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Molecular and Cellular Biology, January 2006, p. 535-547, Vol. 26, No. 2
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.2.535-547.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Regulation of Sexual Dimorphism: Mutational and Chemogenetic Analysis of the Doublesex DM Domain{dagger}

Wei Zhang, Biaoru Li, Rupinder Singh, Uma Narendra, Lingyang Zhu,{ddagger} and Michael A. Weiss*

Case Western Reserve School of Medicine, Department of Biochemistry, 10900 Euclid Avenue, Cleveland, Ohio 44106-4935

Received 28 June 2005/ Returned for modification 12 September 2005/ Accepted 21 October 2005

Doublesex (dsx) is a transcription factor in Drosophila that regulates somatic sexual differentiation. Male- and female-specific splicing isoforms of DSX share a novel DNA-binding domain, designated the DM motif. Broadly conserved among metazoan sex-determining factors, the DM domain contains a nonclassical zinc module and binds in the DNA minor groove. Here, we characterize the DM motif by site-directed and random mutagenesis using a yeast one-hybrid (Y1H) system and extend this analysis by chemogenetic complementation in vitro. The Y1H system is based on a sex-specific Drosophila enhancer element and validated through studies of intersexual dsx mutations. We demonstrate that the eight motif-specific histidines and cysteines engaged in zinc coordination are each critical and cannot be interchanged; folding also requires conserved aliphatic side chains in the hydrophobic core. Mutations that impair DNA binding tend to occur at conserved positions, whereas neutral substitutions occur at nonconserved sites. Evidence for a specific salt bridge between a conserved lysine and the DNA backbone is obtained through the synthesis of nonstandard protein and DNA analogs. Together, these results provide molecular links between the structure of the DM domain and its function in the regulation of sexual dimorphism.

* Corresponding author. Mailing address: Department of Biochemistry, 10900 Euclid Ave., Cleveland, OH 44106-4935. Phone: (216) 368-5991. Fax: (216) 368-3419. E-mail: michael.weiss{at}case.edu.

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/.

{ddagger} Present address: Array BioPharma, 3200 Walnut St., Boulder, CO 80301.

Molecular and Cellular Biology, January 2006, p. 535-547, Vol. 26, No. 2
0022-538X/06/$08.00+0     doi:10.1128/MCB.26.2.535-547.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Yang, Y., Zhang, W., Bayrer, J. R., Weiss, M. A. (2008). Doublesex and the Regulation of Sexual Dimorphism in Drosophila melanogaster: STRUCTURE, FUNCTION, AND MUTAGENESIS OF A FEMALE-SPECIFIC DOMAIN. J. Biol. Chem. 283: 7280-7292 [Abstract] [Full Text]  


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