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. 2015 Jun 22;6:7307. doi: 10.1038/ncomms8307

Figure 1. Leukemia-associated ASXL1 truncation mutations cooperate with BAP1 to promote deubiquitination of H2AK119Ub.

Figure 1

(a) ASXL1 interacts with BAP1 to form a deubiquitinase complex that acts on H2AK119Ub. ASXL1 is the regulatory subunit of this complex and BAP1 is the deubiquitinase. The ubiquitin-carboxyl hydrolase (UCH) domain of BAP1 is at its N terminus. ASXL1 has a C-terminal atypical PHD Zinc-finger, a putative N-terminal DNA-binding domain and three PRRs that are thought to facilitate protein–protein interactions. Shown below are three cancer-associated ASXL1 mutations and two ASXL1 truncations, ASXL1(1–1305), and ASXL1(1–479), which we have employed in our studies. (b) 293T cells were mock transfected or transfected with mammalian expression vectors encoding BAP1, full-length ASXL1, or both. Expression of ASXL1 and BAP1 was confirmed by western blotting carried out on nuclear lysates. Acid-extracted histones were probed with antibodies against the indicated proteins and histone modifications. As shown, co-transfection of ASXL1+BAP1 results in marked reduction in levels of H2AK119Ub but not H2BK120Ub. (c,d) HEK293T cells were co-transfected with mammalian expression vectors encoding BAP1 with full-length ASXL1 or ASXL1 truncations/mutations as indicated. Western blottings to examine expression of ASXL1 and ASXL1 mutations are shown in Supplementary Fig. 1c. (c) Cells were fixed 48 h post transfection, permeabilized and stained with anti-ASXL1 (red) or anti-FLAG (red) and anti-H2AK119Ub (green) antibodies. Scale bar, 10 μm. (d) Nuclear lysates and acid-extracted histones prepared from transfected cells were subjected to western blotting.