Regulation of sexual dimorphism: mutational and chemogenetic analysis of the doublesex DM domain.
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.
Zhang
Wei
W
Case Western Reserve School of Medicine, Department of Biochemistry, 10900 Euclid Ave., Cleveland, Ohio 44106-4935, USA.
Li
Biaoru
B
Singh
Rupinder
R
Narendra
Uma
U
Zhu
Lingyang
L
Weiss
Michael A
MA
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000502:physiology; D029721:Drosophila Proteins / Q000235:genetics* / Q000502:physiology; D004331:Drosophila melanogaster / Q000235:genetics / Q000502:physiology*; D004742:Enhancer Elements, Genetic; D005260:Female; D006639:Histidine / Q000235:genetics; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012727:Sex Characteristics; D015032:Zinc / Q000737:chemistry
D004268:DNA-Binding Proteins; C070369:DSX protein, Drosophila; D029721:Drosophila Proteins; D006639:Histidine; D015032:Zinc; D003545:Cysteine