A Critical Role Of Notch2 In EBNA2-mediated CBF1 And HES1 Expression: Implication In EB Viral Infection

Notch is a transmembrane receptor. Notch receptors are activated by interaction with membrane bound ligands, which in vertebrates are designated Jagged / Delta. Notch receptors have at least three sites (S1 to S3), where they are proteolytically cleaved . During maturation, the 300 kDa precursor protein is cleaved by a furine-like convertase at S1; Ligand-receptor interactions induce two further cleavages which removes most of the Notch extracellular domain (NEC) at S2 and releases Notch intracellular domain (Notch-IC) at S3. After cleavage, Notch-IC is translocated to the nucleus, where it interacts with RBP-J(or CBF1) and modulates gene expression. In mammals four Notch-genes (Notch 1-4) have been described, encoding receptors with a single transmembrane domain. Several mammalian target genes have been described, which are induced through the interaction of Notch-IC with RBP-J, like the hairy enhancer of split (HES or Hes ) and hairy-related transcription factor (HRT or Hey)Epstein-Barr virus (EBV) establishes lifelong persistent infections in humans by latently infecting B cells, with a potential development of both lymphoid and epithelial tumors. Recent studies have demonstrated that immortalization of B cells infected by Epstein-Barr virus (EBV) depends on the virally encoded Epstein-Barr virus nuclear antigen 2 (EBNA2) protein ,a essential mediator for B cell transformation. Although there is less homologue between Notch receptor and EBNA2 genes, they share several biochemical and functional properties, such as interaction with RBP-J kappa (or CBF1) a nuclear component of the Notch signaling pathway. However, it is not clear whether Notch interacts with EBNA2 in driving the downstream signaling.To gain insight into the Notch2 receptor- and EBNA2-operated expression of CBF1 and HES1, in the present study, Notch 2 or/and EBNA2 gain- or loss-of-function approaches were employed to analyze the CBF1 and HES1 promoter activity in vitro using HeLa cell and Cos7 cell.We observed that overexpression of either active form of Notch2 (N2IC) and EBNA2 up-regulated CBF1 promoter expression. The N2IC-mediated CBF1 expression was blocked by CBF1 promoter mutation (3XMT); However, EBNA2-mediated CBF1 expression was partially blocked by CBF1 mutation (3XMT). Theses results suggest that a set of cis-elements on CBF1 promoter regulated by Notch-IC and EBNA2 is overlapping but not identical. In addition, as in Notch-triggered signaling, EBNA2 might operate its downstream gene expression through CBF-1-mediated replacement of repressor proteins to release their transactivation domains. Meanwhile, we found Notch2 and EBNA2 triggered HES1 expression, suggesting that EBNA2 is able to activate downstream targets of Notch signaling. Interestingly, the EBNA2-mediated CBF1 and HES1 expression was shut down by the Notch2-RNAi.Our data demonstrate that activation of Notch2 is critical for EBNA2-operated signaling, suggesting a possible involvement of Notch signaling (especially endogenous Notch signaling) in EBV infection. The Notch receptor family plays an important role in cell fate determination and vasculogenesis. The presence of multiple Notch ligand receptor combinations suggests that each of the Notch receptors may play a distinctive role. Herein we report a novel mechanism underlying the countervailing regulation of vascular smooth muscle cell (VSMC) growth by Notch-1 and Notch-2.A series of experimental approaches were applied including transient transfection in different VSMC, quantitative real-time reverse transcription-PCR (QRTPCR), [³H] thymidine incorporation, Western blotting, p27^KIP1 promoter analysis, HES1 site-directed mutagenesis analysis, and chromatin immunoprecipitation (ChIP). Meanwhile we generated A7r5 stable cell lines for forced expression of the constitutively active Notch1 or Notch2 intracellular domain (N1-IC) or (N2-IC), and HES1 site-directed mutagenesis of p27^KIP1 promoter.We observed that Nl-IC stable cell line inhibited VSMC growth in association with increased expression of the cell-cycle inhibitor p27^KIP1. In contrast, N2-IC stable cell line stimulated VSMC growth and induced a decrease in p27^KIP1 levels. A deletion mapping analysis of about 3.5 kb of the 5' promoter region of the p27^KIP1 promoter indicated that a cis-element located between -774 to -549 bp is critical for the differential regulation of p27^KIP1 transcriptional activity by Nl-IC and N2-IC and contained putative HES1 binding sites. Indeed, HES1 expression was up-regulated by N1-IC but not by N2-IC stimulation. Moreover the essential mediator role of HES1 in conferring differential response of N1-IC vs N2-IC on p27^KIP1 gene transcription was confirmed by a series of experiments that included site-directed mutagenesis, chromatin immunoprecipitation and RNAi-mediated knockdown of HES1. Furthermore, knockdown of HES1 led to a potent attenuation of both N1-IC-induced growth arrest as well as N2-IC-stimulated VSMC growth.Taken together, these results indicate that Notch-1 and Notch-2 receptors differentially govern one of the critical downstream effectors of the Notch transcriptional cascade HES1 and thereby induce diametric effects on VSMC growth regulation.