Epstein-Barr virus immortalization: Notch2 interacts with CBF1 and blocks differentiation

Abstract

EBNA2 is essential for immortalization of B cells by Epstein-Barr virus. EBNA2 is tethered to responsive promoters through a cellular factor, CBF1. CBF1 also binds to the activated form of mammalian Notch1, providing a linkage between EBNA2 function and Notch signalling. However, Notch2 is the predominant form expressed in spleen. The degree to which these Notch homologs are functionally convergent is not known. We present evidence that Notch2 also signals through CBF1. As is the case for Notch1, Notch2 interacted with the minimal repression domain of CBF1 and was targeted to CBF1 through the intracellular, subtransmembrane domain. Additional characterization suggested that the interaction domain of Notch may be bipartite. The intracellular domain of Notch2 (Notch2IC) located to the nucleus. This activated form of Notch2 transactivated expression of a target gene containing upstream CBF1 binding sites. The use of CBF1 mutants carrying amino acid substitutions in the transcriptional repression domain revealed that activation of gene expression by Notch2 is also based on masking of CBF1-mediated repression. Targeting of Notch1 and targeting of Notch2 were found to be identical and distinguishable from targeting by EBNA2. Mutation of CBF1 at codons 249 to 251 abolished interaction with both Notch proteins but not with EBNA2. In a biological examination of Notch2 function in muscle cells, Notch2IC activated endogenous HES-1 gene expression and blocked muscle cell differentiation. Overall, the data imply that at least a subset of the intracellular events following signalling in cells expressing Notch2 are common to those in Notch1-expressing cells. The concept that EBNA2 functions by mimicking Notch signalling is therefore viable whether cells are expressing Notch1 or Notch2.