Abstract
The activation of H2B K120 monoubiquitylation (H2BK120ub) by H2B S112 GlcNAcylation (H2BS112GlcNAc) has an important role in regulating transcriptional activation, yet its mechanism remains unclear. Here we chemically synthesized H2BS112GlcNAc-modified nucleosomes and quantitatively evaluated how H2BS112GlcNAc stimulates ubiquitylation by RNF20/RNF40–RAD6A E3–E2 enzymes. Cryo-electron microscopy determination of a chemically trapped RNF20/RNF40–RAD6A–Ub–H2BS112GlcNAc nucleosome complex revealed that the H2BS112GlcNAc moiety interacts with the E2 enzyme RAD6A but not the E3 ligase RNF20/RNF40. Mutagenesis and kinetics analyses demonstrated that H2BS112GlcNAc allosterically stimulates ubiquitin transfer from the RAD6A~Ub thioester to H2B K120 by enhancing the nucleophilicity of H2B K120. Structure‒activity relationship analysis further identified the essential roles of the C2 N-acetyl group and the β-configuration of C1 on the H2BS112GlcNAc moiety. These findings provide the structural evidence of histone posttranslational modification crosstalk involving O-GlcNAcylation and reveal how O-GlcNAcylation can allosterically stimulate enzyme activity through substrate modification.
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The cryo-EM maps and atomic model of RNF20/RNF40–RAD6A in complex with H2BS112GlcNAc nucleosome were deposited to the EM Data Bank and Protein Data Bank under accession codes EMD-62509 and PDB 9KQO, respectively. The three different conformational maps were deposited as additional maps to the EM Data Bank under accession code EMD-62509. Source data are provided with this paper.
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Acknowledgements
This study was supported by the National Key R&D Program of China (2025YFA1310000/2023YFA0915300 to M.P.), the National Natural Science Foundation of China (22207065 to Y.L., 22137005, 92253302, 22227810, T2488301 to L.L., 22277073 to M.P., 32501108 to H.A.), the Shanghai Natural Science Foundation (25ZR1402193 to H.A.), the Shanghai Frontiers Science Center of Drug Target Identification and Delivery (ZXWH2170101 to H.A.), the Shanghai Rising-Star Program (22QA1404900 to M.P.), the Shanghai Pilot Program for Basic Research - Shanghai Jiao Tong University (21TQ1400224 to M.P.), the Taishan Scholars Program (tsqn202306268 to Y.L.) and the New Cornerstone Investigator Program (to L.L.). Z.D. acknowledges support from the Shuimu Tsinghua Scholars Program and National Facility for Translational Medicine (Shanghai). We acknowledge the Tsinghua University Branch of China National Center for Protein Sciences for cryo-EM data screening and collection.
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Conceptualization, H.A., Z.D., M.P. and L.L. Design of experiments, Z.D., H.A. and L.L. Synthesis of glycosylated histone H2B variants, H.A. and S.T. Synthesis of ubiquitylation intermediate mimic, Z.D., H.A., L.Z. and S.T. Synthesis of glycobased amino acid building blocks, Y.L. and X.D. Cloning and protein purification, Y.D., Z.D., M.S., L.Z., S.T., Q.S. and Z.T. Cryo-EM sample preparation, Z.D. Cryo-EM data processing and model building, H.A. and Z.D. Biochemical assays, Z.D. and Y.D. Visualization and figure preparation, Z.D. Writing—original draft, H.A. and Z.D. Writing—review and editing, all authors. Funding and supervision, H.A., M.P. and L.L.
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Extended data
Extended Data Fig. 1 Structure and synthetic intermediates of H2BS112GlcNAc.
a, Chemical structure of O-GlcNAc modified on H2B S112 residue. The distance from the H2B S112 αC atom to the C4-OH hydrogen of O-GlcNAc is 8.5 Å based on its atomic model. b, In our reported structure of the RNF20/RNF40–RAD6A–nucleosome complex (PDB: 8IEJ), a semi-transparent green spherical with a radius of 8.5 Å is centered on the H2B S112 αC atom. Within this structural model, O-GlcNAc modified on H2B S112 may potentially interact with RNF20 or RAD6A. c, Representative RP-HPLC trace (214 nm) of purified 1. d, Representative ESI-MS spectra and deconvoluted MS spectra of purified 1. e, Representative RP-HPLC trace (214 nm) of crude 2, crude 3, and purified 3. f, Representative ESI-MS spectra and deconvoluted MS spectra of crude 2. g, Representative ESI-MS spectra and deconvoluted MS spectra of purified 3. h, RP-HPLC monitoring of peptide hydrazide ligation from 1 and 3 to generate 4. Peaks of 3 and 4 are indicated. i, Representative RP-HPLC trace (214 nm) of purified 4. j, Representative ESI-MS spectra and deconvoluted MS spectra of purified 4.
Extended Data Fig. 2 Design and synthesis of the nucleosomal H2B ubiquitylation mimic SP1 complex.
a, Schematic representation of H2B ubiquitylation by human RNF20/RNF40–RAD6A complex on H2BS112GlcNAc nucleosome. The heterodimeric RNF20/RNF40 RING domains bind RAD6A~Ub thioester non-covalently (indicated by a dashed gray arrow) to form the tetrahedron transition state. b, Schematic representation of the engineered mimic (SP1) of H2B ubiquitylation intermediate. The RNF20 RING domain is covalently fused with RAD6A by SICC strategy. The covalent linker and the non-covalent interactions between RNF20 RING and RAD6A are indicated as a solid green line and a dashed gray arrow, respectively. c, Amino acid sequence of histone H2B K120C mutant. The ligation sites A58 and A97, the glycosylation site S112, and the conjugating site K120C are indicated by red, light sea green, and orange solid circles, respectively. d, Convergent synthetic route of probe 17.
Extended Data Fig. 3 Mass spectrometry of probe 17 synthetic intermediates.
a–l, Representative ESI-MS spectra and deconvoluted MS spectra of synthetic intermediates 6 (a), 8 (b), 7 (c), 9 (d), 10 (e), 11 (f), 12 (g), UbMesNa (h), 13 (i), 14 (j), 15 (k), and 16 (l) shown in Extended Data Fig. 2.
Extended Data Fig. 4 Cryo-EM data processing for RNF20/RNF40–RAD6A–NCPGlcNAc complex.
a, Representative micrograph from the dataset. b, CTF estimation of the micrograph shown in a. c, Representative 2D averages. d, Data processing flowchart. e, The final reconstruction colored by local resolution. f, Euler angle distributions of the final reconstruction. g, Fourier shell correlation curve calculated from final refinement. The resolution at FSC = 0.143 cutoff was highlighted. h, Fourier shell correlation curve for model-map correlation shown at FSC = 0.5.
Extended Data Fig. 5 Sample cryo-EM densities and conformational flexibility analysis.
a, Nucleosmal 147-bp DNA density with atomic model fitted. b, Histone octamer density with cartoon model fitted. c, Sample densities of histone H2A, H2B, H3, and H4, and nucleosomal SHL0 DNA with atomic model fitted. d, Cryo-EM density and model of the H2B αC helix (residues 103–125) modified with O-GlcNAc. e–h, RNF20 RING domain (g) and RNF40 RING domain (h) densities p with cartoon model fitted. i, Conformational flexibility analysis of the complex. Cryo-EM maps and models of three distinct states are shown. j, Structural alignment of the three conformational states, highlighting the rotation of the RNF20/RNF40 RING dimer and the movement of ubiquitin.
Extended Data Fig. 6 Biochemical investigations of H2BS112GlcNAc-promoted H2BK120ub generation.
a, SYBR-Gold-stained EMSA gels analyzing the binding affinity of human RNF20/RNF40 to NCPunmod. (top) or NCPGlcNAc (bottom). A red line with circular endpoints indicates the RNF20/RNF40 concentration (1,280 nM) that shifts approximately half of the free nucleosomes (NCPunmod. or NCPGlcNAc) to form complexes. b, Cryo-EM density and models of H2BS112GlcNAc–RAD6A Q93 interaction. c, SDS-PAGE characterization of wild-type (WT) RAD6A and its Q93A/I, K66A/I mutants. d, Representative fluorescent and CBB-stained gel images of in vitro H2B ubiquitylation assay analyzing activities of WT RAD6A and its mutants on NCPunmod. or NCPGlcNAc substrates. e, Quantified data of RAD6A mutant assay shown in d. Data show each replicate and error bars indicate mean ± SD from n = 3 independent replicates. f, Bar graph depicting fold changes in ubiquitylation activity, calculated as the ratio of NCPGlcNAc to NCPunmod. levels (NCPGlcNAc/NCPunmod.), derived from panel e. A two-sided, unpaired Student’s t-test was employed to calculate p-values; ns means not significant (p > 0.05). g, Representative fluorescent and CBB-stained gel images of single-turnover assay analyzing H2B ubiquitylation rates on NCPGlcNAc or NCPunmod. at a defined range of RAD6A concentrations. h,i Representative fluorescent and CBB-stained gel images of single-turnover assay analyzing H2B ubiquitylation rates on NCPGlcNAc or NCPunmod. at a pH range of 6.0–9.0 (h) or 7.0–7.5 (i). j, Representative fluorescent and CBB-stained gel images of single-turnover assay analyzing ubiquitin transfer from RAD6A~Ub thioester to nucleosomal H2B K120. k, Quantified data of single-turnover assay shown in j. Data shows the results from n = 2 independent replicates. The histone H4 bands in the CBB-stained gel image were used as the loading control in d and g–k. Assays were performed as n = 3 independent replicates in g–i.
Extended Data Fig. 7 Chemically synthesized monoglycosylated H2B variants were incorporated into nucleosomes.
a, Amino acid sequence of histone H2B. The ligation sites A58 and A97, and the glycosylation site S112 are indicated by red and light sea green solid circles, respectively. b, Convergent synthetic route of monoglycosylated H2B variants (G-H2B). The chemical structures of O-D-pyranoses studied here are shown at the bottom. c, Size-exclusion chromatograms of H2A/G-H2B histone dimers. d, Representative CBB-stained gel image of purified H2A/G-H2B histone dimers. e, Representative CBB-stained SDS-PAGE (top) and SYBR-Gold-stained native PAGE (bottom) gel images of purified H2B-monoglycosylated NCPs for biochemical assays. Two independent PAGE analyses in d and e were performed with similar results.
Extended Data Fig. 8 Mass spectrometries of monoglycosylated H2B synthetic intermediates.
a–e, Representative ESI-MS spectra and deconvoluted MS spectra of synthetic intermediates of H2BS112-β-GlcN (a), H2BS112-β-Glc (b), H2BS112-β-GalNAc (c), H2BS112-α-GlcNAc (d), and H2BS112-β-Xyl (e).
Extended Data Fig. 9 Structure-activity relationship analysis of the O-linked GlcNAcylation on H2B using synthetic H2B-monoglycosylated NCP substrates.
a, Representative fluorescent and CBB-stained gel images of in vitro H2B ubiquitylation assay analyzing activities of RNF20/RNF40–RAD6A on different H2B-monoglycosylated NCP substrates. b, Representative fluorescent and CBB-stained gel images of single-turnover assay analyzing H2B ubiquitylation rates on different H2B-monoglycosylated NCP substrates at a defined range of RAD6A concentrations. c, Representative fluorescent and CBB-stained gel images of single-turnover assay analyzing H2B ubiquitylation rates on different H2B-monoglycosylated NCP substrates at a pH range of 6.0–8.5. Assays were performed as n = 3 independent replicates in a–c.
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Deng, Z., Tao, S., Du, Y. et al. Allosteric activation of RNF20/RNF40–RAD6A-mediated H2BK120 monoubiquitylation by H2BS112 GlcNAcylation. Nat Chem Biol (2026). https://doi.org/10.1038/s41589-025-02109-6
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DOI: https://doi.org/10.1038/s41589-025-02109-6
