1. Volume 54, Issue 3, Pages 392-406 (May 2014)
Autodeubiquitination Protects the Tumor Suppressor BAP1 from Cytoplasmic Sequestration Mediated by the Atypical Ubiquitin Ligase UBE2O 
Nazar Mashtalir, Salima Daou, Haithem Barbour, Nadine N. Sen, Jessica Gagnon, Ian Hammond-Martel, Haider H. Dar, Marc Therrien, El Bachir Affar 
Molecular Cell 
Volume 54, Issue 3, Pages (May 2014)
DOI: /j.molcel
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2. Molecular Cell 2014 54, 392-406DOI: (10.1016/j.molcel.2014.03.002)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1
UBE2O Interacts with and Ubiquitinates BAP1, and This Effect Is Actively Counteracted by BAP1 Autodeubiquitination
(A) Purified BAP1 complexes were used for western blot.
(B) HeLa nuclear extract was resolved by glycerol gradient and used for western blot.
(C) 293T cells were cotransfected with 2 μg of HA-Ub, 2 μg of Myc-UBE2O (wild-type or CD), and 1 μg of Flag-BAP1 (wild-type or C91S) expression vectors, and cell extracts were used for immunoprecipitation.
(D) Top, schema representing the human UBE2O with secondary structure prediction (α helixes are in red, and β strands are in yellow). The main predicted domains are indicated as CR1 (conserved region 1), CR2 (conserved region 2), UBC (ubiquitin conjugating), and CTR (C-terminal region). Bottom, schema representing UBE2O deletion mutants used for the ubiquitination assay.
(E) 293T cells were cotransfected with 2 μg of HA-Ub, 2 μg of Myc-UBE2O fragments, and 1 μg of C91S expression vectors, and cell extracts were used for western blot.
See also Figure S1.
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4. Figure 2 Characterization of UBE2O-Mediated Ubiquitination of BAP1
(A) In vitro ubiquitination reaction with bead-immobilized BAP1 or C91S complexes incubated with immunopurified UBE2O. Fractions were used for western blot detection of components of the BAP1 complex.
(B) In vitro ubiquitination reaction using the BAP1 complex inhibited with NEM.
(C) The C91S complex was used for in vitro reaction with UBE2O and either unmodified or methylated ubiquitin.
(D) 293T cells were cotransfected with 2 μg of wild-type ubiquitin or K0 mutant ubiquitin, 1 μg of BAP1 or C91S, and 2 μg of empty vector or His-UBE2O expression vectors, and cell lysates were used for immunoprecipitation.
(E) 293T cells were cotransfected with 1 μg of HA-Ub, 2 μg of BAP1 or C91S, and 1 μg of His-UBE2O expression vectors. Cells were treated with DMSO or 20 μM MG132 for 8 hr before immunoprecipitation.
(F) MCF7 cells were transfected with control or UBE2O siRNA for 96 hr, treated with 20 μg/ml of cycloheximide, and analyzed by western blot. Asterisk indicates nonspecific bands.
See also Figure S2.
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5. Figure 3 BAP1 NLS Is Required for Its Ubiquitination by UBE2O
(A) Schematic representation of GST-BAP1 fragments used for pull-down assay with His-UBE2O.
(B) 293T cells were cotransfected with 2 μg of HA-Ub, 2 μg of His-UBE2O, and 1 μg of Myc-CTD-NLS expression vectors, and cell extracts were used for immunoprecipitation.
(C) U2OS cells were cotransfected with 2 μg of shControl, shUBE2O#1, or shUBE2O#2; 1 μg of HA-Ub, and 0.5 μg of Myc-CTD-NLS expression vectors, and cell extracts were used for western blot.
(D) Purified complexes of BAP1 or BAP1 ΔCTD were used for western blot detection of components of BAP1 complex.
(E) 293T cells were cotransfected with 2 μg of HA-Ub, 2 μg of His-UBE2O, and 1 μg of Myc-BAP1 mutants expression vectors, and cell extracts were used for immunoprecipitation. Asterisk indicates nonspecific bands.
See also Figure S3.
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6. Figure 4
Ubiquitination of BAP1 NLS by UBE2O Promotes Its Cytoplasmic Sequestration
(A) 293T cells were cotransfected with 2 μg of HA-Ub, 2 μg of Myc-UBE2O, and 1 μg of Flag-C91S per dish, and cell extracts from 40 dishes were used for Flag immunoprecipitation. The indicated bands were excised for MS analysis.
(B) Table of ubiquitination events detected by MS.
(C) Cells were cotransfected with 2 μg of HA-Ub, 2 μg of His-UBE2O, and 1 μg of Myc-tagged BAP1 mutants expression vectors, and cell extracts were used for immunoprecipitation.
(D) U2OS cells were cotransfected with either 2 μg of empty vector, Myc-UBE2O, or Myc-UBE2O CD and 3 μg of either Flag-HA-BAP1 or Flag-HA-C91S expression vectors and were used for immunofluorescence analysis. Representative cell counts for BAP1 subcellular localization are shown. C, cytoplasmic; N, nuclear.
(E) U2OS cells were transfected with Flag-HA-BAP1 mutants expression vectors and used for immunofluorescence analysis as in (D).
See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
BAP1 Intramolecular Interaction Promotes Autodeubiquitination, a Mechanism Disrupted by Cancer Mutations
(A) Predicted model of BAP1 UCH/CTD interface based on UCH37 crystal structure (PDB ID code 3IHR). Note that NLS and NORS are not present in UCH37 and thus are not included in the model.
(B) Schematic representation of the mutants used for the complementation assay and the deletion mutants used in the context of full-length BAP1.
(C) 293T cells were cotransfected with 2 μg of the indicated GFP fusion constructs and 2 μg of Myc-CTD-NLS expression vectors, and cell extracts were used for immunoprecipitation.
(D) 293T cells were cotransfected with 2 μg of HA-Ub and either 2 μg of empty vector or His-UBE2O, 1 μg of the indicated GFP fusion constructs, and 1 μg of Myc-CTD-NLS expression vectors.
(E) 293T cells were cotransfected with 2 μg of HA-Ub, 2 μg of His-UBE2O, and 1 μg of Myc-BAP1 mutant expression vectors.
(F) Purified BAP1 complexes were analyzed by silver stain and western blotting. Densitometric ratios between UBE2O and BAP1 indicate its relative abundance in the complexes. The histogram shows two independent experiments.
(G) In vitro nucleosome deubiquitination reaction with purified BAP1 complexes. Samples were incubated at 37°C for the indicated times.
(H) 293T cells were cotransfected with 2 μg of HA-Ub, 2 μg of His-UBE2O expression vectors, 1 μg of indicated GFP fusion constructs, and 1 μg of Myc-tagged constructs.
(I) 293T cells were cotransfected with 2 μg of HA-Ub, 2 μg of His-UBE2O, and 1 μg of Myc-tagged BAP1 mutant expression vectors.
(J) U2OS cell lines stably expressing Flag-HA-BAP1 and its mutant forms were used for immunofluorescence analysis.
See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
UBE2O Shuttles between the Nucleus and Cytoplasm and Promotes BAP1 Cytoplasmic Localization during Adipocyte Differentiation
(A) Effect of kinase inhibition on nuclear localization of UBE2O. U2OS cells were transfected with 4 μg Myc-UBE2O or Myc-UBE2O CD expression vectors and then treated with a panel of kinase inhibitors for 24 hr prior harvesting for IF. + and − indicate the relative intensity in the nuclear staining of UBE2O.
(B) Representative images of UBE2O localization in U2OS cells treated with CDK inhibitors.
(C) Effects of siRNA depletion of UBE2O, BAP1 on cell proliferation, determined by MTT assay. siRNA for OGT is used as a control of decreased proliferation. Note that the values are relative to the nontarget control siRNA for each time point. Data are represented as mean ± SD for a representative experiment.
(D and E) Effects of overexpression of UBE2O wild-type or catalytic dead form on the differentiation of 3T3-L1. Immunodetection of differentiation markers (D) and oil red O staining (E). YY1 is used as a loading control.
(F) Localization of BAP1 and mutants during adipocyte differentiation. The BAP1 NLST1 corresponds to a mutant in which the NLS region of BAP1 which includes the UBE2O-binding motif is replaced with the T large antigen NLS. The BAP1 K/R mutant is mutated in the UBE2O-ubiquitination sites of the NLS.
See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7 UBE2O Targets a Subset of Chromatin-Associated Proteins
(A) In vitro ubiquitination reaction with the purified UBE2O. Samples were incubated for the indicated times.
(B) 293T cells were cotransfected with 2 μg of HA-Ub and 2 μg of indicated Myc-UBE2O mutants, and cell extracts were used for immunoprecipitation.
(C) Sequence alignment between the UBE2O/BAP1 NLS and a subset of identified UBE2O substrates. Hydrophobic amino acids are in red, and polar amino acids are in blue. A large N-terminal extension containing the NLS of CDT1 is not shown.
(D) 293T cells were cotransfected with 2 μg of HA-Ub and 1 μg of Flag-CXXC1 or 2 μg of Flag-INO80, and either 2 μg of empty vector, Myc-UBE2O, or Myc-UBE2O CD expression vectors, and cell extracts were used for immunoprecipitation.
(E) U2OS cells were cotransfected with 0.5 μg of HA-Ub and either 1 μg of empty vector, Myc-UBE2O, Myc-UBE2O CD and 1 μg of either Flag-INO80 or Flag-CXXC1 expression vectors and used for immunofluorescence analysis. Representative cell counts are shown.
See also Figure S7.
Molecular Cell  , DOI: ( /j.molcel )
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