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

Abstract

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.

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

This work was supported in part by a grant from the National Institutes of Health to B. Baker (GM037731-20, subcontract GM037731-19S1 to M.A.W.). R.S. was supported in part by an NIH training grant in molecular oncology at the Case School of Medicine (CA059366).

We thank B. Baker for advice and encouragement, N. B. Phillips and W. Yang for discussion, J. Bayrer and K. Huang for assistance with figures, and E. Collins and S. Price for preparation of the manuscript.

This is a contribution from the Cleveland Center for Structural Biology.