Abstract
Repression of gene expression by protein complexes of the Polycomb group is a fundamental mechanism that governs embryonic development and cell-type specification1,2,3. The Polycomb repressive deubiquitinase (PR-DUB) complex removes the ubiquitin moiety from monoubiquitinated histone H2A K119 (H2AK119ub1) on the nucleosome4, counteracting the ubiquitin E3 ligase activity of Polycomb repressive complex 1 (PRC1)5 to facilitate the correct silencing of genes by Polycomb proteins and safeguard active genes from inadvertent silencing by PRC1 (refs. 6,7,8,9). The intricate biological function of PR-DUB requires accurate targeting of H2AK119ub1, but PR-DUB can deubiquitinate monoubiquitinated free histones and peptide substrates indiscriminately; the basis for its exquisite nucleosome-dependent substrate specificity therefore remains unclear. Here we report the cryo-electron microscopy structure of human PR-DUB, composed of BAP1 and ASXL1, in complex with the chromatosome. We find that ASXL1 directs the binding of the positively charged C-terminal extension of BAP1 to nucleosomal DNA and histones H3–H4 near the dyad, an addition to its role in forming the ubiquitin-binding cleft. Furthermore, a conserved loop segment of the catalytic domain of BAP1 is situated near the H2A–H2B acidic patch. This distinct nucleosome-binding mode displaces the C-terminal tail of H2A from the nucleosome surface, and endows PR-DUB with the specificity for H2AK119ub1.
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Data availability
The cryo-EM density maps have been deposited in the Electron Microscopy Data Bank (EMDB) with accession numbers EMD-34431 and EMD-34432 for the chromatosomal and nucleosomal complexes, respectively. Constituent and consensus maps for the chromatosomal complex have been deposited with accession numbers EMD-35179, EMD-35180, EMD-35181 and EMD-35182. The atomic coordinates for the structure of PR-DUB bound to the chromatosome have been deposited in the PDB with the accession code 8H1T. The coordinates for the structures of Drosophila Caly in complex with DEUBAD of ASX (PDB ID: 6HGC), H2AK15-ubiquitnated NCP in complex with the BRCA1–BARD complex (PDB ID: 7E8I), the H2BK120-ubiqutinated NCP in complex with the SAGA DUB module (PDB ID: 4ZUX), the UCH-L5 complex with DEUBAD of RPN13 and ubiquitin (PDB ID: 4UEL) and the chromatosome with linker histone H1.4 (PDB ID: 7K5Y) were downloaded from the RCSB Protein Data Bank (https://www.rcsb.org). The RNA-seq data have been deposited in the Genome Sequence Archive at the National Genomics Data Center, China National Center for Bioinformation–Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA006013) and are publicly accessible at https://ngdc.cncb.ac.cn/gsa. Two public RNA-seq datasets with the accession numbers GSE162739 and GSE161995 were used in this study. The list of differentially expressed genes (DEGs) defined in a previous study7 was downloaded from ref. 7 (https://www.sciencedirect.com/science/article/pii/S1097276521005001?via%3Dihub#app2), with the original file name of 1-s2.0-S1097276521005001-mmc3.xlsx. The list of DEGs defined in a previous study8. was downloaded from the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE161995), with the original file name of GSE161995_BAP1ff_cnRNA_seq_DESeq2_Table_GEO.txt. A threshold of Padj < 0.05 and fold change > 1.5 was used to extract the DEGs. Source data are provided with this paper.
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Acknowledgements
We thank B. Zhu, X. Huang, L. Chen, X. Li and other staff members at the Center for Biological Imaging of the Institute of Biophysics (IBP), Chinese Academy of Sciences (CAS), for support in cryo-EM data collection; X. Yu at the Protein Science Core Facility of IBP for help with MALS analyses; Y. Gu for participation at an early stage of the study; and J. Chen for logistic support. The study was supported in part by grants from the Chinese Ministry of Science and Technology (2019YFA0508900), the Natural Science Foundation of China (31991162, 92153302 and 91853204), the K. C. Wong Education Foundation (GJTD-2020-06), the Youth Innovation Promotion Association (2018125) and the Strategic Priority Research Program (XDB37010100) of CAS.
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R.-M.X. & B.Z. designed and supervised the project. G.L. contributed experimental designs and advised on enzymatic activity assays. X.Z. advised on cryo-EM analysis. C.T. advised on the synthesis of ubiquitinated histones. W.G. performed protein expression and purification and cryo-EM sample preparation, analysed the structure and performed biochemical experiments. C.Y. prepared cryo-EM samples, collected data and solved and analysed the structures. J.L. analysed the cellular effects of BAP1 mutations. Z.Y. supervised the cryo-EM sample preparation and fluorescent polarization experiments. X.L. advised on enzymatic activity assays. Y.Z. performed bioinformatic analysis. C.-P.L. helped with structural model building. Y.L. contributed to the analysis of cellular effects of BAP1 mutations. W.G., C.Y. and R.-M.X. wrote the manuscript, and all authors read and edited the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Cryo-EM sample preparation.
a, Elution profile of size-exclusion column chromatography (SEC) of the BAP1–ASXL1(1–378) complex. Peak 1 was eluted close to the void volume of the column. b, Coomassie-stained SDS–PAGE analysis of fractions from SEC shown in (a). M, molecular weight marker; values are labelled on the left. Peak 2 fractions were collected for assembling complex with NCP. c, Western blot analyses of DUB activities of the wild-type and the C91S mutant BAP1 complexes with ASXL1(1–378). Same amount of the 187-bp DNA H2AK119ub1 chromatosome substrate was used in each reaction. From left to right, lane 1, no enzyme added; lanes 2–7 and lanes 8–13, successive twofold dilutions of the wild-type and C91S BAP1 enzyme complexes, respectively, detected with anti-H2A (top panel) and anti-His tag (bottom panel) antibodies. d, SEC-MALS analysis of BAP1 C91S–ASXL1(1–378) complex at the concentration of 2.2 mg/ml. Calculated molar mass = 164∓8.2 kDa, which is far below the 244.2 kDa calculated molecular mass of a dimer of the BAP1–ASXL1(1–378) complex. The data were analysed by the ASTRA software. e, GoldView-stained native PAGE shows the reconstituted NCP, NCP in a 600 mM NaCl buffer, at which point H1 was added and dialysed to zero salt to assemble the chromatosome. f, GoldView-stained native PAGE of fractions after GraFix. The chromatosome alone was used as a control. Fractions 12–14, highlighted in the red box, were used for cryo-EM sample preparation.
Extended Data Fig. 2 Structure determination of the BAP1–ASXL1(1–378)–H1–NCP(H2AK119ub1) complex.
a, A representative electron micrograph low-pass-filtered to 15 Å from 21,466 micrographs. b, Selected reference-free 2D class average. c, Workflow of cryo-EM data processing. d, The 3.0-Å global refinement map of the full complex (left), and the 3.9-Å local refinement BAP1–ASXL1(1–378)–ubiquitin subcomplex (right). For convenience of presentation, a composite map was constructed by combining indicated global and focused refinement maps. e, Angular distribution of the full complex particles in the final reconstructions. f, Gold-standard Fourier correlation of the BAP1–ASXL1(1–378)–H1–NCP(H2AK119ub1) complex.
Extended Data Fig. 3 EM density maps of various components or regions of the PR-DUB–substrate complex.
a, 601 nucleosomal DNA. b, The SHL 0 region of nucleosomal DNA. c, Human histone octamer. Two H3s are coloured in blue and light blue, H4s in pale cyan and pale green, H2As in yellow-orange and bright orange, and H2Bs in pink and light pink. d, The globular domain of linker histone H1.4. e, Human BAP1. f, The DEUBAD domain of ASXL1. g, The ubiquitin moiety of H2AK119ub1. h, Selected regions of core histones. i, BAP1 regions. j, The DEUBAD domain of ASXL1. k, The C-terminal region of the ubiquitin moiety of H2AK119ub1. Atomic models are shown in a stick representation, and the maps are contoured at 5σ~7σ.
Extended Data Fig. 4 Cryo-EM analysis of the BAP1–ASXL1(1–378)–NCP(H2AK119ub1) complex without H1.
a, A representative electron micrograph low-pass-filtered to 15 Å from 6,269 micrographs. b, Selected reference-free 2D class average. c, 2D class averages of two BAP1–ASXL1 complexes bound to NCP in higher contrast. The red arrows indicate the density for the bound BAP1–ASXL1 complexes. d, Workflow of cryo-EM data processing. e, Alignment of the EM densities for the BAP1–ASXL1(1–378)–NCP(H2AK119ub1) complexes with (blue) and without (grey) H1. f, 3D classification map of two BAP1–ASXL1(1–378) complexes bound to the nucleosome.
Extended Data Fig. 5 BAP1 residues in distinct regions involved in H2AK119 deubiquitination and nucleosome binding.
a, Sequence alignment of the CTE region of BAP1 orthologues. The RRRQ (aa 699–702) CTE finger motif is boxed in dashed lines. b, Alignment of the RRSRR motif (aa 56–60) in the β2–α2 loop of BAP1. c, Alignment of the sequences encompassing the α8–β6 loop of BAP1, Caly and UCH-L5. Above the sequence, BAP1 secondary structure elements are depicted, and period signs denote whole 10th residues of BAP1. d–f, Quantification of catalytic activities of western blot measurements in Figs. 2d, g and h, respectively. The values for indicated wild-type and BAP1 mutant complexes measured at 5 and 40 min in Fig. 2d,h are shown with clear and grey-shaded bars, respectively. The wild-type values (at 40 min) were set to unity, and all values were derived from three replicates, and represented as mean ± s.e.m. P values, displayed at the top of the bar charts, denote the results of an unpaired two-tailed t-test. g, Fluorescence polarization measurements of binding affinities of the BAP1–ASXL1 complexes carrying indicated BAP1 (56–60 mut) or ASXL1 (T262W, 1–330) mutations to the NCP(H2AK119ub1) assembled with 167-bp 601 DNA containing a 5’-FAM fluorophore. The data were analysed in the same way as in Fig. 2e (n = 3 biologically independent experiments; mean ± s.e.m.). h, Western blot analysis of free histone H2AK119 deubiquitination by wild-type and indicated BAP1 mutants in complex with ASXL1(1–378). A ‘−’ sign indicates a mock reaction without enzyme added. Reactions were monitored at the indicated time points, and detected with anti-His tag (top) or anti-H2A (bottom) antibodies.
Extended Data Fig. 6 Gene set enrichment analysis.
a,b, Upregulated (a) and downregulated (b) genes in Bap1 KO ES cells versus wild-type ES cells from a previous study7 with respect to the global transcriptional changes observed in our Bap1 KO + EV ES cells versus wild-type ES cells. c,d, Upregulated (c) and downregulated (d) genes analysed in a previous study 8 relative to the global transcriptional changes we observed.
Extended Data Fig. 7 Gene expression analysis of Bap1 mutants.
a, Percentage of rescued BAP1-regulated genes (differentially expressed genes between Bap1 KO + EV ES cells and wild-type ES cells) in the indicated wild-type and mutant BAP1 rescue cells. b, Expression correlation among the 14 RNA-seq samples of mouse ES cells on BAP1-regulated genes (differentially expressed genes between Bap1 KO + EV and wild-type R1). There are two replicates for each cell line. Pearson correlation coefficients are indicated.
Extended Data Fig. 8 Cellular DUB activities and changes in gene expression for BAP1 CTE cancer-associated mutations.
a, Western blot detection of the levels of ubH2A and BAP1 in Bap1 KO cells rescued with the indicated BAP1 mutant plasmids. Quantification of the western blot results is represented in a bar chart of mean ± s.e.m. (n = 4 biologically independent experiments). P values, displayed at the top of the bar chart, denote the results of an unpaired two-tailed t-test. b, Heat maps of transcriptional levels of BAP1-regulated genes in wild-type and indicated rescue cell lines. c, Percentage of rescued BAP1-regulated genes (differentially expressed genes between Bap1 KO and wild-type ES cells) in the indicated mutant BAP1 rescue cells. d, Expression correlation among the 10 RNA-seq samples.
Extended Data Fig. 9 Quantification of cellular DUB activities and RNA-seq analysis for non-CTE BAP1 cancer-associated mutations.
a, Quantification of western blot detection of H2AK119ub and BAP1, as shown in Fig. 3h, in Bap1 KO cells transfected with an empty vector (EV), or plasmids re-expressing wild-type or the indicated BAP1 mutants. Bar charts represent plots of mean ± s.e.m. (n = 4 biologically independent experiments). P values, displayed at the top of the bar chart, denote the results of an unpaired two-tailed t-test. b, Heat maps of transcriptional levels of BAP1-regulated genes in wild-type and indicated rescue cell lines. There are two replicates for each cell line. c, Percentage of rescued BAP1-regulated genes (differentially expressed genes between Bap1 KO and wild-type ES cells) in each indicated mutant BAP1 rescue cells. d, Expression correlation among the 10 RNA-seq samples.
Extended Data Fig. 10 Structure and function of ASXL1 domains and the effect of H1 on H2AK119 deubiquitination.
a, Domain structures of ASXL1–ASXL3 and alignment of their N-terminal sequences encompassing HARE-HTH and DEUBAD domains. b, Left, schematic representation of ASXL1 constructs used in the deubiquitinase assay. Wavy lines represent a 2×(GGGGS) linker. Middle, western blot analysis of nucleosomal H2AK119 deubiquitination of BAP1 complexes with the indicated ASXL1 fragments. H3 lanes are sample-processing controls. Right, quantification of catalytic activities based on western blot measurements (n = 3 biologically independent experiments, mean ± s.e.m.). P values, displayed at the top of the bar chart, denote the results of an unpaired two-tailed t-test. No adjustment was made for multiple comparisons. c, A BAP1–ASXL1(1–378) complex modelled to the proximal DNA side (left) of the chromatosome based on the observed binding mode in the distal DNA side (right) sterically clashes with H1 (see inset).
Supplementary information
Supplementary Information
This file contains the sequences for mutagenesis primers and nucleosomal DNA (Supplementary Table 1) and the uncropped blots and FACS gating strategy (Supplementary Figs. 1 and 2).
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Ge, W., Yu, C., Li, J. et al. Basis of the H2AK119 specificity of the Polycomb repressive deubiquitinase. Nature 616, 176–182 (2023). https://doi.org/10.1038/s41586-023-05841-y
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DOI: https://doi.org/10.1038/s41586-023-05841-y
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