Figure 6

When targeted to a heterologous promoter, Mab21l2 behaves as a strong transcriptional repressor. (A) An in vitro-translated chimeric protein containing the GAL4 DNA binding domain fused to the N-terminus of MAB21L2 was used in an electrophoretic mobility shift assay. An end-labeled ds-DNA containing the GAL4 nucleotide binding site (5XUAS) was incubated in native conditions with in vitro translated GAL4-MAB21L2 (G-M) and separated electrophoretically by nondenaturing PAGE. As negative controls, we used free oligonucleotides (F) and rabbit reticulocyte lysates (RRL). As a positive control we used a previously tested GAL4 fusion protein, GAL4-D9NT (G-D9) [40]. The arrow indicates a bandshift specific for GAL4-MAB21L2 and absent in negative controls. u: unbound. (B) 5XUAS-luc transcription is clearly downregulated in cells transfected with GAL4-MAB21L2 (G-M) vs. untransfected COS7 cells or same cells transfected with GAL4 DBD, G-D) alone (*: p = 0.0019). No downregulation of 5XUAS-luc activity was observed in COS7 cells transfected with a fusion of GAL4 and the N-terminal domain of the HOXB3 protein, which possesses no transcriptional activation or repression activity [24] (ns: not significant). Statistical analysis was conducted using the T test method (two tails).