1α binds to PAR directly in vitro. His-PDHE1α was used to pull down a commerical PAR protein (20 pmol) in vitro. f, Schematic of the HA-tagged full-length (FL) and deletion mutants of PDHE1α used in this study (top panel). HeLa cells were transiently transfected with these plasmids encoding HA-tagged PDHE1α FL and mutants. Cells were irradiated with 3 Gy IR, then lysed immediately. The cell lysates were incubated with anti-HA affinity matrix and the immunoprecipitates were blotted with specific antibodies (bottom panel). g, Sequence alignment of potential PDHE1α PAR-binding motifs and PARylation sites of different species; key residues are shown in red. A schematic of PDHE1α PAR-binding motif (blue) and PARylation site (green) mutations is shown below the alignment. h, Validation of the PDHE1α PAR-binding motif at potential sites predicted by sequence alignment. HeLa cells were transiently transfected with the above-mentioned HA-tagged PDHE1α plasmids harboring PAR-binding site deletion-mutants and immunoprecipitated with a HA antibody for analysis by western blot/dot blot (for PAR) using specific antibodies.

Source data

Extended Data Fig. 5 PARylation of PDHE1α by PARP1 is essential for its recruitment to DNA damage sites.

a-b, Western blot showing PDHE1α PARylation and the effect of individal potential PARylation site mutation (a) and combined mutations (b) on chromatin accumulation. PDHE1α-deficient HeLa cells were transfected with PDHE1α WT and a mutant construct, irradiated with 3 Gy IR and different fractions were harvested immediatedly. The cell lysates were subjected to immunoprecipitation using anti-HA agarose and analysed by western blotting. c, Dynamics of PDHE1α accumulation at DNA damage sites monitored by laser micro-irradiation-coupled live-cell imaging of HeLa cells expressing NLS-fused PDHE1α-WT or PDHE1α-6A constructs. GFP signal accumulation intensity at DSB stripes was quantified from three independent replicates (n = 20 for each group), the curve shows the mean ± s.e.m. Scale bars, 5 μm.

Source data

Extended Data Fig. 6 PDHE1α PARylation is required for chromatin accessibility after DNA damage.

a, PDHE1α-deficient HeLa cells were reconstituted with PAR-binding site mutated or deleted constructs, and the cells were irradiated with 5 Gy IR and harvested for PDHE1α enzyme activity assay using a commercial assay kit. n = 3 biological replicates were collected. The statistical analyses were performed using a two-tailed student’s t-test, and data shows the mean ± s.d. b, IF staining showing the PARylated PDHE1α is required for pan-Ac signal accumulation on DSB sites. HeLa-PDHE1α-KO cells rescued with NLS-GFP-fused WT and PARylation-defective PDHE1α constructs were laser micro-irradiated and immuno-labeled with γH2AX and pan-Ac antibodies; nuclei were counterstained with DAPI. The pan-Ac intensity at DSBs was quantified (n >30). Scale bars, 5 μm. c, Quantification of γH2AX foci to confirm that similar amounts of DSBs were induced in the WT and all PDHE1α-mutant groups. The data representative of three independent replicates where data were collected from 100 cells per condition. d, MNase sensitivity assay to evaluate chromatin condensation. WT and PARylation-defective-construct-overexpressing PDHE1α-KO cells were treated with or without etoposide for 1 h. The gel image was captured by BioRad ChemiDoc XRS+, and the intensity of each nucleosome was calculated by Image J. e, HeLa cells and PDHE1α-KO cells were irradiated with 3 Gy IR followed by recovery for 1 h. The cellular chromatin fractions were extracted and analyzed by western blotting using antibodies against the indicated proteins. f, Accumulation of RFP-tagged 53BP1 and RPA70 at damage stripes on PDHE1α-deficient cells monitored by laser micro-irradiation-coupled live-cell imaging. RFP signal accumulation intensity at DSB stripes was quantified from three independent experiments (n = 20 cells were collected each group), the curve shows the mean ± s.e.m. Scale bars, 5 μm.

Source data

Extended Data Fig. 7 PDHE1α PARylation coordinated with its enzyme activity is required for chromatin accessibility after DNA damage.

a, GFP-tagged enzymatic mutated PDHE1α overexpressing cells monitored by laser micro-irradiation-coupled live-cell imaging. RFP signal accumulation intensity at DSB stripes was quantified from three independent experiments (n = 20 cells were collected each group), the curve shows the mean ± s.e.m. Scale bars, 5 μm. b, Western blot showing that overexpression of PDHE1α PAR-binding defective mutants have no effect on total protein levels of the incidated DSB repair factors in whole cell lysates extracted from HeLa cells. c, PDHE1α-deficient HeLa cells were reconstituted with enzyme-dead constructs and subjected to 5 Gy IR. The cells were then collected for PDHE1α enzyme activity assay using a commercial assay kit. n = 3 biological replicates were collected. The statistical analyses were performed using a two-tailed student’s t-test, and data shows the mean ± s.d. d, Accumulation of GFP-tagged enzymatic mutated PDHE1α at DNA damage sites monitored by laser micro-irradiation-coupled live-cell imaging of HeLa cells. GFP signal accumulation intensity at DSB stripes was quantified from three independent experiments (n = 15, 16, 12 and 9 cells for WT, R302C, K313R and R378H group were collected respectively), the curve shows the mean ± s.e.m. Scale bars, 5 μm. e, Western blot showing that overexpression of PDHE1αenzymatic defective mutants have no effect on total protein levels of the incidated DSB repair factors in whole cell lysates extracted from HeLa cells.

Source data

Extended Data Fig. 8 PDHE1α recruited to chromatin facilitates DNA damage repair and maintains genome integrity.

a, Comet assay showing the DSB repair efficiency in WT and PDHE1α-KO HeLa cells treated with etoposide (10 μM). Representative images are shown in the left panel. Relative tail moments of ≥100 cells were quantified using ImageJ. The center lines of the box-and-whisker plots show the medians, box limits indicate the 25th and 75th percentiles and whiskers extend 1.5x the interquartile range from the 25th and 75th percentiles. The median values of each group are listed under the boxplots. Scale bars, 50 μm. b, Representative gating strategy used to analyze DNA repair efficiency (left) and flow cytometry data for the DR-U2OS and EJ5-U2OS reporter assays shown in Fig. 6B. c, IF staining showing the kinetics of γH2AX foci formation and disappearance from WT and PDHE1α-deficient HeLa cells treated with etoposide (10 μM). The Representative images of γH2AX foci are shown (left), and the number of foci (≥100 cells) was quantified (right) from three independent experiments. Scale bars, 5 μm.

Source data

Extended Data Fig. 9 NLS-fused, but not NES-fused PDHE1α facilitates DNA damage repair and maintains genome integrity.

a, Immunostaining showing the intracellular distribution of WT/NLS/NES-fused PDHE1α in HeLa cells. The cells were overexpressed with the above constructs and subjected to immunofluorescence using the specific antibody. b, Mitochondrial OXPHOS detection in WT HeLa and PDHE1α-overexpressing cells. Cells were seeded on black-bottom 96 well plate. The cells were treated as indicated and subjected to OCR detection using commercial kit. The data was collected from 8 independent replicates in each condition. c, Comet assay showing the DSB repair efficiency of PDHE1α-KO HeLa cells stably expressing WT, NLS and NES-fused PDHE1α constructs. Cells were treated with 3 Gy IR. Representative images are shown in the left panel. Relative tail moments of ≥100 cells were quantified using ImageJ. The center lines of the box-and-whisker plots show the medians, box limits indicate the 25th and 75th percentiles and whiskers extend 1.5x the interquartile range from the 25th and 75th percentiles. The median values of each group are listed under the boxplots. Scale bars, 50 μm. d, Flow cytometric analysis showing the HR and NHEJ repair efficiency of DR-U2OS and EJ5-U2OS cells transfected with the indicated plasmids. Rescue efficiencies were determined by western blotting (bottom). n = 3 biological replicates, data are mean ± s.d. The statistical analyses of d and e were performed using a two-tailed student’s t-test. e, Chromosome aberration analysis showing the genome stability of PDHE1α-KO HeLa cells stably expressing WT, NLS and NES-fused PDHE1α constructs. Arrowheads denote chromosome breaks. Data in e represents three independent replicates and a total of 60 cells per group were collected and counted, the data shows the mean ± s.d.; Scale bars, 5 μm.

Source data

Extended Data Fig. 10 PDHE1α is a critical mediator of cellular resistance to DNA-damaging treatment.

a, Representative images of colony-formation corresponding to Fig. 7c. b, Genotyping of the Pdha1f/f and Pdha1f/f; CMV cre mice. The DNA from mice tail was extracted and subjected to PCR amplification using specific primers. c, Average body weight of mice post-IR (5 Gy). The body weight of each mouse was monitored every 4 days, and the curve was generated from the average weight of 10 mice per group. Data are mean ± s.d. d, The villi heights and average number of crypts per millimeter of the small intestine length were counted and plotted from 10 mice and each mice collected 2 views for analysis. The statistical analyses were performed using a two-tailed student’s t-test, and data shows the mean ± s.d. e, The intensity of indicated DNA damage markers (7K) were quantified. n = 3 biological replicates from the 10 mice was collected and analyzed using a two-tailed student’s t-test, data are mean ± s.d. f, PDHE1α-KO cells stably expressing PAR-binding-defective constructs were inoculated subcutaneously into nude mice. The average volume of each tumor and mouse body weight were monitored at the indicated time-points and used to plot the growth curve. g, PDHE1α-KO cells stably expressing PARylation-defective constructs were inoculated subcutaneously into nude mice, and the tumor site was irradiated every 5 days. The average body weight of each mouse was monitored at the indicated time-points. Data in f and g were collected from 8 mice of each group. h, H-scores of the indicated markers were quantified for three individual tumors collected from the 8 mice in each group. The statistical analyses were performed using a two-tailed student’s t-test, and data shows the mean ± s.d.

Source data

Supplementary information

Supplementary Information

All replicated original blots.

Reporting Summary

Supplementary Table 1

Key resources table.

Supplementary Table 2

Proteomics results.

Source data

Source Data Fig. 1

Unprocessed western blots.

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Unprocessed western blots.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Unprocessed western blots.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Unprocessed western blots.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Unprocessed western blots.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 6

Unprocessed western blots.

Source Data Fig. 6

Statistical source data.

Source Data Fig. 7

Statistical source data.

Source Data Extended Data Fig. 1

Unprocessed western blots.

Source Data Extended Data Fig. 1

Statistical source data.

Source Data Extended Data Fig. 2

Unprocessed western blots.

Source Data Extended Data Fig. 2

Statistical source data.

Source Data Extended Data Fig. 3

Unprocessed western blots.

Source Data Extended Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 4

Unprocessed western blots.

Source Data Extended Data Fig. 5

Unprocessed western blots.

Source Data Extended Data Fig. 5

Statistical source data.

Source Data Extended Data Fig. 6

Unprocessed western blots.

Source Data Extended Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 7

Unprocessed western blots.

Source Data Extended Data Fig. 7

Statistical source data.

Source Data Extended Data Fig. 8

Statistical source data.

Source Data Extended Data Fig. 9

Unprocessed western blots.

Source Data Extended Data Fig. 9

Statistical source data.

Source Data Extended Data Fig. 10

Statistical source data.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, J., Chen, F., Tian, Y. et al. PARylated PDHE1α generates acetyl-CoA for local chromatin acetylation and DNA damage repair. Nat Struct Mol Biol 30, 1719–1734 (2023). https://doi.org/10.1038/s41594-023-01107-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41594-023-01107-3

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing