Truncation Mutations in the Transactivation Region of PAX6 Result in Dominant-Negative Mutants*
- From the Department of Biochemistry and Molecular Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
Abstract
PAX6 is a transcription factor with two DNA-binding domains (paired box and homeobox) and a proline-serine-threonine (PST)-rich transactivation domain. PAX6 regulates eye development in animals ranging from jellyfish toDrosophila to humans. Heterozygous mutations in the humanPAX6 gene result in various phenotypes, including aniridia, Peter’s anomaly, autosomal dominant keratitis, and familial foveal dysplasia. It is believed that the mutated allele ofPAX6 produces an inactive protein and aniridia is caused due to genetic haploinsufficiency. However, several truncation mutations have been found to occur in the C-terminal half ofPAX6 in patients with Aniridia resulting in mutant proteins that retain the DNA-binding domains but have lost most of the transactivation domain. It is not clear whether such mutants really behave as loss-of-function mutants as predicted by haploinsufficiency. Contrary to this theory, our data showed that these mutants are dominant-negative in transient transfection assays when they are coexpressed with wild-type PAX6. We found that the dominant-negative effects result from the enhanced DNA binding ability of these mutants. Kinetic studies of binding and dissociation revealed that various truncation mutants have 3–5-fold higher affinity to various DNA-binding sites when compared with the wild-type PAX6. These results provide a new insight into the role of mutant PAX6 in causing aniridia.
Footnotes
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↵* This work was supported by National Institutes of Health Grants EY09675, EY10608, and CA16672 and Texas Advanced Research Program Grant 000015-046.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 Biochemistry and Molecular Biology, Box 117, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030. Tel.: 713-792-2690; Fax: 713-790-0329; E-mail: saunders{at}odin.mdacc.tmc.edu.
- Abbreviations:
- PAGE
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polyacrylamide gel electorphoresis
- EMSA
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electrophoretic mobility shift assay
- C/EBP
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cAMP responese element-binding protein
- luc
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luciferase
- aa
-
amino acid(s).
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- Received April 10, 1998.
- Revision received June 11, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
