Functional consequences of the G208fsX mutation.

(A) Schematic representation of the G208fsX mutation in the DNA-binding domain (DBD) with the frame shift sequences depicted with hatched bars. (B) EMSA analysis of the binding of nuclear extracts from COS7 cells transfected with PU.1 wild-type (wt; lane 2) or G208fsX (mut; lane 4) to the PU.1 binding site in the M-CSF receptor. ss indicates that PU.1 wild-type binding was supershifted with carboxyl terminal-specific PU.1 antiserum (lane 3); C, competition of wild-type binding with 100-fold excess of unlabeled oligonucleotide (lane 5); and p, labeled probe alone (lane 1). As in Figure 3C, the complex migrating more slowly than wild-type PU.1, and which does not react with the anti-PU.1 antibody, has been observed previously in EMSA using this probe.20 (C) COS7 cells were transfected with PU.1 wild-type (wt) or the PU.1 mutant G208fsX (mut) together with either AML1 or the pcDNA3 vector (V). The ability to activate the PU.1 site in the M-CSF receptor promoter was measured. Synergy represents the ratio of the activity seen 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 and indicated to the right of each bar. (D) c-Jun–deficient F9 cells were transfected with PU.1 wild-type (wt) or the G208fsX mutant (mut) 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 synergy was determined as described for panel C.