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

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.

果蝇的神经命运特化受到原神经基因（proneural genes）的产物所促进，例如achaete-scute复合体（achaete–scute complex）的编码产物，同时被分裂增强子复合体（Enhancer of split, E(spl)）、hairy基因（hairy）以及extramacrochaetae基因（extramacrochaetae）的产物所拮抗。由于上述所有蛋白均携带螺旋-环-螺旋（helix-loop-helix, HLH）二聚化结构域，本研究采用酵母双杂交系统（yeast two-hybrid system）探究了它们之间潜在的两两相互作用。该系统的可靠性通过其能够精准复现已被报道的Da（Daughterless）、无刚毛蛋白Ac（Achaete）、刚毛蛋白Sc（Scute）与Extramacrochaetae蛋白（extramacrochaetae）之间的相互作用得以验证。本研究证实，7种E(spl)碱性HLH蛋白之间可形成同源或异源二聚体，且具有不同的结合偏好性。进一步研究发现，E(spl)蛋白家族中的一个亚类可与Da形成异源二聚体，另一个亚类可与原神经蛋白形成异源二聚体，还有一个亚类可同时与二者结合，这表明E(spl)家族内部存在功能特化。Hairy蛋白（Hairy）与本次检测的所有HLH蛋白均无相互作用，但可与非HLH蛋白Groucho蛋白（Groucho）结合；Groucho蛋白本身可与所有E(spl)碱性HLH蛋白结合，但无法与任何原神经蛋白或Da结合。本研究通过定点诱变与缺失突变（site-specific and deletion mutagenesis）技术，探究了部分相互作用的结构基础。