Truncated mammalian Notch1 activates CBF1/RBPJk-repressed genes by a mechanism resembling that of Epstein-Barr virus EBNA2.
The Notch/Lin-12/Glp-1 receptor family participates in cell-cell signaling events that influence cell fate decisions. Although several Notch homologs and receptor ligands have been identified, the nuclear events involved in this pathway remain incompletely understood. A truncated form of Notch, consisting only of the intracellular domain (NotchIC), localizes to the nucleus and functions as an activated receptor. Using both an in vitro binding assay and a cotransfection assay based on the two-hybrid principle, we show that mammalian NotchIC interacts with the transcriptional repressor CBF1, which is the human homolog of Drosophila Suppressor of Hairless. Cotransfection assays using segments of mouse NotchIC and CBF1 demonstrated that the N-terminal 114-amino-acid region of mouse NotchIC contains the CBF1 interactive domain and that the cdc10/ankyrin repeats are not essential for this interaction. This result was confirmed in immunoprecipation assays in which the N-terminal 114-amino-acid segment of NotchIC, but not the ankyrin repeat region, coprecipitated with CBF1. Mouse NotchIC itself is targeted to the transcriptional repression domain (aa179 to 361) of CBF1. Furthermore, transfection assays in which mouse NotchIC was targeted through Gal4-CBF1 or through endogenous cellular CBF1 indicated that NotchIC transactivates gene expression via CBF1 tethering to DNA. Transactivation by NotchIC occurs partially through abolition of CBF1-mediated repession. This same mechanism is used by Epstein-Barr virus EBNA2. Thus, mimicry of Notch signal transduction is involved in Epstein-Barr virus-driven immortalization.
Hsieh
J J
JJ
Department of Pharmacology and Molecular Sciences, The Johns Hopkins University of Medicine, Baltimore, Maryland 21205, USA.
Henkel
T
T
Salmon
P
P
Robey
E
E
Peterson
M G
MG
Hayward
S D
SD
D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D029721:Drosophila Proteins; D004331:Drosophila melanogaster; D019309:Epstein-Barr Virus Nuclear Antigens; D005656:Fungal Proteins / Q000235:genetics*; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D051880:Receptors, Notch; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D015398:Signal Transduction
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; D008565:Membrane Proteins; C497759:N protein, Drosophila; D051880:Receptors, Notch; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins