Fig. 6
PR-DUBs oligomerisation enables efficient nucleosome recruitment. a Electrophoretic mobility shift assays comparing the ability of wild-type Calypsono tail–ASX and Calypso–ASX complexes to bind nucleosomes at varying protein concentrations (0.5, 1, 2, 4, 8 μM). Recombinant nucleosomes reconstituted with a 220 bp Widom 601 DNA sequence labelled at the 5′ with an IRDye®700 were used to visualise nucleosomes. b Electrophoretic mobility shift assays testing the binding of wild type and L340A Calypso–ASX proteins to nucleosomes at increased protein concentrations (as for panel a). Bands corresponding to the shifted nucleosome particle (indicated as shifted NCP) were quantified. Quantitated triplicates corresponding to three independent experiments are shown on the right, with a line passing through the mean. c Electrophoretic mobility shift assays comparing the ability of wild type and L635A BAP1–ASXL1 complexes to bind nucleosomes at different protein concentrations (0.1, 0.2, 0.5, 1, and 2 μM). Recombinant nucleosomes were reconstituted as outlined in a. Experiments were quantified as described in b. d Schematic representation of the mechanisms that underpin PR-DUB recruitment and activity on nucleosomes. In the absence of the C-terminal positively charged tails (left), the oligomeric PR-DUB complex cannot be recruited to nucleosomes, resulting in a total loss of activity and maintenance of the H2AK119Ub mark. The 1:1 PR-DUB complex (middle), despite the presence of the C-terminal tail, has reduced affinity for nucleosomes and hence limited ability to deubiquitinate H2AK119Ub. Only a PR-DUB complex that bears two C-terminal tails and that has full ability to form a bidentate complex (right) can be efficiently recruited to nucleosomes, thereby resulting in increased activity. e Scale model for bidentate recruitment of the Calypso–ASX complex to H2AK119Ub nucleosomes. The Calypso dimer and ubiquitin are shown in surface representation, while the ASX Deubad domains are shown as cartoon; the surface of the nucleosome particle is represented as electrostatic potential. The C-terminal positively charged tails of the two Calypso molecules are shown in blue. Black arrows indicate the distances between the two Calypso active sites as well as between the two mono-ubiquitinated Histone 2A Lys119 residues of the nucleosome particle
