Fig. 3

Effect of Smad proteins on the transcriptional activity of mutated DLX proteins found in autistic individuals. A, Schematic representation of the location of the DLX2 and DLX5 mutations identified in autistic individuals. Note that all mutations occur in regions of homology between DLX proteins. The DLX2 serine insertion (Ser7) is in the DllA domain conserved in the DLX2/3/5 subgroup (green boxes), the DLX2 glutamic acid to lysine (Glu-Lys) mutation is in a conserved region flanking the homeodomain of the DLX2/3/5 subgroup (blue boxes), while the DLX2 alanine to threonine (Ala-Thr) and both DLX5 serine to proline (Ser-Pro) and serine to arginine (Ser-Arg) mutations occur in a proline-rich domain conserved in all six mammalian DLX proteins (yellow boxes). B, Expression of wild type (WT) and mutant DLX proteins. COS cells were transfected with plasmids expressing the indicated proteins, followed by Western blot analysis with an anti-FLAG antibody. C, Transient transcription assays. COS cells were transfected with a LacZ reporter plasmid driven by the Dlx5/6 enhancer-i in the absence or presence of the indicated combinations of proteins. All DLX2 mutants behaved like wild type DLX2 in these assays in the absence or presence of Smad proteins. By contrast, Smad co-expression did not enhance DLX5 Ser-Pro-mediated transcriptional activity, while the DLX5 Ser-Arg mutant exhibited an impaired transcriptional interaction with Smad2. Results are shown relative to the activity of the reporter alone and represent the means ± the S.D. of at least four experiments performed in duplicates (*, p < 0.05 using one-way analysis of variance with a Dunnett’s post test)