The activity of the Epstein–Barr virus BamHI W promoter in B cells is dependent on the binding of CREB/ATF factors

Helen Kirby1, Alan Rickinson1 and Andrew Bell1

CRC Institute for Cancer Studies, The University of Birmingham, Edgbaston, Birmingham B15 2TA, UK1

Author for correspondence: Andrew Bell. Fax +44 121 414 4486. e-mail a.i.bell{at}bham.ac.uk


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
The programme of Epstein–Barr virus (EBV) gene expression that leads to virus-induced growth transformation of resting B lymphocytes is initiated through activation of the BamHI W promoter, Wp. The factors regulating Wp, and the basis of its preferential activity in B cells, remain poorly understood. Previous work has identified a B cell-specific enhancer region which is critical for Wp function and which contains three binding sites for cellular factors. Here we focus on one of these sites and show, using bandshift assays, that it interacts with three members of the CREB/ATF family of cell transcription factors, CREB1, ATF1 and ATFa. A mutation which abrogates the binding of these factors reduces Wp reporter activity specifically in B cell lines, whereas a mutation which converts the site to a consensus CREB-binding sequence maintains wild-type promoter function. Furthermore Wp activity in B cell, but not in non-B cell, lines could be inhibited by cotransfection of expression plasmids expressing dominant negative forms of CREB1 and ATF1. Increasing the basal activity of CREB/ATF proteins in cells by treatment with protein kinase A or protein kinase C agonists led to small increases in Wp activity in B cell lines, but did not restore promoter activity in non-B cell lines up to B cell levels. We conclude that CREB/ATF factors are important activators of Wp in a B cell environment but require additional B cell-specific factors in order to mediate their effects.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Epstein–Barr virus (EBV), a lymphotropic herpesvirus with potent B cell-transforming properties, is aetiologically linked to a number of human malignancies and is the causative agent of infectious mononucleosis, a self-limiting lymphoproliferative disease (Rickinson & Kieff, 1996 ). The experimental infection of resting B cells in vitro leads to the outgrowth of EBV-transformed lymphoblastoid cell lines (LCLs). Such LCLs proliferate indefinitely in culture as the result of the coordinate action of a limited set of viral gene products (Kieff, 1996 ); these include the six nuclear antigens (EBNAs 1, 2, 3A, 3B, 3C and LP), three latent membrane proteins (LMP1, LMP2A and LMP2B), products of the highly spliced BamHI A transcripts and two small non-polyadenylated RNAs (EBERs 1 and 2). The initiating event in this growth transformation pathway is the activation of a virus promoter, Wp, located in the BamHI W repeat region of the EBV genome (Sample et al., 1986 ; Woisetschlaeger et al., 1990 ; Alfieri et al., 1991 ). This promoter drives the initial expression of EBNA2, a transactivator which, together with a second protein, EBNA-LP, is responsible for the induction of the remaining latent genes (Abbot et al., 1990 ; Fhraeus et al., 1990 ; Wang et al., 1990 ; Sung et al., 1991 ; Woisetschlaeger et al., 1991 ; Zimber-Strobl et al., 1991 ; Jin & Speck, 1992 ; Rooney et al., 1992 ; Harada & Kieff, 1997 ; Nitsche et al., 1997 ).

Although there is increasing understanding of the mechanisms of action of these viral transforming proteins, the cellular factors which trigger the activation of Wp and which therefore initiate the transformation process remain poorly characterized. There is clearly some degree of B cell specificity to this process since the transforming programme of viral antigen expression is not activated when the virus is introduced experimentally into other cell types (Li et al., 1992 ). In this context, a number of studies using Wp reporter constructs have suggested that Wp is optimized for expression in B cells; such reports have also identified a number of regulatory sequences that are critical for promoter activity, including a B cell-specific enhancer (Ricksten et al., 1988 ; Jansson et al., 1992 ; Nilsson et al., 1993 ; Bell et al., 1998 ). In particular, we recently characterized a number of novel Wp regulatory elements including a promoter proximal sequence termed UAS1, which confers preferential activity in B cells, and which contains three adjacent binding sites for cellular factors (Bell et al., 1998 ). One of these binding elements (site A) contains the motif 5' TTACGTAA 3', which is similar to the cyclic AMP response element (CRE) (Montminy et al., 1986 ). Such CRE motifs can interact with a family of related DNA-binding proteins which include the CRE-binding proteins (CREBs), activating transcription factors (ATFs) and CRE modulator proteins (CREMs) (reviewed by Meyer & Habener, 1993 ); all of these factors share both a conserved basic and a leucine zipper domain (bZIP structure) which are important for DNA binding and dimerization respectively (Johnson & McKnight, 1989 ; Landschulz et al., 1989 ). Since these CREB/ATF factors provide a possible mechanism for the regulation of Wp, we have characterized the CREB/ATF factors which bind to the promoter sequence and investigated the contribution of these factors to Wp activity in different cell types.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Cell lines and culture.
A number of cell lines were used throughout this work for transient assays and the preparation of nuclear extracts. Ramos and DG75 are EBV-negative Burkitt’s lymphoma (BL) cell lines of B cell origin, IB4 and X50-7 are EBV-transformed LCLs, Jurkat and CEM are T leukaemic cell lines, and K562 is a proerythroleukaemic cell line. All cells were propagated as suspension cultures in growth medium containing RPMI 1640 supplemented with 10% (v/v) selected foetal calf serum, 2 mM glutamine and 100 mg/l gentamicin.

{blacksquare} Plasmid constructs.
The Wp reporter vector Wp440/GL2, in which the expression of a luciferase reporter gene is driven by Wp sequences from -440 to +175 (relative to the Wp RNA startsite), has been described previously (Bell et al., 1998 ). The MutA and ConA substitutions (shown in Fig. 1A) were introduced into this Wp fragment by site-directed mutagenesis using an appropriate oligonucleotide and the Sculptor mutagenesis system (Amersham Pharmacia). A second luciferase reporter, pCRE-LUC (Stratagene), containing four copies of a consensus CRE, was also used as a reference CREB-dependent promoter in control experiments. The dominant negative CREB and ATF mutants described in this work were constructed from the full-length cDNAs for the rat CREB{alpha} and human ATF1 proteins, which were kindly provided by Helen Hurst (ICRF Oncology Unit, Hammersmith Hospital, London, UK). The K-CREB cDNA, which contains an Arg to Leu substitution at codon 301 (Walton et al., 1992 ), was generated by site-directed mutagenesis; the cDNAs encoding the truncated proteins 254-CREB (containing CREB residues 254–341) and 181-ATF (containing ATF1 residues 181–271) were constructed by PCR amplification of the corresponding full-length cDNA. cDNA fragments encoding wild-type and mutant proteins were inserted into the expression vector pcDNA3 (InVitrogen) such that they could be expressed from either the phage T7 promoter (for the purposes of in vitro transcription and translation) or the CMV IE promoter (for expression in mammalian cells).



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Fig. 1. A consensus CRE can functionally substitute for site A sequences. (A) Schematic illustration of Wp showing the location of three main regulatory elements, UAS1–3, and the position of several binding sites for known and unknown cellular factors which are critical for Wp activity. Inset below the figure is the nucleotide sequence of the site A probe (Wp sequences -102 to -77) containing a CRE-like motif (boxed). The sequences of the MutA and ConA competitor oligonucleotides are also shown and the base substitutions in each are shaded. (B) Effect of the MutA and ConA changes on factor binding. The figure shows the results of a bandshift experiment in which the protein–DNA complexes obtained by incubation of a radiolabelled site A probe with DG75 nuclear extract were challenged by the addition of different competitor sequences. Lane 1 indicates probe A without the addition of nuclear extract; lane 2, probe A incubated with DG75 nuclear extract; lanes 3 and 4, probe and nuclear extract in the presence of 100- and 1000-fold excesses of unlabelled site A competitor, respectively; lanes 5 and 6, probe and nuclear extract in the presence of 100- and 1000-fold excesses of the MutA competitor, respectively; lanes 7 and 8, probe and nuclear extract in the presence of 100- and 1000-fold excesses of the ConA competitor, respectively. (C) Effect of the MutA and ConA substitutions on Wp activity. The figure shows the mean results (with standard deviations) from three independent experiments in which different B and non-B cell lines were transfected with a luciferase reporter carrying the indicated Wp promoter sequences. The bars indicate luciferase expression measured in whole cell extracts, which have been normalized for differences in transfection efficiency using a constitutively expressed {beta}-galactosidase gene.

 
{blacksquare} Transient transfections and reporter gene assays.
Wp reporter activity was assayed by quantifying luciferase expression in whole cell lysates prepared from cells transiently transfected with the indicated reporter constructs; the results in Figs 1 and 4 have been normalized for differences in transfection efficiency by using a cotransfected CMV-{beta}gal plasmid, as described previously (Bell et al., 1998 ). To examine the effects of forskolin and phorbol 12-myristate 13-acetate (PMA) on Wp activity (see Fig. 5), transfected cells were grown for 18 h in complete growth medium before being divided equally into three cultures, supplemented with DMSO (control), 10 µM forskolin or 25 ng/ml PMA, and then grown for another 24 h prior to harvesting.



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Fig. 4. Dominant negative mutants of CREB and ATF1 can suppress CREB/ATF-dependent promoter activity. (A) Inhibition of CRE-LUC expression. The figure shows the mean results of two independent experiments in which DG75 cells were cotransfected with the CRE-LUC reporter in the presence of the indicated amounts of an expression vector encoding K-CREB, 254-CREB and 181-ATF. (B) Inhibition of Wp expression. The figure shows the results of three independent experiments in which DG75 cells were cotransfected with a Wp luciferase reporter (Wp440) in the presence of increasing amounts of K-CREB, 254-CREB and 181-ATF expression vectors. (C, D) Dominant negative CREB/ATF proteins specifically suppress Wp activity in B cells. The figures show the results of two independent experiments in which DG75 (panel C) or K562 cells (panel D) were cotransfected with a luciferase reporter carrying either the wild-type Wp sequences (Wp440) or a derivative carrying the MutA substitution, and 10 µg expression vector encoding the indicated dominant negative protein. In each case, the heights of the bars indicate luciferase expression determined in whole cell extracts and have been normalized for differences in transfection efficiency.

 


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Fig. 5. Induction of Wp by the PKC activator PMA and the PKA activator forskolin. The figure shows the mean results (with standard deviations) from four independent experiments in which the cultures of the EBV-negative BL cell line DG75 and the T cell line Jurkat were transiently transfected with the indicated Wp luciferase reporter and then exposed to either PMA (25 ng/ml) or forskolin (10 µM). The bars indicate the luciferase expression determined in whole cell lysates.

 
{blacksquare} Bandshift assays.
All oligonucleotides were purchased from Alta Bioscience, The University of Birmingham. The preparation of nuclear extracts, probe labelling and in vitro DNA binding assays have all been described previously (Bell et al., 1998 ). Where indicated, competitor DNA fragments (1 pmol) and specific antisera (2 µl) were added prior to the addition of radiolabelled probe. The following antisera were purchased from Santa Cruz Biotechnology: a monoclonal antibody (MAb) reactive against ATF1, CREB1 and CREM1 (25C10G), MAbs against CREB1 (24H4B) and ATF1 (C41-5.1); a polyclonal antibody to CREB2 (C20); a polyclonal antibody to ATF2 (C-19). Antiserum against ATFa was a generous gift of Bruno Chatton (University of Strasbourg, France). In vitro translated (IVT) proteins for bandshifts were prepared using the T7 TNT coupled wheat germ extract system (Promega). For experiments involving heterodimers between wild-type and mutant subunits, the appropriate IVT proteins were mixed in the indicated ratios, heated to 50 °C for 10 min to allow subunit dissociation, and then cooled to room temperature (Hurst et al., 1991 ).


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Binding of cellular factors to site A is critical for Wp activity in B cells
Fig. 1(A) is a schematic illustration of Wp, indicating the position of a number of regulatory elements within the promoter. Although upstream sequences designated UAS2 and UAS3 are required for optimal activity, the promoter proximal region UAS1 is the most important determinant of the preferential activity of Wp in B cells. While UAS1 contains three binding sites (A, B and C) for cellular factors, the present work focuses on the CRE-like motif 5' TTACGTAA 3' within site A. We have previously described a mutation within this sequence, designated MutA (Fig. 1A, line 2), which is sufficient to abrogate the binding of cellular factors to this site in a bandshift assay (Bell et al., 1998 ). We reasoned that if this CRE-like motif does indeed bind CREB/ATF factors, then a mutation which alters this motif to a consensus CRE should not affect this interaction. To test this, we synthesized a new sequence, ConA (Fig. 1A, line 3), in which the two non-canonical bases in site A have been mutated to the corresponding nucleotides in the consensus CRE. We then compared the ability of the MutA and ConA sequences to compete for the binding of cellular factors to a site A probe by means of a bandshift assay. The results in Fig. 1(B) show that the formation of site A-specific complexes could not be inhibited by the addition of an oligonucleotide carrying the MutA sequence (Fig. 1B, lanes 5 and 6) even at the highest concentration of competitor; by contrast, the ConA sequence competed as effectively as the wild-type site A oligonucleotide (Fig. 1B, cf. lanes 3, 4 and 7, 8), implying that the proteins bound to site A do belong to the CREB/ATF family.

We next used transient transfection experiments to compare the effects of the ConA and MutA changes on the activity of a Wp luciferase reporter construct (Fig. 1C). In the two B cell lines, Ramos and DG75, Wp itself shows high activity; introduction of the ConA substitution did not significantly alter this activity whereas the MutA substitution, which abolishes factor binding, reduced luciferase expression by 12-fold and 9-fold, respectively. These results strongly suggest that the binding of cellular factors to site A is critical for Wp activation in B cells. We also carried out parallel assays in the erythroleukaemic cell line K562, where base-line Wp activity is reported to be much lower and there is only a marginal contribution from the UAS1 region (Bell et al., 1998 ). In the present experiments, Wp activity in K562 was only marginally reduced by the MutA substitution; furthermore, the low Wp activity detected in these cells could not be improved by replacing the site A sequences with a consensus CRE (Fig. 1C). Similar low level activities of Wp itself, and of the ConA and MutA constructs, were also observed in two further non-B cell lines, CEM and Jurkat (data not shown).

Identification of CREB/ATF proteins that interact with site A
To identify which members of the CREB/ATF family are responsible for Wp activation in B cells, we performed bandshift experiments in which a labelled site A probe was incubated with B cell nuclear extract in the presence of specific antisera directed against different CRE-binding proteins (Fig. 2A). In the first instance we used a monoclonal antiserum raised against the conserved bZIP domain of ATF1, CREB1 and CREM1; this resulted in the loss of three complexes and the appearance of several supershifted species (Fig. 2A, lane 2), demonstrating that site A can interact with multiple CREB/ATF factors. Formation of two of these complexes was specifically inhibited by the addition of an ATF1 antibody (lane 3), while the third was supershifted in the presence of a CREB1 antibody (lane 4). We also tried antisera raised to other bZIP factors that have been reported to bind to CRE sequences, either as homodimers or as heterodimers with CREB1 and ATF1. While the addition of antibodies to CREB2 (lane 5), ATF2 (lane 6), C/EBP and c-Jun (not shown) did not affect either the formation or mobility of any of these site A-specific complexes, the presence of an ATFa antibody (lane 8) partially inhibited the formation of a complex which migrated with a slightly lower mobility than the CREB1 complex.



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Fig. 2. Identification of CREB/ATF proteins bound to site A. (A) The figure shows the results of a bandshift experiment in which a radiolabelled site A probe was incubated with DG75 nuclear extract in the presence of antibodies directed against different CREB/ATF proteins. Lanes 1 and 7, no antibody control; lane 2, MAb that recognizes the bZIP domain of CREB1/ATF1/CREM1; lane 3, ATF1-specific antibody; lane 4, CREB1-specific antibody; lane 5, CREB2-specific antibody; lane 6, ATF2-specific antibody; lane 8, ATFa-specific antibody. The positions of specific CREB/ATF complexes are marked by the arrows. (B) Protein–DNA complexes obtained by incubating a site A probe with K562 nuclear extract; antibodies were added in the same order as described in the legend to panel (A).

 
To examine the possibility that the lack of function of site A in non-B cells reflects the absence of one or more of these CREB/ATF factors, we next extended our supershift analysis to include other cell types. Fig. 2(B) show the results of an experiment in which the site A probe was incubated with K562 nuclear extract; it is clear that the pattern of protein–DNA complexes using this nuclear extract is identical to that seen in the presence of B cell factors. Similar ATF1-, CREB1- and ATFa-containing complexes were also detected using nuclear extracts prepared from the EBV-positive BL cell line Akata, the EBV-transformed LCLs IB4 and X50-7, and the T cell lines Jurkat and CEM (H. Kirby, unpublished observations), suggesting that the CREB/ATF proteins which interact with site A are ubiquitously expressed.

Design and testing of dominant negative forms of CREB1 and ATF1
To further examine the role of CREB/ATF factors in Wp activation, we generated dominant negative forms of these proteins designed to disrupt the function of the endogenous factors; these mutants are illustrated in Fig. 3(A). Our first strategy was to exploit a previously described mutant, known as K-CREB (Walton et al., 1992 ), containing a single amino acid substitution within the basic DNA-binding domain which disrupts DNA-binding activity; the results of the bandshift experiment shown in Fig. 3(B) confirm that IVT K-CREB is unable to bind to the site A probe (lanes 4–6) whereas IVT wild-type CREB1 does bind and the complex is shifted by a CREB1-specific MAb (lanes 1–3). K-CREB reportedly inhibits the activity of endogenous CREB/ATF factors by sequestering them as non-binding heterodimers (Walton et al., 1992 ; Yang et al., 1996 ; Ying et al., 1997 ). This was confirmed in our system by an experiment in which increasing amounts of IVT K-CREB were added to a binding reaction containing the site A probe and a constant amount of IVT wild-type CREB protein; the results (Fig. 3B, lanes 7–10) clearly show that K-CREB inhibited the formation of the CREB1–site A complex. The binding of ATF1 to site A was also inhibited by K-CREB in a similar fashion (data not shown).



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Fig. 3. DNA-binding properties of K-CREB, 254-CREB and 181-ATF. (A) Schematic illustration of the domain structure of CREB/ATF proteins. The figure shows the Arg-301 to Leu substitution within K-CREB, and the structure of two truncated proteins, 254-CREB and 181-ATF. (B) K-CREB disrupts DNA binding by wild-type CREB1 through the formation of inactive heterodimers. The figure shows the protein–DNA complexes obtained by incubating the indicated IVT proteins with a radiolabelled site A probe. Lanes 1–3, 1 µl IVT CREB1 alone; lanes 4–6, 1 µl IVT K-CREB alone; lanes 7–10, 2 µl IVT CREB1 incubated with 0, 2, 4 and 10 µl IVT K-CREB respectively. (C) 254-CREB can form mixed heterodimers with wild-type CREB1. Lanes 1–3, 1 µl IVT CREB1 alone; lanes 4–6, 2 µl IVT 254-CREB alone; lanes 7–10, 2 µl IVT CREB1 incubated with 0, 2, 4 and 10 µl IVT 254-CREB respectively. (D) 181-ATF can form mixed heterodimers with wild-type ATF1. Lanes 1–3, 1 µl IVT ATF1 alone; lanes 4–6, 1 µl IVT 181-ATF alone; lanes 7–10, 1 µl IVT ATF1 incubated with 0, 1, 2 and 5 µl IVT 181-ATF respectively. The positions of the different homodimeric and heterodimeric complexes are marked by arrows.

 
Our second strategy was to construct truncated forms of CREB1 and ATF1 (254-CREB and 181-ATF respectively) which contained the minimal sequences required for dimerization and DNA binding (Fig. 3A); such mutants might be expected to inhibit CREB/ATF-dependent gene expression due to the lack of the N-terminal transactivation domain. When the relevant IVT proteins were tested in bandshift assays using the site A probe, the results (Fig. 3C, lanes 4–6; 3D, lanes 4–6) indicated that both 254-CREB and 181-ATF retain the ability to bind DNA. Note that the CREB1-specific MAb used in these experiments could recognize both the wild-type and truncated versions of the protein (Fig. 3C, lanes 2 and 5) whereas the ATF1-specific MAb only detected the full-length ATF1 (Fig. 3D, lane 3). The results in Fig. 3(C), lanes 8–10, also indicate that coincubation of IVT 254-CREB and IVT wild-type CREB1 can lead to the formation of mixed heterodimers which appear as complexes of intermediate mobility between those containing either homodimers of 254-CREB or wild-type CREB1. In parallel experiments, we also demonstrated that truncated 181-ATF can form mixed heterodimers with wild-type ATF1 subunits (Fig. 3D, lanes 8–10). Increasing the proportion of 254-CREB or 181-ATF species relative to the wild-type protein significantly reduced the formation of CREB1 or ATF1 homodimers.

Expression of dominant negative forms of CREB1 and ATF1 abrogates Wp activity in B cells
Having validated the DNA-binding properties of the dominant negative proteins in a cell-free system, we then asked whether these mutants could interfere with CREB/ATF-dependent reporter gene expression in cells. As a first step we expressed K-CREB, 254-CREB or 181-ATF by transient transfection (using the relevant empty expression vector as a control) and looked for any effects of the proteins on the activity of a cotransfected reporter construct, pCRE-LUC, which contains four copies of a consensus CRE upstream of a minimal promoter. The data in Fig. 4(A) show that each of the dominant negative mutants could abrogate expression of this reference CREB-dependent promoter in a dose-dependent manner in the DG75 B cell line. We then used a similar cotransfection assay to test the ability of these mutants to interfere with the activity of a Wp reporter construct. The data in Fig. 4(B) show that K-CREB, 254-CREB and 181-ATF reduced Wp activity to 20, 25 and 22%, respectively, of the values observed in DG75 cells cotransfected with Wp and the empty expression vector. These results clearly underline the importance of CREB/ATF factors for Wp promoter activity in B cells. To ensure that these effects were dependent on factor binding to site A, we repeated the experiment using a Wp reporter carrying a mutated site A; the results in Fig. 4(C) show that the mutants were again effective inhibitors of wild-type Wp activity in DG75 cells, but did not inhibit luciferase expression from the MutA construct. Finally, we examined the effects of the dominant negative mutants on Wp activity in the non-B cell line K562, where previous work had shown that the binding of CREB/ATF factors did not contribute significantly to Wp activation. As expected, expression of K-CREB, 254-CREB and 181-ATF failed to suppress luciferase expression either from wild-type Wp or the MutA construct in K562 cells (Fig. 4D).

CREB/ATF-dependent activation of Wp mediated by intracellular signalling pathways
Since the above results strongly implicated CREB/ATF factors in Wp activation, we examined if Wp activity could be enhanced by stimulating intracellular signalling pathways known to influence CREB/ATF activity (Delmas et al., 1994 ). The best described of these pathways involves protein kinase A (PKA), which is reported to induce CREB/ATF activity in a variety of cell lines (De Cesare et al., 1999 ), and protein kinase C (PKC), which a recent report suggests to be the principal mediator of CREB/ATF activation in B cells (Xie & Rothstein, 1995 ). To analyse if either of these pathways could play a role in regulating Wp, we studied the effect of the PKC activator PMA and of the PKA activator forskolin. The B cell line DG75 and the T cell line Jurkat were transiently transfected with a Wp reporter construct (or with a MutA construct as a control) and cultured in normal medium for 18 h, then split and exposed either to PMA or forskolin, or kept in normal medium as a control; a further 24 h later, promoter activity was determined in induced versus non-induced cultures by luciferase assay (Fig. 5). In DG75 cells, addition of PMA resulted in a 2·5-fold increase in Wp activity; this induction was, at least in part, dependent on CREB/ATF binding to site A since in the same experiments there was only a 1·7-fold increase in expression from the (much less active) MutA construct. Again in DG75 cells, forskolin mediated a smaller 1·6-fold increase in Wp activity but this appeared to be independent of CREB/ATF since a similar -fold increase was observed using the MutA reporter. These results are consistent with the view that PKC-mediated activation of CREB/ATF factors in B cells can indeed upregulate Wp activity through an interaction involving site A.

Extending the approach to non-B cells allowed us to ask whether a PKA- and/or PKC-induced activation of CREB/ATF factors could restore Wp activity to the levels regularly seen in B cell lines. In fact this was not the case; the -fold increases observed over the low Wp basal levels remained small. Thus PMA mediated a 2-fold increase in activity both of the wild-type and of the MutA reporter constructs, implying that this effect was independent of site A, whereas forskolin mediated a 1·7-fold increase in Wp activity compared to a 1·3-fold increase for MutA. The PMA and forskolin treatments were nevertheless clearly mediating CREB/ATF activation in these experiments since in parallel assays of the same type, the reference CREB-dependent reporter pCRE-LUC showed 5- to 10-fold increases in activity (data not shown).


   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
Activation of Wp, the first viral promoter to be expressed following infection of a resting B cell, is a critical step in the EBV-driven growth transformation process. It is therefore important to identify the cellular and viral factors that influence Wp activity and to understand why Wp is preferentially active in B cells as opposed to other cell types. To date, few cellular proteins have been directly implicated in Wp regulation; these include the factors YY1, which can mediate Wp activation in a wide variety of cell types through interaction with a second regulatory sequence UAS2 (Bell et al., 1998 ), and possibly NF{kappa}B (Sugano et al., 1997 ). The EBV-encoded viral antigen EBNA1 has also been reported to upregulate Wp activity, but presumably this can only occur in EBV-infected B cells which are already expressing the EBV latent proteins (Puglielli et al., 1996 ).

In an effort to gain a better understanding of Wp regulation, we recently examined the contribution of different upstream sequences to the overall promoter activity in different cell lines (Bell et al., 1998 ). While two sequences, designated UAS2 and UAS3, activated Wp activity in a broad range of cell types, we found that a third promoter proximal sequence, UAS1, was B cell-specific and was the key determinant of the increased activity of Wp in B cell lines. Of three adjacent binding sites (sites A, B and C) that contribute to UAS1 function, the present work addressed regulation through site A and followed up our earlier observation that this site contained a sequence closely related to the consensus binding site for CREB/ATF factors (Benbrook & Jones, 1994 ). Altering two critical nucleotides in site A, so as to destroy this homology (MutA sequence), blocked complex formation at this site in a bandshift assay (Fig. 1B), and when introduced into the Wp reporter led to a 9- to 12-fold fall in promoter activity in B cells (Fig. 1C). By contrast, two nucleotide changes which converted the motif to an exact match with the consensus CRE (ConA sequence) did not affect factor binding or promoter activity. These findings provide strong circumstantial evidence that activation of Wp through site A is mediated by members of the CREB/ATF family.

Detailed analysis of the different site A-specific complexes formed in bandshift assays identified the factors ATF1, CREB1 and ATFa (Fig. 2). The two distinct ATF1-containing complexes revealed using an ATF1-specific antibody (Fig. 2, lane 3) likely correspond to ATF1 homodimers since they were unaffected by the addition of antibodies to other bZIP proteins, such as CREB1 and CREM1, known to associate with ATF1 (Hurst et al., 1991 ; Rehfuss et al., 1991 ); the different mobilities of these two ATF1-containing complexes possibly reflect differences in phosphorylation since this is known to influence the migration of ATF1 species in a bandshift assay (Masson et al., 1993 ). The CREB1-containing complex (Fig. 2, lane 4) is likely to involve a CREB1 homodimer as this complex was also unaffected by the addition of antisera to other CREB/ATF factors. We also identified a fourth complex containing ATFa, a recent addition to the CREB/ATF family (Gaire et al., 1990 ; Chatton et al., 1993 ). It is known that ATFa can associate with a distinct subset of bZIP factors which includes c-Jun (Chatton et al., 1994 ), but antibody shift experiments gave no evidence of ATFa/c-Jun heterodimers bound to site A.

Independent evidence for the involvement of CREB/ATF factors in Wp regulation was provided by subsequent experiments in which the expression of dominant negative CREB and ATF mutants suppressed Wp activity in a dose-dependent manner (Fig. 4B). Importantly, these transdominant effects required the presence of the site A sequence within the promoter, indicating that this repression was not the result of some non-specific inhibition of Wp activity (Fig. 4C). Note that the CREB/ATF mutant proteins used in these experiments have different properties. K-CREB, originally described by Walton et al. (1992) , is unable to bind DNA as a homodimer but can sequester partner subunits as non-binding heterodimers (Fig. 3B). Because K-CREB will only heterodimerize with other bZIP proteins that have a similar leucine zipper structure (Foulkes et al., 1991 ; Hurst et al., 1991 ), this mutant will disrupt the function of ATF1, CREM1 and CREB1 itself, but not factors such as c-Jun and ATF2. The two remaining mutants, 254-CREB and 181-ATF, are truncated proteins which can bind normally to DNA but are unable to activate transcription due to lack of an activation domain; such proteins can exert a dominant negative effect through occupation of a CRE site within the target promoter thereby blocking access to all endogenous CRE-binding factors. In addition, the results in Fig. 3(C, D) suggest that these latter mutants may also suppress CREB/ATF-dependent activation indirectly through the formation of mixed heterodimers. Overall, our studies demonstrated that K-CREB was as effective as 254-CREB and 181-ATF at suppressing Wp activity in B cell lines (Fig. 4B), consistent with the view that CREB1 and ATF1 are among the most important factors influencing Wp activation via site A. Interestingly, these dominant negative mutants only reduced Wp activation up to 5-fold in B cell lines whereas mutations destroying site A (MutA construct) caused a 9- to 12-fold reduction; this implies that the transdominant effects never completely eliminated activation of Wp by endogenous CREB/ATF proteins.

Two other examples of EBV promoters that are regulated by CREB/ATF factors have been previously described. The promoter for latent membrane protein 1 (LMP1p) contains a CRE motif within the promoter proximal element (Fhraeus et al., 1994 ) which can recruit CREB1/ATF1 heterodimers, resulting in direct LMPp activation, or an ATF2–c-Jun complex which by itself has little effect but is required for EBNA2-mediated activation of the promoter (Sjöblom et al., 1998 ); we see no evidence for the latter complexes binding Wp. The promoter for lytic cycle regulator BZLF1 (Zp) has also been shown to interact with multiple CREB/ATF complexes in both B cell (Wang et al., 1997 ) and epithelial cell (MacCallum et al., 1999 ) nuclear extracts. However a notable difference between these promoters and Wp is that LMP1p and Zp reporter constructs have low basal activities in B cell lines which can be induced through the overexpression of CREB/ATF factors (Sjöblom et al., 1998 ; Wang et al., 1997 ). This is not true in the case of Wp, suggesting that the factors which bind site A are not limiting for the transcription of this promoter; indeed, overexpression of CREB1, ATF1 or ATFa in B cell lines does not further increase the activity of a Wp reporter construct (H. Kirby, unpublished observations).

The final set of experiments was prompted by the observation that the CREB/ATF factors binding to site A in bandshift assays are clearly present in both B cell and non-B cell extracts (Fig. 2), yet this binding only leads to a functional activation of the promoter in B cells (Fig. 1C, Fig. 4C, D). One possible explanation for these apparently contradictory results is that the relevant CREB/ATF factors are constitutively activated in B cells but not in other cell types. We therefore studied the effects of adding inducers of CREB/ATF activity to B and non-B cell lines following transient transfection with Wp reporters (Fig. 5). In the B cell line DG75, PMA (a PKC agonist) did bring about a small increase in Wp activity through an effect mediated by site A, whereas forskolin (a PKA agonist) did not. These results are consistent with reports that CREB1 activity in B cells is dependent upon phosphorylation by PKC rather than PKA (Xie & Rothstein, 1995 ; Xie et al., 1996 ); furthermore, the modest increase observed even with this PKC agonist is in line with reported values for CREB/ATF-dependent activation of the bcl-2 promoter in B cells in similar transient assays (Wilson et al., 1996 ). We infer that the activation state of CREB/ATF factors is not limiting for Wp activity in B cell lines though the situation in resting cells (i.e. the natural target of EBV infection) remains to be determined. When parallel experiments were conducted in the Jurkat T cell line, where basal Wp activity is much lower, any increases in Wp activity observed were again small. Most importantly, we did not observe a restoration of Wp activity up to the levels seen in B cell lines. The preferential activity of Wp in B cells therefore appears unlikely to be due to a difference in availability or in the activation state of CREB/ATF factors capable of interacting with site A. More likely is the possibility that the lineage specificity of Wp is determined by the, as yet, unidentified factors that bind at other regulatory sites within UAS1.


   Acknowledgments
 
This work was funded by a programme grant awarded by the Cancer Research Campaign (CRC), London, UK, and a CRC funded studentship to H.K. We would like to thank Dr Helen Hurst (ICRF Oncology Unit, Hammersmith Hospital, London, UK) for kindly providing us with the {alpha}CREB and ATF1 cDNAs and for many helpful discussions. We are also grateful to Dr Bruno Chatton (University of Strasbourg, France) for providing the ATFa antibody and ATFa expression vectors.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 28 July 1999; accepted 12 January 2000.