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. 2021 Aug 25;10:e65872. doi: 10.7554/eLife.65872

Figure 7. Nucleoporin-98 (NUP98)-HBO1 fusion transforms myeloid progenitors through interaction with MLL.

(A) Structural requirement of NUP98-HBO1 fusion. Various NUP98 fusion constructs were examined for the transformation of myeloid progenitors (along with an HBO1 construct) as in Figure 1A. Relative Hoxa9 expression in first round colonies was analyzed. Asterisk indicates that the qPCR probe detected a human HOXA9 coding sequence included in the NUP98-HOXA9 construct in addition to endogenous murine Hoxa9. Zn: zinc finger; MYST: MYST HAT domain; HD: homeodomain; FG: phenylalanine-glycine repeat. (B) Association with MLL by NUP98-HBO1. The chromatin fraction of virus-packaging cells transiently expressing the indicated transgenes was subjected to immunoprecipitation (IP)-western blotting. Endogenous MLLC fragment was detected by a specific anti-MLL antibody. (C) Structural requirement of various NUP98 fusions for leukemic transformation. Indicated NUP98 fusion constructs were examined for the transformation of myeloid progenitors as in Figure 1A. ING: inhibitor of growth domain of ING4 and ING5; NuA4: histone acetyltransferase subunit NuA4; EPcA-I/II: enhancer of polycomb A domains I and II. (D) Association with MLL by various NUP98 fusions. IP-western blotting was performed as in (B). (E) Requirement of MLL for NUP98 fusion-immortalized cells. sgRNA competition assays for Kmt2a were performed on NUP98-HOXA9- and NUP98-HBO1-immortalized cells as in Figure 2F. (F) Effects of pharmacologic inhibition of MENIN-MLL interaction on NUP98 fusion-immortalized cells. NUP98-HBO1- and NUP98-HOXA9-immortalized cells were cultured in the presence of 10 μM of MI-2–2 MENIN-MLL interaction inhibitor, and their proliferation was monitored every 3 days. MLL-AF10-immortalized cells were also analyzed as a positive control. VC: vehicle control.

Figure 7.

Figure 7—figure supplement 1. Functional differences between nucleoporin-98 (NUP98)-HOXA9 and NUP98-HBO1.

Figure 7—figure supplement 1.

(A) Targeting ability to the HOXA9-binding site. Virus-packaging cells transiently expressing indicated transgenes were subjected to chromatin immunoprecipitation (ChIP)-qPCR analysis. The percent input values of each probe (mean ± SD of PCR triplicates) are shown. A HOXA9-binding site was previously identified at the HOXA7 locus (Shima et al., 2017). The CD4 locus was analyzed as a negative control. (B) Effects of pharmacologic inhibition of MYST HATs on NUP98 fusion-immortalized cells. NUP98-HBO1- and NUP98-HOXA9-ICs were cultured in the presence of 10 μM of WM1119 MYST HAT inhibitor and their proliferation was monitored every 3 days. MLL-AF10-immortalized cells were also analyzed as the positive control. VC: vehicle control.