A transactivation domain in the HOX N-terminus is essential for transformation. (A) Design and expression of HOX N-terminal deletion derivatives. Immunoblotting was done with an anti-HA antibody detecting an engineered epitope tag. (B) Structure and expression of GAL4-HOX constructs. To avoid interference with GAL4-based DNA binding, only the respective HOX N-terminus without the homeodomain was fused to GAL4. Western blotting was performed in lysates of 293T cells transfected with the respective construct as indicated. An anti-GAL4 antibody was used for detection. (C) Structure-function correlation for N-terminal HOXA1 mutants. Left panel: Transforming activity of HOXA1 deletion clones as determined in replating assays. Data are mean ± SD of third round colony numbers representing 3 biologic replicates. P values were determined by Student t test. Right panel: Transactivation by corresponding GAL4-HOX fusions. REH cells were electroporated with 0.1 μg of a standard GAL4-SV40 minimal promoter vector (pGL3) and 0.9 μg of a GAL4-HOX expression plasmid. Luciferase values are given as mean ± SD of triplicates. Background values achieved with empty expression vector were set to 1 unit. (D) Transformation and transactivation determined for HOXA9 derivatives in analogy to the experiments shown in panel C.