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J. Biol. Chem., Vol. 283, Issue 11, 7280-7292, March 14, 2008

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Doublesex and the Regulation of Sexual Dimorphism in Drosophila melanogaster

STRUCTURE, FUNCTION, AND MUTAGENESIS OF A FEMALE-SPECIFIC DOMAIN^*

From the Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106

The DSX (Doublesex) transcription factor regulates somatic sexual differentiation in Drosophila. Female and male isoforms (DSX^F and DSX^M) are formed due to sex-specific RNA splicing. DNA recognition, mediated by a shared N-terminal zinc module (the DM domain), is enhanced by a C-terminal dimerization element. Sex-specific extension of this element in DSX^F and DSX^M leads to assembly of distinct transcriptional preinitiation complexes. Here, we describe the structure of the extended C-terminal dimerization domain of DSX^F as determined by multidimensional NMR spectroscopy. The core dimerization element is well ordered, giving rise to a dense network of interresidue nuclear Overhauser enhancements. The structure contains dimer-related UBA folds similar to those defined by x-ray crystallographic studies of a truncated domain. Whereas the proximal portion of the female tail extends helix 3 of the UBA fold, the distal tail is disordered. Ala substitutions in the proximal tail disrupt the sex-specific binding of IX (Intersex), an obligatory partner protein and putative transcriptional coactivator; IX-DSX^F interaction is, by contrast, not disrupted by truncation of the distal tail. Mutagenesis of the UBA-like dimer of DSX^F highlights the importance of steric and electrostatic complementarity across the interface. Two temperature-sensitive mutations at this interface have been characterized in yeast model systems. One weakens a network of solvated salt bridges, whereas the other perturbs the underlying nonpolar interface. These mutations confer graded gene-regulatory activity in yeast within a physiological temperature range and so may provide novel probes for genetic analysis of a sex-specific transcriptional program in Drosophila development.

Received for publication, October 23, 2007 , and in revised form, December 6, 2007.

The atomic coordinates and NMR-derived restraints (code 2jz0 and 2jz1) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported in part by a subcontract (to M. A. W.) from National Institutes of Health Grant GM037731 (to B. Baker). This is a contribution from the Cleveland Center for Structural Biology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2 and Figs. S1-S7.

¹ Both authors contributed equally to this work.

² Present address: Division of Cancer Biology, Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins School of Medicine, Baltimore, MD 21231.

³ Predoctoral fellow of the Medical Scientist Training Program at the Case School of Medicine (Grant T32 GM07250). Present address: Dept. of Pediatrics, University of California, San Francisco, CA 94143.

⁴ To whom correspondence should be addressed. E-mail: michael.weiss{at}case.edu.

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