Figure 2.
Figure 2. PAF1 is necessary for MLL-ENL function and can be recruited by MLL and ENL. (A) Schematic overview of the MLL-ENL fusion protein. A CxxC domain critical for MLL-ENL function has been previously identified as PAF1-binding site.22,23 Two mutations that abrogate PAF1 interaction (R1153A and a deletion of the CxxC domain from aa1258 to the breakpoint) are indicated. In patient samples, either nearly full length ENL or only the C-terminal coiled-coil can be found fused to MLL. (B) Expression and activity of combinatorial MLL-ENL fusion derivatives. Fusions of wt-MLL or the 2 PAF1-binding defective mutants MLL∆1258 and MLL R1153A with either full-length ENL or only the ENL C-terminal domain were assembled in retroviral vectors. The correct expression of the corresponding flag-tagged proteins was ascertained by western blot in extracts of retroviral packaging cell lines. Biological activity was assessed by transduction of primary hematopoietic precursors isolated from murine bone marrow, followed by a standard replating assay. The figure shows stained colonies of cells after 2 replatings that were transduced with MLL constructs as labeled. 10000 cells were plated for each well. (C) Evaluation of colony-forming cell numbers. Colony-forming assays, as above, were conducted in triplicate with averages and standard deviations given for each combination. The ability of each construct to bind PAF1 is indicated above the respective column. Colony numbers are given in relation to MLL-ENL. (D) Transplantation assays. Primary hematopoietic cells were transduced with MLL R1153A-ENLfull, which recruits PAF1 exclusively through the ENL moiety or with MLL-ENL and ENL as controls. Infected cells were selected and injected into sublethally irradiated syngenic recipients. The Kaplan-Meier plot gives survival times in days after transplant. Dot plots indicate spleen weights and white blood cell counts of diseased animals including statistical evaluation by 2-tailed t test. (E) Expression of key MLL-ENL target genes Hoxa9 and Meis1 is dependent on PAF1 binding. Primary hematopoietic precursor cells were transduced with wt and PAF1 defective MLL ENLfull/ENL-C combinations, as above. RNA was harvested after the first round of replating at a time point when all cells had not yet exhausted their replating potential. Hoxa9 and Meis1 RNA concentrations were determined by qPCR and normalized to β-actin expression. Shown are averages and standard deviations of PCR triplicates in relation to expression levels reached in cells transduced with MLL-ENLfull. (F) Histone H2B ubiquitination at Hoxa9 and Meis1 loci. Cells transduced and harvested as described for panel D were subject to crosslinking and H2Bub-specific chromatin immunoprecipitation. Material from the Hoxa9 and Meis1 loci was quantified with primers for the respective region by qPCR. Results are plotted as before. *P < .05. CFC, colony-forming cell; WBC, white blood cells.

PAF1 is necessary for MLL-ENL function and can be recruited by MLL and ENL. (A) Schematic overview of the MLL-ENL fusion protein. A CxxC domain critical for MLL-ENL function has been previously identified as PAF1-binding site.22,23  Two mutations that abrogate PAF1 interaction (R1153A and a deletion of the CxxC domain from aa1258 to the breakpoint) are indicated. In patient samples, either nearly full length ENL or only the C-terminal coiled-coil can be found fused to MLL. (B) Expression and activity of combinatorial MLL-ENL fusion derivatives. Fusions of wt-MLL or the 2 PAF1-binding defective mutants MLL∆1258 and MLL R1153A with either full-length ENL or only the ENL C-terminal domain were assembled in retroviral vectors. The correct expression of the corresponding flag-tagged proteins was ascertained by western blot in extracts of retroviral packaging cell lines. Biological activity was assessed by transduction of primary hematopoietic precursors isolated from murine bone marrow, followed by a standard replating assay. The figure shows stained colonies of cells after 2 replatings that were transduced with MLL constructs as labeled. 10000 cells were plated for each well. (C) Evaluation of colony-forming cell numbers. Colony-forming assays, as above, were conducted in triplicate with averages and standard deviations given for each combination. The ability of each construct to bind PAF1 is indicated above the respective column. Colony numbers are given in relation to MLL-ENL. (D) Transplantation assays. Primary hematopoietic cells were transduced with MLL R1153A-ENLfull, which recruits PAF1 exclusively through the ENL moiety or with MLL-ENL and ENL as controls. Infected cells were selected and injected into sublethally irradiated syngenic recipients. The Kaplan-Meier plot gives survival times in days after transplant. Dot plots indicate spleen weights and white blood cell counts of diseased animals including statistical evaluation by 2-tailed t test. (E) Expression of key MLL-ENL target genes Hoxa9 and Meis1 is dependent on PAF1 binding. Primary hematopoietic precursor cells were transduced with wt and PAF1 defective MLL ENLfull/ENL-C combinations, as above. RNA was harvested after the first round of replating at a time point when all cells had not yet exhausted their replating potential. Hoxa9 and Meis1 RNA concentrations were determined by qPCR and normalized to β-actin expression. Shown are averages and standard deviations of PCR triplicates in relation to expression levels reached in cells transduced with MLL-ENLfull. (F) Histone H2B ubiquitination at Hoxa9 and Meis1 loci. Cells transduced and harvested as described for panel D were subject to crosslinking and H2Bub-specific chromatin immunoprecipitation. Material from the Hoxa9 and Meis1 loci was quantified with primers for the respective region by qPCR. Results are plotted as before. *P < .05. CFC, colony-forming cell; WBC, white blood cells.

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