Krüppel (Kr), a segmentation gene of Drosophila, encodes a protein sharing structural features of the DNA-binding "finger motif" of TFIIIA, a Xenopus transcription factor. Low-stringency hybridization of the Kr finger coding sequence revealed multiple copies of homologous DNA sequences in the genomes of Drosophila and other eukaryotes. Molecular analysis of one Kr-homologous DNA clone identified a developmentally regulated gene. Its product, a finger protein, relates to Kr by the invariant positioning of crucial amino acid residues within the finger repeats and by a stretch of seven amino acids connecting the finger loops, the "H/C link." This H/C link is conserved in several nuclear and chromosome-associated proteins of Drosophila and other eukaryotic organisms including mammals. Our results demonstrate a new subfamily of evolutionarily conserved nuclear and possibly DNA-binding proteins that again relate to a Drosophila segmentation gene as in the case of the homeo domain.

Despite high conservation of the Notch pathway, its repression appears diverse between organisms. In Drosophila, a high-affinity complex forms between the CSL orthologue Su(H) and Hairless, which is analyzed in great detail in vitro and in vivo. Drosophila Hairless is shown to bind CBF1 and inhibit Notch transcriptional output in mammalian cells.

No abstract

Notch is the receptor for a conserved signaling pathway that regulates numerous cell fate decisions during development [1]. Signal transduction involves the presenilin-dependent intracellular processing of Notch and the nuclear translocation of the intracellular domain of Notch, NICD [2-6]. NICD associates with Suppressor of Hairless [Su(H)], a DNA binding protein, and Mastermind (Mam), a transcriptional coactivator [7-9]. In the absence of Notch signaling, Su(H) acts as a transcriptional repressor [10, 11]. Repression by Su(H) is relieved by the activation of Notch [12-16]. In the Drosophila embryo, this transcriptional switch from repression to activation is important for patterning the expression of the single-minded (sim) gene along the dorsoventral axis [12]. Here, we investigate the mechanisms by which Su(H) inhibits the expression of Notch target genes in Drosophila. We show that Hairless, an antagonist of Notch signaling [17-19], is required to repress the transcription of the sim gene. Hairless forms a DNA-bound complex with Su(H). Furthermore, it directly binds the Drosophila C-terminal Binding Protein (dCtBP), which acts as a transcriptional corepressor. The dCtBP binding motif of Hairless is essential for the function of Hairless in vivo. We propose that Hairless mediates transcriptional repression by Su(H) via the recruitment of dCtBP.

Notch is a conserved signaling pathway that specifies cell fates in metazoans. Receptor-ligand interactions induce changes in gene expression, which is regulated by the transcription factor CBF1/Su(H)/Lag-1 (CSL). CSL interacts with coregulators to repress and activate transcription from Notch target genes. While the molecular details of the activator complex are relatively well understood, the structure-function of CSL-mediated repressor complexes is poorly defined. In Drosophila, the antagonist Hairless directly binds Su(H) (the fly CSL ortholog) to repress transcription from Notch targets. Here, we determine the X-ray structure of the Su(H)-Hairless complex bound to DNA. Hairless binding produces a large conformational change in Su(H) by interacting with residues in the hydrophobic core of Su(H), illustrating the structural plasticity of CSL molecules to interact with different binding partners. Based on the structure, we designed mutants in Hairless and Su(H) that affect binding, but do not affect formation of the activator complex. These mutants were validated in vitro by isothermal titration calorimetry and yeast two- and three-hybrid assays. Moreover, these mutants allowed us to solely characterize the repressor function of Su(H) in vivo.

The Notch signalling pathway regulates cell-cell communication in higher eukaryotes. Cellular differentiation and tissue development relies on correct intercellular communication, accounting for the high interest in the Notch signalling pathway. Together with mastermind and CSL (CBF-1, Suppressor of Hairless, lag-2) DNA-binding proteins, Notch forms a complex that mediates transcriptional activation of the respective target genes. This activation is strictly controlled, and deregulation causes extreme developmental defects. In Drosophila, the stringency of the control system is given by the general Notch-antagonist Hairless. Hairless assembles in a repressor complex on Notch target genes, which involves Suppressor of Hairless and two corepressors, Groucho and C-terminal binding protein. In mammals, CBF-1 recruits corepressors on its own. In addition Hairless recruits also other proteins. One example is the Pros26.4 AAA-ATPase which specifically destabilises Hairless resulting in a novel positive regulation of Notch signalling. By inhibition of Notch, Hairless not only regulates cellular differentiation but also has anti-apoptotic functions. Moreover, many genetic interactions imply a cross-talk between Hairless and the EGF-receptor pathway, which might act independently of Notch. Surprisingly, no Hairless homologue has been identified in mammals so far, despite the high degree of conservation of other components of the pathway. This discrepancy might be resolved in the future, once all components of the repressor-complex in the different species have been identified. In conclusion, Hairless is a central component of the regulation of the Notch signalling pathway in Drosophila, and is hence essential for cell differentiation and tissue development in the fly.

Cell fate choices during metazoan development are driven by the highly conserved Notch signalling pathway. Notch receptor activation results in release of the Notch intracellular domain (NICD) that acts as transcriptional co-activator of the DNA-binding protein CSL. In the absence of signal, a repressor complex consisting of CSL bound to co-repressors silences Notch target genes. The Drosophila repressor complex contains the fly CSL orthologue Suppressor of Hairless [Su(H)] and Hairless (H). The Su(H)-H crystal structure revealed a large conformational change within Su(H) upon H binding, precluding interactions with NICD. Based on the structure, several sites in Su(H) and H were determined to specifically engage in complex formation. In particular, three mutations in Su(H) were identified that affect interactions with the repressor H but not the activator NICD. To analyse the effects these mutants have on normal fly development, we introduced these mutations into the native Su(H) locus by genome engineering. We show that the three H-binding deficient Su(H) alleles behave similarly. As these mutants lack the ability to form the repressor complex, Notch signalling activity is strongly increased in homozygotes, comparable to a complete loss of H activity. Unexpectedly, we find that the abundance of the three mutant Su(H) protein variants is altered, as is that of wild type Su(H) protein in the absence of H protein. In the presence of NICD, however, Su(H) mutant protein persists. Apparently, Su(H) protein levels depend on the interactions with H as well as with NICD. Based on these results, we propose that in vivo levels of Su(H) protein are stabilised by interactions with transcription-regulator complexes.

Background Polycomb (PcG) and trithorax (trxG) genes encode proteins involved in the maintenance of gene expression patterns, notably Hox genes, throughout development. PcG proteins are required for long-term gene repression whereas TrxG proteins are positive regulators that counteract PcG action. PcG and TrxG proteins form large complexes that bind chromatin at overlapping sites called Polycomb and Trithorax Response Elements (PRE/TRE). A third class of proteins, so-called “Enhancers of Trithorax and Polycomb” (ETP), interacts with either complexes, behaving sometimes as repressors and sometimes as activators. The role of ETP proteins is largely unknown.Methodology/Principal FindingsIn a two-hybrid screen, we identified Cyclin G (CycG) as a partner of the Drosophila ETP Corto. Inactivation of CycG by RNA interference highlights its essential role during development. We show here that Corto and CycG directly interact and bind to each other in embryos and S2 cells. Moreover, CycG is targeted to polytene chromosomes where it co-localizes at multiple sites with Corto and with the PcG factor Polyhomeotic (PH). We observed that corto is involved in maintaining Abd-B repression outside its normal expression domain in embryos. This could be achieved by association between Corto and CycG since both proteins bind the regulatory element iab-7 PRE and the promoter of the Abd-B gene.Conclusions/SignificanceOur results suggest that CycG could regulate the activity of Corto at chromatin and thus be involved in changing Corto from an Enhancer of TrxG into an Enhancer of PcG.