DNA Binding by the Male and Female Doublesex Proteins of Drosophila melanogaster

Abstract

To provide a base for understanding the mechanisms of these several transcriptional functions, we have been investigating the physical properties of DSX proteins. Previous investigations show that DSX M and DSX F are identical over their first 397 amino acids, which include a DNA binding domain (5, 6). The proteins differ because the dsx transcript is spliced differently in males and females. This alternative splicing produces only one difference between the two proteins, a 152-amino acid carboxyl terminus in DSX M and a completely different 30-amino acid carboxyl terminus in DSX F . DSX proteins have two protein oligomerization domains: one co-localized with the DNA binding domain, and the other localized at the splice boundary and requiring both sex-specific and sex-nonspecific sequences for the oligomerization activity (7). Both proteins have highly extended shapes, forming dimers at low protein concentrations and higher order oligomers at higher protein concentrations (8). Although the equilibria between protein oligomers and DNA are complex, the fundamental DNA binding species of each protein is a dimer (8).In this paper we investigate the DNA binding properties of purified DSX M and DSX F dimers. We find that DSX M and DSX F affinities for short DNA oligomers are indistinguishable whether those oligomers contain a specific tight binding site or nonspecific sites. The association and dissociation rates as well as the dependence on monovalent and divalent cations are also indistinguishable between the male and female proteins. These results indicate that the region common to the two proteins is the predominant determinant when dimers bind to individual DNA sites. We infer that the sex-specific oligomerization domain is likely to play roles in binding cooperativity when the proteins bind to multiple DNA sites or when DSX proteins interact with different regulatory proteins, for example, the tissue specifying interaction with the bZIP protein mentioned above. In this investigation we also observed that the dimer-DNA complex dissociation has a first order dependence upon free DNA concentration. We infer that direct transfer between DNAs occurs when DSX proteins are searching for specific sites in the many short open DNA regions of chromatin.

The Gene doublesex of the Fruit Fly Anastrepha obliqua (Diptera, Tephritidae)

“…Among the dipterans, it is higher among the tephritids A. obliqua, B. oleae, and B. tryoni. The number of amino acids and the similarity of the OD1 and OD2 domains are very high among all species, as expected, since these domains endow the Dsx proteins with the capacity to interact with other proteins and with DNA (An et al 1996;Cho and Wensink 1997).…”

Regulation of Sexual Dimorphism: Mutational and Chemogenetic Analysis of the Doublesex DM Domain

“…Such binding is strengthened by strong C-terminal dimer contacts (16). An AD-DM fusion protein exhibits significant Y1H activity in accord with its autonomous high-affinity DNA-binding activity (15,52,68). Because its expression is less toxic to Saccharomyces cerevisiae than was an intact AD-DSX F fusion protein, the AD-DM construct is employed as a template for mutagenesis.…”

“…DSX M and DSX F contain identical DM domains (25) and exhibit similar DNA-binding properties (15,17). Minor-groove targeting of the DSX DM domain is proposed to allow simultaneous binding of major-groove factors to composite DNA enhancer elements (68).…”

Masculinization of XX Drosophila transgenic flies expressing the Ceratitis capitata DoublesexM isoform

“…We generated 20 Drosophila transgenic lines expressing a Ccdsx M cDNA from the hsp70 heat shock promoter (hsp70::CcDSX M ). When raised at 25°C, males and females, showed wild type anal and genital B A faces for protein-protein and protein-DNA interactions (An et al, 1996, Cho andWensink, 1997). The Ccdsx sex-specific transcripts share a common region composed of two exons, but differ for the presence of a female-and a male-specific exons (Fig.…”