Michelle J. West

Translational induction of the c-myc oncogene via activation of the FRAP/TOR signalling pathway

Previous studies on the regulation of c-myc have focused on the transcriptional control of this proto-oncogene. We have investigated the signalling pathways involved under circumstances in which there is a translational up-regulation in the levels of c-myc protein. We have demonstrated an up to tenfold serum-dependent increase of c-myc protein levels in Epstein-Barr virus immortalized B-cell lines 2–4 h after disruption of cellular aggregates, which is not accompanied by an equivalent increase in mRNA. Overall protein synthesis rates only increased threefold suggesting that the c-myc message was being selectively translated. We observed increases in the phosphorylation of p70 and p85 S6 kinases and of initiation factor eIF-4E binding protein 1 (4E-BP1) 1–2 h after stimulation, suggesting activation of the FRAP/TOR signalling pathway. The increased phosphorylation of 4E-BP1 led to a decrease in its association with eIF-4E and an increase in its association with the eIF-4G component of the eIF-4F initiation complex. The signalling inhibitors rapamycin and wortmannin blocked the phosphorylation of 4E-BP1 and abolished the translational component of the c-myc response. Our data suggest that dissociation of eIF-4E from 4E-BP1, leading to an increase in the formation of the eIF-4F initiation complex, relieves the translation repression imposed on the c-myc mRNA by its structured 5′UTR.

Repression of the HIV-1 5′ LTR promoter and inhibition of HIV-1 replication by using engineered zinc-finger transcription factors

Zinc finger domains are small DNA-binding modules that can be engineered to bind desired target sequences. Functional transcription factors can be made from these DNA-binding modules, by fusion with an appropriate effector domain. In this study, eight three-zinc-finger proteins (ZFPs) that bound HIV-1 sequences in vitro were engineered into transcription repressors by linking them to the Krüppel-associated box (KRAB) repressor domain (KOX1). One protein, ZFP HIVB-KOX, which bound to a 9-bp region overlapping two Sp1 sites, was found to repress a Tat-activated 5′ LTR cellular HIV-reporter assay to almost basal levels. A related six-finger protein, HIVBA′-KOX, was made to target all three Sp1 sites in the 5′ LTR promoter and efficiently inhibited both basal and Tat-activated transcription in unstimulated and mitogen-stimulated T cells. In contrast, a combination of two unlinked three-finger ZFPs, HIVA′-KOX and HIVB-KOX, which bind over the same region of DNA, resulted in less effective repression. Finally, HIVBA′-KOX was tested for its capacity to block viral replication in a cellular infection assay using the HIV-1 HXB2 strain. This ZFP was found to inhibit HIV-1 replication by 75% compared with control constructs, thus demonstrating the potential of this approach for antiviral therapy.

Activation of Human Immunodeficiency Virus Transcription in T Cells Revisited: NF-κB p65 Stimulates Transcriptional Elongation

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) is able to establish a persistent latent infection during which the integrated provirus remains transcriptionally silent. Viral transcription is stimulated by NF-κB, which is activated following the exposure of infected T cells to antigens or mitogens. Although it is commonly assumed that NF-κB stimulates transcriptional initiation alone, we have found using RNase protection assays that, in addition to stimulating initiation, it can also stimulate elongation from the HIV-1 long terminal repeat. When either Jurkat or CCRF/CEM cells were activated by the mitogens phorbol myristate acetate and phytohemagglutinin, elongation, as measured by the proportion of full-length transcripts, increased two- to fourfold, even in the absence of Tat. Transfection of T cells with plasmids carrying the different subunits of NF-κB demonstrated that the activation of transcriptional elongation is mediated specifically by the p65 subunit. It seems likely that initiation is activated because of NF-κBs ability to disrupt chromatin structures through the recruitment of histone acetyltransferases. To test whether p65 could stimulate elongation under conditions where it did not affect histone acetylation, cells were treated with the histone deacetylase inhibitor trichostatin A. Remarkably, addition of p65 to the trichostatin A-treated cell lines resulted in a dramatic increase in transcription elongation, reaching levels equivalent to those observed in the presence of Tat. We suggest that the activation of elongation by NF-κB p65 involves a distinct biochemical mechanism, probably the activation of carboxyl-terminal domain kinases at the promoter.

Recruitment of TFIIH to the HIV LTR is a rate-limiting step in the emergence of HIV from latency

Latently infected cells rapidly initiate HIV transcription after exposure to signals that induce NF‐κB. To investigate the role of TFIIH during HIV reactivation in vivo, we developed a population of Jurkat cells containing integrated, but transcriptionally silent, HIV proviruses. Surprisingly, the HIV promoter in unactivated Jurkat T cells is partially occupied and carries Mediator containing the CDK8 repressive module, TFIID and RNAP II that is hypophosphorylated and confined to the promoter region. Significantly, the promoter is devoid of TFIIH. Upon stimulation of the cells by TNF‐α, NF‐κB and TFIIH are rapidly recruited to the promoter together with additional Mediator and RNAP II, but CDK8 is lost. Detailed time courses show that the levels of TFIIH at the promoter fluctuate in parallel with NF‐κB recruitment to the promoter. Similarly, recombinant p65 activates HIV transcription in vitro and stimulates phosphorylation of the RNAP II CTD by the CDK7 kinase module of TFIIH. We conclude that the recruitment and activation of TFIIH represents a rate‐limiting step for the emergence of HIV from latency.

EBV EBNA 2 stimulates CDK9-dependent transcription and RNA polymerase II phosphorylation on serine 5

EBNA 2 is one of only five viral genes essential for the infection and immortalization of human B cells by the cancer-associated virus Epstein-Barr virus (EBV). EBNA 2 activates cellular and viral transcription and associates with components of the basal transcription apparatus and a number of coactivators. We provide the first evidence to show that the mechanism of transcriptional activation by EBNA 2 also involves phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (pol II). We found that transcriptional activation by EBNA 2 was inhibited by a dominant-negative mutant of the pol II CTD kinase, CDK9, and by low concentrations of the CDK9 inhibitor 5, 6-dichloro-1-β-D-ribofuranosylbenzimidazole. Moreover, using chromatin immunoprecipitation assays we demonstrated that EBNA 2 stimulates both pol II recruitment and pol II phosphorylation on serine 5 of the CTD in vivo. These results identify a new step in the transcription cycle that is subject to regulation by a key EBV-encoded transcription factor and highlight CDK9 inhibitors as potential anti-EBV agents.

Modulation of enhancer looping and differential gene targeting by Epstein-Barr virus transcription factors directs cellular reprogramming.

Epstein-Barr virus (EBV) epigenetically reprogrammes B-lymphocytes to drive immortalization and facilitate viral persistence. Host-cell transcription is perturbed principally through the actions of EBV EBNA 2, 3A, 3B and 3C, with cellular genes deregulated by specific combinations of these EBNAs through unknown mechanisms. Comparing human genome binding by these viral transcription factors, we discovered that 25% of binding sites were shared by EBNA 2 and the EBNA 3s and were located predominantly in enhancers. Moreover, 80% of potential EBNA 3A, 3B or 3C target genes were also targeted by EBNA 2, implicating extensive interplay between EBNA 2 and 3 proteins in cellular reprogramming. Investigating shared enhancer sites neighbouring two new targets (WEE1 and CTBP2) we discovered that EBNA 3 proteins repress transcription by modulating enhancer-promoter loop formation to establish repressive chromatin hubs or prevent assembly of active hubs. Re-ChIP analysis revealed that EBNA 2 and 3 proteins do not bind simultaneously at shared sites but compete for binding thereby modulating enhancer-promoter interactions. At an EBNA 3-only intergenic enhancer site between ADAM28 and ADAMDEC1 EBNA 3C was also able to independently direct epigenetic repression of both genes through enhancer-promoter looping. Significantly, studying shared or unique EBNA 3 binding sites at WEE1, CTBP2, ITGAL (LFA-1 alpha chain), BCL2L11 (Bim) and the ADAMs, we also discovered that different sets of EBNA 3 proteins bind regulatory elements in a gene and cell-type specific manner. Binding profiles correlated with the effects of individual EBNA 3 proteins on the expression of these genes, providing a molecular basis for the targeting of different sets of cellular genes by the EBNA 3s. Our results therefore highlight the influence of the genomic and cellular context in determining the specificity of gene deregulation by EBV and provide a paradigm for host-cell reprogramming through modulation of enhancer-promoter interactions by viral transcription factors.

RNA Polymerase II Stalling Promotes Nucleosome Occlusion and pTEFb Recruitment to Drive Immortalization by Epstein-Barr Virus

Epstein-Barr virus (EBV) immortalizes resting B-cells and is a key etiologic agent in the development of numerous cancers. The essential EBV-encoded protein EBNA 2 activates the viral C promoter (Cp) producing a message of ∼120 kb that is differentially spliced to encode all EBNAs required for immortalization. We have previously shown that EBNA 2-activated transcription is dependent on the activity of the RNA polymerase II (pol II) C-terminal domain (CTD) kinase pTEFb (CDK9/cyclin T1). We now demonstrate that Cp, in contrast to two shorter EBNA 2-activated viral genes (LMP 1 and 2A), displays high levels of promoter-proximally stalled pol II despite being constitutively active. Consistent with pol II stalling, we detect considerable pausing complex (NELF/DSIF) association with Cp. Significantly, we observe substantial Cp-specific pTEFb recruitment that stimulates high-level pol II CTD serine 2 phosphorylation at distal regions (up to +75 kb), promoting elongation. We reveal that Cp-specific pol II accumulation is directed by DNA sequences unfavourable for nucleosome assembly that increase TBP access and pol II recruitment. Stalled pol II then maintains Cp nucleosome depletion. Our data indicate that pTEFb is recruited to Cp by the bromodomain protein Brd4, with polymerase stalling facilitating stable association of pTEFb. The Brd4 inhibitor JQ1 and the pTEFb inhibitors DRB and Flavopiridol significantly reduce Cp, but not LMP1 transcript production indicating that Brd4 and pTEFb are required for Cp transcription. Taken together our data indicate that pol II stalling at Cp promotes transcription of essential immortalizing genes during EBV infection by (i) preventing promoter-proximal nucleosome assembly and ii) necessitating the recruitment of pTEFb thereby maintaining serine 2 CTD phosphorylation at distal regions.

Downregulation of Integrin Receptor-Signaling Genes by Epstein-Barr Virus EBNA 3C via Promoter-Proximal and -Distal Binding Elements

ABSTRACT 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.

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 Burkitts lymphoma. DOI: http://dx.doi.org/10.7554/eLife.18270.001

A single amino acid in EBNA-2 determines superior B lymphoblastoid cell line growth maintenance by Epstein-Barr virus type 1 EBNA-2

ABSTRACT Sequence differences in the EBNA-2 protein mediate the superior ability of type 1 Epstein-Barr virus (EBV) to transform human B cells into lymphoblastoid cell lines compared to that of type 2 EBV. Here we show that changing a single amino acid (S442D) from serine in type 2 EBNA-2 to the aspartate found in type 1 EBNA-2 confers a type 1 growth phenotype in a lymphoblastoid cell line growth maintenance assay. This amino acid lies in the transactivation domain of EBNA-2, and the S442D change increases activity in a transactivation domain assay. The superior growth properties of type 1 EBNA-2 correlate with the greater induction of EBV LMP-1 and about 10 cell genes, including CXCR7. In chromatin immunoprecipitation assays, type 1 EBNA-2 is shown to associate more strongly with EBNA-2 binding sites near the LMP-1 and CXCR7 genes. Unbiased motif searching of the EBNA-2 binding regions of the differentially regulated cell genes identified an ETS-interferon regulatory factor composite element motif that closely corresponds to the sequences known to mediate EBNA-2 regulation of the LMP-1 promoter. It appears that the superior induction by type 1 EBNA-2 of the cell genes contributing to cell growth is due to their being regulated in a manner different from that for most EBNA-2-responsive genes and in a way similar to that for the LMP-1 gene. IMPORTANCE The EBNA-2 transcription factor plays a key role in B cell transformation by EBV and defines the two EBV types. Here we identify a single amino acid (Ser in type 1 EBV, Asp in type 2 EBV) of EBNA-2 that determines the superior ability of type 1 EBNA-2 to induce a key group of cell genes and the EBV LMP-1 gene, which mediate the growth advantage of B cells infected with type 1 EBV. The EBNA-2 binding sites in these cell genes have a sequence motif similar to the sequence known to mediate regulation of the EBV LMP-1 promoter. Further detailed analysis of transactivation and promoter binding provides new insight into the physiological regulation of cell genes by EBNA-2.