Epstein-Barr Virus Nuclear Antigen 3 (EBNA3) Proteins Regulate EBNA2 Binding to Distinct RBPJ Genomic Sites

Abstract

Latent infection of B lymphocytes by Epstein-Barr virus (EBV) in vitro results in their immortalization into lymphoblastoid cell lines (LCLs); this latency program is controlled by the EBNA2 viral transcriptional activator, which targets promoters via RBPJ, a DNA binding protein in the Notch signaling pathway. Three other EBNA3 proteins (EBNA3A, EBNA3B, and EBNA3C) interact with RBPJ to regulate cell gene expression. The mechanism by which EBNAs regulate different genes via RBPJ remains unclear. Our chromatin immunoprecipitation with deep sequencing (ChIP-seq) analysis of the EBNA3 proteins analyzed in concert with prior EBNA2 and RBPJ data demonstrated that EBNA3A, EBNA3B, and EBNA3C bind to distinct, partially overlapping genomic locations. Although RBPJ interaction is critical for EBNA3A and EBNA3C growth effects, only 30 to 40% of EBNA3-bound sites colocalize with RBPJ. Using LCLs conditional for EBNA3A or EBNA3C activity, we demonstrate that EBNA2 binding at sites near EBNA3A-or EBNA3C-regulated genes is specifically regulated by the respective EBNA3. To investigate EBNA3 binding specificity, we identified sequences and transcription factors enriched at EBNA3A-, EBNA3B-, and EBNA3C-bound sites. This confirmed the prior observation that IRF4 is enriched at EBNA3A-and EBNA3C-bound sites and revealed IRF4 enrichment at EBNA3B-bound sites. Using IRF4-negative BJAB cells, we demonstrate that IRF4 is essential for EBNA3C, but not EBNA3A or EBNA3B, binding to specific sites. These results support a model in which EBNA2 and EBNA3s compete for distinct subsets of RBPJ sites to regulate cell genes and where EBNA3 subset specificity is determined by interactions with other cell transcription factors. IMPORTANCE Epstein-Barr virus (EBV) latent gene products cause human cancers and transform B lymphocytes into immortalized lymphoblastoid cell lines in vitro.EBV nuclear antigens (EBNAs) and membrane proteins constitutively activate pathways important for lymphocyte growth and survival. An important unresolved question is how four different EBNAs (EBNA2, -3A, -3B, and -3C) exert unique effects via a single transcription factor, RBPJ. Here, we report that each EBNA binds to distinct but partially overlapping sets of genomic sites. EBNA3A and EBNA3C specifically regulate EBNA2's access to different RBPJ sites, providing a mechanism by which each EBNA can regulate distinct cell genes. We show that IRF4, an essential regulator of B cell differentiation, is critical for EBNA3C binding specificity; EBNA3A and EBNA3B specificities are likely due to interactions with other cell transcription factors. EBNA3 titration of EBNA2 transcriptional function at distinct sites likely limits cell defenses that would be triggered by unchecked EBNA2 prooncogenic activity. E pstein-Barr virus (EBV) is a herpesvirus that infects over 90% of the population by adulthood. Primary EBV infection usually presents as a nonspecific illness in early childhood but often manifests as infectious mononucleosis in adolescents (1). Thereafter, EBV es...

Current Progress in EBV-Associated B-Cell Lymphomas

“…ChIP-seq analysis shows that EBNA2 and EBNA3s (EBNA3A, EBNA3B, and EBNA3C) can target multiple cellular genes through cell-specific regulation of long-range enhancer-promoter interactions [56]. Another study indicated that while these four latent antigens can competitively bind to RBPJκ at its repressive sites to control cellular genes expression, EBNA3s are more likely to interact with other transcription factors [57]. For example, IRF4 is essential for EBNA3C to associate with specific sites on viral and cellular DNA [16, 55, 57, 58].…”

“…Another study indicated that while these four latent antigens can competitively bind to RBPJκ at its repressive sites to control cellular genes expression, EBNA3s are more likely to interact with other transcription factors [57]. For example, IRF4 is essential for EBNA3C to associate with specific sites on viral and cellular DNA [16, 55, 57, 58]. A recent study identified a number of host dependency factors in BL and LCLs using CRISPR/Cas9 loss-of-function screen [59].…”

The Epstein-Barr Virus Regulome in Lymphoblastoid Cells

“…EBNALP binds preferentially to promoters than enhancers, and co-activates with EBNA2 by removing transcription repressors, including N-CoR, from EBNA2 (Harada and Kieff, 1997; Ling et al, 2005; Portal et al, 2011; Portal et al, 2013). EBNA3A and 3C can be tethered to DNA through cell TFs including IRF4 and BATF (Banerjee et al, 2013; Jiang et al, 2014; Schmidt et al, 2015; Wang et al, 2015). EBNA3A and 3C repress both p14 ARF and p16 INK4A , thereby preventing cell senescence (Maruo et al, 2006; Skalska et al, 2010).…”

Core binding factor (CBF) is required for Epstein-Barr virus EBNA3 proteins to regulate target gene expression

“…Indeed, EBNA3 ChIP-seq experiments have been performed previously and thousands of EBNA3 binding sites have been identified (24,27–29). These studies have been very informative, each an improvement on the previous, as reagents and resources have become available.…”