A network of interacting transcriptional regulators involved in Drosophila neural fate specification revealed by the yeast two-hybrid system

Abstract

Neural fate specification in Drosophila is promoted by the products of the proneural genes, such as those of the achaete-scute complex, and antagonized by the products of the Enhancer of split [E(spl)] complex, hairy, and extramacrochaetae. As all these proteins bear a helix-loop-helix (HLH) dimerization domain, we investigated their potential pairwise interactions using the yeast two-hybrid system. The fidelity of the system was established by its ability to closely reproduce the already documented interactions among Da, Ac, Sc, and Extramacrochaetae. We show that the seven E(spl) basic HLH proteins can form homo- and heterodimers inter-se with distinct preferences. We further show that a subset of E(spl) proteins can heterodimerize with Da, another subset can heterodimerize with proneural proteins, and yet another with both, indicating specialization within the E(spl) family. Hairy displays no interactions with any of the HLH proteins tested. It does interact with the non-HLH protein Groucho, which itself interacts with all E(spl) basic HLH proteins, but with none of the proneural proteins or Da. We investigated the structural requirements for some of these interactions by site-specific and deletion mutagenesis.

Figure 1

Examples of Drosophila HLH proteins. Schematic depiction of Drosophila HLH proteins belonging to different classes. Da is an E-protein and Sc is an activator, encoded by a gene in the ASC. Emc is a negative regulator that lacks a basic domain. Hairy and E(spl)Mγ are two members of the Hairy/E(spl) class of negative regulators. Structural motifs are marked: b, basic domain; HLH, helix-loop-helix domain; LZ, putative leucine zipper; AD, conserved acidic domain among the ASC proteins; Or, Orange domain; and W, terminal WRPW tetrapeptide conserved among Hairy/E(spl) class members.

Figure 2

Western blot analysis of fusion constructs. (A) Extracts from LexA construct bearing cells probed with an anti-LexA antibody. (B) B42 extracts probed with an anti-HA epitope tag antibody. ΔW: B42-m5ΔW. Sizes of markers in kDa are indicated on the side of each blot. Proteins of approximately expected sizes were observed as the major bands.

Figure 3

Summary of interactions. Interactions are indicated by arrows connecting the proteins involved. Proteins grouped together display qualitatively identical interactions with other proteins in this study. ProN, proneural proteins, typified by Ac and Sc. All E(spl) proteins (in box) interact with each other and with Gro. Thick arrows in the box indicate the strongest interactions within the E(spl) family.