TY - JOUR
T1 - Regulation of sexual dimorphism
T2 - Mutational and chemogenetic analysis of the doublesex DM domain
AU - Zhang, Wei
AU - Li, Biaoru
AU - Singh, Rupinder
AU - Narendra, Uma
AU - Zhu, Lingyang
AU - Weiss, Michael A.
PY - 2006/1
Y1 - 2006/1
N2 - Doublesex (dsx) is a transcription factor in Drosophila that regulates somatic sexual differentiation. Male- and female-speciftc 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-speciftc 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.
AB - Doublesex (dsx) is a transcription factor in Drosophila that regulates somatic sexual differentiation. Male- and female-speciftc 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-speciftc 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.
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U2 - 10.1128/MCB.26.2.535-547.2006
DO - 10.1128/MCB.26.2.535-547.2006
M3 - Article
C2 - 16382145
AN - SCOPUS:30644477195
SN - 0270-7306
VL - 26
SP - 535
EP - 547
JO - Molecular and Cellular Biology
JF - Molecular and Cellular Biology
IS - 2
ER -