Epstein-Barr Virus Nuclear Antigen 3C Inhibits Expression of COBLL1 and the ADAM28-ADAMDEC1 Locus via Interaction with the Histone Lysine Demethylase KDM2B.

Epstein-Barr virus nuclear antigen 3C (EBNA3C) is a well-defined repressor of host gene expression in B cells transformed by Epstein-Barr virus (EBV) that cooperates with various cellular factors. It is established that EBNA3C interacts with the cellular factor RBPJ (RBP-Jκ or CBF1) through two distinct motifs: the TFGC motif, also called the homology domain (HD) motif, and the VWTP motif. In this study, we investigated the role of each motif in EBNA3C transcriptional repression activity by using two novel recombinant viruses with single RBPJ interaction motifs mutated (EBNA3C HDmut and EBNA3C W227S). Infection of primary B cells with either of these recombinant EBVs led to the successful establishment of lymphoblastoid cell lines (LCLs). Gene expression analysis showed that full repression of EBNA3C target genes is not achieved by EBNA3C HDmut compared to that with EBNA3C W227S or the EBNA3C wild type (WT). Focusing on the well-characterized EBNA3C-repressed genes COBLL1, ADAM28, and ADAMDEC1, we investigated the mechanism of EBNA3C-mediated transcriptional repression. Chromatin immunoprecipitation (ChIP) analysis indicated that EBNA3C HDmut is still able to recruit Polycomb proteins BMI1 and SUZ12 to COBLL1 as efficiently as EBNA3C WT does, leading to the full deposition of the repressive histone mark H3K27me3. However, we found that the activation-associated chromatin mark H3K4me3 is highly enriched at EBNA3C target genes in LCLs expressing EBNA3C HDmut. We show here that EBNA3C interacts with the histone lysine demethylase KDM2B and that this interaction is important for H3K4me3 removal and for the EBNA3C-mediated repression of COBLL1 and the ADAM28-ADAMDEC1 locus.IMPORTANCE EBV is a virus associated with human cancers and is well known for its ability to transform B lymphocytes into continuously proliferating lymphoblastoid cell lines. EBNA3C is considered an oncoprotein and has been shown to be essential for B cell transformation by EBV. EBNA3C is well characterized as a viral transcription factor, but very little is known about its mechanisms of action. In the present study, we demonstrate that removal of the activating histone mark H3K4me3 and deposition of the repressive mark H3K27me3 by EBNA3C on COBLL1 are achieved by at least two distinct mechanisms. Furthermore, we discovered that EBNA3C interacts with the lysine demethylase KDM2B and that this interaction is important for its transcriptional repressive function. The findings in this study provide new insights into the mechanism used by the oncoprotein EBNA3C to repress cellular target genes.

Epstein-Barr virus nuclear protein EBNA3C directly induces expression of AID and somatic mutations in B cells.

Activation-induced cytidine deaminase (AID), the enzyme responsible for induction of sequence variation in immunoglobulins (Igs) during the process of somatic hypermutation (SHM) and also Ig class switching, can have a potent mutator phenotype in the development of lymphoma. Using various Epstein-Barr virus (EBV) recombinants, we provide definitive evidence that the viral nuclear protein EBNA3C is essential in EBV-infected primary B cells for the induction of AID mRNA and protein. Using lymphoblastoid cell lines (LCLs) established with EBV recombinants conditional for EBNA3C function, this was confirmed, and it was shown that transactivation of the AID gene (AICDA) is associated with EBNA3C binding to highly conserved regulatory elements located proximal to and upstream of the AICDA transcription start site. EBNA3C binding initiated epigenetic changes to chromatin at specific sites across the AICDA locus. Deep sequencing of cDNA corresponding to the IgH V-D-J region from the conditional LCL was used to formally show that SHM is activated by functional EBNA3C and induction of AID. These data, showing the direct targeting and induction of functional AID by EBNA3C, suggest a novel role for EBV in the etiology of B cell cancers, including endemic Burkitt lymphoma.

EBNA3C Directs Recruitment of RBPJ (CBF1) to Chromatin during the Process of Gene Repression in EBV Infected B Cells.

It is well established that Epstein-Barr virus nuclear antigen 3C (EBNA3C) can act as a potent repressor of gene expression, but little is known about the sequence of events occurring during the repression process. To explore further the role of EBNA3C in gene repression-particularly in relation to histone modifications and cell factors involved-the three host genes previously reported as most robustly repressed by EBNA3C were investigated. COBLL1, a gene of unknown function, is regulated by EBNA3C alone and the two co-regulated disintegrin/metalloproteases, ADAM28 and ADAMDEC1 have been described previously as targets of both EBNA3A and EBNA3C. For the first time, EBNA3C was here shown to be the main regulator of all three genes early after infection of primary B cells. Using various EBV-recombinants, repression over orders of magnitude was seen only when EBNA3C was expressed. Unexpectedly, full repression was not achieved until 30 days after infection. This was accurately reproduced in established LCLs carrying EBV-recombinants conditional for EBNA3C function, demonstrating the utility of the conditional system to replicate events early after infection. Using this system, detailed chromatin immunoprecipitation analysis revealed that the initial repression was associated with loss of activation-associated histone modifications (H3K9ac, H3K27ac and H3K4me3) and was independent of recruitment of polycomb proteins and deposition of the repressive H3K27me3 modification, which were only observed later in repression. Most remarkable, and in contrast to current models of RBPJ in repression, was the observation that this DNA-binding factor accumulated at the EBNA3C-binding sites only when EBNA3C was functional. Transient reporter assays indicated that repression of these genes was dependent on the interaction between EBNA3C and RBPJ. This was confirmed with a novel EBV-recombinant encoding a mutant of EBNA3C unable to bind RBPJ, by showing this virus was incapable of repressing COBLL1 or ADAM28/ADAMDEC1 in newly infected primary B cells.