Antecedent chromatin organization determines cGAS recruitment to ruptured micronuclei.

Micronuclei (MN) are cytosolic bodies that sequester acentric fragments or mis-segregated chromosomes from the primary nucleus. Spontaneous rupture of the MN envelope allows recognition by the viral receptor cyclic GMP-AMP synthase (cGAS), initiating interferon signaling downstream of DNA damage. Here, we demonstrate that MN rupture is permissive but not sufficient for cGAS localization. Chromatin characteristics such as histone 3, lysine 79 dimethylation (H3K79me2) are present in the nucleus before DNA damage, retained in ruptured MN, and regulate cGAS recruitment. cGAS is further responsive to dynamic intra-MN processes occurring prior to rupture, including transcription. MN chromatin tethering via the nucleosome acidic patch is necessary for cGAS-dependent interferon signaling. Our data suggest that both damage-antecedent nuclear chromatin status and MN-contained chromatin organizational changes dictate cGAS recruitment and the magnitude of the cGAS-driven interferon cascade. Our work defines MN as integrative signaling hubs for the cellular response to genotoxic stress.

Regulation of B cell receptor signalling by Epstein-Barr virus nuclear antigens.

The cancer-associated Epstein-Barr virus (EBV) latently infects and immortalises B lymphocytes. EBV latent membrane protein 2A and EBV-encoded microRNAs are known to manipulate B cell receptor signalling to control cell growth and survival and suppress lytic replication. Here, we show that the EBV transcription factors EBNA2, 3A, 3B and 3C bind to genomic sites around multiple B cell receptor (BCR) pathway genes, regulate their expression and affect BCR signalling. EBNA2 regulates the majority of BCR pathway genes associated with binding sites, where EBNA3 proteins regulate only 42% of targets predicted by binding. Both EBNA2 and 3 proteins predominantly repress BCR pathway gene expression and target some common genes. EBNA2 and at least one EBNA3 protein repress the central BCR components CD79A and CD79B and the downstream genes BLNK, CD22, CD72, NFATC1, PIK3CG and RASGRP3. Studying repression of CD79B, we show that EBNA2 decreases transcription by disrupting binding of Early B cell Factor-1 to the CD79B promoter. Consistent with repression of BCR signalling, we demonstrate that EBNA2 and EBNA3 proteins suppress the basal or active BCR signalling that culminates in NFAT activation. Additionally, we show that EBNA2, EBNA3A and EBNA3C expression can result in reductions in the active serine 473 phosphorylated form of Akt in certain cell contexts, consistent with transcriptional repression of the PI3K-Akt BCR signalling arm. Overall, we identify EBNA2, EBNA3A and EBNA3C-mediated transcription control of BCR signalling as an additional strategy through which EBV may control the growth and survival of infected B cells and maintain viral latency.

Pumilio directs deadenylation-associated translational repression of the cyclin-dependent kinase 1 activator RGC-32.

Response gene to complement-32 (RGC-32) activates cyclin-dependent kinase 1, regulates the cell cycle and is deregulated in many human tumours. We previously showed that RGC-32 expression is upregulated by the cancer-associated Epstein-Barr virus (EBV) in latently infected B cells through the relief of translational repression. We now show that EBV infection of naïve primary B cells also induces RGC-32 protein translation. In EBV-immortalised cell lines, we found that RGC-32 depletion resulted in cell death, indicating a key role in B cell survival. Studying RGC-32 translational control in EBV-infected cells, we found that the RGC-32 3'untranslated region (3'UTR) mediates translational repression. Repression was dependent on a single Pumilio binding element (PBE) adjacent to the polyadenylation signal. Mutation of this PBE did not affect mRNA cleavage, but resulted in increased polyA tail length. Consistent with Pumilio-dependent recruitment of deadenylases, we found that depletion of Pumilio in EBV-infected cells increased RGC-32 protein expression and polyA tail length. The extent of Pumilio binding to the endogenous RGC-32 mRNA in EBV-infected cell lines also correlated with RGC-32 protein expression. Our data demonstrate the importance of RGC-32 for the survival of EBV-immortalised B cells and identify Pumilio as a key regulator of RGC-32 translation.

MYC activation and BCL2L11 silencing by a tumour virus through the large-scale reconfiguration of enhancer-promoter hubs.

Lymphomagenesis in the presence of deregulated MYC requires suppression of MYC-driven apoptosis, often through downregulation of the pro-apoptotic BCL2L11 gene (Bim). Transcription factors (EBNAs) encoded by the lymphoma-associated Epstein-Barr virus (EBV) activate MYC and silence BCL2L11. We show that the EBNA2 transactivator activates multiple MYC enhancers and reconfigures the MYC locus to increase upstream and decrease downstream enhancer-promoter interactions. EBNA2 recruits the BRG1 ATPase of the SWI/SNF remodeller to MYC enhancers and BRG1 is required for enhancer-promoter interactions in EBV-infected cells. At BCL2L11, we identify a haematopoietic enhancer hub that is inactivated by the EBV repressors EBNA3A and EBNA3C through recruitment of the H3K27 methyltransferase EZH2. Reversal of enhancer inactivation using an EZH2 inhibitor upregulates BCL2L11 and induces apoptosis. EBV therefore drives lymphomagenesis by hijacking long-range enhancer hubs and specific cellular co-factors. EBV-driven MYC enhancer activation may contribute to the genesis and localisation of MYC-Immunoglobulin translocation breakpoints in Burkitt's lymphoma.

Downregulation of integrin receptor-signaling genes by Epstein-Barr virus EBNA 3C via promoter-proximal and -distal binding elements.

Epstein-Barr virus (EBV) establishes a persistent latent infection in B lymphocytes and is associated with the development of numerous human tumors. Epstein-Barr nuclear antigen 3C (EBNA 3C) is essential for B-cell immortalization, has potent cell cycle deregulation capabilities, and functions as a regulator of both viral- and cellular-gene expression. We performed transcription profiling on EBNA 3C-expressing B cells and identified several chemokines and members of integrin receptor-signaling pathways, including CCL3, CCL4, CXCL10, CXCL11, ITGA4, ITGB1, ADAM28, and ADAMDEC1, as cellular target genes that could be repressed by the action of EBNA 3C alone. Chemotaxis assays demonstrated that downregulation of CXCL10 and -11 by EBNA 3C is sufficient to reduce the migration of cells expressing the CXCL10 and -11 receptor CXCR3. Gene repression by EBNA 3C was accompanied by decreased histone H3 lysine 9/14 acetylation and increased histone H3 lysine 27 trimethylation. In an EBV-positive cell line expressing all latent genes, we identified binding sites for EBNA 3C at ITGB1 and ITGA4 and in a distal regulatory region between ADAMDEC1 and ADAM28, providing the first demonstration of EBNA 3C association with cellular-gene control regions. Our data implicate indirect mechanisms in CXCL10 and CXCL11 repression by EBNA 3C. In summary, we have unveiled key cellular pathways repressed by EBNA 3C that are likely to contribute to the ability of EBV-immortalized cells to modulate immune responses, adhesion, and B-lymphocyte migration to facilitate persistence in the host.

Upregulation of the cell-cycle regulator RGC-32 in Epstein-Barr virus-immortalized cells.

Epstein-Barr virus (EBV) is implicated in the pathogenesis of multiple human tumours of lymphoid and epithelial origin. The virus infects and immortalizes B cells establishing a persistent latent infection characterized by varying patterns of EBV latent gene expression (latency 0, I, II and III). The CDK1 activator, Response Gene to Complement-32 (RGC-32, C13ORF15), is overexpressed in colon, breast and ovarian cancer tissues and we have detected selective high-level RGC-32 protein expression in EBV-immortalized latency III cells. Significantly, we show that overexpression of RGC-32 in B cells is sufficient to disrupt G2 cell-cycle arrest consistent with activation of CDK1, implicating RGC-32 in the EBV transformation process. Surprisingly, RGC-32 mRNA is expressed at high levels in latency I Burkitt's lymphoma (BL) cells and in some EBV-negative BL cell-lines, although RGC-32 protein expression is not detectable. We show that RGC-32 mRNA expression is elevated in latency I cells due to transcriptional activation by high levels of the differentially expressed RUNX1c transcription factor. We found that proteosomal degradation or blocked cytoplasmic export of the RGC-32 message were not responsible for the lack of RGC-32 protein expression in latency I cells. Significantly, analysis of the ribosomal association of the RGC-32 mRNA in latency I and latency III cells revealed that RGC-32 transcripts were associated with multiple ribosomes in both cell-types implicating post-initiation translational repression mechanisms in the block to RGC-32 protein production in latency I cells. In summary, our results are the first to demonstrate RGC-32 protein upregulation in cells transformed by a human tumour virus and to identify post-initiation translational mechanisms as an expression control point for this key cell-cycle regulator.