Deubiquitinating activity, but not HCF-1 binding, is required for BAP1 function in thymocytes. Deubiquitinating activity, but not HCF-1 binding, is required.

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Deubiquitinating activity, but not HCF-1 binding, is required for BAP1 function in thymocytes. Deubiquitinating activity, but not HCF-1 binding, is required for BAP1 function in thymocytes. (A) Coimmunoprecipitation (IP) of BAP1 with various members of the BAP1 core complex and with members of the PRCs in thymocytes of C57BL/6 mice. (B) Western blot showing protein levels of EZH2, HCF-1, and OGT in 4-OHT–treated Bap1fl/flRosa26CreERT2 and Bap1wt/wtRosa26CreERT2 DN3 cells isolated from OP9-DL1 cocultures at day 14. (C) Bap1fl/flRosa26CreERT2 and Bap1wt/wtRosa26CreERT2 bone marrow progenitor cells were transduced with empty vector, full-length BAP1 (WT), HCF-1 interaction mutant BAP1 (4A), or catalytically dead BAP1 (C91A). Transduced cells were then cultured with OP9-DL1 stromal cells, and BAP1 deletion was induced at day 2 as described in Fig. 3. Staining profiles of GFP+ CD45+ lineage− cells at day 18 are shown. Data are representative of at least three biological replicates in two independent experiments. (D) Proportion of DP cells within the GFP+ CD45+ lineage− gate at day 20 of coculture (n = 3). (E) Histogram profiles of global H2AK119ub and H3K27me3 levels in WT and KO DN3 cells (CD45+ lineage− CD3− TCRβ− CD4− CD8− CD44− CD25+) from OP9-DL1 cocultures at day 13. (F) Histone mass spectrometry of BAP1 WT and KO DN3 cells sorted from OP9-DL1 cultures at day 14. Heat maps depicting the relative abundance of each posttranslational modification normalized to WT abundance levels are shown. Teresita L. Arenzana et al. Sci. Immunol. 2018;3:eaal1953 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works