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. 2003 Oct;23(19):6901-8.
doi: 10.1128/MCB.23.19.6901-6908.2003.

EBNA-1, a bifunctional transcriptional activator

Gregory Kennedy  1 , Bill Sugden
Affiliations

EBNA-1, a bifunctional transcriptional activator

Gregory Kennedy et al. Mol Cell Biol. 2003 Oct.

Abstract

Transient-transfection assays have been used to identify transcription factors, and genetic analyses of these factors can allow a dissection of their mechanism of activation. Epstein-Barr nuclear antigen 1 (EBNA-1) has been shown to activate transcription from transfected templates, but its ability to activate transcription from nuclear templates has been controversial. We have established cells with integrated EBNA-1-responsive templates and have shown that EBNA-1 activates transcription from these chromatin-embedded templates dose dependently. A mutational analysis of EBNA-1 has identified a domain required for transcriptional activation of integrated templates, but not of transfected templates. The ability of EBNA-1 to activate transcription from both integrated and transfected templates can be inhibited by a derivative of EBNA-1 lacking the amino acids required for activation from integrated templates. EBNA-1's mode of activating transfected templates is therefore genetically distinct from that acting on integrated templates.

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Figures

FIG. 1.
FIG. 1.
Characterization of integrated template DNAs in clones of BJAB cells. (A) Structures of the two templates, FR-TK-luciferase and TK-luciferase, introduced into BJAB cells. The positions and sequences of the two primers used to characterize the templates and the sizes of their products generated by PCR are also shown. ORF, open reading frame. (B) Agarose gel resolving PCR products of DNAs isolated from two clones of BJAB cells into which FR-TK-luciferase (clones 2-12 and 2-14) was introduced and two clones into which TK-luciferase (clones 3-6 and 3-33) was introduced. The gel also includes the products derived by amplifying known amounts of the parental plasmids to serve as size markers and to permit estimates of the number of template molecules integrated into each cell.
FIG. 2.
FIG. 2.
Transcription of integrated templates induced by EBNA-1 is dose dependent. (A) Structures of retroviral vectors to establish EBNA-1's dose-dependent induction of transcription. The control vector expresses β-galactosidase (LacZ), and both it and the vector expressing EBNA-1 also express enhanced GFP (eGFP), whose translation is mediated by an internal ribosomal entry site (IRES). LTR, long terminal repeat. (B) Fluorescence-activated cell sorter profiles reflecting the sorting of the infected cells as a function of the level of their expression of eGFP and the level of expression of EBNA-1 as measured by Western blotting in one set of infected, sorted cells 48 h following infection. (C) Mean RLU obtained in two independent experiments performed in duplicate were normalized by setting the RLU output from cells infected with LacZ virus expressing low GFP to 1. The differences in RLU in cells with integrated FR-TK-luciferase infected with a retrovirus expressing EBNA-1 and sorted for different levels of expression of eGFP are statistically significant (P < 0.05; Jonckhere-Terpstra test). The error bars indicate standard errors of the means.
FIG. 3.
FIG. 3.
Mutational analysis of EBNA-1 identifies a transcriptional activation domain within LR1. (A) Derivatives of EBNA-1 depicted schematically. EBNA-1 has two highly charged regions within its amino terminus, LR1 (shaded boxes) and LR2. The nuclear localization signal of EBNA-1 is found adjacent to a putative flexible linker domain or JD and is represented in all derivatives by the hatched boxes. The Gly-Gly-Ala repeats span ∼225 residues in the B95-8 strain of EBV. The derivative used in these studies contains only three Gly-Gly-Ala repeats. wt, wild type. (B) Western blot demonstrating expression levels of the various derivatives relative to that of wild-type EBNA-1. The membrane was simultaneously probed with antibodies to EBNA-1 and β-actin, which served as a loading control. The relative levels of expression of the transfected derivatives of EBNA-1 were corrected for loading error, and OD units obtained from ImageQuant analysis are represented as OD units per transfected cell at the bottom of each lane. (C) Mean luciferase results corrected for transfection efficiency obtained from at least three independent transfections performed in duplicate are depicted graphically. The increases in transcription over empty vector (mean increase [n-fold] over BJAB cells lacking integrated FR-TK-luciferase, 10; average number of RLU, 5,425 ± 231) mediated by wt-EBNA-1 (mean increase [n-fold] over empty vector, 26 [P = 5.3 × 10−6]; average number of RLU, 85,000 ± 3,780), Δ359-369 (mean increase [n-fold] over empty vector, 21 [P = 0.009]; average number of RLU, 95,900 ± 17,800), shuffled JD (mean increase [n-fold] over empty vector, 56 [P = 0.05]; average number of RLU, 116,000 ± 10,700), and 2×LR1 (mean increase [n-fold] over empty vector, 46.7 [P = 0.007]; average number of RLU, 21,2000 ± 31,000) are significant. The apparent increases in transcription over wild-type EBNA-1 mediated by the shuffled JD and 2×LR1 derivatives are not statistically significant (P > 0.05). The derivatives Δ65-89 (mean increase [n-fold] over empty vector, 1.5 [P = 0.85]; average number of RLU, 6,510 ± 491), 2×LR2 (mean increase [n-fold] over empty vector, 2.5 [P = 0.21]; average number of RLU, 9,169 ± 1,070), and dnE1 (mean increase [n-fold] over empty vector, 2.4 [P = 0.33]; average number of RLU, 8,957 ± 1,800) were found to have no effect on transcription from the integrated template.
FIG. 4.
FIG. 4.
The ability of EBNA-1 to activate transcription from transfected templates is independent of its ability to activate transcription from integrated templates. (A) The mean CAT activities obtained in three independent experiments performed in duplicate normalized to the CAT activity obtained in cells transfected with FR-TK-CAT and an empty vector control are depicted graphically. The CAT activity obtained from cells transfected with a vector encoding wild-type (wt) EBNA-1 was 150-fold higher than that obtained from cells transfected with a control plasmid (P = 0.002). The CAT activity obtained from cells transfected with a vector encoding Δ65-89 was 24-fold higher than that obtained from cells transfected with a control plasmid (P = 0.001). The derivative 2×LR2 was also found to increase transcription from the transfected template 20-fold over cells transfected with a control plasmid (P = 0.002), while the dnE1 derivative had no effect on transcription from this template (P = 0.3). (B) The Δ65-89 derivative of EBNA-1 activates transcription from transfected templates in a dose-dependent fashion (P = 0.003; Jonckhere-Terpstra test). BJAB cells were transfected with 50 ng of a plasmid encoding FR-TK-luciferase with increasing amounts of plasmids encoding either wild-type EBNA-1 or Δ65-89. Luciferase activity was assayed 48 h posttransfection and is represented on the y axis as induction (n-fold) over cells transfected with a vector encoding FR-TK-luciferase in the absence of EBNA-1. The error bars indicate standard errors of the means.
FIG. 5.
FIG. 5.
The derivative of EBNA-1 with amino acids 65 to 89 deleted inhibits wild-type EBNA-1's transcription function in a dominant-negative manner. (A) Graphic representation of the results of three independent experiments performed in duplicate using BJAB cells stably transfected with FR-TK-luciferase plus 2 μg of a vector encoding EBNA-1 and either no vector or increasing amounts of a vector encoding Δ65-89. (B) Graphic representation of the results of three independent experiments performed in duplicate in which BJAB cells were transiently transfected with a plasmid encoding FR-TK-luciferase along with 3 μg of one encoding EBNA-1 and no vector or increasing amounts of the vector encoding Δ65-89. The decrease in luciferase activity mediated by Δ65-89 is statistically significant in both panels (P ≤ 0.004; Jonckhere-Terpstra test). The error bars indicate standard errors of the means.
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