FIGURE 7.
Intramolecular interaction in BAP1 is required to create an ASXM-inducible CUBI. A, schematic representation of the different BAP1 mutants generated for in vitro experiments done in B. B, in vitro DUB reaction of nucleosomal H2A using His-BAP1 or its mutant forms (8 ng, 2 pm) in the presence or absence of MBP-ASXM2 (10 ng, 2,8 pm). C and D, in vitro deubiquitination assay of nucleosomal histone H2A using purified FLAG-HA BAP1, BAP1ΔCTD1, or BAP1ΔCC2 complexes. BAP1ΔHBM was used as a control because HCF-1 is not required for BAP1 DUB activity. E, in vivo DUB activity of BAP1ΔCTD is abolished due to the lack of interaction with ASXL1/2. FLAG-H2A (0.2 μg) expression construct was co-expressed in 293T cells with either Myc-BAP1 (1 μg) or Myc-BAP1 ΔCTD (1 μg) with or without Myc-ASXL1 (4 μg) or Myc-ASXL2 (6 μg) expression constructs. Three days post-transfection, cells were harvested for immunoblotting. YY1 is used as a loading control. F, His-BAP1 mutants (1.6 μg, 20 nm) and MBP-ASXM2 (2 μg, 30 nm) were subjected to GST-ubiquitin pulldown assay followed by immunoblotting.