Epstein-Barr virus (EBV) persists in human B-cells by maintaining its chromatinized episomes within the nucleus. We have previously shown that cellular factor Poly ADP-ribose polymerase 1 (PARP1) ...binds the EBV genome, stabilizes CTCF binding at specific loci, and that PARP1 enzymatic activity correlates with maintaining a transcriptionally active latency program. To better understand PARP1's role in regulating EBV latency, here we functionally characterize the effect of PARP enzymatic inhibition on episomal structure through in situ HiC mapping, generating a complete 3D structure of the EBV genome. We also map intragenomic contact changes after PARP inhibition to global binding of chromatin looping factors CTCF and cohesin across the EBV genome. We find that PARP inhibition leads to fewer total unique intragenomic interactions within the EBV episome, yet new chromatin loops distinct from the untreated episome are also formed. This study also illustrates that PARP inhibition alters gene expression at the regions where chromatin looping is most effected. We observe that PARP1 inhibition does not alter cohesin binding sites but does increase its frequency of binding at those sites. Taken together, these findings demonstrate that PARP has an essential role in regulating global EBV chromatin structure and latent gene expression.
CTLA4 is a cell surface receptor on T cells that functions as an immune checkpoint molecule to enforce tolerance to cognate antigens. Anti-CTLA4 immunotherapy is highly effective at reactivating ...T-cell responses against melanoma, which is postulated to be due to targeting CTLA4 on T cells. Here, we report that CTLA4 is also highly expressed by most human melanoma cell lines, as well as in normal human melanocytes. Interferon-γ (IFNG) signaling activated the expression of the human CTLA4 gene in a melanocyte and melanoma cell-specific manner. Mechanistically, IFNG activated CTLA4 expression through JAK1/2-dependent phosphorylation of STAT1, which bound a specific gamma-activated sequence site on the CTLA4 promoter, thereby licensing CBP/p300-mediated histone acetylation and local chromatin opening. In melanoma cell lines, elevated baseline expression relied upon constitutive activation of the MAPK pathway. Notably, RNA-seq analyses of melanoma specimens obtained from patients who had received anti-CTLA4 immunotherapy (ipilimumab) showed upregulation of an IFNG-response gene expression signature, including CTLA4 itself, which correlated significantly with durable response. Taken together, our results raise the possibility that CTLA4 targeting on melanoma cells may contribute to the clinical immunobiology of anti-CTLA4 responses.
These findings show that human melanoma cells express high levels of the immune checkpoint molecule CTLA4, with important possible implications for understanding how anti-CTLA4 immunotherapy mediates its therapeutic effects.
.
Background and aims
Epstein–Barr virus (EBV)-associated gastric carcinoma (EBVaGC) is the most common EBV-associated cancer and accounts for ~ 10% of all gastric cancers (GC). Epstein–Barr virus ...nuclear antigen 1 (EBNA1), which is critical for the replication and maintenance of the EBV latent genome, is consistently expressed in all EBVaGC tumors. We previously developed small molecule inhibitors of EBNA1. In this study, we investigated the efficacy and selectivity of an EBNA1 inhibitor in cell-based and animal xenograft models of EBV-positive and EBV-negative gastric carcinoma.
Methods
We tested the potency of an EBNA1 inhibitor, VK-1727, in vitro and in xenograft studies, using EBV-positive (SNU719 and YCCEL1) and EBV-negative (AGS and MKN74) GC cell lines. After treatment, we analyzed cell viability, proliferation, and RNA expression of EBV genes by RT-qPCR.
Results
Treatment with VK-1727 selectively inhibits cell cycle progression and proliferation in vitro. In animal studies, treatment with an EBNA1 inhibitor resulted in a significant dose-dependent decrease in tumor growth in EBVaGC xenograft models, but not in EBV-negative GC xenograft studies. Gene expression analysis revealed that short term treatment in cell culture tended towards viral gene activation, while long-term treatment in animal xenografts showed a significant decrease in viral gene expression.
Conclusions
EBNA1 inhibitors are potent and selective inhibitors of cell growth in tissue culture and animal models of EBV-positive GC. Long-term treatment with EBNA1 inhibitors may lead to loss of EBV in mouse xenografts. These results suggest that pharmacological targeting of EBNA1 may be an effective strategy to treat patients with EBVaGC.
Epstein-Barr Virus (EBV) is a human gamma-herpesvirus that is widespread worldwide. To this day, about 200,000 cancer cases per year are attributed to EBV infection. EBV is capable of infecting both ...B cells and epithelial cells. Upon entry, viral DNA reaches the nucleus and undergoes a process of circularization and chromatinization and establishes a latent lifelong infection in host cells. There are different types of latency all characterized by different expressions of latent viral genes correlated with a different three-dimensional architecture of the viral genome. There are multiple factors involved in the regulation and maintenance of this three-dimensional organization, such as CTCF, PARP1, MYC and Nuclear Lamina, emphasizing its central role in latency maintenance.
The Epstein Barr virus (EBV) infects almost 95% of the population worldwide. While typically asymptomatic, EBV latent infection is associated with several malignancies of epithelial and lymphoid ...origin in immunocompromised individuals. In latently infected cells, the EBV genome persists as a chromatinized episome that expresses a limited set of viral genes in different patterns, referred to as latency types, which coincide with varying stages of infection and various malignancies. We have previously demonstrated that latency types correlate with differences in the composition and structure of the EBV episome. Several cellular factors, including the nuclear lamina, regulate chromatin composition and architecture. While the interaction of the viral genome with the nuclear lamina has been studied in the context of EBV lytic reactivation, the role of the nuclear lamina in controlling EBV latency has not been investigated. Here, we report that the nuclear lamina is an essential epigenetic regulator of the EBV episome. We observed that in B cells, EBV infection affects the composition of the nuclear lamina by inducing the expression of lamin A/C, but only in EBV+ cells expressing the Type III latency program. Using ChIP-Seq, we determined that lamin B1 and lamin A/C bind the EBV genome, and their binding correlates with deposition of the histone repressive mark H3K9me2. By RNA-Seq, we observed that knock-out of lamin A/C in B cells alters EBV gene expression. Our data indicate that the interaction between lamins and the EBV episome contributes to the epigenetic control of viral gene expression during latency, suggesting a restrictive function of the nuclear lamina as part of the host response against viral DNA entry into the nucleus.
Epstein-Barr Virus (EBV) can establish latent infections with distinct gene expression patterns referred to as latency types. These different latency types are epigenetically stable and correspond to ...different promoter utilization. Here we explore the three-dimensional conformations of the EBV genome in different latency types. We employed Chromosome Conformation Capture (3C) assay to investigate chromatin loop formation between the OriP enhancer and the promoters that determine type I (Qp) or type III (Cp) gene expression. We show that OriP is in close physical proximity to Qp in type I latency, and to Cp in type III latency. The cellular chromatin insulator and boundary factor CTCF was implicated in EBV chromatin loop formation. Combining 3C and ChIP assays we found that CTCF is physically associated with OriP-Qp loop formation in type I and OriP-Cp loop formation in type III latency. Mutations in the CTCF binding site located at Qp disrupt loop formation between Qp and OriP, and lead to the activation of Cp transcription. Mutation of the CTCF binding site at Cp, as well as siRNA depletion of CTCF eliminates both OriP-associated loops, indicating that CTCF plays an integral role in loop formation. These data indicate that epigenetically stable EBV latency types adopt distinct chromatin architectures that depend on CTCF and mediate alternative promoter targeting by the OriP enhancer.
Epstein-Barr virus (EBV), which is associated with multiple human tumors, persists as a minichromosome in the nucleus of B lymphocytes and induces malignancies through incompletely understood ...mechanisms. Here, we present a large-scale functional genomic analysis of EBV. Our experimentally generated nucleosome positioning maps and viral protein binding data were integrated with over 700 publicly available high-throughput sequencing data sets for human lymphoblastoid cell lines mapped to the EBV genome. We found that viral lytic genes are coexpressed with cellular cancer-associated pathways, suggesting that the lytic cycle may play an unexpected role in virus-mediated oncogenesis. Host regulators of viral oncogene expression and chromosome structure were identified and validated, revealing a role for the B cell-specific protein Pax5 in viral gene regulation and the cohesin complex in regulating higher order chromatin structure. Our findings provide a deeper understanding of latent viral persistence in oncogenesis and establish a valuable viral genomics resource for future exploration.
Display omitted
► The EBV transcriptome and epigenome were analyzed by systems approaches ► Viral lytic genes are coexpressed with cellular cancer-associated pathways ► B cell-specific Pax5 protein regulates viral oncoprotein expression ► CTCF-cohesin mediate long-distance DNA interactions important for latency regulation
Latent membrane protein 1 (LMP1) is the major transforming protein of Epstein-Barr virus (EBV) and is critical for EBV-induced B-cell transformation in vitro. Poly(ADP-ribose) polymerase 1 (PARP1) ...regulates accessibility of chromatin, alters functions of transcriptional activators and repressors, and has been directly implicated in transcriptional activation. Previously we showed that LMP1 activates PARP1 and increases Poly(ADP-ribos)ylation (PARylation) through PARP1. Therefore, to identify targets of LMP1 that are regulated through PARP1, LMP1 was ectopically expressed in an EBV-negative Burkitt's lymphoma cell line. These LMP1-expressing cells were then treated with the PARP inhibitor olaparib and prepared for RNA sequencing. The LMP1/PARP targets identified through this RNA-seq experiment are largely involved in metabolism and signaling. Interestingly, Ingenuity Pathway Analysis of RNA-seq data suggests that hypoxia-inducible factor 1-alpha (HIF-1α) is an LMP1 target mediated through PARP1. PARP1 is acting as a coactivator of HIF-1α-dependent gene expression in B cells, and this co-activation is enhanced by LMP1-mediated activation of PARP1. HIF-1α forms a PARylated complex with PARP1 and both HIF-1α and PARP1 are present at promoter regions of HIF-1α downstream targets, leading to accumulation of positive histone marks at these regions. Complex formation, PARylation and binding of PARP1 and HIF-1α at promoter regions of HIF-1α downstream targets can all be attenuated by PARP1 inhibition, subsequently leading to a buildup of repressive histone marks and loss of positive histone marks. In addition, LMP1 switches cells to a glycolytic 'Warburg' metabolism, preferentially using aerobic glycolysis over mitochondrial respiration. Finally, LMP1+ cells are more sensitive to PARP1 inhibition and, therefore, targeting PARP1 activity may be an effective treatment for LMP1+ EBV-associated malignancies.
The principal understanding of the Poly(ADP-ribose) polymerase (PARP) regulation of genomes has been focused on its role in DNA repair; however, in the past few years, an additional role for PARPs ...and PARylation has emerged in regulating viral-host interactions. In particular, in the context of DNA virus infection, PARP1-mediated mechanisms of gene regulations, such as the involvement with cellular protein complexes responsible for the folding of the genome into the nucleus, the formation of chromatin loops connecting distant regulatory genomic regions, and other methods of transcriptional regulation, provide additional ways through which PARPs can modulate the function of both the host and the viral genomes during viral infection. In addition, potential viral amplification of the activity of PARPs on the host genome can contribute to the pathogenic effect of viral infection, such as viral-driven oncogenesis, opening the possibility that PARP inhibition may represent a potential therapeutic approach to target viral infection. This review will focus on the role of PARPs, particularly PARP1, in regulating the infection of DNA viruses.
The establishment and maintenance of Epstein-Barr Virus (EBV) latent infection requires distinct viral gene expression programs. These gene expression programs, termed latency types, are determined ...largely by promoter selection, and controlled through the interplay between cell-type specific transcription factors, chromatin structure, and epigenetic modifications. We used a genome-wide chromatin-immunoprecipitation (ChIP) assay to identify epigenetic modifications that correlate with different latency types. We found that the chromatin insulator protein CTCF binds at several key regulatory nodes in the EBV genome and may compartmentalize epigenetic modifications across the viral genome. Highly enriched CTCF binding sites were identified at the promoter regions upstream of Cp, Wp, EBERs, and Qp. Since Qp is essential for long-term maintenance of viral genomes in type I latency and epithelial cell infections, we focused on the role of CTCF in regulating Qp. Purified CTCF bound approximately 40 bp upstream of the EBNA1 binding sites located at +10 bp relative to the transcriptional initiation site at Qp. Mutagenesis of the CTCF binding site in EBV bacmids resulted in a decrease in the recovery of stable hygromycin-resistant episomes in 293 cells. EBV lacking the Qp CTCF site showed a decrease in Qp transcription initiation and a corresponding increase in Cp and Fp promoter utilization at 8 weeks post-transfection. However, by 16 weeks post-transfection, bacmids lacking CTCF sites had no detectable Qp transcription and showed high levels of histone H3 K9 methylation and CpG DNA methylation at the Qp initiation site. These findings provide direct genetic evidence that CTCF functions as a chromatin insulator that prevents the promiscuous transcription of surrounding genes and blocks the epigenetic silencing of an essential promoter, Qp, during EBV latent infection.
PDF
