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Lymphomas driven by Epstein-Barr virus nuclear antigen-1 (EBNA1) are dependant upon Mdm2.

ABSTRACT: Epstein-Barr virus (EBV)-associated Burkitt's lymphoma is characterised by the deregulation of c-Myc expression and a restricted viral gene expression pattern in which the EBV nuclear antigen-1 (EBNA1) is the only viral protein to be consistently expressed. EBNA1 is required for viral genome propagation and segregation during latency. However, it has been much debated whether the protein plays a role in viral-associated tumourigenesis. We show that the lymphomas which arise in EµEBNA1 transgenic mice are unequivocally linked to EBNA1 expression and that both C-Myc and Mdm2 deregulation are central to this process. Tumour cell survival is supported by IL-2 and there is a skew towards CD8-positive T cells in the tumour environment, while the immune check-point protein PD-L1 is upregulated in the tumours. Additionally, several isoforms of Mdm2 are upregulated in the EµEBNA1 tumours, with increased phosphorylation at ser166, an expression pattern not seen in Eµc-Myc transgenic tumours. Concomitantly, E2F1, Xiap, Mta1, C-Fos and Stat1 are upregulated in the tumours. Using four independent inhibitors of Mdm2 we demonstrate that the EµEBNA1 tumour cells are dependant upon Mdm2 for survival (as they are upon c-Myc) and that Mdm2 inhibition is not accompanied by upregulation of p53, instead cell death is linked to loss of E2F1 expression, providing new insight into the underlying tumourigenic mechanism. This opens a new path to combat EBV-associated disease.

SUBMITTER: AlQarni S

PROVIDER: S-EPMC6054874 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Lymphomas driven by Epstein-Barr virus nuclear antigen-1 (EBNA1) are dependant upon Mdm2.

AlQarni Sana S Al-Sheikh Yazeed Y Campbell Donald D Drotar Mark M Hannigan Adele A Boyle Shelagh S Herzyk Pawel P Kossenkov Andrew A Armfield Kate K Jamieson Lauren L Bailo Mariarca M Lieberman Paul M PM Tsimbouri Penelope P Wilson Joanna B JB

Oncogene 20180425 29

Epstein-Barr virus (EBV)-associated Burkitt's lymphoma is characterised by the deregulation of c-Myc expression and a restricted viral gene expression pattern in which the EBV nuclear antigen-1 (EBNA1) is the only viral protein to be consistently expressed. EBNA1 is required for viral genome propagation and segregation during latency. However, it has been much debated whether the protein plays a role in viral-associated tumourigenesis. We show that the lymphomas which arise in EµEBNA1 transgenic ...[more]

PMID: 29691476

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Project description:The Epstein-Barr Virus (EBV) Nuclear Antigen 1 (EBNA1) protein is required for the establishment of EBV latent infection in proliferating B-lymphocytes. EBNA1 is a multifunctional DNA-binding protein that stimulates DNA replication at the viral origin of plasmid replication (OriP), regulates transcription of viral and cellular genes, and tethers the viral episome to the cellular chromosome. EBNA1 also provides a survival function to B-lymphocytes, potentially through its ability to alter cellular gene expression. To better understand these various functions of EBNA1, we performed a genome-wide analysis of the viral and cellular DNA sites associated with EBNA1 protein in a latently infected Burkitt lymphoma B-cell line. Chromatin-immunoprecipitation (ChIP) combined with massively parallel deep-sequencing (ChIP-Seq) was used to identify cellular sites bound by EBNA1. Sites identified by ChIP-Seq were validated by conventional real-time PCR, and ChIP-Seq provided quantitative, high-resolution detection of the known EBNA1 binding sites on the EBV genome at OriP and Qp. We identified at least one cluster of unusually high-affinity EBNA1 binding sites on chromosome 11, between the divergent FAM55 D and FAM55B genes. A consensus for all cellular EBNA1 binding sites is distinct from those derived from the known viral binding sites, suggesting that some of these sites are indirectly bound by EBNA1. EBNA1 also bound close to the transcriptional start sites of a large number of cellular genes, including HDAC3, CDC7, and MAP3K1, which we show are positively regulated by EBNA1. EBNA1 binding sites were enriched in some repetitive elements, especially LINE 1 retrotransposons, and had weak correlations with histone modifications and ORC binding. We conclude that EBNA1 can interact with a large number of cellular genes and chromosomal loci in latently infected cells, but that these sites are likely to represent a complex ensemble of direct and indirect EBNA1 binding sites.

Project description:Epstein-Barr virus nuclear antigen (EBNA) leader protein (EBNALP) is one of the first viral genes expressed upon B-cell infection. EBNALP is essential for EBV-mediated B-cell immortalization. EBNALP is thought to function primarily by coactivating EBNA2-mediated transcription. Chromatin immune precipitation followed by deep sequencing (ChIP-seq) studies highlight that EBNALP frequently cooccupies DNA sites with host cell transcription factors (TFs), in particular, EP300, implicating a broader role in transcription regulation. In this study, we investigated the mechanisms of EBNALP transcription coactivation through EP300. EBNALP greatly enhanced EP300 transcription activation when EP300 was tethered to a promoter. EBNALP coimmunoprecipitated endogenous EP300 from lymphoblastoid cell lines (LCLs). EBNALP W repeat serine residues 34, 36, and 63 were required for EP300 association and coactivation. Deletion of the EP300 histone acetyltransferase (HAT) domain greatly reduced EBNALP coactivation and abolished the EBNALP association. An EP300 bromodomain inhibitor also abolished EBNALP coactivation and blocked the EP300 association with EBNALP. EBNALP sites cooccupied by EP300 had significantly higher ChIP-seq signals for sequence-specific TFs, including SPI1, RelA, EBF1, IRF4, BATF, and PAX5. EBNALP- and EP300-cooccurring sites also had much higher H3K4me1 and H3K27ac signals, indicative of activated enhancers. EBNALP-only sites had much higher signals for DNA looping factors, including CTCF and RAD21. EBNALP coactivated reporters under the control of NF-κB or SPI1. EP300 inhibition abolished EBNALP coactivation of these reporters. Clustered regularly interspaced short palindromic repeat interference targeting of EBNALP enhancer sites significantly reduced target gene expression, including that of EP300 itself. These data suggest a previously unrecognized mechanism by which EBNALP coactivates transcription through subverting of EP300 and thus affects the expression of LCL genes regulated by a broad range of host TFs.IMPORTANCE Epstein-Barr virus was the first human DNA tumor virus discovered over 50 years ago. EBV is causally linked to ∼200,000 human malignancies annually. These cancers include endemic Burkitt lymphoma, Hodgkin lymphoma, lymphoma/lymphoproliferative disease in transplant recipients or HIV-infected people, nasopharyngeal carcinoma, and ∼10% of gastric carcinoma cases. EBV-immortalized human B cells faithfully model key aspects of EBV lymphoproliferative diseases and are useful models of EBV oncogenesis. EBNALP is essential for EBV to transform B cells and transcriptionally coactivates EBNA2 by removing repressors from EBNA2-bound DNA sites. Here, we found that EBNALP can also modulate the activity of the key transcription activator EP300, an acetyltransferase that activates a broad range of transcription factors. Our data suggest that EBNALP regulates a much broader range of host genes than was previously appreciated. A small-molecule inhibitor of EP300 abolished EBNALP coactivation of multiple target genes. These findings suggest novel therapeutic approaches to control EBV-associated lymphoproliferative diseases.

Dataset Information

Lymphomas driven by Epstein-Barr virus nuclear antigen-1 (EBNA1) are dependant upon Mdm2.

Publications

Lymphomas driven by Epstein-Barr virus nuclear antigen-1 (EBNA1) are dependant upon Mdm2.

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