Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P51532 (transcriptional activator)
 6,546 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Epstein-Barr virus (EBV) transforms resting B cells in vitro very efficiently. The nuclear viral protein EBV nuclear antigen 2 (EBNA2) is absolutely required for this process and also acts as a transcriptional activator of cellular and viral genes. As shown previously, EBNA2 transactivates the promoters of the viral latent membrane proteins. It interacts indirectly with an EBNA2-responsive cis element of the terminal protein 1 (TP1) promoter. To identify the sequences mediating EBNA2 transactivation of the bidirectional promoter region driving expression of the latent membrane proteins LMP and TP2 in opposite directions, we assayed the effects of EBNA2 on the activities of promoter deletion and site-directed mutants of TP2 and LMP promoter luciferase reporter gene constructs by cotransfections into EBNA2-negative Burkitt's lymphoma cells. We were able to delineate an 80-bp EBNA2-responsive region (EBNA2RE) between -232 and -152 relative to the LMP RNA start site which could also mediate EBNA2-dependent activation on a heterologous promoter. Sequences of 20 and 32 bp located at the 5' and 3' ends, respectively, of the EBNA2RE were both essential for EBNA2 responsiveness. Full transactivation of the LMP and TP2 promoters seemed to require 20 bp of 5' adjacent sequences in addition to the 80-bp element. Electrophoretic mobility shift assays revealed specific protein-DNA complexes formed at the EBNA2RE. Oligonucleotides from -181 to -152 and -166 to -132 relative to the LMP RNA start site visualized one B-cell and one B-cell-plus-HL60-specific retarded protein-DNA complex, respectively. Additionally, an oligonucleotide from -253 to -210 revealed two specific protein-DNA complexes with nuclear extracts from different B and non-B cells, suggesting also the binding of ubiquitously expressed proteins on the EBNA2RE. Thus, these experiments defined a 80-bp cis element sufficient for conferring EBNA2 inducibility and demonstrated specific interactions of cellular proteins at DNA sequences within the EBNA2RE, which are critical for transactivation by EBNA2.
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PMID:Identification and characterization of an Epstein-Barr virus nuclear antigen 2-responsive cis element in the bidirectional promoter region of latent membrane protein and terminal protein 2 genes. 793 76

The EBNA-2 protein is essential for the establishment of a latent Epstein-Barr virus (EBV) infection and for B-cell immortalization. EBNA-2 functions as a transcriptional activator that modulates viral latency gene expression as well as the expression of cellular genes, including CD23. We recently demonstrated that EBNA-2 transactivation of the EBV latency C promoter (Cp) is dependent on an interaction with a cellular DNA-binding protein, CBF1, for promoter targeting. To determine whether targeting via CBF1 is a common mechanism for EBNA-2-mediated transactivation, we have examined the requirements for activation of the cellular CD23 promoter. Binding of CBF1 to a 192-bp mapped EBNA-2-responsive region located at position -85 bp to -277 bp upstream of the CD23 promoter was detected in electrophoretic mobility shift assays. The identity of the bound protein as CBF1 was established by showing that the bound complex was competed for by the CBF1 binding site from the EBV Cp, that the bound protein could be supershifted with a bacterially expressed fusion protein' containing amino acids 252 to 425 of EBNA-2 but was unable to interact with a non-CBF1-binding EBNA-2 mutant (WW323SR), and that in UV cross-linking experiments, the Cp CBF1 binding site and the CD23 probe bound proteins of the same size. The requirement for interaction with CBF1 was demonstrated in a transient cotransfection assay in which the multimerized 192-bp CD23 response region was transactivated by wild-type EBNA-2 but not by the WW323SR mutant. Reporter constructions carrying multimerized copies of the 192-bp CD23 response region or multimers of the CBF1 binding site from the CD23 promoter were significantly less responsive to EBNA-2 transactivation than equivalent constructions carrying a multimerized region from the Cp or multimers of the CBF1 binding site from the Cp. Direct binding and competition assays using 30-mer oligonucleotide probes representing the individual CBF1 binding sites indicated that CBF1 bound less efficiently to the CD23 promoter and the EBV LMP-1 promoter sites than to the Cp site. To investigate the basis for this difference, we synthesized a series of oligonucleotides carrying mutations across the CBF1 binding site and used these as competitors in electrophoretic mobility shift assays. The competition experiments indicated that a central core sequence, GTGGGAA, common to all known EBNA-2-responsive elements, is crucial for CBF1 binding. Flanking sequences on either side of this core influence the affinity for CBF1.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:EBNA-2 upregulation of Epstein-Barr virus latency promoters and the cellular CD23 promoter utilizes a common targeting intermediate, CBF1. 805 21

The Epstein-Barr virus protein EBNA2 acts as a transcriptional activator of cellular and viral genes and plays a crucial role in the immortalization of human primary B-cells by EBV. We have shown previously that EBNA2 transactivates the promoters of the latent membrane antigens LMP, TP1 and TP2. The promoter of the TP1 gene was chosen as a model system to study the molecular mechanism of EBNA2 mediated transactivation. To identify an EBNA2 dependent cis-acting element, various TP1 promoter-reporter gene constructs were transfected in the absence and presence of an EBNA2 expression vector into the established B-cell line BL41-P3HR1. We were able to delineate an 81 bp EBNA2 responsive region between -258 and -177 relative to the TP1 RNA start site. The element worked in either orientation and could mediate EBNA2 dependent transactivation on a heterologous promoter. Electrophoretic mobility shift assays revealed three specific protein-DNA complexes formed with sequences of the EBNA2 responsive element. Two of these were not cell type specific, but the third was detected only in EBNA2 positive cell extracts. Gel-shift analysis in the presence of EBNA2 specific monoclonal antibodies revealed that EBNA2 is a component of the third complex. Thus, these experiments demonstrate that EBNA2 interacts with an EBNA2 responsive cis-element of the TP1 promoter.
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PMID:The Epstein-Barr virus nuclear antigen 2 interacts with an EBNA2 responsive cis-element of the terminal protein 1 gene promoter. 838 49

The Epstein-Barr virus (EBV) EBNA2 protein is a transcriptional activator that regulates viral and cellular gene expression and is also essential for EBV-driven immortalization of B lymphocytes. The EBNA2-responsive enhancer in the viral latency C promoter (Cp) binds two cellular factors, CBF1 and CBF2. The precise role of the CBF2 protein for Cp enhancer function is presently unclear. CBF2 does not appear to interact with EBNA2 and binds just downstream of CBF1 between positions -339 and -368 in the Cp EBNA2 enhancer. Within this region an 8-bp sequence, CAGTGCGT, can be found, and a similar sequence is also located downstream of CBF1 binding sites in other EBNA2-responsive promoters. Previous studies have indicated that mutations and methylation in this sequence affect EBNA2 responsiveness. To investigate the requirements for CBF2 binding, we synthesized a series of oligonucleotides carrying double transversion mutations spanning both the conserved core sequence and outside flanking sequences. Surprisingly, mutations outside of the conserved core sequence in 4 bases immediately flanking the 5' end, GGTT, had the most deleterious effect on CBF2 binding. Mutations in the conserved core had a gradient effect, with those near the 5' end having the most deleterious effects on CBF2 binding. In addition, the affinities of CBF2 for binding to the LMP-1, LMP-2, and CD23 promoters were also measured. These promoters contain the conserved core but lack the 5' flanking GGTT motif and bound CBF2 weakly or not at all. Using Cp reporter plasmids containing CBF2 mutant binding sites, we were also able to show that at lower doses of EBNA2, Cp transactivation required a functional CBF2 binding site but that higher doses of EBNA2 transactivated CBF2 mutant promoters to 40% of wild-type levels. These assays indicate that CBF2 is important for EBNA2-mediated transactivation of the viral latency Cp. In addition, CBF2 activity was found to be associated with two polypeptides of 27 and 33 kDa.
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PMID:Characterization of the CBF2 binding site within the Epstein-Barr virus latency C promoter and its role in modulating EBNA2-mediated transactivation. 942 Feb 75

Epstein-Barr virus nuclear antigen 2 (EBNA2) is required for EBV-mediated immortalization of primary human B cells and is a direct transcriptional activator of viral and cellular genes. The prototype EBNA2 protein contains a unique motif in which 43 out of 45 amino acids are prolines (polyproline region [PPR]). Previous genetic analysis has shown that deletion of the PPR resulted in viruses unable to immortalize B cells, although the protein did appear transcriptionally functional (R. Yalamanchili, S. Harada, and E. Kieff, J. Virol. 70:2468-2473, 1996). The PPR's uniqueness and requirement for immortalization make it an attractive therapeutic target. However, the role of this highly unusual motif for immortalization remains enigmatic. We have recently developed a transcomplementation assay that allows both genetic and functional analyses of EBNA2 in EBV-mediated immortalization maintenance (A. V. Gordadze, R. Peng, J. Tan, G. Liu, R. Sutton, B. Kempkes, G. W. Bornkamm, and P. D. Ling, J. Virol. 75:5899-5912, 2001). Surprisingly, we found that DeltaPPR-EBNA2 was able to support B-cell proliferation similar to that of wild-type EBNA2 in this assay, indicating that deletion of the PPR from EBNA2 does not result in a loss of function required for immortalization maintenance. Further analysis of this mutant EBNA2 revealed that it consistently activated the viral LMP1 and LMP2A promoters severalfold better than wild-type EBNA2 in transient cotransfection assays. In addition, one striking difference between lymphoblastoid cell lines expressing wild-type EBNA2 from those expressing DeltaPPR-EBNA2 is that the latter cells have significantly reduced EBV genomic levels. The data are consistent with a model in which lower EBNA2 target gene dosage may be selected for in DeltaPPR-EBNA2-dependent cell lines to compensate for hyperactive stimulation of viral genes, such as LMP-1, which is cytostatic for B cells when overexpressed. It is conceivable that the hyperactivity rather than the loss of function, as hypothesized previously, could be responsible for the inability of recombinant DeltaPPR-EBNA2 EBVs to immortalize B cells.
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PMID:The EBNA2 polyproline region is dispensable for Epstein-Barr virus-mediated immortalization maintenance. 1207 34

Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2), a direct transcriptional activator of viral and cellular genes, is required for EBV-induced B-cell transformation. The functional role of conserved regions within the amino terminus of the protein preceding the poly-proline region has yet to be fully characterized. Thus, we tested whether the EBNA2 amino-terminal 30 amino acid residues, containing evolutionarily conserved region 1, are required for stimulating viral and cellular gene expression necessary for B-cell transformation in a viral transcomplementation assay. We found that these residues are required for its ability to induce LMP-1 expression in lymphoblastoid cell lines (LCLs), to stimulate LMP-1 promoter reporter plasmids in transient-cotransfection assays, and to rescue LCL growth following inactivation of endogenous wild-type EBNA2 protein. Deletion of amino acid residues 3 to 30 also impaired its ability to self-associate in coimmunoprecipitation assays. These data indicate that EBNA2 residues 3 to 30 comprise an essential domain required for induction of LMP-1 expression and, consequently, for maintenance of the immortalized phenotype of LCLs. The ability to self-associate into dimers or multimers conferred by this domain may be an important mechanism for these effects.
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PMID:EBNA2 amino acids 3 to 30 are required for induction of LMP-1 and immortalization maintenance. 1504 8