Epstein-Barr Virus Proteins EBNA3A and EBNA3C Together Induce Expression of the Oncogenic MicroRNA Cluster miR-221/miR-222 and Ablate Expression of Its Target p57KIP2

Bazot, Quentin; Paschos, Kostas; Skalska, Lenka; Kalchschmidt, Jens; Parker, Gillian A.; Allday, Martin J.

doi:10.1371/journal.ppat.1005031

Cited by 37 publications

(53 citation statements)

References 91 publications

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“…( B ) Two clones of an EBNA3A-regulatable cell line with EBNA3A under control of 4-hydroxy-tamoxifen (HT) (Bazot et al, 2015). qPCR of BFL-1 mRNA is shown when EBNA3A is removed by withdrawal from HT.…”

Section: Resultsmentioning

confidence: 99%

Epstein-Barr virus ensures B cell survival by uniquely modulating apoptosis at early and late times after infection

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Dai

Bazot

et al. 2017

eLife

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Latent Epstein-Barr virus (EBV) infection is causally linked to several human cancers. EBV expresses viral oncogenes that promote cell growth and inhibit the apoptotic response to uncontrolled proliferation. The EBV oncoprotein LMP1 constitutively activates NFκB and is critical for survival of EBV-immortalized B cells. However, during early infection EBV induces rapid B cell proliferation with low levels of LMP1 and little apoptosis. Therefore, we sought to define the mechanism of survival in the absence of LMP1/NFκB early after infection. We used BH3 profiling to query mitochondrial regulation of apoptosis and defined a transition from uninfected B cells (BCL-2) to early-infected (MCL-1/BCL-2) and immortalized cells (BFL-1). This dynamic change in B cell survival mechanisms is unique to virus-infected cells and relies on regulation of MCL-1 mitochondrial localization and BFL-1 transcription by the viral EBNA3A protein. This study defines a new role for EBNA3A in the suppression of apoptosis with implications for EBV lymphomagenesis.DOI: http://dx.doi.org/10.7554/eLife.22509.001

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Section: Resultsmentioning

confidence: 99%

Epstein-Barr virus ensures B cell survival by uniquely modulating apoptosis at early and late times after infection

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Dai

Bazot

et al. 2017

eLife

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“…Although EBNA3s bind an RBPJ domain that is distinct from the EBNA2/ICN binding site, they nevertheless limit EBNA2 activation by competing for RBPJ binding (15)(16)(17)(18). In LCLs and Burkitt lymphoma tumor cells, the EBNA3 proteins have been shown to regulate distinct but extensively overlapping sets of cell genes (19)(20)(21)(22)(23)(24)(25)(26). EBNA3 proteins have been implicated in the pathogenesis of Burkitt lymphoma and in attenuating an antiproliferative DNA damage response during EBV transformation of primary B lymphocytes (19,(27)(28)(29).…”

Section: Epstein-barr Virus (Ebv) Latent Gene Products Cause Human Camentioning

confidence: 99%

Epstein-Barr Virus Nuclear Antigen 3 (EBNA3) Proteins Regulate EBNA2 Binding to Distinct RBPJ Genomic Sites

Wang

Welch

et al. 2016

J Virol

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Latent infection of B lymphocytes by Epstein-Barr virus (EBV) in vitro results in their immortalization into lymphoblastoid cell lines (LCLs); this latency program is controlled by the EBNA2 viral transcriptional activator, which targets promoters via RBPJ, a DNA binding protein in the Notch signaling pathway. Three other EBNA3 proteins (EBNA3A, EBNA3B, and EBNA3C) interact with RBPJ to regulate cell gene expression. The mechanism by which EBNAs regulate different genes via RBPJ remains unclear. Our chromatin immunoprecipitation with deep sequencing (ChIP-seq) analysis of the EBNA3 proteins analyzed in concert with prior EBNA2 and RBPJ data demonstrated that EBNA3A, EBNA3B, and EBNA3C bind to distinct, partially overlapping genomic locations. Although RBPJ interaction is critical for EBNA3A and EBNA3C growth effects, only 30 to 40% of EBNA3-bound sites colocalize with RBPJ. Using LCLs conditional for EBNA3A or EBNA3C activity, we demonstrate that EBNA2 binding at sites near EBNA3A-or EBNA3C-regulated genes is specifically regulated by the respective EBNA3. To investigate EBNA3 binding specificity, we identified sequences and transcription factors enriched at EBNA3A-, EBNA3B-, and EBNA3C-bound sites. This confirmed the prior observation that IRF4 is enriched at EBNA3A-and EBNA3C-bound sites and revealed IRF4 enrichment at EBNA3B-bound sites. Using IRF4-negative BJAB cells, we demonstrate that IRF4 is essential for EBNA3C, but not EBNA3A or EBNA3B, binding to specific sites. These results support a model in which EBNA2 and EBNA3s compete for distinct subsets of RBPJ sites to regulate cell genes and where EBNA3 subset specificity is determined by interactions with other cell transcription factors. IMPORTANCE Epstein-Barr virus (EBV) latent gene products cause human cancers and transform B lymphocytes into immortalized lymphoblastoid cell lines in vitro.EBV nuclear antigens (EBNAs) and membrane proteins constitutively activate pathways important for lymphocyte growth and survival. An important unresolved question is how four different EBNAs (EBNA2, -3A, -3B, and -3C) exert unique effects via a single transcription factor, RBPJ. Here, we report that each EBNA binds to distinct but partially overlapping sets of genomic sites. EBNA3A and EBNA3C specifically regulate EBNA2's access to different RBPJ sites, providing a mechanism by which each EBNA can regulate distinct cell genes. We show that IRF4, an essential regulator of B cell differentiation, is critical for EBNA3C binding specificity; EBNA3A and EBNA3B specificities are likely due to interactions with other cell transcription factors. EBNA3 titration of EBNA2 transcriptional function at distinct sites likely limits cell defenses that would be triggered by unchecked EBNA2 prooncogenic activity. E pstein-Barr virus (EBV) is a herpesvirus that infects over 90% of the population by adulthood. Primary EBV infection usually presents as a nonspecific illness in early childhood but often manifests as infectious mononucleosis in adolescents (1). Thereafter, EBV es...

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“…They do not bind DNA directly, but, via contacts with cellular cofactors, are largely targeted to chromatin at gene promoters and/or distal regulatory elements [ 12 – 15 ]. Often, regulation of transcription involves long-distance chromatin interactions (chromosome ‘looping’) between promoter and enhancer elements mediated by EBNA3 proteins [ 16 – 19 ].…”

Section: Introductionmentioning

confidence: 99%

EBV epigenetically suppresses the B cell-to-plasma cell differentiation pathway while establishing long-term latency

et al. 2017

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Mature human B cells infected by Epstein-Barr virus (EBV) become activated, grow, and proliferate. If the cells are infected ex vivo, they are transformed into continuously proliferating lymphoblastoid cell lines (LCLs) that carry EBV DNA as extra-chromosomal episomes, express 9 latency-associated EBV proteins, and phenotypically resemble antigen-activated B-blasts. In vivo similar B-blasts can differentiate to become memory B cells (MBC), in which EBV persistence is established. Three related latency-associated viral proteins EBNA3A, EBNA3B, and EBNA3C are transcription factors that regulate a multitude of cellular genes. EBNA3B is not necessary to establish LCLs, but EBNA3A and EBNA3C are required to sustain proliferation, in part, by repressing the expression of tumour suppressor genes. Here we show, using EBV-recombinants in which both EBNA3A and EBNA3C can be conditionally inactivated or using virus completely lacking the EBNA3 gene locus, that—after a phase of rapid proliferation—infected primary B cells express elevated levels of factors associated with plasma cell (PC) differentiation. These include the cyclin-dependent kinase inhibitor (CDKI) p18INK4c, the master transcriptional regulator of PC differentiation B lymphocyte-induced maturation protein-1 (BLIMP-1), and the cell surface antigens CD38 and CD138/Syndecan-1. Chromatin immunoprecipitation sequencing (ChIP-seq) and chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) indicate that in LCLs inhibition of CDKN2C (p18INK4c) and PRDM1 (BLIMP-1) transcription results from direct binding of EBNA3A and EBNA3C to regulatory elements at these loci, producing stable reprogramming. Consistent with the binding of EBNA3A and/or EBNA3C leading to irreversible epigenetic changes, cells become committed to a B-blast fate <12 days post-infection and are unable to de-repress p18INK4c or BLIMP-1—in either newly infected cells or conditional LCLs—by inactivating EBNA3A and EBNA3C. In vitro, about 20 days after infection with EBV lacking functional EBNA3A and EBNA3C, cells develop a PC-like phenotype. Together, these data suggest that EBNA3A and EBNA3C have evolved to prevent differentiation to PCs after infection by EBV, thus favouring long-term latency in MBC and asymptomatic persistence.

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Epstein-Barr Virus Proteins EBNA3A and EBNA3C Together Induce Expression of the Oncogenic MicroRNA Cluster miR-221/miR-222 and Ablate Expression of Its Target p57KIP2

Cited by 37 publications

References 91 publications

Epstein-Barr virus ensures B cell survival by uniquely modulating apoptosis at early and late times after infection

Epstein-Barr virus ensures B cell survival by uniquely modulating apoptosis at early and late times after infection

Epstein-Barr Virus Nuclear Antigen 3 (EBNA3) Proteins Regulate EBNA2 Binding to Distinct RBPJ Genomic Sites

EBV epigenetically suppresses the B cell-to-plasma cell differentiation pathway while establishing long-term latency

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