Molecular Consequences of a Frameshifted DLX3 Mutant Lead ma to Tricho-Dento-Osseous Svndrome.

The homeodomain protein Distal-less-3 (Dlx3) plays a crucial role during embryonic development. This transcription factor is known to be essential for placental formation and to be involved in skin and skeletal organogenesis. In humans, a frameshift mutation in the coding sequence of the DLX3 gene results in an ectodermal dysplasia called Tricho-Dento-Osseous syndrome (TDO). The main features of this autosomal dominant disorder are defects in hair, teeth, and bone. To investigate the functional alterations caused by the mutated DLX3TDO isoform ex vivo, we used tetracycline-inducible osteoblastic and keratinocyte cell lines and calvarial derived osteoblasts in which the expression of DLX3WT and/or DLX3TDO could be regulated and monitored. Immunocytochemical analysis revealed that both DLX3WT and DLX3TDO recombinant proteins are targeted to the nucleus. However, as demonstrated by electrophoresis mobility shift assay, DLX3TDO is not able to bind to the canonical Dlx3 binding site. Furthermore, we demonstrate that the frameshifted C-terminal domain in DLX3TDO is accountable for the loss of DNA binding activity because the C-terminal domain in DLX3WT is not required for DNA binding activity. Although DLX3TDO alone cannot bind to a Dlx3 responsive element, when DLX3WT and DLX3TDO are co-expressed they form a complex that can bind DNA. Concomitant with the inability to bind DNA, DLX3TDO has a defective transcriptional activity. Moreover, the transcriptional activity of DLX3WT is significantly reduced in the presence of the mutated isoform, indicating that DLX3TDO has a dominant negative effect on DLX3WT transcriptional activity. [ABSTRACT FROM AUTHOR]