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Structural and mechanistic basis for nucleosomal H2AK119 deubiquitination by single-subunit deubiquitinase USP16

Abstract

Epigenetic regulators have a crucial effect on gene expression based on their manipulation of histone modifications. Histone H2AK119 monoubiquitination (H2AK119Ub), a well-established hallmark in transcription repression, is dynamically regulated by the opposing activities of Polycomb repressive complex 1 (PRC1) and nucleosome deubiquitinases including the primary human USP16 and Polycomb repressive deubiquitinase (PR-DUB) complex. Recently, the catalytic mechanism for the multi-subunit PR-DUB complex has been described, but how the single-subunit USP16 recognizes the H2AK119Ub nucleosome and cleaves the ubiquitin (Ub) remains unknown. Here we report the cryo-EM structure of USP16–H2AK119Ub nucleosome complex, which unveils a fundamentally distinct mode of H2AK119Ub deubiquitination compared to PR-DUB, encompassing the nucleosome recognition pattern independent of the H2A–H2B acidic patch and the conformational heterogeneity in the Ub motif and the histone H2A C-terminal tail. Our work highlights the mechanism diversity of H2AK119Ub deubiquitination and provides a structural framework for understanding the disease-causing mutations of USP16.

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Fig. 1: Cryo-EM structure of USP16 bound to H2AK119Ub nucleosomes.
Fig. 2: Interactions between USP16 and nucleosomes.
Fig. 3: Interactions between USP16 and Ub.
Fig. 4: USP16 interacts with the nucleosome in an H2A–H2B independent manner.
Fig. 5: Conformational plasticity of the H2A tail and Ub in H2AK119Ub deubiquitination.
Fig. 6: Structural mapping of USP16 mutants in cancer and mode comparison of USP16 and PR-DUB on H2AK119Ub nucleosomes.

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Data availability

The cryo-EM maps and atomic model of USP16–H2AK119Ub nucleosome complex have been deposited in the Electron Microscopy Data Bank and PDB under accession codes EMD-37503 and 8WG5, respectively. The ubiquitin and unmodified nucleosome structures are available under PDB accession codes 1UBQ and 7XD1, respectively. The predicted structural model of full-length human USP16 is available in the AlphaFold Protein Structure Database under accession code AF-Q9Y5T5-F1. Newly created materials from this study may be requested from the corresponding authors. Source data are provided with this paper.

References

  1. Wang, H. B. et al. Role of histone H2A ubiquitination in polycomb silencing. Nature 431, 873–878 (2004).

    Article  CAS  PubMed  Google Scholar 

  2. Cao, R., Tsukada, Y. & Zhang, Y. Role of Bmi-1 and Ring1A in H2A ubiquitylation and Hox gene silencing. Mol. Cell 20, 845–854 (2005).

    Article  CAS  PubMed  Google Scholar 

  3. Ismail, I. H., Andrin, C., McDonald, D. & Hendzel, M. J. BMI1-mediated histone ubiquitylation promotes DNA double-strand break repair. J. Cell Biol. 191, 45–60 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Ginjala, V. et al. BMI1 is recruited to DNA breaks and contributes to DNA damage-induced H2A ubiquitination and repair. Mol. Cell. Biol. 31, 1972–1982 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Tamburri, S. et al. Histone H2AK119 mono-ubiquitination is essential for polycomb-mediated transcriptional repression. Mol. Cell 77, 840–856 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Barbour, H., Daou, S., Hendzel, M. & Affar, E. Polycomb group-mediated histone H2A monoubiquitination in epigenome regulation and nuclear processes. Nat. Commun. 11, 5947 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Uckelmann, M. & Sixma, T. K. Histone ubiquitination in the DNA damage response. DNA Repair 56, 92–101 (2017).

    Article  CAS  PubMed  Google Scholar 

  8. Xiao, X. et al. Histone H2A ubiquitination reinforces mechanical stability and asymmetry at the single-nucleosome level. J. Am. Chem. Soc. 142, 3340–3345 (2020).

    Article  CAS  PubMed  Google Scholar 

  9. Fierz, B., Kilic, S., Hieb, A. R., Luger, K. & Muir, T. W. Stability of nucleosomes containing homogenously ubiquitylated H2A and H2B prepared using semisynthesis. J. Am. Chem. Soc. 134, 19548–19551 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Zhao, J. C. et al. RYBP/YAF2-PRC1 complexes and histone H1-dependent chromatin compaction mediate propagation of H2AK119ub1 during cell division. Nat. Cell Biol. 22, 439–452 (2020).

    Article  CAS  PubMed  Google Scholar 

  11. Cooper, S. et al. Jarid2 binds mono-ubiquitylated H2A lysine 119 to mediate crosstalk between Polycomb complexes PRC1 and PRC2. Nat. Commun. 7, 13661 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Kasinath, V. et al. JARID2 and AEBP2 regulate PRC2 in the presence of H2AK119ub1 and other histone modifications. Science 371, eabc3393 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Zhang, Z. et al. Role of remodeling and spacing factor 1 in histone H2A ubiquitination-mediated gene silencing. Proc. Natl Acad. Sci. USA 114, E7949–E7958 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. McBride, M. J. et al. The nucleosome acidic patch and H2A ubiquitination underlie mSWI/SNF recruitment in synovial sarcoma. Nat. Struct. Mol. Biol. 27, 836–845 (2024).

    Article  Google Scholar 

  15. Tong, Z. et al. Synovial sarcoma X breakpoint 1 protein uses a cryptic groove to selectively recognize H2AK119Ub nucleosomes. Nat. Struct. Mol. Biol. 31, 300–310 (2024).

    Article  CAS  PubMed  Google Scholar 

  16. Jeusset, L. M. P. & McManus, K. J. Developing targeted therapies that exploit aberrant histone ubiquitination in cancer. Cells 8, 165 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Cao, J. & Yan, Q. Histone ubiquitination and deubiquitination in transcription, DNA damage response and cancer. Front. Oncol. 2, 26 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Joo, H. Y. et al. Regulation of cell cycle progression and gene expression by H2A deubiquitination. Nature 449, 1068–1072 (2007).

    Article  CAS  PubMed  Google Scholar 

  19. Xu, Y. et al. Ubp-M serine 552 phosphorylation by cyclin-dependent kinase 1 regulates cell cycle progression. Cell Cycle 12, 3219–3227 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Yang, W. et al. The histone H2A deubiquitinase Usp16 regulates embryonic stem cell gene expression and lineage commitment. Nat. Commun. 5, 3818 (2014).

    Article  CAS  PubMed  Google Scholar 

  21. Gu, Y. et al. The histone H2A deubiquitinase Usp16 regulates hematopoiesis and hematopoietic stem cell function. Proc. Natl Acad. Sci. USA 113, E51–E60 (2016).

    Article  CAS  PubMed  Google Scholar 

  22. Adorno, M. et al. Usp16 contributes to somatic stem-cell defects in Down’s syndrome. Nature 501, 380–384 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Reinitz, F. et al. Inhibiting USP16 rescues stem cell aging and memory in an Alzheimer’s model. eLife 11, e66037 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Souroullas, G. P. & Sharpless, N. E. Down’s syndrome link to ageing. Nature 501, 325–326 (2013).

    Article  CAS  PubMed  Google Scholar 

  25. Xu, J. C., Dawson, V. L. & Dawson, T. M. Usp16: key controller of stem cells in Down syndrome. EMBO J. 32, 2788–2789 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Volk, M., Maver, A., Lovrecic, L., Juvan, P. & Peterlin, B. Expression signature as a biomarker for prenatal diagnosis of trisomy 21. PLoS ONE 8, e74184 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Kong, X. D., Liu, N., Xu, X. J., Zhao, Z. H. & Jiang, M. Screening of human chromosome 21 genes in the dorsolateral prefrontal cortex of individuals with Down syndrome. Mol. Med. Rep. 11, 1235–1239 (2015).

    Article  CAS  PubMed  Google Scholar 

  28. Scheuermann, J. C. et al. Histone H2A deubiquitinase activity of the Polycomb repressive complex PR-DUB. Nature 465, 243–247 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Sahtoe, D. D., van Dijk, W. J., Ekkebus, R., Ovaa, H. & Sixma, T. K. BAP1/ASXL1 recruitment and activation for H2A deubiquitination. Nat. Commun. 7, 10292 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Foglizzo, M. et al. A bidentate polycomb repressive-deubiquitinase complex is required for efficient activity on nucleosomes. Nat. Commun. 9, 3932 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  31. De, I. et al. Structural basis for the activation of the deubiquitinase calypso by the polycomb protein ASX. Structure 27, 528–536.e4 (2019).

    Article  CAS  PubMed  Google Scholar 

  32. Ge, W. et al. Basis of the H2AK119 specificity of the Polycomb repressive deubiquitinase. Nature 616, 176–182 (2023).

    Article  CAS  PubMed  Google Scholar 

  33. Thomas, J. F. et al. Structural basis of histone H2A lysine 119 deubiquitination by Polycomb repressive deubiquitinase BAP1/ASXL1. Sci. Adv. 9, eadg9832 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Morgan, M. T. et al. Structural basis for histone H2B deubiquitination by the SAGA DUB module. Science 351, 725 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Atanassov, B. S. et al. ATXN7L3 and ENY2 coordinate activity of multiple H2B deubiquitinases important for cellular proliferation and tumor growth. Mol. Cell 62, 558–571 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Hicke, L., Schubert, H. L. & Hill, C. P. Ubiquitin-binding domains. Nat. Rev. Mol. Cell Biol. 6, 610–621 (2005).

    Article  CAS  PubMed  Google Scholar 

  37. Pai, M. T. et al. Solution structure of the Ubp-M BUZ domain, a highly specific protein module that recognizes the C-terminal tail of free ubiquitin. J. Mol. Biol. 370, 290–302 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Ai, H. S. et al. Examination of the deubiquitylation site selectivity of USP51 by using chemically synthesized ubiquitylated histones. Chem. Bio. Chem. 20, 221–229 (2019).

    Article  CAS  PubMed  Google Scholar 

  39. Fang, G. M. et al. Protein chemical synthesis by ligation of peptide hydrazides. Angew. Chem. Int. Ed. 50, 7645–7649 (2011).

    Article  CAS  Google Scholar 

  40. Qi, Y. K., Ai, H. S., Li, Y. M. & Yan, B. H. Total chemical synthesis of modified histones. Front. Chem. 6, 19 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Ai, H. S. et al. H2B Lys34 ubiquitination induces nucleosome distortion to stimulate Dot1L activity. Nat. Chem. Biol. 18, 972–980 (2022).

    Article  CAS  PubMed  Google Scholar 

  42. Tweedie-Cullen, R. Y., Reck, J. M. & Mansuy, I. M. Comprehensive mapping of post-translational modifications on synaptic, nuclear and histone proteins in the adult mouse brain. J. Proteome Res. 8, 4966 (2009).

    Article  CAS  PubMed  Google Scholar 

  43. Chu, G. C., Zhao, R., Wu, X. W., Shi, J. & Li, Y. M. One-pot synthesis of a bis-thio-acetone linked ubiquitinated histones using 1,3-dibromoacetone. J. Org. Chem. 85, 15631–15637 (2020).

    Article  CAS  PubMed  Google Scholar 

  44. Deng, Z. et al. Mechanistic insights into nucleosomal H2B monoubiquitylation mediated by yeast Bre1-Rad6 and its human homolog RNF20/RNF40-hRAD6A. Mol. Cell 83, 3080–3094.e14 (2023).

    Article  CAS  PubMed  Google Scholar 

  45. Magnusson, A. O. et al. nanoDSF as screening tool for enzyme libraries and biotechnology development. FEBS J. 286, 184–204 (2019).

    Article  CAS  PubMed  Google Scholar 

  46. Wen, J., Lord, H., Knutson, N. & Wikström, M. Nano differential scanning fluorimetry for comparability studies of therapeutic proteins. Anal. Biochem. 593, 113581 (2020).

    Article  CAS  PubMed  Google Scholar 

  47. Hu, M. et al. Crystal structure of a UBP-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde. Cell 111, 1041–1054 (2002).

    Article  CAS  PubMed  Google Scholar 

  48. Hu, M. et al. Structure and mechanisms of the proteasome-associated deubiquitinating enzyme USP14. EMBO J. 24, 3747–3756 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Ye, Y., Scheel, H., Hofmann, K. & Komander, D. Dissection of USP catalytic domains reveals five common insertion points. Mol. Biosyst. 5, 1797–1808 (2009).

    Article  CAS  PubMed  Google Scholar 

  50. Tong, Z., Ai, H. & Xu, Z. et al. Synovial sarcoma X breakpoint 1 protein uses a cryptic groove to selectively recognize H2AK119Ub nucleosomes. Nat. Struct. Mol. Biol. 31, 300–310 (2024).

    Article  CAS  PubMed  Google Scholar 

  51. McGinty, R. K. & Tan, S. Recognition of the nucleosome by chromatin factors and enzymes. Curr. Opin. Struct. Biol. 37, 54–61 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. McGinty, R. K. & Tan, S. Principles of nucleosome recognition by chromatin factors and enzymes. Curr. Opin. Struct. Biol. 71, 16–26 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Wang, H. B. et al. Structure of the transcription coactivator SAGA. Nature 577, 717–720 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Barbera, A. J. et al. The nucleosomal surface as a docking station for Kaposi’s sarcoma herpesvirus LANA. Science 311, 856–861 (2006).

    Article  CAS  PubMed  Google Scholar 

  55. Gersch, M. et al. Mechanism and regulation of the Lys6-selective deubiquitinase USP30. Nat. Struct. Mol. Biol. 24, 920–930 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. McGinty, R. K., Henrici, R. C. & Tan, S. Crystal structure of the PRC1 ubiquitylation module bound to the nucleosome. Nature 514, 591–596 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Ai, H. S. et al. Synthetic E2-Ub-nucleosome conjugates for studying nucleosome ubiquitination. Chem 9, 1221–1240 (2023).

    Article  CAS  Google Scholar 

  58. Zhou, X. et al. Exploring genomic alteration in pediatric cancer using ProteinPaint. Nat. Genet. 48, 4–6 (2015).

    Article  Google Scholar 

  59. Abaan, O. D. et al. The exomes of the NCI-60 Panel: a genomic resource for cancer biology and systems pharmacology. Cancer Res. 73, 4372–4382 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Seshagiri, S. et al. Recurrent R-spondin fusions in colon cancer. Nature 488, 660–664 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Giannakis, M. et al. Genomic correlates of immune-cell infiltrates in colorectal carcinoma. Cell Rep. 15, 857–865 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Bonilla, X. et al. Genomic analysis identifies new drivers and progression pathways in skin basal cell carcinoma. Nat. Genet. 48, 398–406 (2016).

    Article  CAS  PubMed  Google Scholar 

  63. Jung, S. H. et al. Distinct genomic profiles of gestational choriocarcinoma, a unique cancer of pregnant tissues. Exp. Mol. Med. 52, 2046–2054 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Pickering, C. R. et al. Mutational landscape of aggressive cutaneous squamous cell carcinoma. Clin. Cancer Res. 20, 6582–6592 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Uckelmann, M. et al. USP48 restrains resection by site-specific cleavage of the BRCA1 ubiquitin mark from H2A. Nat. Commun. 9, 229 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  66. Zhang, Z. et al. USP49 deubiquitinates histone H2B and regulates cotranscriptional pre-mRNA splicing. Genes Dev. 27, 1581–1595 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Reyes-Turcu, F. E. et al. The ubiquitin binding domain ZnF UBP recognizes the C-terminal diglycine motif of unanchored ubiquitin. Cell 124, 1197–1208 (2006).

    Article  CAS  PubMed  Google Scholar 

  68. Hard, R. L., Liu, J., Shen, J., Zhou, P. & Pei, D. HDAC6 and Ubp-M BUZ domains recognize specific C-terminal sequences of proteins. Biochemistry 49, 10737–10746 (2010).

    Article  CAS  PubMed  Google Scholar 

  69. Zheng, J. et al. The pleiotropic ubiquitin-specific peptidase 16 and its many substrates. Cells 12, 886 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Sen Nkwe, N. et al. A potent nuclear export mechanism imposes USP16 cytoplasmic localization during interphase. J. Cell Sci. 133, jcs239236 (2020).

    Article  CAS  PubMed  Google Scholar 

  71. Ai, H. et al. Chemical synthesis of post-translationally modified H2AX reveals redundancy in interplay between histone phosphorylation, ubiquitination, and methylation on the binding of 53BP1 with nucleosomes. J. Am. Chem. Soc. 144, 18329–18337 (2022).

    Article  CAS  PubMed  Google Scholar 

  72. Xu, T. H. et al. Structure of nucleosome-bound DNA methyltransferases DNMT3A and DNMT3B. Nature 586, 151–155 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Lei, J. L. & Frank, J. Automated acquisition of cryo-electron micrographs for single particle reconstruction on an FEI Tecnai electron microscope. J. Struct. Biol. 150, 69–80 (2005).

    Article  PubMed  Google Scholar 

  74. Li, X. M. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10, 584–590 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Zhang, K. Gctf: real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  77. van Heel, M. & Schatz, M. Fourier shell correlation threshold criteria. J. Struct. Biol. 151, 250–262 (2005).

    Article  PubMed  Google Scholar 

  78. Pettersen, E. F. et al. UCSF chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  79. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D. 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank the National Key R&D Program of China (grant nos. 2022YFC3401500 for L.L. and 2023YFA0915300 for M.P.) for financial support. This study was supported by the National Natural Science Foundation of China (grant nos. 22137005, 92253302, 22227810 for L.L. and 22277073 for M.P.). This work has been supported by the XPLORER prize and the New Cornerstone Science Foundation. M.P. thanks the Shanghai Rising-Star Program (grant no. 22QA1404900) and Shanghai Pilot Program for Basic Research—Shanghai Jiao Tong University (grant no. 21TQ1400224). M.P. was also supported by the Fundamental Research Funds for the Central University. H.A. thanks the funding from China Postdoctoral Science Foundation (grant nos. 2022TQ0170 and 2022M720075) and the National Facility for Translational Medicine (Shanghai). We acknowledge the Tsinghua University Branch of China National Center for Protein Sciences (Beijing) for cryo-EM data collection.

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Authors and Affiliations

Authors

Contributions

L.L. and M.P. supervised the project. H.A. proposed the idea, designed the experiments and analyzed the results. H.A. and Z.H. cloned the plasmids, expressed the proteins (USP16 variants, BAP1–ASXL1 and histones) and reconstituted the nucleosomes. G.-C.C. synthesized the histone H2AK119Ub with native isopeptide bond. H.A. and Q.S. synthesized the histone H2AK119Ub with DBA linkage. H.A. prepared the cryo-EM samples, collected the cryo-EM data and solved the cryo-EM structure of USP16 bound to H2AK119Ub nucleosomes. H.A., Z.H. and Z.D. conducted the biochemical experiments including SEC–MALS, USP16 protein BS3 crosslinking experiment, Ub-AMC hydrolysis, Ub-PA crosslinking, nucleosomal deubiquitination activity and EMSA binding assay. Z.H. conducted the nanoDSF analysis. H.A. wrote the manuscript. H.A. and Z.D. collated the experimental data and prepared the figures and tables. H.A., Z.D., Z.H., M.P. and L.L. revised the manuscript. H.A., Z.H., Z.D., G.-C.C., Q.S., Z.T., J.-B.L., M.P. and L.L. read and analyzed the manuscript.

Corresponding authors

Correspondence to Man Pan or Lei Liu.

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Nature Structural & Molecular Biology thanks Haibo Wang, Hengbin Wang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Sara Osman was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Biochemical characterization of the USP16 enzymatic activity.

a, SDS-PAGE analysis of the purified USP16 protein. The USP16 purity was conducted at least three times with similar results. b, USP16 crosslinking reaction with Ub-PA at the indicated time of 30, 60 and 90 minutes, a higher shift band was observed with the addition of Ub-PA. The experiment was conducted twice with similar results. c, Ub-AMC hydrolysis experiment in the presence of USP16 or blank buffer. d, USP16 deubiquitination activity on H2AK119Ub nucleosomes at 0, 2, 5, 30 minutes, the decrease of H2AUb and increase of H2A and Ub were observed. The experiment was conducted three times with similar results. e-f, EMSA results of USP16 on unmodified nucleosome and non-cleavable DBA-linked H2AK119Ub nucleosomes. The experiment was conducted twice with similar results. g, SEC-MALS result of USP16, the molecular weight of the major peak is 108 kDa. h, The SEC result of BS3-crosslinked USP16, the USP16 was crosslinked with BS3 and stopped with Tris buffer, then concentrated for SEC analysis. i, SDS-PAGE analysis of the fractions in h, highlighted in light orange. The former peak in SEC appears to be over-crosslinked dimer at the molecular weight between 150~250 kDa. The later major peak is the USP16 monomer at the molecular weight at around of 100 kDa. The BS3-crosslinking experiment in h-i was conducted twice with similar results.

Source data

Extended Data Fig. 2 Cryo-EM process of USP16-H2AK119Ub nucleosome complex.

a, Representative image collected on 300 kV Titan Krios. b, 2D classification of the USP16-H2AK119Ub nucleosome complex. c, Cryo-EM data processing workflow. d-e, Local resolution of the USP16-H2AK119Ub nucleosome complex and USP16-Ub estimated by ResMap. f, The angle distribution of USP16-H2AK119Ub nucleosome complex. g. Resolution estimation of the overall complex and USP16-Ub at a resolution of 3.05 Å and 3.77 Å, respectively.

Extended Data Fig. 3 Representative local cryo-EM map of USP16-H2AK119Ub nucleosome complex.

a, Cryo-EM maps of nucleosomal DNA over atomic coordinate. b, Cryo-EM maps of histone core over atomic coordinate. c-e, Cryo-EM maps of USP16 CD1 domain, USP16 CD2 domain and Ub. f, Representative regions of histone H2A helix (46-72), H2B helix (104-124), H3 helix (85-114) and H4 helix (49-76). g, Local resolution of SHL0 DNA. h, Local resolution of USP16 CD1 domain in helix (206-215) and helix (278-292). i, Local resolution of CD2 domain in sheet (691-698) and sheet (813-822). j, Local resolution of Ub C-terminal residue (69-76).

Extended Data Fig. 4 The effect of DNA on USP16 activity and binding.

a, EMSA result of human USP16(FL) with 147 bp 601-positioning sequence DNA. The DNA shifted with the increased USP16 concentration. The concentration of USP16 was two-fold diluted starting at 9.2 μM, and the 147 bp DNA was kept at a constant concentration of 50 nM. b, The DNA inhibitory experiment of USP16 deubiquitination activity on H2AK119Ub nucleosomes. With the gradual increased concentration of 147 bp DNA, the H2AK119Ub cleavage was hindered. All the experiments were performed independently twice with similar results.

Source data

Extended Data Fig. 5 Structural comparison of USP16, USP14 and USP7.

a-f, Alignment of catalytic domain of USP14 or USP7 to USP16-H2AK119Ub nucleosome complex structure. A specific insertion of the helix-turn-helix motif exists exclusively in USP16 (circled in red dashed lines in panel a), but was a flexible loop in USP14 (circled in red dashed lines in panel b) and a short linker in USP7 (circled in red dashed lines in panel c). USP16 interacts with both DNA SHL 0 and SHL 6 (panel d). The USP14 would clash with DNA around SHL 6.5 (panel e) and USP7 would clash with DNA SHL0 (panel f). g-i, Comparison of the USP16, USP14 and USP7 in complex with Ub. Ub regions including the I36 patch, I44 patch and Ub C-tail were conservatively engaged by the three deubiquitinases. j-l, Interaction details of Ub C-terminal tail with USP16, USP14 and USP7. m-o, Interaction details of the Ub I36 patch with USP16, USP14 and USP7.

Extended Data Fig. 6 Sequence alignment of USP16 in different species.

The sequence of USP16 species including human, mouse, bovine and zebrafish were aligned in UniProt (https://www.uniprot.org/align) and colored by online website ESPript-3.0 (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). The more conservative the residue, the redder the box that frames them. The conserved USP16 residues that interact with Ub, nucleosomal DNA or histones were labelled underlined and highlighted in yellow.

Extended Data Fig. 7 EMSA results of USP16 on unmodified nucleosome and H2A–H2B acidic patch-mutated nucleosomes.

In the centre, the representative acidic residues of the nucleosome H2A–H2B acidic patch have been labelled with side chains and colored in orange. The EMSA results of the indicated nucleosome mutant was in the vicinity. The USP16 was diluted twofold starting at 2.3 μM and the nucleosome was kept at a constant concentration of 50 nM. USP16 EMSA experiments with independent mutated nucleosome samples were performed once.

Source data

Extended Data Fig. 8 Structural comparison of Ub recognition by USP16 and PR-DUB.

a, Recognition of Ub by USP16 CD1 and CD2 domains, left, the interaction details, right, the recognition model. b, Recognition of Ub by BAP1 and ASXL1, left, the interaction details, right, the recognition model. c, Specific interaction involved in Ub D58/N60 by USP16 but not engaged by BAP1. d, Specific interaction involved in Ub E24 and D39 by BAP1/ASXL1 but not engaged by USP16.

Extended Data Fig. 9 Conformational plasticity of H2AK119Ub in different complex structures.

Six conformations of the H2AK119Ub nucleosome in different binding proteins were aligned, including the H2AK119Ub writer PRC1 (PDB: 8GRM), the H2AK119Ub modification reader proteins SSX1 (PDB: 8HQY), JARID2 (PDB: 6WKR) and AEBP2 (PDB: 6WKR), the H2AK119Ub modification eraser BAP1/ASXL1 (PDB: 8H1T) and USP16 (this work). The separated H2AK119Ub conformations were surrounded.

Extended Data Fig. 10 USP16 mutations found in cancer.

a, USP16 mutations across the polypeptide chain, different kinds of mutations were labeled with different colors, the figure was layout from the online ProteinPaint website (https://proteinpaint.stjude.org/). b, Cancer types summary of the USP16 mutations, the panel was laid out from the cBioPortal website (https://www.cbioportal.org/).

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Structure–effect relationship statistics of cancer-associated USP16 mutations.

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Ai, H., He, Z., Deng, Z. et al. Structural and mechanistic basis for nucleosomal H2AK119 deubiquitination by single-subunit deubiquitinase USP16. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01342-2

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