Constitutively active human Notch1 binds to the transcription factor CBF1 and stimulates transcription through a promoter containing a CBF1-responsive element.
Notch is a transmembrane receptor that plays a critical role in cell fate determination. In Drosophila, Notch binds to and signals through Suppressor of Hairless. A mammalian homologue of Suppressor of Hairless, named CBF1 (or RBPJk), is a ubiquitous transcription factor whose function in mammalian Notch signaling is unknown. To determine whether mammalian Notch can stimulate transcription through a CBF1-responsive element (RE), we cotransfected a CBF1-RE-containing chloramphenicol acetyltransferase reporter and N1(deltaEC), a constitutively active form of human Notch1 lacking the extracellular domain, into DG75, COS-1, HeLa, and 293T cells, which all contain endogenous CBF1. N1(deltaEC) dramatically increased chloramphenicol acetyltransferase activity in these cells, indicating functional coupling of Notch1 and CBF1. The activity was comparable to that produced by the Epstein-Barr virus protein EBNA2, a well-characterized, potent transactivator of CBF1. To test whether CBF1 and Notch1 interact physically, we tagged CBF1 with an epitope from the influenza virus hemagglutinin or with the N-terminal domain of gal4, and transfected the tagged CBF1 plus N1(deltaEC) into COS-1 cells. Cell lysates were immunoprecipitated and immunoblotted with several anti-Notch1 antibodies [to detect N1(deltaEC)] or with antibodies to hemagglutinin or gal4 (to detect CBF1). Each immunoprecipitate contained a complex of N1(deltaEC) and CBF1. In summary, we find that the truncated, active form of human Notch1, N1(deltaEC), binds CBF1 and activates transcription through a CBF1-RE-containing promoter. We conclude that CBF1 is a critical downstream protein in the human Notch1 signaling pathway.
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Division of Hematology/Oncology, Children's Hospital, Boston, MA 02115, USA.
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D000818:Animals; D000956:Antigens, Viral / Q000096:biosynthesis / Q000378:metabolism; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001665:Binding Sites; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D004330:Drosophila; D029721:Drosophila Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006367:HeLa Cells; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000096:biosynthesis / Q000378:metabolism; D006801:Humans; D008322:Mammals; D008565:Membrane Proteins / Q000096:biosynthesis / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D051881:Receptor, Notch1; D011956:Receptors, Cell Surface; D051880:Receptors, Notch; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D016385:TATA Box; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D000956:Antigens, Viral; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C064042:CBF1 protein, S cerevisiae; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; D005656:Fungal Proteins; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D008565:Membrane Proteins; C497759:N protein, Drosophila; C497761:NOTCH1 protein, human; D051881:Receptor, Notch1; D011956:Receptors, Cell Surface; D051880:Receptors, Notch; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D015500:Chloramphenicol O-Acetyltransferase