EBNA2 and Notch signalling in Epstein-Barr virus mediated immortalization of B lymphocytes.

Epstein-Barr virus (EBV) has the ability to immortalize B cells. A viral key protein for immortalization is the transactivator EBNA2 that controls expression of several viral and cellular genes. EBNA2 is tethered to promoters by interacting with the cellular repressor RBP-J. This resembles the physiological activation of RBP-J-repressed promoters by activated Notch receptors (Notch-IC). Since EBNA2 and Notch-IC have been shown to be partially interchangeable in regard to activation of target genes in B cell lines and modulation of differentiation processes it is conceivable that EBNA2 is a biological equivalent of an activated Notch receptor.

Activation of the Notch-regulated transcription factor CBF1/RBP-Jkappa through the 13SE1A oncoprotein.

Signaling through the Notch pathway controls cell growth and differentiation in metazoans. Following binding of its ligands, the intracellular part of the cell surface Notch1 receptor (Notch1-IC) is released and translocates to the nucleus, where it alters the function of the DNA-binding transcription factor CBF1/RBP-Jkappa. As a result, CBF1/RBP-Jkappa is converted from a repressor to an activator of gene transcription. Similarly, the Epstein Barr viral oncoprotein EBNA2, which is required for B-cell immortalization, activates genes through CBF1. Moreover, the TAN-1 and int-3 oncogenes represent activated versions of Notch1 and Notch4, respectively. Here, we show that the adenoviral oncoprotein 13S E1A also binds to CBF1/RBP-Jkappa, displaces associated corepressor complexes, and activates CBF1/RBP-Jkappa-dependent gene expression. Our results suggest that the central role of the Notch-CBF1/RBP-Jkappa signaling pathway in cell fate decisions renders it susceptible to pathways of viral replication and oncogenic conversion.

Activated Notch1 can transiently substitute for EBNA2 in the maintenance of proliferation of LMP1-expressing immortalized B cells.

Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) and latent membrane protein 1 (LMP1) are essential for immortalization of human B cells by EBV. EBNA2 and activated Notch transactivate genes by interacting with the cellular transcription factor RBP-Jkappa/CBF1. Therefore, EBNA2 can be regarded as a functional homologue of activated Notch. We have shown previously that the intracellular domain of Notch1 (Notch1-IC) is able to transactivate EBNA2-regulated viral promoters and to induce phenotypic changes in B cells similar to those caused by EBNA2. Here we investigated whether Notch1-IC can substitute for EBNA2 in the maintenance of B-cell proliferation. Using an EBV-immortalized lymphoblastoid cell line in which EBNA2 function can be regulated by estrogen, we demonstrate that murine Notch1-IC, in the absence of functional EBNA2, is unable to maintain LMP1 expression and to maintain cell proliferation. However, in a lymphoblastoid cell line expressing LMP1 independently of EBNA2, murine Notch1-IC can transiently maintain proliferation after EBNA2 inactivation. After 4 days, cell numbers do not increase further, and cells in the G2 phase of the cell cycle start to die. In contrast to EBNA2, murine Notch1-IC is unable to upregulate the expression of the c-myc gene in these cells.

Activated Notch1 modulates gene expression in B cells similarly to Epstein-Barr viral nuclear antigen 2.

Both Epstein-Barr viral nuclear antigen 2 (EBNA2) and activated Notch transactivate genes by interacting with the transcription factor RBP-Jkappa. The viral protein EBNA2 may hence be regarded as a functional equivalent of an activated Notch receptor. Until now, nothing has been known about the physiological role of Notch signaling in B cells. Here we investigated whether activated Notch can induce the same phenotypic changes as EBNA2 in Burkitt's lymphoma cells. An estrogen receptor fusion protein of the intracellular part of mouse Notch 1 (mNotch1-IC), mimicking in the presence of estrogen a constitutively active Notch receptor, was stably transfected into the Burkitt's lymphoma cell lines BL41-P3HR1 and HH514. Northern blot analysis revealed that the LMP2A gene is induced by Notch-IC in the presence of estrogen, whereas increased expression of LMP1 could be detected only if cycloheximide was simultaneously added. Concerning the cellular genes regulated by EBNA2, Notch-IC was able to upregulate CD21 but not CD23 expression. Immunoglobulin mu (Igmu) expression, which is downregulated by EBNA2, was also negatively regulated by Notch-IC. Similarly to EBNA2, Notch-IC was able to repress c-myc expression, which is under the control of the immunoglobulin heavy-chain locus in Burkitt's lymphoma cells with a t(8;14) translocation. The data show that Notch-IC is able to participate in gene regulation in B cells.

Activated mouse Notch1 transactivates Epstein-Barr virus nuclear antigen 2-regulated viral promoters.

Epstein-Barr virus nuclear antigen 2 (EBNA2) is essential for B-cell immortalization by EBV, most probably by its ability to transactivate a number of cellular and viral genes. EBNA2-responsive elements (EBNA2REs) have been identified in several EBNA2-regulated viral promoters, each of them carrying at least one RBP-Jkappa recognition site. RBP-Jkappa recruits EBNA2 to the EBNA2RE and, once complexed to EBNA2, is converted from a repressor into an activator. An activated form of the cellular receptor Notch also interacts with RBP-Jkappa, providing a link between EBNA2 and Notch signalling. To determine whether activated Notch is able to transactivate EBNA2-responsive viral promoters, we performed cotransfection experiments with activated mouse Notch1 (mNotch1-IC) and luciferase constructs of the BamHI C, LMP1, and LMP2A promoters. We present here evidence that mNotch1-IC transactivates viral promoters known to be regulated by EBNA2. As shown for EBNA2, mutations or deletions of the RBP-Jkappa sites diminish or eliminate mNotch1-IC-mediated transactivation of the promoters, pointing to an essential role for Notch-RBP-Jkappa interaction. In addition to RBP-Jkappa, other cellular factors may bind within the EBNA2REs of viral promoters. While some factors appear to play an important role in both EBNA2- and mNotch1-IC-mediated transactivation, others are only important for the activity of either EBNA2 or mNotch1-IC. We could observe specific mNotch1-IC-responsive regions, thereby throwing more light upon which cofactors interact with EBNA2 and mNotch1-IC, thus enabling them to become functionally transactivators in vivo.

Functional replacement of the intracellular region of the Notch1 receptor by Epstein-Barr virus nuclear antigen 2.

The intracellular region (RAMIC) of the mouse Notch1 receptor interacts with RBP-J/CBF-1, which binds to the DNA sequence CGTGGGAA and suppresses differentiation by transcriptional activation of genes regulated by RBP-J. Epstein-Barr virus nuclear antigen 2 (EBNA2) is essential for immortalization of human B cells by the virus. EBNA2 is a pleiotropic activator of viral and cellular genes and is targeted to DNA at least in part by interacting with RBP-J. We found that EBNA2 and the Notch1 RAMIC compete for binding to RBP-J, indicating that their interaction sites on RBP-J overlap at least partially. EBNA2 and Notch1 RAMIC transactivated the same set of viral and host promoters, i.e., the EBNA2 response element of the Epstein-Barr virus TP1 and the HES-1 promoter. Furthermore, EBNA2 functionally replaced the Notch1 RAMIC by suppressing differentiation of C2C12 myoblast progenitor cells.

RBP-L, a transcription factor related to RBP-Jkappa.

RBP-Jkappa is a sequence-specific DNA binding protein which plays a central role in signalling downstream of the Notch receptor by physically interacting with its intracellular region. Although at least four Notch genes exist in mammals, it is unknown whether each Notch requires a specific downstream signalling molecule. Here we report isolation and characterization of a mouse RBP-Jkappa-related gene named RBP-L that is expressed almost exclusively in lung, in contrast to the ubiquitous expression of RBP-Jkappa. For simplicity, we propose to call RBP-Jkappa RBP-J. The RBP-L protein bound to a DNA sequence almost identical to that of RBP-J. Surprisingly, RBP-L did not interact with any of the known four mouse Notch proteins. Although we found that RBP-L and EBNA-2 cooperated in transcriptional activation, they did not show significantly strong protein-protein interaction that can be detected by several in vivo and in vitro assays. This is again in contrast to physical association of RBP-J with EBNA-2. Several models to explain functional interaction between RBP-L and EBNA-2 are discussed.

Both Epstein-Barr viral nuclear antigen 2 (EBNA2) and activated Notch1 transactivate genes by interacting with the cellular protein RBP-J kappa.

The Epstein-Barr viral nuclear antigen 2 (EBNA2) plays a key role during establishment and maintenance of B cell immortalization after Epstein-Barr virus (EBV) infection. EBNA2 acts as a transactivator of cellular and viral genes. We studied two EBNA2 regulated viral promoters (TP1 promoter and LMP/TP2 promoter) in detail to learn more about the molecular mechanisms of EBNA2-mediated transactivation. In both promoters we could identify at least one binding site for the cellular repressor protein RBP-J kappa. EBNA2 is tethered to the EBNA2 responsive promoter elements by interaction with this cellular protein. Although necessary, the binding of RBP-J kappa is not sufficient for EBNA2-mediated transactivation. At least two further cellular proteins, which are different in the studied promoters are important for efficient transactivation. The identification of RBP-J kappa as central mediator of EBNA2 transactivation suggested an interference of EBNA2 with the highly conserved Notch receptor signal transduction pathway. We could show that an activated form of the Notch receptor can transactivate a reporter construct containing a hexamer of the two RBP-J kappa binding sites of the TP1 promoter supporting the idea that EBNA2 acts as a functional equivalent of an activated Notch receptor.

c-myc expression is activated by the immunoglobulin kappa-enhancers from a distance of at least 30 kb but not by elements located within 50 kb of the unaltered c-myc locus in vivo.

50 kb of contiguous DNA sequences covering the human c-myc coding region and approximately 20 kb of flanking upstream and downstream sequences were cloned onto a prokaryotic F-factor derived plasmid, which also contains a selectable marker and the plasmid origin of DNA replication oriP of Epstein Barr virus (EBV). Since these plasmids replicate extrachromosomally after stable transfection into EBV-positive B-cell lines, the gene regulation of c-myc can be analysed independent from chromosomal integration positions. Despite the presence of all known c-myc regulatory elements on these constructs, expression from the stably transfected c-myc gene was barely detectable in either cell line. Hypermethylation of these plasmids could be excluded as a mechanism for the lack of gene expression. Insertion of the immunoglobulin kappa-intron and 3' enhancers, however, activated c-myc transcription, when placed adjacent to or separated from the c-myc promoters by as far as 30 kb. These results indicate that transcription of c-myc in vivo requires additional and still unidentified control elements located outside this 50 kb fragment, and experimentally demonstrate long range enhancer function in vivo.

Variable pause positions of RNA polymerase II lie proximal to the c-myc promoter irrespective of transcriptional activity.

Transcriptional activation of the c-myc proto-oncogene is mediated by the transition of promoter proximal, paused RNA polymerase II (pol II) into a processive transcription mode. Using a transcription assay which allows the high resolution mapping of transcriptional complexes in intact nuclei, we have characterized the promoter proximal pause positions of pol II. Pol II paused in a nucleosome-free region close to the transcription start site as well as further downstream, between positions +17 and +52. These pause positions were detected in both transcriptionally active and inactive c-myc genes. Pharmacological inhibition of the C-terminal phosphorylation of the large subunit of pol II did not affect the paused transcription complexes, but had an inhibitory effect on transcription of nucleosomal DNA downstream of position +150. The different properties of pol II proximal and distal to the promoter suggest a model in which c-myc transcription is regulated by the activation of promoter bound polymerases.

The role of immunoglobulin kappa elements in c-myc activation.

Burkitt's lymphoma cells are characterized by chromosomal translocations involving the proto-oncogene c-myc on chromosome 8 and one of the immunoglobulin gene loci on chromosome 2, 14 or 22. The translocated c-myc allele is transcriptionally activated, shows a preferential usage of promoter P1 over P2 (promoter shift) and lacks the ability to retain the transcription complex at the P2 promoter. In order to define the elements of the immunoglobulin kappa gene involved in deregulation of c-myc in a t(2;8) translocation, we designed constructs consisting of c-myc and different parts of the immunoglobulin kappa gene locus. Chromatin analysis of these stably transfected constructs revealed DNase I hypersensitive sites within the c-myc 5' region characteristic for an actively transcribed c-myc gene and three sites within the immunoglobulin kappa locus corresponding to the matrix attachment region, the intron and 3' enhancers, respectively. These three regulatory elements were necessary and sufficient for maximal c-myc activation and formation of the promoter shift. Kinetic nuclear run on experiments were performed to study the distribution of transcription complexes on c-myc exon 1 on constructs with and without the immunoglobulin kappa regulatory elements. The absence of a pausing polymerase complex at the c-myc P2 promoter could be demonstrated for constructs consisting of c-myc and the two kappa enhancers. Therefore the two enhancers are sufficient to relief the elongational block at the P2 promoter, however, the matrix attachment region is additionally required for maximal c-myc activation observed in Burkitt's lymphoma cells.

The Spi-1/PU.1 and Spi-B ets family transcription factors and the recombination signal binding protein RBP-J kappa interact with an Epstein-Barr virus nuclear antigen 2 responsive cis-element.

Epstein-Barr virus (EBV) immortalizes resting human B cells very efficiently in vitro. The EBV nuclear protein EBNA2 is absolutely required for this process. It also activates transcription of cellular, as well as viral, genes. It is assumed that EBNA2 contributes to B cell immortalization by its transactivating potential, since its transforming and transactivating functions could not be separated. Mutational analysis of the 80 bp EBNA2 responsive cis-element within the viral bidirectional LMP/TP2 promoter region identified two sequence elements, which are both essential for transactivation by EBNA2. These sequences harbour putative consensus binding sites for Spi-1 oncoprotein and recombination signal binding protein RBP-J kappa, the homologue of Drosophila Suppressor of Hairless. Electrophoretic mobility shift assays demonstrated the high affinity binding of Spi-1 and Spi-B, both members of the Ets family of transcription factors, to one sequence element. The other element bound RBP-J kappa with low affinity. In addition, co-transfections showed that the replacement of the Spi-1/Spi-B binding site in the bi-directional LMP/TP2 promoter by the analogous SV40 Spi-1 responsive element did not impair its function on EBNA2-mediated transactivation. It is concluded that the transcriptional regulators Spi-1 and Spi-B as well as RBP-J kappa play an essential role in transactivating the LMP/TP2 promoter by EBNA2 and therefore in the immortalization of B cells by EBV.

Crucial sequences within the Epstein-Barr virus TP1 promoter for EBNA2-mediated transactivation and interaction of EBNA2 with its responsive element.

EBNA2 is one of the few genes of Epstein-Barr virus which are necessary for immortalization of human primary B lymphocytes. The EBNA2 protein acts as a transcriptional activator of several viral and cellular genes. For the TP1 promoter, we have shown previously that an EBNA2-responsive element (EBNA2RE) between -258 and -177 relative to the TP1 RNA start site is necessary and sufficient for EBNA2-mediated transactivation and that it binds EBNA2 through a cellular factor. To define the critical cis elements within this region, we cloned EBNA2RE mutants in front of the TP1 minimal promoter fused to the reporter gene for luciferase. Transactivation by EBNA2 was tested by transfection of these mutants in the absence and presence of an EBNA2 expression vector into the established B-cell line BL41-P3HR-1. The analysis revealed that two identical 11-bp motifs and the region 3' of the second 11-bp motif are essential for transactivation by EBNA2. Methylation interference experiments indicated that the same cellular factor in the absence of EBNA2 binds either one (complex I) or both (complex III) 11-bp motifs with different affinities, giving rise to two different specific protein-DNA complexes within the left-hand 54 bp of EBNA2RE. A third specific complex was shown previously to be present only in EBNA2-expressing cells and to contain EBNA2. Analysis of this EBNA2-containing complex revealed the same protection pattern as for complex III, indicating that EBNA2 interacts with DNA through binding of the cellular protein to the 11-bp motifs. Mobility shift assays with the different mutants demonstrated that one 11-bp motif is sufficient for binding the cellular factor, whereas for binding of EBNA2 as well as for efficient transactivation by EBNA2, both 11-bp motifs are required.

Epstein-Barr virus nuclear antigen 2 exerts its transactivating function through interaction with recombination signal binding protein RBP-J kappa, the homologue of Drosophila Suppressor of Hairless.

Epstein-Barr virus nuclear antigen 2 (EBNA-2) plays a crucial role in B cell immortalization by Epstein-Barr virus (EBV), most probably by its ability to transactivate several cellular and viral genes. Recently, we showed that EBNA-2 interacts with the TP1 promoter of EBV through a cellular protein. In this report we provide evidence that this protein is recombination signal binding protein (RBP)-J kappa, highly conserved in evolution, and originally isolated by its ability to bind to the J kappa-type V(D)J recombination signal sequence. To identify the cellular protein interacting with the TP1 promoter, we performed electrophoretic mobility shift assays using binding sequences of known transcription factors, that carry partial homology to the crucial sequences of the EBNA-2 responsive element (EBNA-2RE), as competitor. Competition assays revealed the RBP-J kappa recognition site as a very efficient competitor of cellular TP1 promoter binding protein. In parallel, we purified the protein to homogeneity from Raji cells by two ion-exchange columns and affinity purification using the EBNA-2RE coupled to magnetic beads. Affinity purified fractions separated on SDS-PAGE revealed a single predominant band after silver staining which was recognized by anti-RBP-J kappa monoclonal antibody. These purified fractions exhibited binding specificity for EBNA-2RE and EBNA-2. In vitro-translated murine RBP-2 cDNA reacted with EBNA-2RE and EBNA-2 in the same fashion as the affinity purified protein. The interaction between RBP-J kappa and EBNA-2 is a prerequisite for EBNA-2-mediated transactivation of the TP1 promoter.

Activation of pausing RNA polymerases by nuclear run-on experiments.

The nuclear run-on transcription assay is the only approach to measure the transcriptional activity of a given gene in its genuine structural and regulatory cellular context. However, serious problems in the interpretation of results can arise from the artificial activation of paused RNA polymerases during the transcription reaction, leading to false results with regard to the level and mode of gene regulation in vivo. We have used the example of the human proto-oncogene c-myc, which has previously been reported to be regulated by premature termination of transcription, to describe the problems and pitfalls in the interpretation of nuclear run-on experiments. We show here that activation of paused, elongation-incompetent polymerases in nuclear run-on experiments produces a strong transcription signal on c-myc exon 1 in cells which do not express c-myc steady-state RNA.

Absence of a paused transcription complex from the c-myc P2 promoter of the translocation chromosome in Burkitt's lymphoma cells: implication for the c-myc P1/P2 promoter shift.

We have shown recently that pausing of RNA polymerase II (pol II) at the transcription start site regulates expression from the P2 promoter of the proto-oncogene c-myc. RNAs initiated at the P2 promoter usually contribute > 80% to steady-state c-myc RNA levels in normal cells. In Burkitt's lymphoma (BL) cells c-myc is chromosomally translocated to an immunoglobulin (Ig) gene and preferentially transcribed from the upstream P1 promoter. We have studied the activity of c-myc promoters in two BL cell lines with high expression of P1 RNA. Kinetic nuclear run-on experiments show that the initiation rate at the c-myc P1 promoter in BL2 and BL60 cells is not increased compared with control BJAB cells, whereas the number of paused polymerases at the P2 promoter is greatly diminished. The translocation c-myc gene of BL60 cells was cloned and stably transfected into the BL cell line Raji. The transfected c-myc gene regained the ability to form a paused transcription complex at the c-myc P2 promoter. The data suggest that a paused polymerase at the c-myc P2 promoter impedes transcription from the upstream P1 promoter on a normal c-myc gene. The c-myc gene on the translocation chromosome in BL cells has lost the ability to retain pol II at the P2 promoter, probably by interaction with elements of the adjacent Ig gene locus.

Early down-regulation of c-myc in dimethylsulfoxide-induced mouse erythroleukemia (MEL) cells is mediated at the P1/P2 promoters.

A block of RNA elongation in exon 1 of the murine c-myc gene has been described for normal mouse fibroblasts, lymphoid and myeloid cell lines and mouse erythroleukemia (MEL) cells. MEL cells differentiate after induction with the chemical agent dimethylsulfoxide (DMSO). The rapid initial down-regulation of c-myc that occurs after treatment with DMSO has been explained by an increase in the block of RNA elongation within the 3' part of c-myc exon 1. In contrast to these reports, we find that down-regulation of c-myc in DMSO-induced MEL cells occurs at the c-myc P1 and P2 promoters. The P1 promoter is repressed by inhibition of initiation, whereas transcription of P2 RNA is blocked by retention of RNA polymerase II at or close to the P2 promoter. The earlier described block of RNA elongation at a run of five thymidines in the 3' part of c-myc exon 1 was not observed.

Hold back of RNA polymerase II at the transcription start site mediates down-regulation of c-myc in vivo.

Premature termination of transcription is assumed to be an important mechanism of c-myc regulation. Induction of terminal differentiation in the promyelocytic leukemia cell line HL60 by dimethyl-sulfoxide (DMSO) is accompanied by a block of RNA elongation within the first exon of the c-myc gene. We have studied the 3'-structure of incompletely elongated transcripts in (i) nuclear RNA of induced and uninduced HL60 cells, (ii) nuclear run-on RNA, and (iii) RNA of in vitro transcribed c-myc constructs. Elongation of c-myc RNA stopped in all three transcriptional systems at similar sites distributed 150-350 bases downstream of the P2 promoter. When HL60 cells were induced to terminal differentiation the short c-myc exon 1 specific RNAs disappeared in nuclear RNA. This implied that RNA polymerase II (pol II) does not continue to transcribe c-myc exon 1 in induced HL60 cells as suggested by earlier nuclear run-on experiments. Therefore, kinetic experiments with small oligonucleotides as probes were performed to determine the start position of pol II on c-myc exon 1 in nuclear run-ons. The results demonstrate that all RNA polymerases are localized at the c-myc P2 promoter in DMSO-treated HL60 cells. Preparation of nuclei for run-on experiments induces a release of pol II from the c-myc P2 promoter leading to the strong nuclear run-on signal on c-myc exon 1. Thus, down-regulation of c-myc in differentiating HL60 cells occurs by retention of pol II at the transcription start site.