Intracellular forms of human NOTCH1 interact at distinctly different levels with RBP-Jkappa in human B and T cells

The cellular transcriptional repressor RBP-J kappa associates with the Epst ein-Barr virus nuclear antigens (EBNAs) determined to be essential for tran sformation of human primary B lymphocytes. It was demonstrated through gene tic analysis that interaction between the viral transactivator EBNA2 and RB P-J kappa is essential for EBV immortalization of primary B lymphocytes, We have shown that the association of RBP-J kappa with intracellular NOTCH1 d iffers significantly in B and T cells. Immunoprecipitation analyses with an tibodies to both the intracellular forms of NOTCH1 and to RBP-J kappa demon strated that little or no RBP-J kappa is associated with NOTCH1 in B cell l ines compared to the RBP-J kappa associated with NOTCH1 in T cell lines and was further demonstrated in human primary lymphocytes. Additionally, EBNA2 can compete with intracellular NOTCH1 for binding to GST-RBP-J kappa in vi tro. Northern blot for the cellular gene hairy enhancer of split (HES1) dem onstrated that HES1 is upregulated in the EBV transformed lymphoblastoid ce lls expressing high levels of EBNA2 and in a T cell line SupT1 overexpressi ng intracellular activated NOTCH1. Hence, EBNAP may be able to compete for the available pool of RBP-J kappa more effectively in human B cells than in T cells and provides a possible explanation for the ability of EBV to pote ntly and efficiently infect and immortalize human B cells.