Figure 4

Mutagenesis of consensus transcription factor binding sequences in the 5'ETO. Erythroid HEL and megakaryocytic MEG-01 cells were transfected with the -729 to -259 bp ETO promoter reporter construct with or without mutations of transcription factor binding sites as indicated (X). The mutations are described in "Materials and Methods". The pGL3/basic and pGL3/SV40-promoter are used as negative and positive control, respectively. Firefly and Renilla luciferase (internal standard) activities were assayed 24 h post-transfection. The luciferase activity of mutated ETO promoter is normalized against luciferase activity of wildtype ETO promoter. Mutation of the GATA -636 site strongly represses transactivation of the ETO promoter reporter. Mutation of the ETS1 -705 binding site increased the luciferase signal twice. Firefly was normalized to Renilla luciferase as internal control for transfection efficiency and the results are given as adjusted Relative Luciferase Units (AdjRLU). Bars represent the mean of results from 3 to 5 separate transfections and the error bars show SEM. **, p < 0.01; ***, p < 0.0001