Sex determination in Drosophila: Biochemical and functional studies of transcription factor doublesex
Doublesex (dsx) and dsx-related genes encode a novel class of transcription factors conserved among metazoans. The members of this family play a critical role in sex differentiation across species from Caenorhabditis elegans to humans. Mutations or deletions of these genes are associated with intersexual abnormalities, including a class of human birth defects termed "9p syndrome". This thesis is focused on the dsx gene from Drosophila melanogaster, the archetype of this class. Sex-specific RNA splicing leads to male and female isoforms, designated DSXM and DSXF. Biochemical and genetic studies of the DM domain and the female-specific C-terminal domain are presented. The importance of individual residues in the DM domain to folding and DNA recognition is probed using a yeast one-hybrid system combined with random mutagenesis. This system is based on a sex-specific Drosophila enhancer element and validated through studies of intersexual dsx mutations. Although key exceptions occur, mutations that impair DNA binding tend to occur at conserved positions whereas neutral substitutions occur at non-conserved sites. Evidence for a specific salt bridge between a conserved lysine and the DNA backbone is obtained through synthesis of non-standard protein and DNA analogs. Structure-function relationships in the female-specific C-terminal domain are investigated by deletion analysis and mutagenesis. The crystal structure of this domain reveals a novel dimeric arrangement of ubiquitin-associated (UBA) folds. Dimerization is mediated by a non-canonical hydrophobic interface extrinsic to the putative Ub-binding surface. Key side chains at this interface, identified by alanine scanning mutagenesis, are conserved among DSX homologs. Although the Intersex (IX) protein is expressed in both male and female Drosophila, genetic studies suggest that IX specifically interacts with DSXF but not DSXM. To define the mechanism of sex-specific recognition between DSXF and IX, both yeast two-hybrid system and co-immunoprecipitation in S2 cells are employed to map the protein-protein interaction surface. It is demonstrated that the dimerization domain (but not the distal portion of the sex-specific tail) is required for DSXF-IX binding. Mutation of solvent-exposed residues in the DSXF UBA domain reveals three residues essential for IX binding. Molecular modeling of this surface suggests that IX binds in a specific groove that spans the dimer interface and crosses contiguous sex-specific and non-specific surfaces. Temperature-sensitive (ts) mutations are of future interest as probes to elucidate the mechanism by which DSXF controls female sex-specific behavior. Two such candidates (V379T and R394A) are thus identified in the DSXF C-terminal dimerization domain (CTD). Screening is based on CTD dimerization because homodimerization of DSXF is necessary for high-affinity DNA recognition, and presumably IX binding. Interestingly, the wild-type side chains of these residues are in close contact. The thermal stability of the variant domains is validated through thermal denaturation studies done by circular dichroism (CD). Allelic replacement of wild-type dsx by designed temperature-sensitive alleles promises to provide definitive evidence for the developmental functions of DSXF in somatic sexual differentiation and courtship behavior.