Molecular Analysis of the Notch Repressor-Complex in Drosophila: Characterization of Potential Hairless Binding Sites on Suppressor of Hairless

Abstract

The Notch signalling pathway mediates cell-cell communication in a wide variety of organisms. The major components, as well as the basic mechanisms of Notch signal transduction, are remarkably well conserved amongst vertebrates and invertebrates. Notch signalling results in transcriptional activation of Notch target genes, which is mediated by an activator complex composed of the DNA binding protein CSL, the intracellular domain of the Notch receptor, and the transcriptional coactivator Mastermind. In the absence of active signalling, CSL represses transcription from Notch target genes by the recruitment of corepressors. The Notch activator complex is extremely well conserved and has been studied in great detail. However, Notch repressor complexes are far less understood. In Drosophila melanogaster, the CSL protein is termed Suppressor of Hairless [Su(H)]. Su(H) functions as a transcriptional repressor by binding Hairless, the major antagonist of Notch signalling in Drosophila, which in turn recruits two general corepressors – Groucho and C-terminal binding protein CtBP. Recently, we determined that the C-terminal domain (CTD) of Su(H) binds Hairless and identified a single site in Hairless, which is essential for contacting Su(H). Here we present additional biochemical and in vivo studies aimed at mapping the residues in Su(H) that contact Hairless. Focusing on surface exposed residues in the CTD, we identified two sites that affect Hairless binding in biochemical assays. Mutation of these sites neither affects binding to DNA nor to Notch. Subsequently, these Su(H) mutants were found to function normally in cellular and in vivo assays using transgenic flies. However, these experiments rely on Su(H) overexpression, which does not allow for detection of quantitative or subtle differences in activity. We discuss the implications of our results.

MAPK-dependent phosphorylation modulates the activity of Suppressor of Hairless in Drosophila

“…1A). These mutations should not affect the DNA binding of Su(H), which involves the NTD and BTD but not the CTD [7], which was confirmed by electro-mobility shift experiments [29,30]: DNA binding or the affinity to the binding site was indistinguishable between wild-type and the Su(H) MAPK-ko or Su(H) MAPK-ac variants (Supplemental Fig. S1A, B).…”

“…As a consequence, the phosphorylation of Su(H) would require higher levels of activated Notch for target gene activation. To date, information on the exact interaction face of the Hairless-Su(H) binding is scarce, leaving the possibility of such a scenario open [30,39].…”

“…DNA binding of Su(H) protein variants was tested by electromobility shift assays using the m8-S1 oligo that contains a single Su(H) binding site as described earlier [29,30]. Su(H) proteins were produced by in vitro translation using the TNT®Coupled Reticulocyte Lysate System (Promega).…”

Structural and functional studies of the RBPJ-SHARP complex reveal conserved corepressor binding site

“…The corepressor Hairless is the major antagonist of Notch signaling in Drosophila and directly binds the CTD of Su(H) (fly CSL ortholog) with high affinity in order to repress Notch target gene transcription in flies 18,34,35 . While there is no sequence similarity between SHARP and Hairless, these two corepressors have evolved to bind the same CTD interface on RBPJ and Su(H) (Figure 3C).…”

“…This activity of CSL is required at all Notch target genes and is conserved in all metazoans. CSL can also function as a transcriptional repressor, by interacting with corepressors such as SHARP 8,9,27 , L3MBTL3 12 , FHL1 11,19 , and RITA1 13,14 in mammals, and Hairless in Drosophila 18,34,35 ; however, its role as a repressor, in particular in mammals, is not well understood. In order to begin to address this gap in our understanding, here we determine the X-ray structure of the RBPJ-SHARP corepressor complex bound to DNA and use a multitude of in vitro and cellular assays to characterize structure based RBPJ and SHARP mutants in order to better understand CSL-corepressor function.…”