Sex-specific gene regulation: The doublesex DM motif is a bipartite DNA-binding domain

Sex-specific gene expression in Drosophila melanogaster is regulated in part by the Doublesex (DSX) transcription factor. Male- and female-specific splicing isoforms share a novel DNA-binding domain, designated the DM motif. This domain is conserved among a newly recognized family of vertebrate transcription factors involved in developmental patterning and sex determination. The DM motif consists of an N-terminal zinc module and a disordered C-terminal tail, hypothesized to fold on specific DNA binding as a recognition α-helix. Truncation of the tail does not perturb the structure of the zinc module but impairs DNA binding and DNA-dependent dimerization. Chemical protein synthesis and alanine scanning mutagenesis are employed to test the contributions of 13 side chains to specific DNA binding. Selected arginine or lysine residues in the zinc module were substituted by norleucine, an isostere that maintains the aliphatic portion of the side chain but lacks a positive charge. Arginine or glutamine residues in the tail were substituted by alanine. Evidence is obtained that both the zinc module and C-terminal tail contribute to a bipartite DNA-binding surface. Conserved arginine and glutamine residues in the tail are required for high affinity DNA recognition, consistent with its proposed role as a nascent recognition α-helix.