Epstein-Barr Virus Latency in B Cells Leads to Epigenetic Repression and CpG Methylation of the Tumour Suppressor Gene Bim

Abstract

In human B cells infected with Epstein-Barr virus (EBV), latency-associated virus gene products inhibit expression of the pro-apoptotic Bcl-2-family member Bim and enhance cell survival. This involves the activities of the EBV nuclear proteins EBNA3A and EBNA3C and appears to be predominantly directed at regulating Bim mRNA synthesis, although post-transcriptional regulation of Bim has been reported. Here we show that protein and RNA stability make little or no contribution to the EBV-associated repression of Bim in latently infected B cells. However, treatment of cells with inhibitors of histone deacetylase (HDAC) and DNA methyltransferase (DNMT) enzymes indicated that epigenetic mechanisms are involved in the down-regulation of Bim. This was initially confirmed by chromatin immunoprecipitation analysis of histone acetylation levels on the Bim promoter. Consistent with this, methylation-specific PCR (MSP) and bisulphite sequencing of regions within the large CpG island located at the 5′ end of Bim revealed significant methylation of CpG dinucleotides in all EBV-positive, but not EBV-negative B cells examined. Genomic DNA samples exhibiting methylation of the Bim promoter included extracts from a series of explanted EBV-positive Burkitt's lymphoma (BL) biopsies. Subsequent analyses of the histone modification H3K27-Me3 (trimethylation of histone H3 lysine 27) and CpG methylation at loci throughout the Bim promoter suggest that in EBV-positive B cells repression of Bim is initially associated with this repressive epigenetic histone mark gradually followed by DNA methylation at CpG dinucleotides. We conclude that latent EBV initiates a chain of events that leads to epigenetic repression of the tumour suppressor gene Bim in infected B cells and their progeny. This reprogramming of B cells could have important implications for our understanding of EBV persistence and the pathogenesis of EBV-associated disease, in particular BL.

EBNA3B-deficient EBV promotes B cell lymphomagenesis in humanized mice and is found in human tumors

“…H These 2 insertions (changing 7 consecutive T residues to 8) are the same, but are included as potential mutations as they occurred in 2 clearly distinct genetic backgrounds (Supplemental Figure 5C). I Described in detail previously (53).…”

Epstein-Barr virus nuclear antigens 3C and 3A maintain lymphoblastoid cell growth by repressing p16 INK4A and p14 ARF expression

“…EBNA3C or EBNA3A have many specific and potentially significant interactions with other transcription factors or modifiers, including PU.1, Spi-B, HDAC1, DP103, prothymosin-α, p300, Nm23-H1, SUMO1, and SUMO3, cyclin A, SCF SKP2 ubiquitin ligase, pRb, Chk2, Mdm2, and MRS18-2, and these interactions could be relevant to CDKN2A p16 INK4A or p14 ARF regulation and LCL growth (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38). Furthermore, EBNA3C upregulates TCL1A and ITGA4, down-regulates JAG1 and NCALD RNAs, and cooperates with EBNA3A in repressing Bim, a proapoptotic Bcl-2 family protein (24,(39)(40)(41). Indeed, EBNA3C and EBNA3A may cooperatively repress the p16 INK4A and p14 ARF tumor suppressors to allow cell cycle progression, as is required for MYC conversion of tissue cells to stem cells, for HPV16-and HPV18-induced cervical cancer, and for other enforced cell proliferations (42)(43)(44).…”

Epstein-Barr virus interactions with the Bcl-2 protein family and apoptosis in human tumor cells

“…Most reports show that BIM is a key regulator of life and death decisions (Anderton et al, 2008;Paschos et al, 2009). Thus, it is not surprising that EBV BHRF1 binds to the BH3-only peptide BIM and interacts strongly with its protein (Flanagan and Letai, 2008).…”

“…What is more, EBNA3C is directly targeted to the BIM promoter (Paschos et al, 2012). Previous research has shown that in the 5′ regulator region of BIM, heritable epigenetic modifications initiated by EBNA3A and EBNA3C play a major role in determining the level of post-transcriptional BIM production expressed in EBV-infected B cells (Paschos et al, 2009). …”