Abstract
The Epstein–Barr virus (EBV) nuclear antigen 3 family of protein is critical for the EBV-induced primary B-cell growth transformation process. Using a yeast two-hybrid screen we identified 22 novel cellular partners of the EBNA3s. Most importantly, among the newly identified partners, five are known to play direct and important roles in transcriptional regulation. Of these, the Myc-interacting zinc finger protein-1 (MIZ-1) is a transcription factor initially characterized as a binding partner of MYC. MIZ-1 activates the transcription of a number of target genes including the cell cycle inhibitor CDKN2B. Focusing on the EBNA3A/MIZ-1 interaction we demonstrate that binding occurs in EBV-infected cells expressing both proteins at endogenous physiological levels and that in the presence of EBNA3A, a significant fraction of MIZ-1 translocates from the cytoplasm to the nucleus. Moreover, we show that a trimeric complex composed of a MIZ-1 recognition DNA element, MIZ-1 and EBNA3A can be formed, and that interaction of MIZ-1 with nucleophosmin (NPM), one of its coactivator, is prevented by EBNA3A. Finally, we show that, in the presence of EBNA3A, expression of the MIZ-1 target gene, CDKN2B, is downregulated and repressive H3K27 marks are established on its promoter region suggesting that EBNA3A directly counteracts the growth inhibitory action of MIZ-1.
Highlights
Epstein–Barr virus (EBV) is a ubiquitous herpesvirus associated with several human cancers, both in immunocompetent individuals (Burkitt’s lymphoma, Hodgkin’s disease (HD), rare T-cell and NK-cell lymphomas, undifferentiated nasopharyngeal carcinoma (NPC), gastric carcinomas) and immuno-compromised individuals [1]
By reciprocal co-immunoprecipitation assays (Figure 1D) we demonstrated that endogenous Myc-interacting zinc finger protein-1 (MIZ-1) and EBNA3A can form a complex under physiological conditions in a relevant cell model––i.e. lymphoblastoid cells immortalized by EBV
In the case of LCL established with wt EBV (LCLwt), the levels of the CDKN2B transcript are induced following treatment with cisplatin––with a pattern similar to that observed in lymphoblastoid cell lines (LCLs) 3A––but the levels remain very low, staying in the range observed in LCL 3A before treatment with cisplatin. These results indicate that EBNA3A efficiently represses CDKN2B expression and that in conditions where this expression is induced by stimuli such as DNA-damage, this repression allows the retention of low levels of CDKN2B transcript compatible, at least to a certain extent, with cell survival
Summary
Epstein–Barr virus (EBV) is a ubiquitous herpesvirus associated with several human cancers, both in immunocompetent individuals (Burkitt’s lymphoma, Hodgkin’s disease (HD), rare T-cell and NK-cell lymphomas, undifferentiated nasopharyngeal carcinoma (NPC), gastric carcinomas) and immuno-compromised individuals (lymphoproliferations and lymphomas, in particular in post-transplant patients) [1]. EBV has the unique capacity to induce growth transformation of resting primary human B-lymphocytes upon their infection, leading to the establishment of lymphoblastoid cell lines (LCLs). In such cell lines, there is no virus production but nine viral proteins––six nuclear antigens (EBNAs 1, 2, 3A, 3B, 3C and LP) and three membrane proteins (LMPs 1, 2A and 2B)––as well as several non-coding RNAs and.
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