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Mechanism of nucleosomal H2A K13/15 monoubiquitination and adjacent dual monoubiquitination by RNF168

Nature Chemical Biology volume 21, pages 668–680 (2025)Cite this article

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Abstract

The DNA damage repair regulatory protein RNF168, a monomeric RING-type E3 ligase, has a crucial role in regulating cell fate and DNA repair by specific and efficient ubiquitination of the adjacent K13 and K15 (K13/15) sites at the H2A N-terminal tail. However, understanding how RNF168 coordinates with its cognate E2 enzyme UbcH5c to site-specifically ubiquitinate H2A K13/15 has long been hampered by the lack of high-resolution structures of RNF168 and UbcH5c~Ub (ubiquitin) in complex with nucleosomes. Here we developed chemical strategies and determined the cryo-electron microscopy structures of the RNF168–UbcH5c~Ub–nucleosome complex captured in transient H2A K13/15 monoubiquitination and adjacent dual monoubiquitination reactions, providing a ‘helix-anchoring’ mode for monomeric E3 ligase RNF168 on nucleosome in contrast to the ‘compass-binding’ mode of dimeric E3 ligases. Our work not only provides structural snapshots of H2A K13/15 site-specific monoubiquitination and adjacent dual monoubiquitination but also offers a near-atomic-resolution structural framework for understanding pathogenic amino acid substitutions and physiological modifications of RNF168.

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Fig. 1: RNF168 efficiently mediates dual monoubiquitination on adjacent H2A K13/15 sites.
Fig. 2: Capturing the complex of RNF168–UbcH5c-mediated nucleosome monoubiquitination using SICC strategy.
Fig. 3: Structural visualization of RNF168–UbcH5c-mediated H2A K13/15 dual monoubiquitination obtained using ACC strategy.
Fig. 4: Capturing the RNF168–UbcH5c complex-mediated nucleosome monoubiquitination using ACC strategy.
Fig. 5: Structural basis of monomeric E3 ligase RNF168 on the H2A–H2B acidic patch.
Fig. 6: Interaction of UbcH5c with nucleosomal DNA SHL 4.5 and cartoon models for nucleosomal H2A ubiquitination mediated by monomeric E3 ligase RNF168.

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Data availability

The cryo-EM maps and atomic model of RNF168–UbcH5c in complex with nucleosomes were deposited to the EM Data Bank and PDB under accession codes EMD-38099 and PDB 8X7I (RNF1681–113–UbcH5c~Ub–nucleosome complex obtained using SICC strategy), EMD-38102 (RNF1681–113–UbcH5c~Ub–nucleosome complex obtained using CC strategy, 200-kV reconstruction), EMD-38100 and PDB 8X7J (RNF1681–113–UbcH5c~Ub–nucleosome complex, no Ub conformation, obtained using ACC strategy), EMD-38101 and PDB 8X7K (RNF1681–113–UbcH5c~Ub–H2A K13Ub–nucleosome complex obtained using ACC strategy), EMD-60066 (RNF1681–113–UbcH5c~Ub–H2A K13Ub–nucleosome complex, two-Ub conformation, obtained using ACC strategy) EMD-39800 (RNF1681–193–UbcH5c~Ub–nucleosome complex). The raw images of the RNF1681–113–UbcH5c~Ub–H2A K13Ub–nucleosome complex were deposited to the EM Public Image Archive under accession code EMPIAR-12143. The RNF168-related cancer missense mutations were derived from the ProteinPaint (https://proteinpaint.stjude.org/), which loads COSMIC data. Source data are provided with this paper.

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Acknowledgements

M.P. was supported by the National Key R&D Program of China (no. 2023YFA0915300), National Natural Science Foundation of China (no. 22277073), Shanghai Rising-Star Program (22QA1404900), Shanghai Pilot Program for Basic Research—Shanghai Jiao Tong University (21TQ1400224) and the Fundamental Research Funds for the Central University. L.Liu was supported by the National Key R&D Program of China (no. 2022YFC3401500), National Natural Science Foundation of China (nos. 22137005, 92253302, 22227810, and T2488301), XPLORER prize and New Cornerstone Investigator Program. C.T. was supported by the National Key R&D Program of China (no. 2022YFC3400502), National Natural Science Foundation of China (nos. 21825703 and 21927814) and Strategic Priority Research Program of Chinese Academy of Sciences (XDB37000000). H.A. thanks the National Facility for Translational Medicine (Shanghai) for funding. We acknowledge the Tsinghua University Branch of China National Center for Protein Sciences (Beijing) for cryo-EM data screening and collection.

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Author notes

  1. These authors contributed equally: Huasong Ai, Zebin Tong, Zhiheng Deng.

Authors and Affiliations

  1. Institute of Translational Medicine, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China

    Huasong Ai, Qingyun Zheng & Man Pan

  2. New Cornerstone Science Laboratory, Tsinghua-Peking Joint Center for Life Sciences, Ministry of Education Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, Center for Synthetic and Systems Biology, Department of Chemistry, Tsinghua University, Beijing, China

    Huasong Ai, Zebin Tong, Zhiheng Deng, Qiang Shi, Shixian Tao, Jiawei Liang, Maoshen Sun, Xiangwei Wu & Lei Liu

  3. School of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, Shanghai, China

    Huasong Ai, Qingyun Zheng, Changlin Tian & Man Pan

  4. School of Pharmaceutical Sciences, Tsinghua University, Beijing, China

    Gaoge Sun & Hang Yin

  5. Howard Hughes Medical Institute, Department of Cell Biology, Harvard Medical School, Boston, MA, USA

    Maoshen Sun

  6. Center for Bioanalytical Chemistry, Hefei National Laboratory of Physical Science at Microscale, University of Science and Technology of China, Hefei, China

    Lujun Liang

  7. College of Pharmaceutical Sciences, Soochow University, Suzhou, China

    Jia-Bin Li

  8. Department of Urology, Zhongnan Hospital of Wuhan University, TaiKang Center for Life and Medical Sciences, School of Pharmaceutical Sciences, Wuhan University, Wuhan, China

    Shuai Gao

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  1. Huasong Ai
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Contributions

H.A., Z.T., Z.D., C.T., L.Liu and M.P. proposed the idea, designed the experiments and analyzed the results. Z.T. and L.Liu designed and optimized the SICC and ACC strategies. Z.T., Z.D., H.A., S.T. and Q.S. cloned the plasmids, expressed the proteins (RNF168, UbcH5c, histones and mutants) and reconstituted the nucleosomes. Z.T., H.A. and Z.D. synthesized the IntN-E2~Ub-conjugated H2A K15C, E2~Ub-conjugated H2A K15C, H2A K15CAT and H2A K13Ub/K15CAT nucleosome probes. H.A., Z.D., Z.T. and S.T. prepared the fluorescently labeled H2A histones. Q.S. and Z.D. synthesized the fluorescently labeled ubiquitinated histone H2A. Z.D. and J.L. synthesized the H2A K13Ubmut/K15 histone. H.A. and Z.D. performed the RNF168–H2A K13/15 selectivity assays and RNF168–UbcH5c mutant activity experiments on unmodified nucleosome substrates or acidic patch mutant nucleosomes. Z.T. performed the RNF168 ubiquitination experiments of Ub chain elongation and Ub mutants on the H2A K15Ub NCP. Z.D. performed the RNF8-related and SET8-related RNF168 ubiquitination experiments and the RNF168 mutant activity tests on ubiquitinated nucleosomes. Z.T. prepared the cryo-EM samples. H.A., Z.T. and Z.D. checked the samples and collected the cryo-EM data. H.A. processed the cryo-EM data, determined the cryo-EM structures and built the atomic models. Z.D. and G.S. conducted the immunofluorescence assay. Z.D., Z.T. and H.A. collated the experimental data and prepared the figure panels and tables. H.A. drafted the manuscript. H.A., Z.D., Z.T., C.T., L.Liu and M.P. revised the manuscript. All authors (H.A., Z.T., Z.D., Q.S., S.T., G.S., J.L., M.S., X.W., Q.Z., L.Liang, H.Y., J.-B.L., S.G., C.T., L.Liu and M.P.) read, discussed and analyzed the manuscript. M.P., L.Liu and C.T. supervised the project.

Corresponding authors

Correspondence to Changlin Tian, Lei Liu or Man Pan.

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Extended data

Extended Data Fig. 1 RNF168 prefers to ubiquitinate H2AK13/15 without polyubiquitin chain linkage preference.

a, In vitro ubiquitination assay to compare the activity of RNF1681–113 and RNF168FL to generate monoubiquitination (H2AUb) and dual-monoubiquitination (H2AUb2) on H2A K13/K15 NCP. b, In vitro ubiquitination assay shows that RNF1681–113 preferentially ubiquitinates H2AK13/15 rather than H2BK120. c, In vitro ubiquitination assay shows that RNF1681–113 together with UbcH5c can generate short ubiquitin chains on H2A K13R/K15 NCP. Note that there is only one lysine (H2A K15) on H2A K13/K15 NCP that can be conjugated with ubiquitin and bands of H2AUb3 were observed, suggesting that at least three ubiquitin were conjugated to H2A K15. The PRC1RING E3 ligase that site-specifically catalyzes nucleosomal H2A K118/K119 monoubiquitination was used as a negative control and it failed to generate significant H2A ubiquitination on H2A K13R/K15 NCP. d. In vitro ubiquitination assay to test the ubiquitin chain elongation activity of RNF1681–113 or RNF168FL on H2A K13R/K15Ub NCP. Note that in this nucleosome substrate, all the lysines on H2A were substituted by arginines so that ubiquitin can only be conjugated to the Ub motif at the H2A K15 site. It was observed that RNF1681–113 or RNF168FL generated H2AUb3, suggesting that no more than two Ub were conjugated to the Ub motif of the H2A K13R/K15Ub NCP and the ubiquitin chain elongation activity of RNF1681–113 or RNF168FL is weak. e,f, In vitro ubiquitination assays to investigate the ubiquitin chain linkage preference of RNF1681–113 on H2A K13R/K15 NCP (e) and H2A K13R/K15Ub NCP (f). These results suggest that RNF1681–113 generates short and mixed ubiquitin chains at H2A K15 sites with no linkage preference in vitro. For all the biochemical results, three independent experiments were performed with similar results.

Source data

Extended Data Fig. 2 Cryo-EM data processing for RNF1681–113/UbcH5c–Ub/NCP complex achieved by SICC strategy.

a, A representative micrograph. The micrographs collected in this dataset show similar particle density and dispersion to the representative micrograph. b, CTF estimation of the micrograph shown in a. c, Representative 2D classifications. d, Data processing flowcharts. e, Euler angle distributions of the final particles for cryo-EM reconstruction. f, Local resolution colored cryo-EM density map. g, Gold standard Fourier shell correlation (FSC) curves showing the overall resolution of 3.27 Å and 3.62 Å for the final density map of RNF168/UbcH5c–Ub/NCP, and RNF168/UbcH5c, respectively.

Extended Data Fig. 3 Cryo-EM data processing for RNF1681–113/UbcH5c–Ub/NCP complex achieved by CC strategy.

a, Schematic presentation of nucleosomal H2A ubiquitination intermediate showing the tetrahedron transition state and two different mimicries. b, Data processing flowcharts. c, Euler angle distributions of the final particles for cryo-EM reconstruction. d, Gold standard Fourier shell correlation (FSC) curves showing the overall resolution of 7.13 Å.

Extended Data Fig. 4 Cryo-EM data processing for RNF1681–193/UbcH5c–Ub/NCP complex achieved by CC strategy.

a, A representative micrograph. The micrographs collected in this dataset show similar particle density and dispersion to the representative micrograph. b, CTF estimation of the micrograph shown in a. c, Representative 2D classifications. d, Data processing flowcharts. e, Euler angle distributions of the final particles for cryo-EM reconstruction of RNF1681–193/UbcH5c/NCP conformation. f, Euler angle distributions of the final particles for cryo-EM reconstruction of RNF1681–193/UbcH5c-Ub/NCP conformation. g, Gold standard Fourier shell correlation (FSC) curves showing the overall resolution of 3.23 Å and 3.59 Å for the final density map of RNF1681–193/UbcH5c/NCP and RNF1681–193/UbcH5c-Ub/NCP conformation, respectively.

Extended Data Fig. 5 Comparison of CC strategy, SICC strategy, and ACC strategies.

a. Cartoon illustration of the CC strategy. E2-Ub is covalently attached to the nucleosome by chemical synthesis, followed by the addition of E3 ligase to form the complex. b. Cartoon illustration of the SICC strategy. Given the basis of the E2-Ub-nucleosome conjugation strategy, the split intein enables protein trans-splicing reaction resulting in the formation of a covalent bond between E3 and E2. A covalent complex of E3-E2-Ub-nucleosome is finally acquired. c. Cartoon illustration of the ACC strategy. Firstly, E3 ligase and E2-Ub are covalently linked by a trans-splicing reaction, and a covalent bond is formed between E2-Ub and nucleosome based on the E3 ligase activity.

Extended Data Fig. 6 Cryo-EM data processing for RNF1681–113/UbcH5c–Ub/H2AK13Ub NCP complex achieved by ACC strategy.

a, A representative micrograph. The micrographs collected in this dataset show similar particle density and dispersion to the representative micrograph. b, Representative 2D classifications. c, Data processing flowcharts. d, Euler angle distributions of the final particles for cryo-EM reconstruction of RNF168/UbcH5c/two Ub conformation. e, Euler angle distributions of the final particles for cryo-EM reconstruction of RNF168/UbcH5c/NCP conformation. f, Gold standard Fourier shell correlation (FSC) curves showing the overall resolution of 3.52 Å and 3.20 Å for the final density map of RNF1681–113/UbcH5c/two Ub conformation and RNF1681–113/UbcH5c/NCP conformation, respectively.

Extended Data Fig. 7 Analysis of the E3-E2 interfaces and canonical closed E2~Ub conformation.

a, Structural alignment of RNF1681–113/UbcH5c–Ub/NCP complex achieved by SICC strategy, and the structure of RNF1681–113/UbcH5c–Ub/H2A K13Ub NCP complex captured by ACC strategy, highlighting the congruence in nucleosome-binding mode and E3 /E2 arrangement. b,c, Close-up views of RNF168/UbcH5c on the nucleosomal acidic patch in the two structures including the RNF1681–113/UbcH5c–Ub/NCP complex by SICC strategy (PDB:8X7I) (b), and the RNF1681–113/UbcH5c–Ub/H2A K13Ub NCP complex by ACC strategy (PDB:8X7K) (c). The Cαs of UbcH5c active center C85 and H2A K15 are depicted as spheres and the distances between them are indicated. d. Overview of interactions of RNF1681–113 and UbcH5c. Rectangle regions indicate closed-up interfaces shown in e and f. e, Close-up view highlighting the RNF168–UbcH5c SPA motif interface. f, Close-up view highlighting the interface centered on the RNF1681–113 linchpin R55 and UbcH5c N92. g. Alignment of four E3/E2–Ub complexes (PDB: 4AUQ, 4AP4, 6TTU, and 8GRM) that contain UbcH5 family E2s with Ub at the close conformation and the current RNF168/UbcH5c structure. The Cαs of UbcH5 E2 residues that play important roles in mediating closed E2~Ub conformation (I88, R90, L97, L104, D116, and D117) and non-covalent ubiquitin binding stimulation (S22) are colored red and blue, respectively. h. In vitro ubiquitination assay using WT-H2A NCP to investigate the effects of UbcH5c mutants on overall nucleosomal H2A ubiquitination pattern. Data show the mean ± SD (bars) from n = 3 independent biological replicates. i. Alignment of our RNF168/UbcH5c in our complex structure with four nucleosomal E3/E2 complexes (PDB: 4R8P, 7JZV, 8IEG, and 8IEJ), highlighting the conserved E3/E2 spatial arrangement and the E3–E2 interface.

Extended Data Fig. 8 Cryo-EM data processing for RNF1681–113/UbcH5c–Ub/NCP complex achieved by ACC strategy.

a, A representative micrograph. The micrographs collected in this dataset show similar particle density and dispersion to the representative micrograph. b, Representative 2D classifications. c, Data processing flowchart. d, Cryo-EM density map at an overall resolution of 6.87 Å representing the closed E2~Ub conformation. e, Local resolution analysis of the 3.39 Å cryo-EM density map. f, Gold standard Fourier shell correlation (FSC) curves showing the overall resolution of 3.39 Å and 3.27 Å for the final density map of RNF1681–113/UbcH5c–Ub/NCP and RNF1681–113/UbcH5c/NCP conformation, respectively.

Extended Data Fig. 9 Sample densities for the cryo-EM reconstructions of RNF168/UbcH5c-mediated nucleosomal H2A ubiquitination.

a–f, Main chain traces of the whole complex (a), DNA (b), histone octamer (c), RNF168 (d), UbcH5c (e), and Ub (f) fit into the cryo-EM density map of the RNF1681–113/UbcH5c–Ub/NCP complex achieved by SICC strategy. g,h, Two views of the representative region of the cryo-EM density map of the RNF1681–113/UbcH5c–Ub/NCP complex achieved by SICC strategy for the RNF168–acidic patch interface with side chains of RNF168 R57, S60, and R63, and some H2A/H2B residues shown. i–n, Main chain traces of the whole complex (i), DNA (j), histone octamer (k), RNF168 (l), UbcH5c (m), and Ub (n) fit into the cryo-EM density map of the RNF1681–113/UbcH5c–Ub/NCP complex achieved by ACC strategy. o and p, Two views of the representative region of the cryo-EM density map of the RNF1681–113/UbcH5c–Ub/NCP complex achieved by ACC strategy for the RNF168–acidic patch interface with side chains of RNF168 S60 and R63, and some H2A/H2B residues shown. q–u, Main chain traces of the whole complex (q), DNA (r), histone octamer (s), RNF168 (t), and UbcH5c (u) fit into the cryo-EM density map of the RNF1681–113/UbcH5c–Ub/H2AK13Ub NCP complex achieved by ACC strategy. v,w, Two views of the representative region of the cryo-EM density map of the RNF1681–113/UbcH5c–Ub/H2AK13Ub NCP complex achieved by ACC strategy for the RNF168–acidic patch interface with side chains of RNF168 R57, S60, and R63, and some H2A/H2B residues were shown.

Extended Data Fig. 10 Biochemical investigations of the role of the structurally observed Ubback in the RNF168-mediated H2A ubiquitination.

a, Models showing the spatial proximity of SHL DNA 3.5 and Ubback and the potential interface of UbcH5c and Ubback. The models are from the RNF168/UbcH5c–Ub/NCP complex achieved by intein-based E2–Ub–nucleosome conjugation strategy. Nucleosomal DNA is present as cryo-EM density surface and Ubback, UbcH5c, and H2A are shown as ribbon models. RNF168, H2B, H3, and H4 are hidden for clarity. The Cαs of Ubback positive residues near the SHL DNA 3.5 (K6, R42, K48, and R72) and Ubback residues near UbcH5c (E34, G35, D39, and R74) are colored red, light blue, and medium blue, respectively. b, Schematic representation of biochemical assays to investigate the effects of Ubback mutations in the second H2A ubiquitination step. Note the Ubback mutations are introduced to the H2A K13Ub/K15 NCP which is the substrate in the 2nd H2A ubiquitination step. c, In vitro ubiquitination assays described in b using H2A K13UbMut/K15 NCP as substrates. Coomassie Brilliant Blue-stained bands of H2AUb (substrate) and H2AUb2 (product) are indicated, and the band of H4 in each lane was used as a control to calculate the H2AUb2 generation efficiency in d. d, Quantified H2AUb2 generation efficiency in c. Combination mutations of Ubback positive residues near the SHL DNA 3.5 (K6D, R42E, K48D, and R72E) and Ubback residues near UbcH5c (D39K and R74E) slightly reduced the second Ub conjugation efficiency at the H2A K15 site on the H2A K13UbMut/K15 NCPs. Data show the mean ± SD (bars) from n = 3 independent biological replicates.

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Ai, H., Tong, Z., Deng, Z. et al. Mechanism of nucleosomal H2A K13/15 monoubiquitination and adjacent dual monoubiquitination by RNF168. Nat Chem Biol 21, 668–680 (2025). https://doi.org/10.1038/s41589-024-01750-x

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