EBV finds a polycomb-mediated, epigenetic solution to the problem of oncogenic stress responses triggered by infection

Abstract

Viruses that establish a persistent infection, involving intracellular latency, commonly stimulate cellular DNA synthesis and sometimes cell division early after infection. However, most cells of metazoans have evolved "fail-safe" responses that normally monitor unscheduled DNA synthesis and prevent cell proliferation when, for instance, cell proto-oncogenes are "activated" by mutation, amplification, or chromosomal rearrangements. These cell intrinsic defense mechanisms that reduce the risk of neoplasia and cancer are collectively called oncogenic stress responses (OSRs). Mechanisms include the activation of tumor suppressor genes and the so-called DNA damage response that together trigger pathways leading to cell cycle arrest (e.g., cell senescence) or complete elimination of cells (e.g., apoptosis). It is not surprising that viruses that can induce cellular DNA synthesis and cell division have the capacity to trigger OSR, nor is it surprising that these viruses have evolved countermeasures for inactivating or bypassing OSR. The main focus of this review is how the human tumor-associated Epstein-Barr virus manipulates the host polycomb group protein system to control - by epigenetic repression of transcription - key components of the OSR during the transformation of normal human B cells into permanent cell lines.

Keywords: B cell transformation; BIM; Epstein–Barr virus; PcG; epigenetic; oncogene-induced senescence; oncogenic stress response; p16INK4a.

FIGURE 1

Activation of cell proto-oncogenes can lead to oncogenic stress responses (OSR). Oncogene “activation” by mutation or constitutive expression at supra-physiological levels can induce aberrant cell division that may become manifest as rapid cell proliferation (hyperproliferation). However higher vertebrates have evolved cell intrinsic “fail-safe” responses to recognize such cells and so block their proliferation or eliminate them completely. These can include the induction of tumor suppressors (ts) such as p16^INK4a that halt the cell cycle and can cause cells to enter a prolonged state of arrest called senescence, or pro-apoptotic proteins such as BIM that can induce programmed cell death (apoptosis). Responses may involve direct activation of the ts genes by oncoproteins or they can result from secondary signaling pathways (DDR) linked to the detection of damaged DNA produced during periods of aberrant DNA replication and cell division.

FIGURE 2

Epitope-tagged EBNA3C associates with the promoter for BIM and genes in the INK4b-ARF-INK4a locus. Schematic representations of (A) approximately 9kb of the BCL2L11 (BIM) promoter and (B) approximately 40 kb including the INK4b-ARF-INK4a locus. Vertical arrows indicate the positions where EBNA3C has been detected by chromatin immunoprecipitation (ChIP) analyses using lymphoblastoid cell lines (LCL) established using EBV expressing an epitope-tagged EBNA3C. These same regions of chromatin are marked by the polycomb (PRC2)-mediated modification H3K27me3 when EBNA3A and EBNA3C are present (adapted from Paschos et al., 2012; Skalska et al., 2013). The BCL2L11 gene transcriptional start site (TSS) and the protein products of INK4b-ARF-INK4a are indicated. A–G in (A) mark the positions of RT-PCR primer sets used in (Paschos et al., 2012).

FIGURE 3

Working hypothesis for the role(s) of EBNA3C and EBNA3A in the PRC2-mediated repression of the BIM promoter. The available data indicate that EBNA3C (and EBNA3A) are recruited to regions proximal to the BCL2L11/BIM transcriptional start site (TSS) in EBV-infected B cells (Paschos et al., 2012; our unpublished data and Figure 2). Irrespective of whether EBNA3C or EBNA3A are expressed in these cells, the PRC2-associated factors RbpA46/48 and JARID2 are present at the locus. Similarly the activation mark H3K4me3 and RNA polymerase II (RNA Pol II) occupy the TSS irrespective of which EBNA3s are expressed. Only when both EBNA3C and EBNA3A are present are core components of the PRC2 complex found at this site and the repressive chromatin mark H3K27me3 is detected across the TSS; concomitantly the level of transcription and BIM expression are reduced. The simultaneous presence of both H3K4me3 and H3K27me3 at the locus define it as a “bivalent” or “poised” domain and is consistent with RNA Pol II always being detected. However only in the absence of either EBNA3C or EBNA3A is RNA Pol II phosphorylated on serine residue 5 (RNA Pol II Ser 5), suggesting that in addition to playing a key role in the recruitment of PRC2 core complex, the presence of EBNA3C and EBNA3A might interfere with serine 5 phosphorylation of RNA Pol II and therefore block the initiation of transcription. Since EBNA3A and EBNA3C can be co-immunoprecipitated from infected B cells and both are necessary for repression of BIM (and p16^INK4a) expression, in this model we assume they are co-localized at these loci. The identity of the factor(s) responsible for targeting EBNA3C and/or EBNA3A to this particular stretch of chromatin is still unknown, as is the mechanism of interaction with PRC2.

FIGURE 4

Events following infection of primary resting B cells by EBV that initiate transformation into continuously proliferating LCLs. (A) During the first 24–48 h post-infection (pi) with a B95.8-derived EBV, cell genes associated with growth and cell cycle are transactivated and their products (e.g., MYC, cyclin D2, cyclin E) drive cells from G0 to G1, to become enlarged, activated and start proliferating. The whole process is driven by the EBV transactivator protein EBNA2, probably assisted by the co-factor EBNA-LP (Sinclair et al., 1994; Spender et al., 1999; Nikitin et al., 2010). During the next 3–4 days cells undergo rounds of rapid cell division (hyperproliferation) and in some cells this results in damage to DNA that can activate the DNA damage response (DDR) and initiate a signaling cascade involving the kinases ATM and CHK2 (Nikitin et al., 2010). If the full complement of nine EBV latency-associated proteins is expressed, the DDR becomes attenuated (in part by EBNA3C) and cells continue to proliferate to produce polyclonal LCLs that have a population doubling time of about 24 h. Early after infection BIM expression is down-regulated, and although the level of p16^INK4a expression increases slightly, this soon reaches a steady state. In both cases we assume that EBNA3A and EBNA3C cooperate by harnessing the polycomb group (PcG) protein system to epigenetically repress (or restrain the transcription of) these ts genes via H3K27me3 (Anderton et al., 2008; Paschos et al., 2012; Skalska et al., 2013). (B) If EBNA3C or EBNA3A are deleted (ΔEBNA3C and ΔEBNA3A) or functionally inactivated in the infecting EBV, beginning about 4–7 days pi, the levels of mRNAs corresponding to p16^INK4a and BIM progressively increase and continue to do so for the next week or two until finally most of the cells arrest and/or die (Skalska et al., 2013 and our unpublished data). The PcG-mediated repression of these two ts genes – in particular p16^INK4a (see text) – is part of a critical countermeasure evolved by EBV to bypass an intrinsic host defense against oncogenic transformation. If primary B cells are p16^INK4a-null, functional EBNA3C is dispensable for the outgrowth of LCLs. This is consistent with p16^INK4a being the dominant barrier to outgrowth and subsequent proliferation of LCLs, and the principal requirement of EBNA3C appears to be to restraining transcription of p16^INK4a (see text for details and Skalska et al., 2013). The precise relationships between DDR, p16^INK4a and EBNA3C/EBNA3A have yet to be defined.