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

Figure 6. MLL-ELL transforms hematopoietic progenitors via association with AF4/ENL/P-TEFb (AEP), but not with EAF1 or p53.

(A) Mutations of ELL selectively abrogate interaction with EAF1. Immunoprecipitation (IP)-western blotting of the chromatin fraction of HEK293TL cells transiently expressing FLAG-tagged GAL4-ELL proteins with or without S600A/K606T substitutions (SA/KT) along with xAF4 and EAF1h was performed as in Figure 4D. (B) Recruitment of exogenously expressed AF4 or EAF1 by ELL mutant proteins. Chromatin immunoprecipitation (ChIP)-qPCR analysis of HEK293TL cells transiently expressing FLAG-tagged GAL4-ELL proteins with or without the SA/KT mutation along with xAF4 and EAF1h was performed as in Figure 4C. (C) Effects of the SA/KT mutation on MLL-ELL-mediated leukemic transformation ex vivo. Various MLL-ELL constructs with or without the SA/KT mutation were examined for the transformation of myeloid progenitors as in Figure 1A. (D) Effects of the SA/KT mutation on MLL-ELL-mediated leukemic transformation in vivo. MLL-ELL or its SA/KT mutant was transduced to c-Kit-positive hematopoietic progenitors and transplanted into syngeneic mice (n = 20). Primary leukemia cells were harvested from the bone marrow and transplanted into secondary recipient mice (n = 20). (E) Enhanced interaction of ELL with AEP mediated by the trithorax homology domain 2 (THD2) domain. IP-western blotting of the chromatin fraction of virus-packaging cells, transiently expressing various HA-tagged MTMT-ELL fusion constructs (with or without the SA/KT mutation), was performed. Endogenous proteins co-purified with MLL-ELL proteins were visualized by specific antibodies.

Figure 6.

Figure 6—figure supplement 1. Roles for EAF1 and p53 in MLL-ELL-mediated transformation 6.

Figure 6—figure supplement 1.

(A) Mutations in ELL selectively abrogated the interaction with p53. Immunoprecipitation (IP)-western blotting of the chromatin fraction of HEK293TL cells transiently expressing FLAG-tagged GAL4-ELL proteins with or without S600A/K606T substitutions (SA/KT) was performed as in Figure 4D. Endogenous AF4 family proteins and p53 co-purified with GAL4-ELL proteins were detected by anti-AF4-, AF5Q31-, and p53-antibodies. (B) Recruitment of endogenous AF4/ENL/P-TEFb complex (AEP) proteins by ELL mutant proteins. Chromatin immunoprecipitation (ChIP)-qPCR analysis of HEK293TL cells transiently expressing FLAG-tagged GAL4-ELL proteins with or without the SA/KT mutation was performed as in Figure 4C. ChIP signals of endogenous ENL and CyclinT1 were detected using the anti-ENL and CyclinT1 antibodies. (C) Protein expression from various MLL fusion constructs. Virus-packaging cells, transiently expressing each MLL fusion construct, were analyzed by western blotting with the indicated antibodies. (D) Requirement of EAF1 and p53 for MLL-ELL-immortalized cells. sgRNA competition assays for Eaf1 and Trp53 were performed as in Figure 2F. (E) Sensitivity to pan MYST HAT inhibitor. Leukemia cells (LCs) for various MLL fusions were cultured in the presence of WM1119, a pan MYST HAT inhibitor, and their relative colony-forming units (mean ± SD of thee biological replicates) were monitored.