THE MOLECULAR-BASIS OF RIEGER-SYNDROME - ANALYSIS OF PITX2 HOMEODOMAIN PROTEIN ACTIVITIES

Rieger syndrome is an autosomal-dominant developmental disorder that i ncludes glaucoma and mild craniofacial dysmorphism in humans. Mutation s in the Pitx2 homeobox gene have been linked to Rieger syndrome. We h ave characterized wild type and mutant Pitx2 activities using electrop horetic mobility shift assays, protein binding, and transient transfec tion assays. Pitx2 preferentially binds the bicoid homeodomain binding site and transactivates reporter genes containing this site. The comb ination of Pitx2 and another homeodomain protein, Pit-1, yielded a syn ergistic 55-fold activation of the prolactin promoter in transfection assays. Addition of Pit-1 increased Pitx2 binding to the bicoid elemen t in electrophoretic mobility shift assays. Furthermore, we demonstrat e specific binding of Pit-1 to Pitx2 in vitro. Thus, wild type Pitx2 D NA binding activity is modulated by protein-protein interactions. We n ext studied two Rieger mutants. A threonine to proline mutation (T68P) in the second helix of the homeodomain retained DNA binding activity with the same apparent K-D and only about a a-fold reduction in the B- max. However, this mutant did not transactivate reporter genes contain ing the bicoid site. The mutant Pitx2 protein binds Pit-1, but there w as no detectable synergism on the prolactin promoter. A second mutatio n (L54Q) in a highly conserved residue in helix 1 of the homeodomain y ielded an unstable protein. Our results provide insights into the pote ntial mechanisms underlying the developmental defects in Rieger syndro me.