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.
Subject(s)
Drosophila Proteins/genetics , Receptors, Notch/genetics , Repressor Proteins/genetics , Transcription Factors/genetics , Amino Acid Sequence , Animals , Animals, Genetically Modified , Binding Sites/genetics , Cells, Cultured , Drosophila Proteins/chemistry , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Eye/growth & development , Eye/metabolism , Female , Gene Expression Regulation, Developmental , Male , Models, Molecular , Molecular Sequence Data , Mutation , Protein Binding , Protein Interaction Domains and Motifs/genetics , Protein Interaction Mapping , Protein Structure, Tertiary , Receptors, Notch/metabolism , Repressor Proteins/chemistry , Repressor Proteins/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolism , Two-Hybrid System TechniquesABSTRACT
In metazoans, the highly conserved Notch pathway drives cellular specification. On receptor activation, the intracellular domain of Notch assembles a transcriptional activator complex that includes the DNA-binding protein CSL, a composite of human C-promoter binding factor 1, Suppressor of Hairless of Drosophila melanogaster [Su(H)], and lin-12 and Glp-1 phenotype of Caenorhabditis elegans. In the absence of ligand, CSL represses Notch target genes. However, despite the structural similarity of CSL orthologues, repression appears largely diverse between organisms. Here we analyze the Notch repressor complex in Drosophila, consisting of the fly CSL protein, Su(H), and the corepressor Hairless, which recruits general repressor proteins. We show that the C-terminal domain of Su(H) is necessary and sufficient for forming a high-affinity complex with Hairless. Mutations in Su(H) that affect interactions with Notch and Mastermind have no effect on Hairless binding. Nonetheless, we demonstrate that Notch and Hairless compete for CSL in vitro and in cell culture. In addition, we identify a site in Hairless that is crucial for binding Su(H) and subsequently show that this Hairless mutant is strongly impaired, failing to properly assemble the repressor complex in vivo. Finally, we demonstrate Hairless-mediated inhibition of Notch signaling in a cell culture assay, which hints at a potentially similar repression mechanism in mammals that might be exploited for therapeutic purposes.
Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Receptors, Notch/metabolism , Repressor Proteins/metabolism , Amino Acid Sequence , Animals , Caenorhabditis elegans Proteins/chemistry , Cells, Cultured , DNA-Binding Proteins/chemistry , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Drosophila melanogaster/growth & development , Drosophila melanogaster/metabolism , Gene Expression Regulation , Immunoglobulin J Recombination Signal Sequence-Binding Protein/chemistry , Larva/genetics , Larva/growth & development , Larva/metabolism , Molecular Sequence Data , Protein Binding , Protein Interaction Domains and Motifs , Receptors, Notch/chemistry , Receptors, Notch/genetics , Repressor Proteins/chemistry , Sequence Deletion , Thermodynamics , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptional Activation , Two-Hybrid System TechniquesABSTRACT
Platelet-derived growth factors (PDGF) and their receptors control cell proliferation, survival, and migration. To test the influence of an oncogenic mutation to embryonic development, a transgenic mouse line expressing PDGFRalpha (D842V) was established and analyzed. Most of the embryos die on embryonic day 12.5 due to massive hemorrhages in the trunk. In mesenchymal cells of mutant animals, proliferation is decreased while apoptosis is increased. Further analyses reveal that the aortic blood vessels are enlarged showing a reduced numbers of vascular smooth muscle cells (vSMC) around the aorta. We hypothesize that the process of aortic wall formation is impaired, leading to subsequent rupture and leakage of the blood vessel resulting in death of the embryos. We speculate that misexpression of PDGFRalpha in SMCs causes failure of vSMC recruitment to the aorta.
