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The molecular basis of Rieger syndrome. Analysis of Pitx2 homeodomain protein activities. J Biol Chem 1998 Aug 07;273(32):20066-72

Date

08/01/1998

Pubmed ID

9685346

DOI

10.1074/jbc.273.32.20066

Scopus ID

2-s2.0-0032493822 (requires institutional sign-in at Scopus site)   122 Citations

Abstract

Rieger syndrome is an autosomal-dominant developmental disorder that includes glaucoma and mild craniofacial dysmorphism in humans. Mutations in the Pitx2 homeobox gene have been linked to Rieger syndrome. We have characterized wild type and mutant Pitx2 activities using electrophoretic mobility shift assays, protein binding, and transient transfection assays. Pitx2 preferentially binds the bicoid homeodomain binding site and transactivates reporter genes containing this site. The combination of Pitx2 and another homeodomain protein, Pit-1, yielded a synergistic 55-fold activation of the prolactin promoter in transfection assays. Addition of Pit-1 increased Pitx2 binding to the bicoid element in electrophoretic mobility shift assays. Furthermore, we demonstrate specific binding of Pit-1 to Pitx2 in vitro. Thus, wild type Pitx2 DNA binding activity is modulated by protein-protein interactions. We next 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 KD and only about a 2-fold reduction in the Bmax. However, this mutant did not transactivate reporter genes containing the bicoid site. The mutant Pitx2 protein binds Pit-1, but there was no detectable synergism on the prolactin promoter. A second mutation (L54Q) in a highly conserved residue in helix 1 of the homeodomain yielded an unstable protein. Our results provide insights into the potential mechanisms underlying the developmental defects in Rieger syndrome.

Author List

Amendt BA, Sutherland LB, Semina EV, Russo AF

Author

Elena V. Semina PhD Chief, Professor in the Ophthalmology and Visual Sciences department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Amino Acid Sequence
Animals
Binding Sites
COS Cells
DNA-Binding Proteins
Genes, Reporter
Genetic Diseases, Inborn
Homeodomain Proteins
Humans
Molecular Sequence Data
Mutation
Nuclear Proteins
Oligodeoxyribonucleotides
Paired Box Transcription Factors
Protein Binding
Transcription Factor Pit-1
Transcription Factors
Transcriptional Activation