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The CSL family protein CBF1 is a nuclear mediator of Notch signaling and has been predicted to contain an N-terminal nuclear localization signal in exon 4. Surprisingly, we found that CBF1 carrying mutations at codon 233 or 249 within exon 7 was restricted to the cytoplasm. In mammalian and yeast two-hybrid assays, these mutations were also associated with a loss of CBF1-mediated transcriptional repression and a severely impaired interaction with the corepressors SMRT and CIR. Overexpression of SMRT rescued the ability of mutant CBF1 to target to the nucleus of transfected cells and similarly rescued nuclear targeting of enhanced green fluorescent protein (EGFP)-CBF1 exons 6 to 9 CBF1(6-9)carrying the codon 233 or 249 mutations. Carboxy-terminally truncated SMRT with amino acids (aa) 1291 to 1495 deleted was unable to rescue the nuclear targeting of mutant EGFP-CBF1(6-9). In yeast two-hybrid assays, the SMRT aa 1291 to 1495 domain interacted with SKIP and SMRT aa 1291 to 1495 colocalized with SKIP within the nuclei of cotransfected cells. Comparison of the intracellular localization of CBF1(6-9) with that of CBF1(5-9) further supported the suggestion that nuclear targeting of CBF1 is dependent on the formation of a CBF1-SMRT-SKIP corepressor complex. These observations suggest that nuclear targeting of CBF1 is itself a component of CBF1-mediated gene regulation and that in the absence of signaling, CBF1 enters the nucleus precommitted to a transcriptional repression function. The activators NotchIC (the intracellular domain of Notch) and Epstein-Barr virus EBNA2 also mediated nuclear targeting of mutant CBF1, consistent with the competition model for activator versus corepressor binding to CBF1.

Notch proteins are transmembrane receptors that mediate intercell communication and direct individual cell fate decisions. The activated intracellular form of Notch, NotchIC, translocates to the nucleus, where it targets the DNA binding protein CBF1. CBF1 mediates transcriptional repression through the recruitment of an SMRT-histone deacetylase-containing corepressor complex. We have examined the mechanism whereby NotchIC overcomes CBF1-mediated transcriptional repression. We identified SKIP (Ski-interacting protein) as a CBF1 binding protein in a yeast two-hybrid screen. Both CBF1 and SKIP are highly conserved evolutionarily, and the SKIP-CBF1 interaction is also conserved in assays using the Caenorhabditis elegans and Drosophila melanogaster SKIP homologs. Protein-protein interaction assays demonstrated interaction between SKIP and the corepressor SMRT. More surprisingly, SKIP also interacted with NotchIC. The SMRT and NotchIC interactions were mutually exclusive. In competition binding experiments SMRT displaced NotchIC from CBF1 and from SKIP. Contact with SKIP is required for biological activity of NotchIC. A mutation in the fourth ankyrin repeat that abolished Notch signal transduction did not affect interaction with CBF1 but abolished interaction with SKIP. Further, NotchIC was unable to block muscle cell differentiation in myoblasts expressing antisense SKIP. The results suggest a model in which NotchIC activates responsive promoters by competing with the SMRT-corepressor complex for contacts on both CBF1 and SKIP.

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.

The Epstein-Barr Virus (EBV) latency C promoter (Cp) is the origin of transcripts for six viral proteins. The promoter is active in lymphoblastoid B-cell lines but silent in many EBV-associated tumors and tumor cell lines. In these latter cell lines, the viral episome is hypermethylated in the vicinity of this promoter. We show that in such a cell line (Rael, a Burkitt's lymphoma line), 5-azacytidine inhibits DNA methyltransferase, brings about demethylation of EBV genomes, activates Cp transcription, and induces the expression of EBNA-2. Investigation of the phenomenon demonstrates the importance of the methylation status of a particular CpG site for the regulation of the Cp: (i) genomic sequencing shows that this site is methylated when the Cp is inactive and is not methylated when the promoter is active; (ii) methylation or transition mutation at this site abolishes complex formation with a cellular binding activity (CBF2) as determined by electrophoretic mobility shift analyses, competition binding analyses, and DNase I footprinting; and (iii) a single C --> T transition mutation at this site is associated with a marked reduction (> 50-fold) of transcriptional activity in a reporter plasmid. Thus, the CBF2 binding activity is shown to be methylation sensitive and crucial to EBNA-2-mediated activation of the Cp.