Our research program is focused on understanding the dynamics of the infection caused by Epstein Barr virus (EBV). Approximately 90% of the world population is infected with EBV. In most individuals most of the time, EBV infection is asymptomatic and EBV+ individuals develop no diseases because the immune system can keep viral gene expression under control. However, when the immune system is not fully functional such as in HIV+ individuals and transplant recipients, then EBV can induced B cell transformation by expressing a small set of viral proteins. Various attempts have been made to target EBV directly, as an alternative to traditional chemotherapy. But EBV+ and EVB- tumors are still treated identically, even though the pathogenic agent is different.

One way in which the infectivity of EBV can be controlled is by changing the expression of EBV genes. Gene expression is modulated by epigenetic modifications. Our work focuses on elucidating how epigenetics contributes to regulating the complex and dynamic gene expression patterns adopted by EBV during latency.

Epigenetics and CTCF in EBV Latency

EBV-induced malignancies have been challenging to target, in large part because EBV establishes a latent infection with complex and dynamic gene expression patterns. These gene expression patterns are referred to as “latency types” that can adapt to diverse host cell-types and immunological responses. This dynamic gene expression allows EBV to escape eradication by immune surveillance and persist as a long-term, latent infection. In a series of studies, our group has explored the role of epigenetics in the regulation of EBV latency. Although a role for epigenetic modifications in EBV has been appreciated for decades, our results over the past years have yielded surprising and important insights into its mechanism. We found that different EBV latency programs correlate with different epigenetic states of the viral episome, and we identified CTCF as the primary cellular factor that regulates these epigenetic patterns. Overall our work shows that: a) the epigenetic regulation of EBV is a dynamic process involving both histone modifications and DNA methylation; b) the cellular factor CTCF is critical for the maintenance of the viral chromatin state of EBV; and c) CTCF regulates the viral promotes. These findings defined the important function of CTCF as a chromatin organizer of the EBV viral genome.

EBV Genome Architecture

In addition to studying the chromatin state of EBV, our team has explored the three-dimensional organization of the EBV genome during latency. Since CTCF promotes chromatin loops in the human genome, we tested the possibility that CTCF acts on the EBV genome in a similar way. By combining 3C assay, ChIP-seq and EBV genetic methods, our group has shown that: a) the EBV genome adopts alternative chromosome conformations during latency; b) CTCF is the critical cellular factor that mediates chromatin loop formation in EBV; c) the viral enhancer at the Ori P region of EBV serves as “chromatin hub” regulating viral promoters through chromatin loops; and d) the disruption of EBV three-dimensional organization impairs viral gene expression. Overall our results have important biological implications since they demonstrate for the first time that viral genomes can also regulate their function by adopting different three-dimensional configurations.

PARP1 and EBV infection

We previously discovered that PARP1 can interact with EBV genome. PARP1 is a host enzyme that post-translationally modifies proteins through the transfer of ADP-ribose from NAD+ onto acceptor proteins. PARP1 is well characterized in its roles in DNA damage and apoptosis, however, in recent years, PARP1 has also been implicated in chromatin modification, transcriptional regulation, and inflammation. Evolutionary analyses have identified other PARP family proteins that likely have evolved a role in host-virus conflicts. Indeed, a body of evidence suggests that PARP1 may serve as a stress sensor—a function that would be especially relevant in regulating herpesvirus lytic reactivation. CTCF is also PARylated by PARP1, subsequently altering its insulator function. Based on the established role of CTCF in EBV latency, the functional interactions of PARP1 and CTCF, and because CTCF and PARP1 are both implicated as viral restriction factors in other herpesviruses, our group has studied the role of PARP1 in regulating the EBV epigenome.

PARP1 and EBV-induced B cell transformation

We have shown for the first time that LMP1 signaling affects important chromatin modifying enzymes, specifically PARP1. PARP1 regulates global gene expression by relaxing chromatin structure and inhibiting the accumulation of repressive histone marks. We discovered that inhibiting PARP1 (1) suppresses malignant transformation in vitro (2) and represses the expression of previously identified LMP1-genetic targets. Thus, our team has explored the hypothesis that PARP1 activity plays a important role in EBV-induced oncogenesis. Current projects in our laboratory aim at establishing PARP inhibitors as a novel target for EBV-induced cancers and at determining the pathogenic mechanisms involved in EBV mediated tumor initiation.

PARP1 and EZH2 functions

Our group previously discovered that inhibition of PARP1 activity dramatically changes the expression levels of hundreds of genes, including genes involved in cancer. We found that increased levels of the Polycomb Repressive Complex 2 (PRC2) catalytic subunit EZH2 are responsible for some effects caused by PARP inhibition. We reported for the first time that (1) PARP1 and EZH2 occupancy negatively correlate across the genome; (2) PARP1 can directly modify EZH2; and (3) PARylation alters the enzymatic activity of EZH2. Based on those data our team has explored the hypothesis that PARP1 and PARylation play an important and underappreciated role in EZH2 activity, and inhibitors of PARP can alter PRC2-mediated gene repression. Current projects in our laboratory aim at establishing PARP1 and PARylation as a novel mechanism of EZH2 regulation and at determining the mechanisms and the functional relevance of PARP-mediated EZH2 inhibition.