Fig. 3.
Fig. 3. The PU.1 mutations G253fsX and G254R identified in AML M4 patient #70 demonstrate decreased DNA binding, transactivation, and synergism with AML1 and c-Jun. / (A) The top left panel represents the one base pair deletion G253fsX; the lower left panel depicts the point mutation G254R. Sequences are shown below the left panels for the mutation (above) and the wild-type (below). The panels on the right are schematic representations of these 2 mutations in the DNA-binding domain (DBD) with the frame shift sequences depicted with hatched bars. (B) Western blot using a FLAG antibody. FLAG-tagged PU.1 wild-type (wt), vector only, which lacks a FLAG-tag (V), or FLAG-tagged G254R, G253fsX, or G208fsX mutants were in vitro translated and run on the SDS gel. Molecular weight markers are shown on the left. (C) Electrophoretic mobility shift assay (EMSA) analyzing DNA binding to the PU.1 site in the M-CSF receptor promoter of in vitro translated proteins encoded by PU.1 wild-type and the PU.1 mutants G253fsX and G254R. The input protein for wild-type and mutant PU.1 proteins is shown in Figure 3B. (Left panel) Binding was supershifted using an antiserum directed against the amino terminus of the PU.1 protein. Consistently, we observed that the complex obtained for PU.1 wild-type with the amino terminal antibody migrated more slowly than the complex containing one of the mutant proteins. (Right panel) Supershift was achieved with an antibody directed against amino acids 251 to 271 of the murine PU.1 protein. X indicates nonspecific binding activity (does not compete with self oligonucleotide); P, labeled probe alone. In both panels, the complex migrating more slowly than wild-type PU.1, which does not react with either antibody, has been observed previously in EMSA using this probe.20 (D, upper panel) COS7 cells were transfected with PU.1 wild-type (wt) or one of the 2 PU.1 mutants identified in AML patient #70 (G253fsX and G254R) together with either AML1 or pcDNA3 vector alone (V). Either 500 ng of a single PU.1 allele or 250 ng each of 2 PU.1 alleles were transfected. The AML1 cofactor CBFβ was present in all transfections in equimolar amounts. The reporter consisted of a luciferase construct with a wild-type PU.1 site.35 The ability to activate the PU.1 site derived from the M-CSF receptor promoter is indicated in luciferase units normalized to wild-type PU.1 ( = 100). Synergy was calculated by the ratio of the activity observed with cotransfected AML1 and PU.1 wild-type divided by the arithmetic addition of AML1 activation alone and PU.1 wild-type activation alone. The same ratio was determined for the PU.1 mutant G254R and indicated to the right of each bar. (Lower panel) The same assay as above, except the reporter consisted of a luciferase construct with the PU.1 site mutated (mut. PU.1 site).35(E) F9 cells that are c-Jun deficient were transfected with PU.1 wild-type (wt) or the 2 PU.1 mutants identified in AML patient #70 (G253fsX and G254R) together with c-Jun or the empty expression vector (V). Empty expression vector was added in all transfections to ensure that equal amounts of DNA were transfected. The ability to activate the PU.1 site in the M-CSF receptor promoter and synergism with c-Jun was determined as described for panel D.

The PU.1 mutations G253fsX and G254R identified in AML M4 patient #70 demonstrate decreased DNA binding, transactivation, and synergism with AML1 and c-Jun.

(A) The top left panel represents the one base pair deletion G253fsX; the lower left panel depicts the point mutation G254R. Sequences are shown below the left panels for the mutation (above) and the wild-type (below). The panels on the right are schematic representations of these 2 mutations in the DNA-binding domain (DBD) with the frame shift sequences depicted with hatched bars. (B) Western blot using a FLAG antibody. FLAG-tagged PU.1 wild-type (wt), vector only, which lacks a FLAG-tag (V), or FLAG-tagged G254R, G253fsX, or G208fsX mutants were in vitro translated and run on the SDS gel. Molecular weight markers are shown on the left. (C) Electrophoretic mobility shift assay (EMSA) analyzing DNA binding to the PU.1 site in the M-CSF receptor promoter of in vitro translated proteins encoded by PU.1 wild-type and the PU.1 mutants G253fsX and G254R. The input protein for wild-type and mutant PU.1 proteins is shown in Figure 3B. (Left panel) Binding was supershifted using an antiserum directed against the amino terminus of the PU.1 protein. Consistently, we observed that the complex obtained for PU.1 wild-type with the amino terminal antibody migrated more slowly than the complex containing one of the mutant proteins. (Right panel) Supershift was achieved with an antibody directed against amino acids 251 to 271 of the murine PU.1 protein. X indicates nonspecific binding activity (does not compete with self oligonucleotide); P, labeled probe alone. In both panels, the complex migrating more slowly than wild-type PU.1, which does not react with either antibody, has been observed previously in EMSA using this probe.20 (D, upper panel) COS7 cells were transfected with PU.1 wild-type (wt) or one of the 2 PU.1 mutants identified in AML patient #70 (G253fsX and G254R) together with either AML1 or pcDNA3 vector alone (V). Either 500 ng of a single PU.1 allele or 250 ng each of 2 PU.1 alleles were transfected. The AML1 cofactor CBFβ was present in all transfections in equimolar amounts. The reporter consisted of a luciferase construct with a wild-type PU.1 site.35 The ability to activate the PU.1 site derived from the M-CSF receptor promoter is indicated in luciferase units normalized to wild-type PU.1 ( = 100). Synergy was calculated by the ratio of the activity observed with cotransfected AML1 and PU.1 wild-type divided by the arithmetic addition of AML1 activation alone and PU.1 wild-type activation alone. The same ratio was determined for the PU.1 mutant G254R and indicated to the right of each bar. (Lower panel) The same assay as above, except the reporter consisted of a luciferase construct with the PU.1 site mutated (mut. PU.1 site).35(E) F9 cells that are c-Jun deficient were transfected with PU.1 wild-type (wt) or the 2 PU.1 mutants identified in AML patient #70 (G253fsX and G254R) together with c-Jun or the empty expression vector (V). Empty expression vector was added in all transfections to ensure that equal amounts of DNA were transfected. The ability to activate the PU.1 site in the M-CSF receptor promoter and synergism with c-Jun was determined as described for panel D.

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