The decision of ectodermal cells to adopt the sensory organ precursor fate in Drosophila is controlled by two classes of basic-helix-loop-helix transcription factors: the proneural Ac and Sc activators promote neural fate, whereas the E(spl) repressors suppress it. We show here that E(spl) proteins m7 and mγ are potent inhibitors of neural fate, even in the presence of excess Sc activity and even when their DNA-binding basic domain has been inactivated. Furthermore, these E(spl) proteins can efficiently repress target genes that lack cognate DNA binding sites, as long as these genes are bound by Ac/Sc activators. This activity of E(spl)m7 and mγ correlates with their ability to interact with proneural activators, through which they are probably tethered on target enhancers. Analysis of reporter genes and sensory organ (bristle) patterns reveals that, in addition to this indirect recruitment of E(spl) onto enhancers via protein-protein interaction with bound Ac/Sc factors, direct DNA binding of target genes by E(spl) also takes place. Irrespective of whether E(spl) are recruited via direct DNA binding or interaction with proneural proteins, the co-repressor Groucho is always needed for target gene repression.

Neurogenesis in all animals is triggered by the activity of a group of basic helix-loop-helix transcription factors, the proneural proteins, whose expression endows ectodermal regions with neural potential. The eventual commitment to a neural precursor fate involves the interplay of these proneural transcriptional activators with a number of other transcription factors that fine tune transcriptional responses at target genes. Most prominent among the factors antagonizing proneural protein activity are the HES basic helix-loop-helix proteins. We have previously shown (1) that two HES proteins of Drosophila, E(spl)m␥ and E(spl)m7, interact with the proneural protein Sc and thereby get recruited onto Sc target genes to repress transcription. Using in vivo and in vitro assays we have now discovered an important dual role for the Sc C-terminal domain. On one hand it acts as a transcription activation domain, and on the other it is used to recruit E(spl) proteins. In vivo, the Sc C-terminal domain is required for E(spl) recruitment in an enhancer context-dependent fashion, suggesting that in some enhancers alternative interaction surfaces can be used to recruit E(spl) proteins. Sc (Scute) and E(spl)m7 (Enhancer of split m7) are two Drosophila bHLH 1 transcription factors, characterized by the basic-helix-loop-helix structural motif, which is responsible for dimerization and DNA binding (2). This large family of transcription factors contains evolutionarily conserved subfamilies, which participate in a variety of biological processes. Sc belongs to the Class II bHLH proteins, more specifically to the achaetescute branch of the proneural family, which also includes vertebrate Ash proteins (achaete-scute homologues). These proteins play a central role in initiating neural development in all metazoans studied so far (3). E(spl)m7, on the other hand, belongs to the HES (Hairy/Enhancer-of-split) family of bHLH proteins, also known as Class VI, which are structurally distinct from the proneural family (4). Although their biological effects encompass a great number of processes, one of their roles is to inhibit neurogenesis; in this context they are expressed downstream of a Notch-mediated signaling pathway termed lateral inhibition (5). HES proteins inhibit neurogenesis by repressing target genes of the proneural proteins or even the expression of proneural genes themselves. Consistent with this biological activity, it has been shown that although proneural proteins are transcriptional activators, HES proteins are repressors. Because of structural differences in their bHLH domains, the two families have distinct target site specificities. Proneural proteins make obligate heterodimers with the ubiquitous bHLH protein Da (Daughterless) and preferentially bind to the so-called E A boxes, CASCTG (6). HES proteins, on the other hand, homodimerize or heterodimerize among themselves and bind preferably to E B or C boxes, CACGTG and CACGCG, respectively (7).A model for neural commitment proposes that genes initiating this process ar...

bHLH-O proteins are a subfamily of the basic-helix-loop-helix transcription factors characterized by an ‘Orange’ protein-protein interaction domain. Typical members are the Hairy/E(spl), or Hes, proteins, well studied in their ability, among others, to suppress neuronal differentiation in both invertebrates and vertebrates. Hes proteins are often effectors of Notch signalling. In vertebrates, another bHLH-O protein group, the Hey proteins, have also been shown to be Notch targets and to interact with Hes. We have studied the single Drosophila Hey orthologue. We show that it is primarily expressed in a subset of newly born neurons, which receive Notch signalling during their birth. Unlike in vertebrates, however, Hey is not expressed in precursor cells and does not block neuronal differentiation. It rather promotes one of two alternative fates that sibling neurons adopt at birth. Although in the majority of cases Hey is a Notch target, it is also expressed independently of Notch in some lineages, most notably the larval mushroom body. The availability of Hey as a Notch readout has allowed us to study Notch signalling during the genesis of secondary neurons in the larval central nervous system.