Interactions among the bHLH domains of the proteins encoded by theEnhancer of split andachaete-scute gene complexes ofDrosophila

TheEnhancer of split andachaete-scute gene complexes [E(spl)-C and AS-C] encode helix-loop-helix proteins required for neurogenesis inDrosophila. Using a heterologous bacterial system, we show that (i) the bHLH domains of the proteins encoded by the two gene complexes differ in their ability to form homo- and/or heterodimers; (ii) the bHLH domains of the E(spl)-C proteins m5, m7 and m8 interact with the bHLH domains of the Ac and Sc proteins. These bHLH domains form an interaction network which may represent the molecular mechanism whereby the competent state of the proneural cells is maintained until the terminal determination to neuroblast occurs. Also, the pattern of interactions of the bHLH domains of certain proteins encoded by the two gene complexes may explain their functional redundancy.     

A functional analysis of the genes Enhancer of split and HLH-m5 during early neurogenesis in Drosophila melanogaster

To assess the functional domains of the proteins encoded by E(spl) and HLH-m5, two genes of the Enhancer of split complex [E(SPL)-C] of Drosophila melanogaster, a number of variants have been made by in vitro mutagenesis, transformed into the germ line of the wild-type, and genetically combined with a chromosomal deletion lacking four of the genes of the E(SPL)-C. All constructs used attenuated the neurogenic phenotype associated with this deletion. However, constructs encoding proteins with truncated carboxy-termini exibited in all cases a higher activity than constructs encoding the full length version of the protein. Neutralization of the basic domain severely reduced, but did not completely abolish the rescuing activity of E(spl), while proteins in which a proline residue within the basic domain had been changed to either threonine or asparagine were slightly less efficient in their rescuing activity than the corresponding wild-type versions. We discuss the possible significance of these results for the function of the protein domains.     

Substitution of basic amino acids in the basic region stabilizes DNA binding by E12 homodimers.

The E2A gene encodes two alternatively spliced products, E12 and E47. The two proteins differ in their basic helix-loop-helix motifs (bHLH), responsible for DNA binding and dimerization. Although both E12 and E47 can bind to DNA as heterodimers with tissue-specific bHLH proteins, E12 binds to DNA poorly as homodimers. An inhibitory domain in E12 has previously been found to prevent E12 homodimers from binding to DNA. By measuring the dissociation rates using filter binding and electrophoretic mobility shift assays, we have shown here that the inhibitory domain interferes with DNA binding by destabilizing the DNA-protein complexes. Furthermore, we have demonstrated that substitution of basic amino acids (not other amino acids) in the DNA-binding domain of E12 can increase the intrinsic DNA-binding activity of E12 and stabilize the binding complexes, thus alleviating the repression from the inhibitory domain. This ability of basic amino acids to stabilize DNA-binding complexes may be of biological significance in the case of myogenic bHLH proteins, which all possess two more basic amino acids in their DNA binding domain than E12. To function as heterodimers with E12, the myogenic bHLH proteins may need stronger DNA binding domains.     

Neural hyperplasia induced by RNA interference with m4/malpha gene activity

The E(spl) complex (E(spl)-C) contains three different classes of genes that are downstream of Notch signaling. The bHLH genes mediate the Notch signal by repressing proneural gene activity, for example during the singularization of mechanosensory organ precursor cells (SOPs). Genes of the second class, the E(spl) m4/malpha family, antagonize this process if overexpressed. Here we show that this is based on dominant-negative effects since RNA interference gives neurogenic phenotypes indistinguishable from E(spl)-C mutations. Furthermore, a third member of the m4/malpha gene family, named bbu/tom, behaves differently with respect to RNA expression patterns, its regulation by Notch signaling and loss of function phenotypes.     

bHLH proteins involved in Drosophila neurogenesis are mutually regulated at the level of stability

Proneural bHLH activators are expressed in all neuroectodermal regions prefiguring events of central and peripheral neurogenesis. Drosophila Sc is a prototypical proneural activator that heterodimerizes with the E-protein Daughterless (Da) and is antagonized by, among others, the E(spl) repressors. We determined parameters that regulate Sc stability in Drosophila S2 cells. We found that Sc is a very labile phosphoprotein and its turnover takes place via at least three proteasome-dependent mechanisms. (i) When Sc is in excess of Da, its degradation is promoted via its transactivation domain (TAD). (ii) In a DNA-bound Da/Sc heterodimer, Sc degradation is promoted via an SPTSS phosphorylation motif and the AD1 TAD of Da; Da is spared in the process. (iii) When E(spl)m7 is expressed, it complexes with Sc or Da/Sc and promotes their degradation in a manner that requires the corepressor Groucho and the Sc SPTSS motif. Da/Sc reciprocally promotes E(spl)m7 degradation. Since E(spl)m7 is a direct target of Notch, the mutual destabilization of Sc and E(spl) may contribute in part to the highly conserved anti-neural activity of Notch. Sc variants lacking the SPTSS motif are dramatically stabilized and are hyperactive in transgenic flies. Our results propose a novel mechanism of regulation of neurogenesis, involving the stability of key players in the process.     

The HLH protein Extramacrochaetae is required for R7 cell and cone cell fates in the Drosophila eye

Notch signaling is one of the most important pathways in development and homeostasis, and is altered in multiple pathologies. Study of Drosophila eye development shows that Notch signaling depends on the HLH protein Extramacrochaetae. Null mutant clones show that extramacrochaetae is required for multiple aspects of eye development that depend on Notch signaling, including morphogenetic furrow progression, differentiation of R4, R7 and cone cell types, and rotation of ommatidial clusters. Detailed analysis of R7 and cone cell specification reveals that extramacrochaetae acts cell autonomously and epistatically to Notch, and is required for normal expression of bHLH genes encoded by the E(spl)-C which are effectors of most Notch signaling. A model is proposed in which Extramacrochaetae acts in parallel to or as a feed-forward regulator of the E(spl)-Complex to promote Notch signaling in particular cellular contexts.     

The <Emphasis Type="Italic">Enhancer of split</Emphasis> and <Emphasis Type="Italic">Achaete-Scute</Emphasis> complexes of Drosophilids derived from simple ur-complexes preserved in mosquito and honeybee

Background  

In Drosophila melanogaster the Enhancer of split-Complex [E(spl)-C] consists of seven highly related genes encoding basic helix-loop-helix (bHLH) repressors and intermingled, four genes that belong to the Bearded (Brd) family. Both gene classes are targets of the Notch signalling pathway. The Achaete-Scute-Complex [AS-C] comprises four genes encoding bHLH activators. The question arose how these complexes evolved with regard to gene number in the evolution of insects concentrating on Diptera and the Hymenoptera Apis mellifera.     

The two Tribolium E(spl) genes show evolutionarily conserved expression and function during embryonic neurogenesis

Tribolium castaneum is a well-characterised model insect, whose short germ-band mode of embryonic development is characteristic of many insect species and differs from the exhaustively studied Drosophila. Mechanisms of early neurogenesis, however, show significant conservation with Drosophila, as a characteristic pattern of neuroblasts arises from neuroectoderm proneural clusters in response to the bHLH activator Ash, a homologue of Achaete–Scute. Here we study the expression and function of two other bHLH proteins, the bHLH-O repressors E(spl)1 and E(spl)3. Their Drosophila homologues are expressed in response to Notch signalling and antagonize the activity of Achaete–Scute proteins, thus restricting the number of nascent neuroblasts. E(spl)1 and 3 are the only E(spl) homologues in Tribolium and both show expression in the cephalic and ventral neuroectoderm during embryonic neurogenesis, as well as a dynamic pattern of expression in other tissues. Their expression starts early, soon after Ash expression and is dependent on both Ash and Notch activities. They act redundantly, since a double E(spl) knockdown (but not single knockdowns) results in neurogenesis defects similar to those caused by Notch loss-of-function. A number of other activities have been evolutionarily conserved, most notably their ability to interact with proneural proteins Scute and Daughterless.