Epstein-Barr Virus-Induced Oncogenesis: Epigenetic Control of LMP1 Expression and LMP1-Mediated Cell Transformation

Abstract

EBV is a human gammaherpesvirus that infects approximately 95% of the population worldwide and is associated with 1% of human cancer incidence. The EBV-genome-encoded latency membrane protein 1 (LMP1) is expressed in nearly all of EBV-associated malignancies and is the major EBV oncogene. Because LMP1 is essential for both establishing a latent EBV infection and promoting tumorigenesis, defining its role in these processes is critical in order to understand EBV-associated cancer development. Due to the importance of epigenetics in regulating EBV gene expression during latency, the goals of this dissertation were two-fold: to define the mechanisms of host epigenetic regulation of LMP1 expression and to establish how LMP1 alters the host epigenome. The host uses its epigenetic machinery to regulate EBV during latency. One mechanism is through the host protein CTCF, which acts as an insulator to prevent the intrusion of heterochromatin into euchromatic regions and prevent DNA methylation within the EBV genome. CTCF also participates in the three-dimensional organization of the EBV episome through the formation of a network of long-range interactions that, in turn, further regulate EBV gene expression. CTCF binds at several key regulatory regions in the EBV genome, including upstream latent gene promoters, with the strongest CTCF binding site in the EBV genome positioned at the LMP1 locus. However, the functional role of CTCF binding in regulating LMP1 has yet to be established. To define the role of CTCF binding at the LMP1 locus we utilized a genetically modified EBV bacmid that contained a site-directed deletion of the CTCF binding site at the LMP1 region. Using EBV-positive B cells we found that CTCF binding at the LMP1 locus is a key regulator of LMP1 expression. Loss of CTCF binding changed the epigenetic profile at the LMP1 locus resulting in the loss of euchromatic and a concomitant gain of heterochromatic histone modifications, as well as an associated increase in DNA methylation near the LMP1 promoter. These epigenetic changes mediated decreased LMP1 expression. Additionally, through chromosome conformation capture (3C) assays, we established that DNA loop formation between the LMP1 loci and the viral enhancer OriP is strictly dependent upon CTCF binding at LMP1. Taken together, these observations suggest an epigenetic mechanism by which the host, through CTCF, contributes to the regulation of LMP1 expression. LMP1 expression is also regulated by structural elements within the EBV genome called terminal repeats (TRs). The number of TRs within an EBV genome varies based upon the circularization of the viral genome, and LMP1 expression is inversely correlated with the number of TRs in epithelial cells. However, the mechanism by which TR number regulates LMP1 expression has yet to be elucidated. We hypothesized that TR number differentially regulates the epigenetic state of the LMP1 promoters to either promote or suppress their activity. By examining the epigenetic state at both LMP1 promoters in isogenic cell lines with different numbers of TRs, we identified differences in histone modifications and DNA methylation at the LMP1 promoters. Furthermore, decreasing the number of TRs eliminated a chromatin loop formed between the LMP1 loci and the viral enhancer OriP. This data suggests that the number of TRs regulates the epigenetic state of LMP1 and ultimately determines promoter usage, which is necessary for LMP1 expression. While the previous work focused on the host mechanisms that regulate LMP1 expression, we also explored if the reverse relationship existed, that is, if EBV hijacks the host epigenetic machinery as a means to alter host gene expression. LMP1 contributes to host cell proliferation and survival through the aberrant activation of biochemical signaling pathways that also modulate epigenetic regulation. Because the PARP1-mediated post-translational modification poly(ADP-ribosyl)ation (PARylation) regulates EBV latency, and LMP1 activates a PARP1 regulator MAPK/ERK, we hypothesized that LMP1 drives the induction of PARP1-mediated PARylation. PARP1 facilitates gene transcription by maintaining a euchromatic state, and as a result, the disruption in epigenetic regulation mediated by LMP1 through PARP1 may also induce changes in host gene expression, including genes that contribute to EBV-mediated tumorigenesis. A panel of EBV-positive cell lines were found to have higher PAR levels than EBV-negative B cells, establishing a relationship between latent EBV infection and cellular PARylation. Because expression of the EBV oncoprotein LMP1 was sufficient to induce cellular PARylation, we explored the model that disruption in cellular PARylation driven by LMP1 expression subsequently promotes epigenetic alterations to elicit changes in host gene expression. PARP inhibition resulted in the accumulation of the repressive histone mark H3K27me3 at a subset of LMP1-regulated genes through induction of EZH2 expression. Inhibition of PARP also suppressed the expression of LMP1-activated genes and LMP1-mediated cellular transformation, demonstrating an essential role for PARP activity in LMP1-induced gene expression and cellular transformation associated with LMP1. This dissertation reveals for the first time the importance of the host protein CTCF and the effect of the number of viral TRs in epigenetically regulating the expression of the oncogenic protein LMP1. This dissertation further establishes a mechanism by which EBV hijacks the host epigenetic machinery and identifies a novel role for LMP1 in driving the expression of tumor-promoting host genes by blocking the incorporation of an inhibitory epigenetic modification through the activation of PARP1, suggesting that PARP1 may be a target for treatment of EBV-associated malignancies.