FIG. 6.
Potential dominant-negative effects of MLL-AFX fusion proteins. (A) MLL-AFX is unable to bind consensus forkhead DNA-binding sites. In vitro-translated proteins (indicated at tops of gel lanes) were incubated with 32P-labeled oligonucleotides encompassing the Fas ligand site corresponding to IGFBP1 IRS (Fas ligand promoter) or a mutated IRS (Am2Bm2) of the IGFBP-1. Reticulocyte lysate (lane 1) was used as a control for nonspecific binding. The arrow indicates specific DNA-binding complexes. (B) MLL-AFX antagonizes FKHRL1-mediated transcriptional activation. An expression construct encoding FKHRL1 was cotransfected into 293 cells with FKHRL1, MLL-AFX, or AFX expression constructs and a reporter gene driven by the Fas ligand promoter (FHRE promoter) or the p27kip-1 promoter (p27kip promoter). The fold induction was corrected for β-galactosidase activity from an internal lacZ control construct in each transfection. (C) Comparable expression levels of MLL-AFX in leukemic (lane 2) and transfected (lane 3) cells. The control (lane 1) consisted of the REH pre-B cell line, which expressed wild-type MLL but not MLL-AFX. Specific bands corresponding to wild-type MLL, MLL-AFX, and FKHRL1 are indicated to the left.