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.

Arbiter of differentiation and death: Notch signaling meets apoptosis

“…Epstein-Barr virus (EBV) immortalizing protein EBNA-2, which is necessary for EBV-induced transformation, mimics Notch-1 and -2 signaling. EBNA-2 binds CBF-1 and converts it into a transcriptional activator, by masking its repressor domain (Hsieh and Hayward, 1995;Hsieh et al, 1996Hsieh et al, , 1997.…”

The Notch Receptor and Its Ligands Are Selectively Expressed During Hematopoietic Development in the Mouse

“…After gentle fixation in 0.5% paraformaldehyde and permeabilization in 70% ethanol, cells were allowed to rehydrate at 37°C before blocking again with anti-CD32 mAb. Cells were then incubated with 0.4 µg of affinity-purified 93-4 antiserum [39] to the intracellular domain of rat Notch1, 1 µg of affinity-purified 93-7 antiserum to the intracellular domain of rat Notch2 [40], or an equivalent amount of normal rabbit IgG. After incubation with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Caltag; Burlingame, CA; http://www.caltag.com), cells were analyzed on a FACScan flow cytometer, using Cell Quest software (Becton Dickinson; Mountain View, CA; http://www.bd.com).…”

Transcriptional profiles in melanocytes from clinically unaffected skin distinguish the neoplastic growth pattern in patients with melanoma

“…Next, among genes with higher expression level in patients with VGP melanoma we found some cellular proliferation regulators: Cyclin D 2 , involved in G 1 /S cell cycle transition; 54 NOTCH2, belonging to the NOTCH genes family and involved in many tumours; 55–58 CSPG2, that can play a role in stimulating proliferation of melanoma cells; 59 FGF5; 60 CITED2; 61 CRIP1; 62 LOX; 63 PRRX1; 64,65 and PRRX2 64 . Finally, we found WNT5B, 66 that seems to be involved in proliferation of some cell types, and WNT5A, that seems to be involved both in cell proliferation 66 and in regulation of cell adhesion and migration 67,68 .…”