Institute for Medical Microbiology and Hygiene, University of Regensburg, Landshuter Strae 22, D-93047 Regensburg, Germany
Correspondence
Fritz Schwarzmann
fritz.schwarzmann{at}gmx.de
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
Present address: MRC, Laboratory of Molecular Biology, Division of Protein and Nucleic Acid Chemistry, Cambridge, UK.
![]() |
MAIN TEXT |
---|
![]() ![]() ![]() ![]() |
---|
To identify HI binding factors we performed yeast one-hybrid assays (Matchmaker One Hybrid System; Clontech). As molecular baits for fishing and screening, we used the central regions of the four HI motifs HI, HI
, HI
and HI
(Schwarzmann et al., 1994
). Five copies of each individual HI sequence were fused in a head-to-tail orientation (HI
, 5'-ACAGATGAACAGATGAACAGATGAACAGATGAACAGATGA-3'; HI
, 5'-ACATATGGACATATGGACATATGGACATATGGACATATG-3'; HI
, 5'- ACAGATGGACAGATGGACAGATGGACAGATGGACAGATGG-3'; HI
, 5'-ACAGAGGAACAGAGGAACAGAGGAACAGAGGAACAGAGGA-3'). These sequences were cloned in both orientations into the reporter plasmids. One of the cDNA clones, which we isolated with an HI
-specific probe, encoded the transcription factor E2-2 (Bain et al., 1993
). E2-2 belongs to the family of E box-binding proteins. Computer analysis detected putative binding sites (5'-CANNTG-3') in the HI motifs HI
, HI
and HI
. No consensus sequence was found in HI
. In vitro translation of the isolated E2-2 cDNA yielded a protein with the expected molecular mass (approximately 75 kDa; Fig. 1
A). However, the recombinant protein did not bind to the published consensus sequences CT1 (Pscherer et al., 1996
) and µE5 (Shen & Kadesch, 1995
) (data not shown). This was thought to be due to incorrect folding or missing modifications, since a monoclonal antibody specific for E2-2 (BD Pharmingen) recognized the in vitro-translated product very inefficiently (Fig. 1B
, lane 4). Therefore, further experiments were performed with E2-2-positive nuclear extracts. Native E2-2 was present in nuclear extracts of the lymphoid cell line BJAB (Fig. 1B
, lane 1) (Ben Bassat et al., 1977
) and in the neuroblastoma cell line NGP (Fig. 1B
, lane 3) but was not detected in the teratocarcinoma cell line Tera 9117 (Fig. 1B
, lane 2).
|
|
To confirm that the slowest-migrating complex formed at HI-associated E boxes, the complex was challenged with binding sites HI, HI
, HI
that had been mutated in their E boxes (HI
*: 5'-CCTCCAACATGCAACTTGCCTCG-3'; HI
*: 5'-CTGTCCACAATACGCTGCTTCCTCC-3'; HI
*: 5'-CCATATGTGGACACTACGACCTGAGC-3') (Fig. 2B
). As shown before, the CT1 site and the µE5 site abolished formation of the complex (Fig. 2B
, lane 9 and 10, respectively). In contrast, the mutated HI sequences did not interfere (Fig. 2B
, lanes 1113) although they still competed with other HI-specific complexes indicated by asterisks. This demonstrated again that the E boxes localized within the HI motifs are specifically bound by proteins.
To demonstrate that the E2-2 protein interacts with the HI-associated E boxes, we performed supershift assays with an E2-2-specific monoclonal antibody. However, the antibody did not work sufficiently well in this type of assay (data not shown). In an alternative experimental approach, H1299 cells, which synthesize very low amounts of E2-2, were transiently transfected with an expression plasmid encoding E2-2 or pUC18 (as a negative control). By creating such an E2-2-positive/negative test system, we wanted to see whether we could unequivocally identify E2-2-specific complexes with HI motifs (Fig. 2C). Using the E2-2-positive extract with the HI motifs HI
, HI
and HI
(Fig. 2C
, lanes 3, 6 and 9, respectively), E2-2-specific complexes were clearly visible (indicated by arrows). These did not form or were significantly less intense with control extract of H1299 cells transfected with pUC18 (Fig. 2C
, lanes 2, 5 and 8). In another shift experiment, the E2-2-specific complex, which formed with the µE5 consensus probe (Fig. 2C
, lanes 12 and 23), could be competed with increasing amounts of HI
, HI
and HI
oligonucleotides (Fig. 2C
, lanes 1722), as well as with a positive control, CT1 (Fig. 2C
, lanes 15 and 16). The EMSA with E2-2-positive/negative extracts clearly demonstrated that E2-2 does bind to the HI-associated E boxes.
Based on EMSA data, we finally performed functional transfection assays with BZLF1 promoter-derived reporter plasmids (Fig. 3). Transfection of the Burkitt's lymphoma cell line DG75 was carried out by electroporation using double pulses: 750 V, 2350
, 25 µF and 100 V, 2310
, 3000 µF (Gene Pulser; Bio-Rad). The nasopharyngeal carcinoma (NPC) epithelial line CNE-L was transfected by calcium phosphate precipitation. To construct reporter plasmids, the regulatory region of BZLF1 was isolated by PCR (primers: 5'-CGGCAAGGAGATCTGTTTAGTG-3' and 5'-GGATCCCTAACGGTACCCCCGG-3'), inserted into the reporter plasmid pGL2basic (Promega) and the E boxes were mutated by site-directed mutagenesis (QuikChange site-directed mutagenesis kit; Stratagene) using the following primers: HI
*: 5'-CCTCCAACATGCAACTTGCCTCG-3' and 5'-CGAGGCAAGTTGCATGTTGGAGG-3'; HI
*: 5'-CTGTCCACAATACGCTGCTTCCTCC-3' and 5'-GGAGGAAGCAGCGTATTGTGGACAG-3'; HI
*: 5'-CCATATGTGGACACTACGACCTGAGC-3' and 5'-GCTCAGGTCGTAGTGTCCACATATGG-3'. The reporter constructs were co-transfected with expression plasmids for BZLF1 protein (Zta) to measure the impact of E box sequences on Zta-mediated transactivation. In the lymphoid cell line (DG75), transactivation ranged from 66- to 290-fold (Fig. 3B
, lanes 610). Mutation of the E boxes within the HI motifs HI
and HI
increased transactivation 2·4-fold compared with the original promoter (Fig. 3B
, lanes 7 and 9). A triple mutation in the three HI motif-associated E boxes showed a cumulative effect and increased transactivation 4·4-fold compared with the original promoter (Fig. 3B
, lane 10), whereas mutation of the HI motif HI
had no stimulating effect (Fig. 3B
, lane 8). In the NPC epithelial line (CNE-L) transactivation rates were quite high (up to 3000-fold) (Fig. 3A
, lane 6). The relatively strong transactivation of the original promoter in the epithelial line and the enhancement of transactivation in the lymphoid cell line after mutation of the E boxes were in accordance with the absence and presence, respectively, of E2-2 in both cell lines. (CNE-L tested negative with EMSA; data not shown.) In contrast to the lymphoid cell line, mutation of the HI-associated E boxes significantly reduced Zta-mediated transactivation in the epithelial line (Fig. 3A
, lanes 710). This indicated that in the latter case the E boxes were not only free of inhibitory factors but were also involved in positive regulation increasing responsiveness to Zta.
|
In the lymphoid cell line, the E2-2 effect was less significant with the original promoter due to the high background expression of E2-2 (Fig. 3B, lane 16). However, overexpression also significantly reduced transactivation (to 62 %) in the case of mutations in HI
(Fig. 3B
, lane 17) and in HI
(Fig. 3B
, lane 19). In the case of the triple E box mutation, E2-2 reduced Zta-mediated transactivation to 43 % (Fig. 3B
, lane 20). A Western blot control of a co-transfection experiment with a Zta-specific antibody ensured that the observed transcriptional repression was not an artefact due to the inhibition of expression of Zta from the transfected vector by E2-2 (Fig. 3C
).
Since the triple mutants still responded to overexpressed E2-2, it seems likely that other E boxes outside the HI motifs confer additional repression. Indeed, three additional E boxes were localized outside the HI sequences at positions -520 to -515 (5'-CACTTG-3'), -344 to -339 (5'-CATTTG-3') and -208 to -203 (5'-CAACTG-3'). Recently, the E box nearest to the start site of transcription was reported to be part of a silencer element (Kraus et al., 2001).
Thus, EMSA experiments and transfection assays have demonstrated that E box-binding proteins, in particular E2-2, interact with the core region of the HI motifs HI, HI
and HI
in the BZLF1 promoter. Mutations of the E boxes within the HI motifs HI
and HI
released transcriptional repression in the lymphoid cell line, demonstrating their role as cell-type-specific repressors. A mutation of the E box in HI
had no influence on transactivation. In contrast to the lymphoid cells, in the epithelial cell line mutation of all three HI-associated E boxes significantly reduced Zta-mediated transactivation, indicating a positive regulatory property in this type of cell.
These data suggest a model where the HI motifs HI and HI
are bi-functional elements. Both inhibitory and stimulatory factors can bind in an exclusive manner to the same HI-associated E boxes. Inhibitory E box-binding factors such as E2-2 are expressed in a cell-type-specific manner in lymphoid cells, since mutations of HI-associated E boxes led to increased transactivation. In this context, the role of the E box in the HI
motif needs to be analysed further. In epithelial cells, such as NPC, the E boxes contribute to enhanced responsiveness to Zta-mediated transactivation by binding (epithelial) cell-type- and differentiation-dependent stimulatory factors.
An alternative model is that the E boxes are situated close to positive regulatory elements with which they interfere. The inhibitory effects result from sterical hindrance or binding of histone deacetylases.
Taking into account the fact that E2-2 and other E box-binding proteins are expressed in a differentiation-dependent manner in lymphoid cells (Church et al., 1985; Ephrussi et al., 1985
; Bain et al., 1993
; O'Riordan & Grosschedl, 1999
) and that virus replication depends on cell differentiation, the HI motifs and the associated E boxes appear to contribute to the stabilization of latency and the control of virus replication in B lymphoid cells via binding of E box-binding proteins.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|
Bain, G., Gruenwald, S. & Murre, C. (1993). E2A and E2-2 are subunits of B-cell-specific E2-box DNA-binding proteins. Mol Cell Biol 13, 35223529.[Abstract]
Becker, J., Leser, U., Marschall, M., Langford, A., Jilg, W., Gelderblom, H., Reichart, P. & Wolf, H. (1991). Expression of proteins encoded by EpsteinBarr virus trans-activator genes depends on the differentiation of epithelial cells in oral hairy leukoplakia. Proc Natl Acad Sci U S A 88, 83328336.[Abstract]
Ben Bassat, H., Goldblum, N., Mitrani, S. & 7 other authors (1977). Establishment in continuous culture of a new type of lymphocyte from a "Burkitt like" malignant lymphoma (line D.G.-75). Int J Cancer 19, 2733.[Medline]
Church, G. M., Ephrussi, A., Gilbert, W. & Tonegawa, S. (1985). Cell-type-specific contacts to immunoglobulin enhancers in nuclei. Nature 313, 798801.[Medline]
Daibata, M., Humphreys, R. E. & Sairenji, T. (1992). Phosphorylation of the EpsteinBarr virus BZLF1 immediate-early gene product ZEBRA. Virology 188, 916920.[CrossRef][Medline]
Decker, L. L., Klamen, L. D. & Thorley-Lawson, D. A. (1996). Detection of the latent form of EpsteinBarr virus DNA in the peripheral blood of healthy individuals. J Virol 70, 32863289.[Abstract]
Ephrussi, A., Church, G. M., Tonegawa, S. & Gilbert, W. (1985). B lineage-specific interactions of an immunoglobulin enhancer with cellular factors in vivo. Science 227, 134140.[Medline]
Flemington, E. & Speck, S. (1990). Identification of phorbol ester response elements in the promoter of EpsteinBarr virus putative lytic switch gene BZLF1. J Virol 64, 12171226.[Medline]
Gutsch, D., Holley Guthrie, E., Zhang, Q. & Kenney, S. (1993). NF-kB can physically interact with the EpsteinBarr virus Z protein and inhibit Z function. In XIII International Herpesvirus Workshop, July 2530. 30.
Katz, D. A., Baumann, R. P., Sun, R., Kolman, J. L., Taylor, N. & Miller, G. (1992).Viral proteins associated with the EpsteinBarr virus transactivator, ZEBRA. Proc Natl Acad Sci U S A 89, 378382.[Abstract]
Kenney, S. C., Holley Guthrie, E., Quinlivan, E. B., Gutsch, D., Zhang, Q., Bender, T., Giot, J. F. & Sergeant, A. (1992). The cellular oncogene c-myb can interact synergistically with the EpsteinBarr virus BZLF1 transactivator in lymphoid cells. Mol Cell Biol 12, 136146.[Abstract]
Kraus, R. J., Mirocha, S. J., Stephany, H. M., Puchalski, J. R. & Mertz, J. E. (2001). Identification of a novel element involved in regulation of the lytic switch BZLF1 gene promoter of EpsteinBarr virus. J Virol 75, 867877.
Miyashita, E. M., Yang, B., Lam, K. M., Crawford, D. H. & Thorley Lawson, D. A. (1995). A novel form of EpsteinBarr virus latency in normal B cells in vivo. Cell 80, 593601.[Medline]
Montalvo, E. A., Shi, Y., Shenk, T. E. & Levine, A. J. (1991). Negative regulation of the BZLF1 promoter of EpsteinBarr virus. J Virol 65, 36473655.[Medline]
Montalvo, E. A., Cottam, M., Hill, S. & Wang, Y.-C. J. (1995). YY1 binds to and regulates cis-acting negative elements in the EpsteinBarr virus BZLF1 promoter. J Virol 69, 41584165.[Abstract]
O'Riordan, M. & Grosschedl, R. (1999). Coordinate regulation of B cell differentiation by the transcription factors EBF and E2A. Immunity 11, 2131.[Medline]
Prang, N., Wolf, H. & Schwarzmann, F. (1995). EpsteinBarr virus lytic replication is controlled by a posttranscriptional mechanism of BZLF1. J Virol 69, 26442648.[Abstract]
Prang, N., Wolf, H. & Schwarzmann, F. (1999). Latency of EpsteinBarr virus is stabilized by antisense-mediated control of the viral immediate-early gene BZLF-1. J Med Virol 59, 512519.[CrossRef][Medline]
Pscherer, A., Dorflinger, U., Kirfel, J., Gawlas, K., Ruschoff, J., Buettner, R. & Schule, R. (1996). The helix-loop-helix transcription factor SEF-2 regulates the activity of a novel initiator element in the promoter of the human somatostatin receptor II gene. EMBO J 15, 66806690.[Abstract]
Ruf, I. K. & Rawlins, D. R. (1995). Identification and characterization of ZIIBC, a complex formed by cellular factors and the ZII site of the EpsteinBarr virus BZLF1 promoter. J Virol 69, 76487657.[Abstract]
Schwarzmann, F., Prang, N., Reichelt, B., Rinkes, B., Haist, S., Marschall, M. & Wolf, H. (1994). Negative cis-acting elements in the distal part of the promoter of EpsteinBarr virus trans-activator gene BZLF1. J Gen Virol 75, 19992006.[Abstract]
Shen, C. P. & Kadesch, T. (1995). B-cell-specific DNA binding by an E47 homodimer. Mol Cell Biol 15, 45184524.[Abstract]
Shimizu, N. & Takada, K. (1994). Analysis of the BZLF1 promoter of EpsteinBarr virus: identification of an anti-immunoglobulin response sequence. J Virol 67, 32403245.
Sinclair, A. J., Brimmell, M. & Farrell, P. J. (1992). Reciprocal antagonism of steroid hormones and BZLF1 in switch between EpsteinBarr virus latent and productive cycle gene expression. J Virol 66, 7077.[Abstract]
Tierney, R. J., Steven, N., Young, L. S. & Rickinson, A. B. (1994). EpsteinBarr virus latency in blood mononuclear cells: analysis of viral gene transcription during primary infection and in the carrier state. J Virol 68, 73747385.[Abstract]
Walling, D. M., Perkins, A. G., Webster-Cyriaque, J., Resnick, L. & Raab-Traub, N. (1994). The EpsteinBarr virus EBNA-2 gene in oral hairy leukoplakia: strain variation, genetic recombination, and transcriptional expression. J Virol 68, 79187926.[Abstract]
Wang, Y. C., Huang, J. M. & Montalvo, E. A. (1997). Characterization of proteins binding to the ZII element in the EpsteinBarr virus BZLF1 promoter: transactivation by ATF1. Virology 227, 323330.[CrossRef][Medline]
Young, L. S., Lau, R., Rowe, M. & 7 other authors (1991). Differentiation-associated expression of the EpsteinBarr virus BZLF1 transactivator protein in oral hairy leukoplakia. J Virol 65, 28682874.[Medline]
Zhang, Q., Gutsch, D. & Kenney, S. (1994). Functional and physical interaction between p53 and BZLF1: implications for EpsteinBarr virus latency. Mol Cell Biol 14, 19291938.[Abstract]
Received 31 July 2002;
accepted 3 December 2002.