Identification and characterization of the SHOX2-SOX5 and SHOX2-SOX6 protein interactions.

<p>A) SHOX2 interacts with SOX5 and SOX6 in the yeast-two hybrid system. The S. cerevisiae strain Y187 was cotransformed with the pGBT9 (BD) and pACT2 (AD) vectors. Interactions were determined by using a β-galactosidase liquid assay with CRPG as substrate. Empty vectors were employed as negative controls. B) SHOX2 interacts with SOX5 and SOX6 in human cells. Nuclear extracts of HEK293 cells overexpressing FLAG:SHOX2 and HA:SOX5 or HA:SOX6 were immunoprecipitated using anti-FLAG-agarose. Western blots (WB) of immunoprecipitates (IP) were probed with SHOX2, SOX5 and SOX6 antibodies. Nuclear extracts corresponding to 10% input were included as protein expression controls and nuclear extract immunoprecipitates of cells transfected only with HA:SOX5 or HA:SOX6 were included as negative controls. The western-blot images clearly show that SOX5 and SOX6 immunoprecipitate only in the presence of SHOX2. C) Characterization of the SHOX2 domains involved in the interaction with SOX6. An scheme of the SHOX2 protein structure showing the amino acid location of the homeodomain (HD) and the OAR domain is shown with the various analysed SHOX2 fragments, indicating their name and the amino acids that they contain. To the right of each fragment, the corresponding yeast two-hybrid results are shown. Y187 cells were cotransformed with SOX6 in the pGBT9 vector and the SHOX2 fragments in the pACT2 vector. Protein interaction percentages were obtained by normalizing the β-galactosidase units of the different SHOX2 fragments to that obtained with full-length SHOX2. Empty vectors were employed as negative controls. D) SHOX2 mutants impair the interaction with SOX6. SHOX2 protein structure scheme showing the localization of the seven missense mutations analyzed. Yeast two-hybrid assay of Y187 cotransformed cells with SOX6 in the pGBT9 vector and the different SHOX2 mutants in the pACT2 vector. Protein interaction percentages were obtained by normalizing the β-galactosidase units of the various SHOX2 mutants to the wildtype SHOX2. Empty vectors were employed as negative controls. E) Characterization of SOX6 domains involved in the interaction with SHOX2 using the yeast two-hybrid assay. SOX6 is schematically drawn showing the amino acid location of two dimerization domains, the first and the second coiled-coils (1st cc and 2nd cc, respectively), and the HMG DNA-binding domain. Depicted below are the SOX6 generated constructs, indicating the name of each fragment and the amino acids that they contain. Y187 cotransformed cells with the different SOX6 fragments in the pGBT9 vector and SHOX2 in the pACT2 vector. The protein interaction percentages were obtained by normalizing the β-galactosidase units of the different SOX6 fragments to that obtained with full-length SOX6. Empty vectors were employed as negative controls.