Figure 1
From: Tumor-derived neomorphic mutations in ASXL1 impairs the BAP1-ASXL1-FOXK1/K2 transcription network
Tumor-derived C-terminally truncated ASXL1 mutants compete with wild-type ASXL1 for binding with BAP1. (A) AML-derived mutations in ASXL1 gene include frameshift mutations (in red), nonsense (in blue), and missense mutations (in green), modified from a previous study by Schnittger et al. NLS, nuclear localization signal; HARE-HTH, HB1, ASXL, restriction endonuclease Helix-Turn-Helix domain; DEUBAD, DEUBiquitinase adaptor domain; PHD, plant homedomain finger. (B) ASXL1 mutation status in K562 and Kasumi-1 cells, and schematic representation of wild-type ASXL1, Y591X and G646Wfs*12 mutations, and the antibody specific to the N-terminal sequence of ASXL1 protein. See also Fig. S1A and S1B. (C) Western blot analysis of the endogenous proteins of full-length and C-terminally truncated mutant ASXL1 in K562 and Kasumi-1 cells. (D) Increased amount of ASXL1N646-Myc were overexpressed in HEK293T cells together with BAP1-Flag, and the interaction between BAP1-Flag and endogenous ASXL1 was determined by Western blot analysis. Relative Flag-BAP1-binding ASXL1 protein levels were quantified. (E) Cells with deletion of mutant ASXL1 allele (referred to as ASXL1+/−) were generated using the CRISPR/Cas9 system in leukemia cells of K562 (left) and Kausmi-1 (right). Co-IP was performed to detect the interaction between BAP1 and ASXL1. (F) Quantification of the protein levels of BAP1-interacting ASXL1 in (E) is shown. (G) Global H2AK119 mono-ubiquitination and H3K27 trimethylation levels in ASXL1+/N590 and ASXL1+/− cells in K562 (left) and ASXL1+/N646 and ASXL1+/− cells in Kausmi-1 (right). Relative H2AK119 mono-ubiquitination was normalized by Histone H2A. Asterisks denote statistical significance with two-tailed Student’s t-test. *P < 0.05 and **P < 0.01 for the indicated comparison