The Molecular Basis of Rieger Syndrome
ANALYSIS OF PITX2 HOMEODOMAIN PROTEIN ACTIVITIES*
- From the Departments of ‡Physiology and Biophysics and¶Pediatrics, University of Iowa, Iowa City, Iowa 52242
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 bicoidelement 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 apparentK D and only about a 2-fold reduction in theB max. 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.
Footnotes
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↵* This work was supported by National Institutes of Health Grants DE09170 with tissue culture support from DK25295 (to A. F. R.) and a NIH Postdoctoral Training Fellowship DK07018 (to B. A. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ To whom correspondence should be addressed: Dept. of Physiology and Biophysics, University of Iowa, Iowa City, IA 52242. Tel.: 319-335-7873; Fax: 319-335-7330; E-mail: brad-amendt{at}uiowa.edu.
- Abbreviations:
- GST
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glutathioneS-transferase
- EMSA
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electrophoretic mobility shift assay
- CMV
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cytomegalovirus
- PCR
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polymerase chain reaction
- TK
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thymidine kinase.
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- Received March 12, 1998.
- Revision received May 18, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
