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Molecular and Cellular Biology, March 2003, p. 1968-1982, Vol. 23, No. 6
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.6.1968-1982.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Dominant Negative Dimerization of a Mutant Homeodomain Protein in Axenfeld-Rieger Syndrome

Irfan Saadi,1 Adisa Kuburas,2 Jamison J. Engle,2 and Andrew F. Russo1,2*

Genetics Program,1 Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 522422

Received 30 May 2002/ Returned for modification 6 August 2002/ Accepted 19 December 2002

Axenfeld-Rieger syndrome is an autosomal-dominant disorder caused by mutations in the PITX2 homeodomain protein. We have studied the mechanism underlying the dominant negative K88E mutation, which occurs at position 50 of the homeodomain. By using yeast two-hybrid and in vitro pulldown assays, we have documented that PITX2a can form homodimers in the absence of DNA. Moreover, the K88E mutant had even stronger dimerization ability, primarily due to interactions involving the C-terminal region. Dimerization allowed cooperative binding of wild-type (WT) PITX2a to DNA containing tandem bicoid sites in a head-to-tail orientation (Hill coefficient, 1.73). In contrast, the WT-K88E heterodimer bound the tandem sites with greatly reduced cooperativity and decreased transactivation activity. To further explore the role of position 50 in PITX2a dimerization, we introduced a charge-conservative mutation of lysine to arginine (K88R). The K88R protein had greatly reduced binding to a TAATCC element and did not specifically bind any other TAATNN motif. Like K88E, K88R formed relatively stronger dimers with WT. As predicted by our model, the K88R protein acted in a dominant negative manner to suppress WT PITX2a activity. These results suggest that the position 50 residue in the PITX2 homeodomain plays an important role in both DNA binding and dimerization activities.

* Corresponding author. Mailing address: Dept. of Physiology and Biophysics, 51 Newton Rd., University of Iowa, Iowa City, IA 52242. Phone: (319) 335-7872. Fax: (319) 335-7330. E-mail: andrew-russo{at}uiowa.edu.

Molecular and Cellular Biology, March 2003, p. 1968-1982, Vol. 23, No. 6
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.6.1968-1982.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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