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. Maleand 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.Sexual dimorphism is regulated by diverse molecular mechanisms (18). In mammals, male development is initiated by a gene on the Y chromosome that is designated Sry (sex-determining region of the Y chromosome) (7,30,41,42). Alternative genetic mechanisms operate in Drosophila melanogaster (and Caenorhabditis elegans), wherein sex is determined by the X-to-autosome ratio, a process linked to X-dosage compensation (Fig. 1A) (19, 59). The sex-determining hierarchy of Drosophila defines an RNA-splicing cascade through the actions of Sex Lethal (SXL) and the TRA factors (18). Doublesex (dsx) functions downstream of this cascade to define one branch of a ramifying pathway (Fig. 1A) (29,59). The sex-specific isoforms of DSX (DSX M and DSX F ; Fig. 1B) regulate most aspects of somatic sexual dimorphism in D. melanogaster. These isoforms share an N-terminal DNA-binding domain, designated the DM motif (Fig. 1C). In this article, we present a mutational analysis of the DSX DM domain to identify key determinants of folding and DNA recognition.The Wilkins-Wolf hypothesis posits that sex-determining pathways have evolved in a reverse order from bottom (most broadly conserved) to top (least conserved) (65, 66). This viewpoint is supported by the ubiquity of DM genes among metazoan sex-determining pathways (35, 59). Such genes encode a newly recognized class of transcription factors (for a review, see reference 59). DM sequences are broadly conserved in metazoans, including nematodes, fish, reptiles, birds, and mammals (Fig. 1D). The deletion of human DM genes on chromosome 9p is associated with intersexual development (6,27,31,49,55,59,63). Although originally described in relation to sex-determining pathways (67), an extended family...

Molecular Cloning of Full-Length Dmrt1 cDNA of Reeves Turtle (Chinemys reevesii)

“…These residues participate in two intertwined Zn 2+ -binding sites [27]. The mutagenesis test in the DM domain of dsx in Drosophila demonstrated that these eight motif-specific histidines and cysteines that are engaged in zinc coordination are all critical and are not interchangeable [26].…”

Direct targets of theD. melanogasterDSXF protein and the evolution of sexual development

“…First, Dam alone was used to measure non-specific methylation (van Steensel and Henikoff, 2000). As the fly genome contains 'transcription factor colocalization hotspots', where transcription factors accumulate through protein-protein interactions (Moorman et al, 2006), a second control was Dam fused to a DSX protein with a mutation in its DNA-binding domain (R91Q) that has lost the ability to bind 2763 RESEARCH ARTICLE DSX targets and the evolution of sex the Yp1 DSX-binding site (Erdman and Burtis, 1993;Zhang et al, 2006). R91Q-Dam should bind to transcription factor hotspots, but not to real DSX-binding sites.…”

Isolation and characterization of oligomerization domain I and II coding regions of doublesex genes in agricultural fruit flies (Diptera: Tephritidae)

“…An amino acid substitution at Y67F was found in both sexes of B. dorsalis, B. correcta, and B. tryoni. This amino acid change was identified as being tolerant of amino acid substitutions (Zhang et al, 2006). Likewise, the amino acid changes at both positions 72 and 101 in OD1 can be different in males and females of B. correcta, because they might be selectively neutral polymorphisms.…”