Structural basis of histone H2A lysine 119 deubiquitination by Polycomb Repressive Deubiquitinase BAP1/ASXL1

Abstract

The maintenance of gene expression patterns during metazoan development is achieved by the actions of Polycomb group (PcG) complexes. An essential modification marking silenced genes is monoubiquitination of histone H2A lysine 119 (H2AK119Ub) deposited by the E3 ubiquitin ligase activity of the non-canonical Polycomb Repressive Complex 1. The Polycomb Repressive Deubiquitinase (PR-DUB) complex cleaves monoubiquitin from histone H2A lysine 119 (H2AK119Ub) to restrict focal H2AK119Ub at Polycomb target sites and to protect active genes from aberrant silencing. BAP1 and ASXL1, subunits that form active PR-DUB, are among the most frequently mutated epigenetic factors in human cancers, underscoring their biological importance. How PR-DUB achieves specificity for H2AK119Ub to regulate Polycomb silencing is unknown, and the mechanisms of most of the mutations in BAP1 and ASXL1 found in cancer have not been established. Here we determine a cryo-EM structure of human BAP1 bound to the ASXL1 DEUBAD domain in complex with a H2AK119Ub nucleosome. Our structural, biochemical, and cellular data reveal the molecular interactions of BAP1 and ASXL1 with histones and DNA that are critical for remodeling the nucleosome and thus establishing specificity for H2AK119Ub. These results further provide a molecular explanation for how >50 mutations in BAP1 and ASXL1 found in cancer can dysregulate H2AK119Ub deubiquitination, providing new insight into understanding cancer etiology.

One sentence summary: We reveal the molecular mechanism of nucleosomal H2AK119Ub deubiquitination by human BAP1/ASXL1.

Fig. 1.. Overview of the structure of BAP1/ASXL1 bound to H2AK119Ub nucleosome.

A) Bar diagram representation of BAP1 and ASXL1 domains. Protein sequences included in this study are shown in grey line; those resolved in the structure in black line; and disordered regions in dashed line. B) Two different views of the model for the BAP1/ASXL1-H2AK119Ub nucleosome complex with key anchor points highlighted. The figure is color coded, depicting BAP1 (purple), ASXL1 (dark blue), Ub (Orange), H2A (yellow), H2B (salmon), H3 (light blue), H4 (green), and DNA (grey).

Fig. 2.. Insights into the mechanism of catalysis by BAP1/ASXL1 and conservation with other deubiquitinases.

(A) Position of Ub engaged with BAP1 and ASXL1 on the nucleosome. (B) BAP1/ASXL1/Ub aligned and superimposed with UCH-L5/RPN13/Ub (PDB ID 4uel) (21)(grey). (C) Hydrophobic interactions between BAP1 and Ub. (D) Electrostatic interactions between BAP1/ASXL1 and Ub. (E) Space-filling representation of our cryo-EM structure with the last observed residue of H2A (H2AP109; shown as blue circled red sphere). (F) Model with space-filling representation of our Cryo-EM structure superposed with wild type (unmodified) nucleosome structure (PDB ID 1kx5) (29) that contains H2A docking domain structured with H2AK119 shown as blue circled red sphere).

Fig. 3.. BAP1/ASXL1 interacts with DNA and acidic patch on the nucleosome.

(A) Overall architecture of the BAP1/ASXL1-H2AK119Ub nucleosome complex with BAP1/ASXL1 anchor points marked (left). Catalytic activity assays on H2AK119Ub nucleosomes (with a DUB-cleavable native gamma-lysine isopeptide linkage) and wild type BAP1/ASXL1, the catalytic inactive mutant of BAP1 (C91S) /ASXL1, and mutations to the nucleosomal anchors (right). (B) Close up view of BAP1/ASXL1 DNA clamp contacting the nucleosome near the DNA dyad. (C) Close up view of the ASXL1 DEUBAD α6 helix, projecting a stretch of basic residues towards a nucleosome DNA exit. (D) Close up view of the BAP1 R-finger interacting with the acidic patch. Quantified results from electromobility shift assay (EMSA) of wild type and mutants of BAP1 DNA clamp (E), ASXL1 DNA exit (F), and BAP1 interaction with the acidic patch (G). Each data point and error bar indicate the mean ± SD from three independent experiments. The standard errors of dissociation constants (Kd) are indicated. Michaelis-Menten curves of BAP1/ASXL1 DNA clamp mutations (H) and the acidic patch mutations (I) on H2AK119Ub nucleosomes. Please see Supplementary Text for extended information on figures 3A, H and I.

Fig. 4.. BAP1/ASXL1-nucleosome contacts are extensively mutated in cancers.

(A) Western blot analysis using the indicated antibodies of total protein extracts from BAP1 WT (E14) or KO mESC with stable expression of WT and various BAP1 mutations (C91S, R699E/R700E; R56E/R59E; R56A/R57A/R59A/R60A). (B) Structure of the BAP1/ASXL1-H2AK119Ub nucleosome complex highlighting three key nucleosome anchors (left) and Ub interactions (right) with cancer mutations shown as colored spheres. Bar diagram showing cancer-associated point mutations and deletions to BAP1 (C) and ASXL1 (D) at nucleosomal anchors and the Ub patch shown in (B) identified in several cancers that can be mechanistically explained by studies presented here. The number of unique cancer types at each interface is shown inside the red spheres.