Activated Mouse Notch1 Transactivates Epstein-Barr Virus Nuclear Antigen 2-Regulated Viral Promoters

Abstract

Epstein-Barr virus nuclear antigen 2 (EBNA2) is essential for B-cell immortalization by EBV, most probably by its ability to transactivate a number of cellular and viral genes. EBNA2-responsive elements (EBNA2REs) have been identified in several EBNA2-regulated viral promoters, each of them carrying at least one RBP-Jκ recognition site. RBP-Jκ recruits EBNA2 to the EBNA2RE and, once complexed to EBNA2, is converted from a repressor into an activator. An activated form of the cellular receptor Notch also interacts with RBP-Jκ, providing a link between EBNA2 and Notch signalling. To determine whether activated Notch is able to transactivate EBNA2-responsive viral promoters, we performed cotransfection experiments with activated mouse Notch1 (mNotch1-IC) and luciferase constructs of the BamHI C, LMP1, and LMP2A promoters. We present here evidence that mNotch1-IC transactivates viral promoters known to be regulated by EBNA2. As shown for EBNA2, mutations or deletions of the RBP-Jκ sites diminish or eliminate mNotch1-IC-mediated transactivation of the promoters, pointing to an essential role for Notch–RBP-Jκ interaction. In addition to RBP-Jκ, other cellular factors may bind within the EBNA2REs of viral promoters. While some factors appear to play an important role in both EBNA2- and mNotch1-IC-mediated transactivation, others are only important for the activity of either EBNA2 or mNotch1-IC. We could observe specific mNotch1-IC-responsive regions, thereby throwing more light upon which cofactors interact with EBNA2 and mNotch1-IC, thus enabling them to become functionally transactivators in vivo.

An auto-regulatory loop for EBV LMP2A involves activation of Notch

“…These data indicate that in HEK293 cells, both RBP-Jκ sites are important for LMP2A auto-regulation. This is in contrast to previous data which demonstrates that the second RBP--IC to activate the minimal LMP2A promoter in B cells (Hofelmayr et al, 1999) and is likely due to the differences in cell types and promoter constructs used.…”

“…These results demonstrate that LMP2A activates the Notch pathway in both B cells and epithelial cells, and exploits this pathway to regulate its own expression. Other groups have shown that Notch-IC can activate EBNA2 responsive promoters, including that of LMP2A, and gel shift analysis has demonstrated that Notch-IC can bind to RBP-Jκ sites in the LMP2A promoter (Hofelmayr et al, 1999). That LMP2A can activate the Notch pathway in vivo provides the missing link in an auto-regulatory loop.…”

“…It has been demonstrated that if the RBP-Jκ sites in the LMP2A minimal promoter are deleted, there is a significant decrease in responsiveness to murine Notch1-IC (Hofelmayr et al, 1999). Similar deletion mutations were generated in the context of the full length LMP2A promoter-firefly luciferase reporter plasmid to examine the requirement of the RBP-Jκ sites for LMP2A auto-regulation.…”

“…It has been shown previously that murine Notch1-IC can bind to and induce transcription from the LMP2A promoter in a reporter assay as well as using the endogenous promoter. The LMP2A promoter contains two consensus RBP-Jκ sites that are important for its activation by both Notch-IC and EBNA2 (Gordadze et al, 2001;Grossman et al, 1994;Hofelmayr et al, 2001;Hofelmayr et al, 1999;Hsieh et al, 1997;Ling et al, 1994;Strobl et al, 2000). In conjunction with these reports, the data from the microarray analysis led to the novel hypothesis that LMP2A constitutively activates the Notch pathway in vivo in order to regulate its own expression in the absence of EBNA2.…”

Epstein‐Barr virus infection and human malignancies

“…It then recruits EBNA2 to activate transcription via its acidic transactivating domain. In fact an activated version of the mouse Notch1 can induce the expression of EBNA2 targets Viruses and cancer which confirms that EBNA2 makes use of the physiological Notch pathway to activate its targets (Hofelmayr et al 1999;Strobl et al 2000). The PU.1 protein, another DNA binding protein has also been suggested to interact with EBNA2 to activate transcription in EBNA2 responsive promoters (Sjoblom et al 1995).…”