Characterization of CSL (CBF-1, Su(H), Lag-1) mutants reveals differences in signaling mediated by Notch1 and Notch2.
Notch is a conserved signaling pathway that plays essential roles during embryonic development and postnatally in adult tissues; misregulated signaling results in human disease. Notch receptor-ligand interactions trigger cleavage of the Notch receptor and release of its intracellular domain (NICD) from the membrane. NICD localizes to the nucleus where it forms a transcriptionally active complex with the DNA-binding protein CSL and the coactivator Mastermind (MAM) to up-regulate transcription from Notch target genes. Previous studies have determined the structure of the CSL-NICD-MAM ternary complex and characterized mutations that affect complex assembly in functional assays. However, as CSL is expressed in all cell types, these studies have been limited to analyzing mutations in NICD and MAM. Here, we describe a novel set of cellular reagents to characterize how mutations in CSL affect its function as a transcriptional activator. Using retrovirally transduced embryonic fibroblasts from a CSL-null mouse, we generated cell lines that express either wild-type or mutant CSL molecules. We then analyzed these mutants for defects in Notch1- (NICD1) or Notch2 (NICD2)-mediated activation from two different transcriptional reporters (HES-1 or 4×CBS). Our results show that mutations targeted to the different domains of CSL display significant differences in their ability to adversely affect transcription from the two reporters. Additionally, a subset of CSL mutants is sensitive to whether NICD1 or NICD2 was used to activate the reporter. Taken together, these studies provide important molecular insights into how Notch transcription complexes assemble at different target genes and promoter arrangements in vivo.
Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, Ohio 45267.
Zhenyu
Z
Yuan
The Wistar Institute, Philadelphia, Pennsylvania 19104.
David R
DR
Friedmann
Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, Ohio 45267.
Bradley D
BD
VanderWielen
Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, Ohio 45267.
Kelly J
KJ
Collins
Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, Ohio 45267. Electronic address: kovallra@ucmail.uc.edu.
Rhett A
RA
Kovall
D000818:Animals; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation; D051881:Receptor, Notch1 / Q000235:genetics / Q000378:metabolism*; D051883:Receptor, Notch2 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics; D015854:Up-Regulation / Q000235:genetics
D004268:DNA-Binding Proteins; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; C430872:MAML1 protein, human; C497761:NOTCH1 protein, human; C497764:NOTCH2 protein, human; C497762:Notch1 protein, mouse; C497765:Notch2 protein, mouse; C497570:RBPJ protein, human; C497571:Rbpj protein, mouse; D051881:Receptor, Notch1; D051883:Receptor, Notch2; D014157:Transcription Factors