Search

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The Notch signaling pathway is instrumental for cell fate decisions. Signals from the Notch receptor are transduced by CSL-type DNA-binding proteins. In Drosophila, this protein is named Suppressor of Hairless [Su(H)]. Together with the intracellular domain of the activated Notch receptor ICN, Su(H) assembles a transcriptional activator complex on Notch target genes. Hairless acts as the major antagonist of the Notch signaling pathway in Drosophila by means of the formation of a repressor complex together with Su(H) and several co-repressors. Su(H) is characterized by three domains, the N-terminal domain NTD, the beta-trefoil domain BTD and the C-terminal domain CTD. NTD and BTD bind to the DNA, whereas BTD and CTD bind to ICN. Hairless binds to the CTD, however, to sites different from ICN. In this work, we have addressed the question of competition and availability of Su(H) for ICN and Hairless binding in vivo. To this end, we overexpressed the CTD during fly development. We observed a strong activation of Notch signaling processes in various tissues, which may be explained by an interference of CTD with Hairless corepressor activity. Accordingly, a combined overexpression of CTD together with Hairless ameliorated the effects, unlike Su(H) which strongly enhances repression when overexpressed concomitantly with Hairless. Interestingly, in the combined overexpression CTD accumulated in the nucleus together with Hairless, whereas it is predominantly cytoplasmic on its own. [ABSTRACT FROM AUTHOR]