PR-DUB maintains the expression of critical genes through FOXK1/2- and ASXL1/2/3-dependent recruitment to chromatin and H2AK119ub1 deubiquitination

BAP1 and ASXL1/2/3 are required for normal cell proliferation and for the expression of a common set of genes. (A) Illustration of the three different PR-DUB complexes and their components. (B) Western blot showing BAP1 levels in wild-type mESCs, Bap1^−/− mESCs, Bap1^−/− + Bap1^WT, and Bap1^−/− + Bap1^MT mESCs. Vinculin (VCL) was used as a loading control. (C) Cell proliferation and growth curves of wild-type mESCs, Bap1^−/− mESCs, Bap1^−/− + Bap1^WT, and Bap1^−/− + Bap1^MT mESCs were performed in three independent biological replicates per condition. (D) Western blots showing BAP1 and ASXL1 levels in wild-type mESCs, Asxl1/2/3^−/− mESCs, Asxl1/2/3^−/− + Asxl1^3xFLAG, and Asxl1/2/3^−/− + hASXL1 mESCs. Vinculin was used as a loading control. (E) Cell proliferation of wild-type mESCs, Asxl1/2/3^−/− mESCs, Asxl1/2/3^−/− + Asxl1^3xFLAG, and Asxl1/2/3^−/− + hASXL1 mESCs. The results present three independent biological replicates per condition. (F) Gene expression analysis of the indicated cell lines. Log₂-normalized mean counts of mapped reads in Bap1^−/− (top) and Asxl1/2/3^−/− (bottom) mESCs versus wild-type mESCs. Only genes up- (blue) and down-regulated (orange) are shown. Down-regulated genes in Bap1^−/− and Asxl1/2/3^−/− mESCs were defined with the following criteria: log₂ fold change ≤ −1, P-value ≤ 0.05, log₂CPM in mESCs ≥ 0.5. Up-regulated genes in Bap1^−/− and Asxl1/2/3^−/− mESCs were defined with the following criteria: log₂ fold change ≥ 1, P value ≤ 0.05, log₂ CPM in KO ≥ 0.5. (G) Euler diagrams demonstrating down-regulated (top) and up-regulated (bottom) genes in common between Bap1^−/− and Asxl1/2/3^−/− mESCs versus wild-type mESCs. Fisher's exact test, (****) P-value < 0.0001.