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Department of Biochemistry, Section for Molecular Genetics, Institute of Medical Biology, University of Tromsø, N-9037 Tromsø, Norway
Correspondence
Ugo Moens
ugom{at}fagmed.uit.no
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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B-responsive genes encoding growth factors, cytokines, transcription factors and cell cycle regulatory proteins. Indeed, a previous study has shown that st-ag enhanced NFB-mediated transcription. This study demonstrates that promoters possessing a consensus TATA box (i.e. TATAAAAG) in the context of either NFB- or Sp1-binding sites are trans-activated by st-ag. Overexpressing the general transcription factor hTAFII130/135, but not hTAFII28 or hTAFII80, stimulated the activity of promoters in a consensus TATA box-dependent mode. Converting the consensus TATA motif into a non-consensus TATA box strongly impaired activation by st-ag and hTAFII130/135. Conversely, mutating a non-consensus TATA motif into the consensus TATA box rendered the mutated promoter inducible by st-ag and hTAFII130/135. Mutation of the TATA box had no effect on TNF
- or RelA/p65-mediated induction of NFB-responsive promoters, indicating a specific st-ag effect on hTAFII130/135. St-ag stimulated the intrinsic transcriptional activity of hTAFII130/135. Substitutions in the conserved HPDKGG motif in the N-terminal region or a mutation that impaired the interaction with protein phosphatase 2A abrogated the ability of st-ag to activate hTAFII130/135-mediated transcription. These results indicate that trans-activation of promoters by st-ag may depend on a consensus TATA motif and suggest that such promoters recruit the general transcription factor hTAFII130/135.
Present address: The National Hospital, Institute of Microbiology, Section for Gene Therapy, N-0027 Oslo, Norway. 
Present address: Department of Pharmacology, Institute of Medical Biology, University of Tromsø, N-9037 Tromsø, Norway.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Arrington & Butel, 2001; Rundell & Parakati, 2001). However, several studies have shown that st-ag augments viral and cellular DNA replication and promotes cell cycle progression in CV-1 cells and human fibroblasts (Shenk et al., 1976; Cicala et al., 1993; Sontag et al., 1993; Howe et al., 1998; Porrás et al., 1999; Whalen et al., 1999). Efficient transformation of growth-arrested cells and enhancement of the transforming activity of limiting concentrations of LT-ag in cell cultures depend on st-ag. Moreover, st-ag may protect against LT-ag-induced apoptosis and a role for st-ag in tumourigenesis in vivo has been demonstrated (Sleigh et al., 1978; Martin et al., 1979; Bikel et al., 1987; Choi et al., 1988; Carbone et al., 1989; Cicala et al., 1993; Howe et al., 1998; Kolzau et al., 1999; Hahn et al., 2002). These observations suggest an important helper function for st-ag in LT-ag-mediated processes.
Numerous proteins assemble on the promoter/enhancer region of genes to accomplish transcription of genes. These proteins include the general transcription factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH and RNA polymerase II. TFIID comprises the TATA box-binding protein (TBP) and about 12 TBP-associated factors (TAFIIs). Although referred to as general transcription factors, distinct TAFII proteins seem to be recruited selectively by specific promoters and are, therefore, essential for the transcription of a subset of genes (Albright & Tjian, 2000; Tsukihashi et al., 2000; Li et al., 2002; and references therein). In addition to these general transcription factors, sequence-specific DNA-binding transcription factors, co-activators and chromatin-remodelling proteins are required to ac2tivate transcription (reviewed by Näär et al., 2001). St-ag has been shown to influence the expression of several viral and cellular genes (reviewed by Moens et al., 1997). The exact mechanism by which st-ag exerts its trans-activation function on gene expression remains elusive, but may involve the activation of signalling pathways, probably through association with and inhibition of the serine/threonine protein phosphatase 2A (PP2A). This inhibition of PP2A by st-ag may subsequently regulate the phosphorylation pattern/activity of sequence-specific DNA-binding transcription factors. Indeed, inhibition of PP2A by st-ag led to the activation of several signalling pathways mediated by mitogen-activated protein kinases, calmodulin-dependent protein kinase IV, phosphatidylinositol 3-kinase/PKC
and Akt/PKB (Sontag et al., 1993, 1997; Frost et al., 1994; Watanabe et al., 1996; Howe et al., 1998; Westphal et al., 1998; Garcia et al., 2000; Yuan et al., 2002). Wild-type, but not mutant, st-ag proteins deficient in PP2A binding can influence the activity of the transcription factors cyclic AMP response element-binding protein (CREB), AP-1, NFB, p53, Sp1, STAT3 and HOX11 (reviewed by Janssens & Goris, 2001; Lacroix et al., 2002). Moreover, st-ag can regulate both serum- and Sp1-responsive promoters in a PP2A-dependent fashion (Frost et al., 1994; Garcia et al., 2000). Whether st-ag can affect the activity of general transcription factors has not been investigated but several studies have demonstrated a TATA-dependent mechanism for transcriptional activation of promoters by other viral proteins. Adenovirus E1A protein exhibits an absolute requirement for a TATA motif, while the varicella-zoster virus immediately-early regulatory protein IE62 trans-activates promoters containing a much broader pattern of TATA sequences. Viral transcriptional activators such as Zta (Epstein–Barr virus) and ICP4 (herpes simplex virus) induce transcription by enhancing or stabilizing TBP binding to the TATA box, again illustrating the importance of the TATA box for virus-induced activation of promoter activity (Perera, 2000; and references therein). The hepatitis B virus pX regulatory protein can increase levels of TBP, which serves to enhance both RNA polymerase I and III promoter activities, while the effects on RNA polymerase II promoters are different. TATA-lacking promoters are generally unaffected by increased levels of TBP, while overexpression of TBP stimulates TATA-containing promoters (Johnson et al., 2000).
Previous studies in NIH 3T3 and CV-1 cells have shown that st-ag can stimulate NFB- and Sp1-dependent transcription and that this induction of transcription was mediated by PKC and its upstream regulator phosphatidylinositol 3-kinase (Sontag et al., 1997; Garcia et al., 2000). Because NFB and Sp1 interact physically with PP2A and st-ag inhibits PP2A, it is generally assumed that a major mechanism by which st-ag induces NFB- and Sp1-dependent transcription relies on prevention of PP2A-mediated dephosphorylation of these transcription factors (Yamashita et al., 1999; Yang et al., 2001; Lacroix et al., 2002). Here, we report that trans-activation of NFB- and Sp1-responsive promoters by st-ag depends on a consensus TATAAAAG TATA motif. Such st-ag responder promoters were also stimulated by the general transcription factor hTAFII130/135. Wild-type st-ag, but not a mutant unable to bind PP2A, or a mutant in the conserved hexapeptide HPDKGG, increased the intrinsic transcriptional activity of hTAFII130/135. These data suggest that hTAFII130/135 is a promoter-specific transcription factor whose activity may be regulated by PP2A-dependent and -independent mechanisms. Transcriptional activation of specific cellular genes by st-ag through preventing PP2A-mediated dephosphorylation of hTAFII130/135 may, therefore, facilitate virus replication or transformation.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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).
Materials.
Recombinant murine TNF was purchased from Alexis Biochemicals. Sonicated salmon sperm and calf thymus DNA were from Amersham Pharmacia. Newborn calf serum was from BioWhittaker. Oligonucleotides were purchased from Eurogentec or Invitrogen.
Plasmids.
The NFB-responsive luciferase reporter plasmids and their corresponding control plasmids BconA-LUC and conA-LUC, TI-3x B-LUC and TI-LUC, 2x B-LUC and 2x B-m1LUC, ENH-TK-LUC and mENH-TK-LUC were kindly provided by E. Sontag (Sontag et al., 1997), X.-F. Wang (Li et al., 1998), C. Scheidereit (Hirano et al., 1998) and F. Bachelerie (Bachelerie et al., 1991), respectively. The plasmids pTAL-LUC and pNFB-LUC(C) were purchased from Clontech, while the plasmid pNFB-LUC(S) was obtained from Stratagene. Expression plasmid for the dominant–negative mutant of IB (pCMV-IBS32/36A) has been described previously and was kindly made available by M. Körner (Ferreira et al., 1998). The GAL4 fusion plasmids pGAL4-p65 and pGAL4-p65(416-550) were kindly provided by B. R. Cullen (Blair et al., 1994). The RelA/p65 expression plasmid was obtained from J. A. Didonato (Zhang et al., 1994), while the plasmid pG5E1bLuc was a kind gift from R. Davis (Seth et al., 1991). The plasmid pCMV5 and the st-ag expression plasmid pCMV5st (wild-type st-ag) were a generous gift from E. Sontag (Sontag et al., 1993). The pMIEP-LUC plasmid, containing the major immediate-early promoter of human cytomegalovirus, and the double mutant pMIEPdm-LUC with non-functional NFB motifs have been described previously (Moens et al., 2001). Plasmid pNFB-fosTATA-LUC was constructed by annealing the double-stranded oligonucleotide 5'-AGCTCGGGAATTTCCGGGATTTCCGGGAATTTCTCATTCATAAAACGCTTGTTATAAAAGCAGTGGCTGCGGCGCCTCGTACTCCAAC-3' (only one strand is given) into the BamHI/HindIII sites of pO-LUC. The resulting plasmid contains three copies of the consensus NFB-binding site linked to the TATA box region of the c-fos promoter. The plasmid was sequenced to verify the insertion. Plasmid pCMVhTAFII130/135 was kindly provided by N. Tanese (Saluja et al., 1998). An expression plasmid for GAL4–hTAFII130/135 fusion protein was prepared by amplifying the hTAFII130/135 cDNA sequences using the pCMVhTAFII130/135 plasmid as template and the primers 5'-CAACAGCGAGGTCGACGAGAAAGTGGG-3' and 5'-GAGCAGCAGTGAAAAGCTTGTCTCACG-3'. The PCR product was digested with SalI/BamHI (restriction sites are underlined) and cloned in the corresponding sites of the GAL4 DNA-binding domain expression plasmid pM (Clontech) to generate pGAL4-hTAFII130/135. The plasmid was sequenced to ensure ligation of the hTAFII130/135 cDNA sequences in the correct reading frame. Expression vectors for hTAFII28 and hTAFII80 were a generous gift from R. G. Roeder (Guermah et al., 2001).
Site-directed mutagenesis.
Site-directed mutagenesis was performed with the QuickChange Site-Directed Mutagenesis kit from Stratagene, according to the instructions of the manufacturer. The TATA box motif in BconA-LUC (see Fig. 3A) was replaced by the TATA element of pNFB-LUC(C) (Fig. 3A) using complementary oligonucleotides (5'-GGGCTGCTCCTCTATATTTTGGGAAGAAAG-3', only one primer is shown; the TATA box is shown in bold). The TATA elements of pTAL-LUC and pNFB-LUC were converted into the TATA element of the BconA promoter using a complementary primer set (5'-GACATGCAAATATAAAAGTTCCGGGGACAC-3', only one primer is given; the converted TATA motif is shown in bold). The st-ag mutants P43L/K45N and P101A were obtained by site-directed mutagenesis applying the primers of the complementary set (5'-GAGTTTCATCTAGATAACGGAGGAGATG-3' and 5'-GCAAACAATGCGCAGAGTGTGCAAAGATGTCTGC-3', respectively, only one primer of the complementary set is shown). All mutations were verified by cycle sequencing using the Big Dye Sequencing kit (Perkin Elmer). Sequencing reactions were analysed on an ABI377 Prism Sequencer (Perkin Elmer).
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). All transfections were performed with cell passages between 130 and 144. Cells were serum-starved (0·2 %) for 18 h before being harvested. The amount of total DNA in each transfection mixture was kept constant (5 µg per well) by adding appropriate control vector DNA and salmon sperm or calf thymus DNA (Amersham Pharmacia). All plasmids were purified with the Qiagen Plasmid Purification kit and 1 µg plasmid was used normally per well. Different plasmid DNA preparations were tested in the transfection studies. Each experiment was performed with three independent parallels and was repeated at least three times to ensure reproducibility of results. Luciferase activity was determined in 20 µl lysate using the Luciferase Assay System kit (Perkin Elmer) and a Luminoscan RT (Labsystems). Co-transfections with a 
-galactosidase reporter plasmid were avoided because expression of st-ag influenced -galactosidase values. Moreover, it is our experience that correction for protein concentrations in each cell lysate had a negligible effect on results (P. A. Olsen, unpublished results).
Western blotting.
Western blotting was performed as described previously (Seternes et al., 1999). GAL4 antibodies were from Santa Cruz Biotechnology (cat #sc-577). Densitometry quantification of the hybridization signals was performed with a LumiImager F1 using LumiAnalyst software (Boehringer Mannheim).
Statistics.
Statistical significance of results was determined by Student's t-test with P<0·01.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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B-responsive promotersB-dependent transcription in both NIH 3T3 and CV-1 cells (Sontag et al., 1997). However, we were not able to repeat these observations using two commercially available NFB reporter gene constructs (pNFB-LUC from Clontech and Stratagene). This prompted us to test whether st-ag could stimulate transcription of other NFB reporter plasmids, including that originally used by E. Sontag and colleagues. Seven different reporter plasmids containing different minimal promoters fused to NFB-binding motifs were co-transfected with an expression plasmid for st-ag in NIH 3T3 cells. The corresponding promoters lacking the NFB motifs were used as controls. None of the promoters lacking NFB-binding motifs were trans-activated by st-ag (results not shown), while only one promoter with NFB-binding sites (BconA-LUC, i.e. the one used by Sontag and colleagues) was induced when st-ag was co-expressed (Fig. 1a). On average, a 4·8-fold increase in luciferase activity was measured in the presence of st-ag. Maximal induction was obtained with the reporter plasmid and the st-ag expression plasmid at a ratio of 1 : 1 (results not shown). The failure of most NFB-responsive promoters to be trans-activated by st-ag was not due to impaired NFB motifs, as these promoters were inducible by co-expression of p65/RelA (Fig. 1b) or after stimulation of transfected cells with TNF (Fig. 1c). Corresponding promoters lacking NFB motifs were not induced by p65/RelA and TNF (data not shown). A synergistic increase in BconA promoter activity was measured when st-ag and RelA/p65 were co-expressed (Fig. 1d). This suggests that st-ag may enhance further the transcriptional potentials of RelA/p65 or that st-ag trans-activates the promoter of the BconA-LUC reporter plasmid through an alternative mechanism.
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B-binding motifs by st-ag is partially mediated by NFB-mediated activation of PKC resulted in IB degradation, subsequent nuclear translocation of NFB and stimulation of expression of NFB-responsive genes (Folgueira et al., 1996; Sontag et al., 1997). As we observed a promoter-selective activation of NFB-responsive promoters by st-ag, we wanted to define whether this activation required an alternative mechanism. Therefore, the dominant–negative IBS32/36A mutant, which is unable to be phosphorylated and proteolytically degraded, was overexpressed (Ferreira et al., 1998). This mutant will retain NFB in the cytoplasm even in the presence of st-ag. Co-expression of IBS32/36A completely abolished RelA/p65-induced luciferase activity from the BconA-LUC reporter plasmid, while st-ag-enhanced NFB-mediated transcription was reduced by 40–50 % (Fig. 2a). The observation that IBS32/36A could not completely block trans-activation of the NFB-responsive promoter by st-ag may suggest an additional mechanism by which st-ag regulates the activity of NFB-directed transcription. It could be imagined that, in addition to promoting nuclear translocation, as shown by Sontag et al. (1997), st-ag stimulated the intrinsic transcriptional potentials of RelA/p65, such as has been demonstrated for other transcription factors (reviewed by Janssens & Goris, 2001). To test this, we examined RelA/p65-mediated transcription in the presence and absence of st-ag by using GAL4 fusion proteins. Cells were co-transfected with the empty expression vector for st-ag (CMV5) or the expression vector for st-ag and with a plasmid encoding GAL4–p65 (full-length) or GAL4–p65(416-550) fusion proteins. The latter encompasses the transactivation domain of RelA/p65. St-ag failed to increase transcription mediated by the GAL4–p65 or GAL4–p65(416-550) fusion proteins, indicating that st-ag does not stimulate the intrinsic transcriptional potentials of NFB (Fig. 2b). Together, these results may suggest that supplementary events or factors, in addition to NFB, are involved in mediating trans-activation of NFB-responsive promoters by st-ag.
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B-mediated transcription
a). Only the st-ag-inducible BconA promoter possessed a TATA motif with perfect identity to the consensus TATA motif, while the other six promoters, which were not trans-activated by st-ag, possessed TATA sequences that diverged from this consensus sequence (Périer et al., 2000). To elucidate the importance of the TATA motif in st-ag-stimulated NFB-mediated transcription, promoter mutation experiments were performed. The TATA motif of the non-st-ag-responsive promoter in pNFB-LUC(C) was replaced with the BconA TATA region and vice versa: the BconA TATA sequence was converted into the TATA motif present in pNFB-LUC(C). These mutations reduced the basal activity of BconA-LUC by almost 50 %, while the mutated NFB-LUC plasmid had a slightly increased basal activity in the absence of st-ag compared to the non-mutated promoter (data not shown). The mutated BconA promoter (BmutconA) was still induced by the st-ag expression plasmid; however, an almost 50 % reduction was measured compared to the wild-type promoter. Interestingly, pNFBmut-LUC with consensus TATA box showed a 4-fold increase in trans-activation by st-ag as compared to the non-mutated pNFB-LUC (Fig. 3b). Neither RelA/p65- nor TNF-induced transcription was affected by mutations in the TATA motif (Fig. 3b). These results clearly indicate the importance of the TATA box motif for st-ag-mediated activation of a promoter. To test experimentally the importance of the TATA motif in the induction of NFB-responsive promoters by st-ag further, we investigated whether a chimeric promoter consisting of the c-fos TATA motif (a consensus TATA motif; Fig. 3a) and three copies of the NFB-binding site could be trans-activated by st-ag. As depicted in Fig. 3(c), this promoter was also induced by st-ag. These results underscore further the importance of the TATA motif in mediating st-ag-induced trans-activation of NFB-responsive promoters.
A Sp-1-responsive promoter with the consensus TATA motif but not with a non-consensus TATA box is also trans-activated by st-ag
The minimal chicken conalbumin (conA) promoter lacking the NFB-binding motifs was not stimulated by st-ag (Fig. 1d). This suggested to us that the TATA box per se is not sufficient to induce activation by st-ag but, in addition to an appropriate TATA motif, an upstream binding motif (e.g. NFB) is required to mediate promoter trans-activation by st-ag. A recent report demonstrated that st-ag could activate Sp1-responsive promoters (Garcia et al., 2000). The authors used promoter constructs containing either the adenovirus major late promoter or the human immunodeficiency virus LTR promoter/enhancer. Both promoters possess a consensus TATA motif (Fig. 3a). This observation indicates that Sp1 motifs in concert with a consensus TATA box sequence may also mediate st-ag-induced trans-activation. This assumption was investigated by testing whether st-ag could activate promoter activity of the plasmid pTAL-LUC. This plasmid contains a single Sp1 motif linked to the same non-consensus TATA box present in pNFB-LUC(C) (Fig. 3a). We also converted the TATA motif in pTAL-LUC into the conA TATA box to generate the reporter plasmid pTALmTATA-LUC. St-ag did not induce transcription from the pTAL-LUC plasmid but changing the TATA box into a consensus TATA motif stimulated luciferase activity about 2-fold in the presence of st-ag (Fig. 4). These results prove that the consensus TATA box combined to specific upstream binding elements can mediate st-ag induction.
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B- or Sp1-binding motifs combined with the conA promoter could be trans-activated by st-ag. The transcription factors NFB and Sp1 have been shown to interact with the general transcription factors hTAFII105 and hTAFII130/135, respectively (Saluja et al., 1998; Yamit-Hezi & Dikstein, 1998). hTAFII105 shares regions of high homology with hTAFII130/135. Interestingly, st-ag has been shown to interact in vitro with dTAFII110, the Drosophila homologue of hTAFII130/135 (Damania & Alwine, 1996). This prompted us to investigate whether hTAFII130/135 is utilized by the Sp1- and NFB-responsive promoters that are activated by st-ag. Therefore, the effect of hTAFII130/135 protein on promoters with consensus and non-consensus TATA boxes was monitored. Overexpression of hTAFII130/135 activated the promoters containing the consensus TATA motif 2- to 5·5-fold, while mutating these promoters into the corresponding promoters with non-consensus TATA sequence reduced trans-activation by hTAFII130/135 (Fig. 5a). These results favour a model in which st-ag induced activation of specific promoters that utilize the general transcription factor hTAFII130/135. The activation of promoters with consensus TATA box by hTAFII130/135 was specific for this general transcription factor because neither hTAFII28 nor hTAFII80 could induce promoters, irrespective of the sequence of the TATA box (Fig. 5b).
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a). The st-ag mutant protein P101A, which fails to bind PP2A (Mungre et al., 1994), did not activate transcription mediated by hTAFII130/135. The st-ag double mutant P43L/K45N, shown previously to be unable to activate the adenovirus E2A promoter but not impaired in PP2A binding (Mungre et al., 1994), did not induce hTAFII130/135-mediated transcription. The differences in activation of hTAFII130/135-mediated transcription by the distinct st-ag variants are not the result of unequal expression levels of the various st-ag proteins, as previous studies have shown that the mutant proteins are expressed with comparable levels and stability as wild-type st-ag (Mungre et al., 1994; Porrás et al., 1996; Watanabe et al., 1996; Howe et al., 1998). Enhanced GAL4–hTAFII130/135-directed transcription in the presence of st-ag was not the result of increased GAL4–hTAFII130/135 protein levels, as co-expression of st-ag hardly (1- to 1·5-fold increase in independent experiments) affected the amount of GAL4–hTAFII130/135 in transfected cells (Fig. 6b).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). It is generally assumed that a major mechanism by which st-ag induces transcription is through inhibition of PP2A-dependent dephosphorylation of transcription factors (reviewed by Janssens & Goris, 2001). Here, we describe a novel mechanism of promoter-specific trans-activation by st-ag. St-ag preferentially activated promoters consisting of a consensus TATAAAAG TATA motif and binding sites for the transcription factor NFB or Sp1. Previous studies had shown that st-ag regulates the activities of the promoter/enhancer of adenovirus E2A and E3 genes, the oncogenes c-fos and junB, the cyclins A and D1 and the proliferating cellular nuclear antigen (PCNA) gene (reviewed by Moens et al., 1997). A closer examination of the TATA boxes in the human c-fos, E3, junB and conA promoters reveals the presence of the TATA consensus motif, supporting the importance of the sequence of the TATA motif. We found that the amplitude of induction of the NFB-responsive promoters with consensus TATA motif (the conA and the chimeric c-fos promoters) by st-ag was comparable to the stimulation reported previously for the phosphoenolpyruvate carboxykinase promoter (Wheat et al., 1994), the cyclin D1 promoter (Watanabe et al., 1996), the cyclin A promoter (Schüchner et al., 2001; Porrás et al., 1996) and the c-fos promoter (Mullane et al., 1998). The consensus TATA promoter fused to a single Sp1 site was only induced 2-fold in our study. This is in agreement with the thymidine kinase promoter. This promoter, which contains a single Sp1 motif and a single CRE motif, was induced to comparable levels (2·5-fold) by st-ag in JEG-3 cells (Watanabe et al., 1996).
The fact that st-ag also affects the activities of the TATA-less cyclin A, cyclin D1 and PCNA promoters (Travali et al., 1989; Herber et al., 1994; reviewed by Moens et al., 1997) may suggest that the flanking regions are important. This observation supports further a plausible importance of the flanking sequences. Converting the consensus TATAAAAGGG motif of the BconA promoter into a non-consensus TATATTTTGG sequence of the NFB-LUC plasmid did not completely abrogate trans-activation by st-ag or hTAFII130/135, as this mutated promoter was still induced 2·5-fold compared to 4- to 5-fold for the unmutated promoter. On the other hand, the promoter of the NFB-LUC plasmid was not responsive to st-ag or hTAFII130/135. This discrepancy may be explained by assuming that sequences flanking the TATA box could be involved in recruiting hTAFII130/135. Indeed, recent studies have demonstrated that TAFIIs are not only recruited through protein–protein interaction with TBP and each other but also that they can bind DNA directly in a sequence-specific mode, thereby contributing to promoter selectivity (Albright & Tjian, 2000; Green, 2001). Moreover, binding of hTAFII130/135 to the flanking sequences may affect the binding of TBP to the TATA box, as was shown recently (Furukawa & Tanese, 2000). Point mutations of the flanking sequences and in the consensus TATA motif may enable the identification of crucial nucleotides required to mediate trans-activation by st-ag.
St-ag-responsive promoters were also activated when hTAFII130/135 was overexpressed and vice versa: st-ag non-responsive promoters were not induced by hTAFII130/135. The exact molecular mechanism(s) underlying the activator effect of st-ag on promoters containing a consensus TATA box combined with a NFB- or Sp1-binding motif remains unknown. St-ag may prevent PP2A-mediated dephosphorylation of hTAFII130/135, as the non-PP2A binding P101A st-ag mutant was unable to stimulate the intrinsic transcriptional activity of hTAFII130/135 and also failed to activate the BconA promoter (result not shown). However, a PP2A-independent mechanism is suggested by the studies with the P43L/K45N double mutant. This mutant st-ag is still able to bind PP2A but failed to stimulate GAL4–hTAFII130/135-mediated transcription and could not activate the BconA promoter (result not shown). Previous studies have demonstrated that this mutant failed to trans-activate the adenovirus E2 and the cyclin A promoters (Mungre et al., 1994; Porrás et al., 1996) but induced the activity of the cyclin D1 promoter with comparable levels as wild-type st-ag (Watanabe et al., 1996). The cyclin A and D1 promoters both lack a TATA box, while a non-consensus TATA motif is present in the adenovirus E2 promoter (Herber et al., 1994). These findings, combined with our observations, add to the diversity of transcriptional regulation by st-ag and require future research to solve the exact mechanisms by which st-ag can trans-activate promoters.
It remains to be established whether the novel mechanism by which st-ag can activate gene expression contributes to a successful SV40 infection or transformation. Prevalence for this mechanism derives from the following observations: SV40 infection induced quiescent cells to re-enter the S phase and re-entering the cell cycle coincided with increased c-fos transcription. St-ag was important for enhanced expression of c-fos (Morike et al., 1988; Glenn & Eckhart, 1990; Ogris et al., 1992). Another study showed that granulocyte macrophage colony-stimulating factor promoter was induced upon polyomavirus replication in haematopoietic cells. The effect of st-ag was, however, not examined (Watanabe et al., 1995). Both the c-fos and the granulocyte macrophage-colony stimulating factor promoter contain a consensus TATA motif and Sp1 (c-fos) or NFB (granulocyte macrophage-colony stimulating factor) binding sites. Finally, this mechanism may also contribute to virus immune evasion. Epstein–Barr virus encodes a protein homologous to cellular IL-10. IL-10 is a negative regulator of IL-12, itself a cytokine that both promotes IFN-
production and influences the development of Th1- and Th2-like cytokine-producing cells, and of TAP, a protein involved in transport and presentation of processed peptide antigens (Ploegh, 1998). The promoter of IL-10 contains a consensus TATAAAAG TATA box and a crucial Sp1 site (Tone et al., 2000; Ma et al., 2001). This makes this gene a putative target for trans-activation by st-ag and a strategy for SV40 to subvert the immune system.
In conclusion, trans-activation of specific promoters by st-ag may rely upon at least two different mechanisms. First, st-ag may prevent PP2A-mediated dephosphorylation of specific transcription factors like CREB, Sp1, NFB, STAT and AP-1 and thereby stimulate the activity of promoters regulated by these transcription factors (reviewed by Janssens & Goris, 2001). In addition, st-ag may stimulate the promoter activity of promoters utilizing the general transcription factor hTAFII130/135. The exact molecular mechanism by which st-ag induces hTAFII130/135-responsive promoters needs to be elucidated.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Apel, I., Yu, C.-L., Wang, T., Dobry, C., Van Antwerp, M. E., Jove, R. & Prochownik, E. V. (1992). Regulation of the junB gene by v-src. Mol Cell Biol 12, 3356–3364.
Arrington, A. S. & Butel, J. S. (2001). SV40 and human tumors. In Human Polyomaviruses. Molecular and Clinical Perspectives, pp. 461–489. Edited by K. Khalili & G. L. Stoner. Chichester: Wiley & Sons.
Bachelerie, F., Alcami, J., Arenzana-Seisdedos, F. & Virelizier, J. L. (1991). HIV enhancer activity perpetuated by NF-B induction on infection of monocytes. Nature 350, 709–712.[CrossRef][Medline]
Bikel, I., Montano, X., Agha, M. E., Brown, M., McCormack, M., Boltax, J. & Livingston, D. M. (1987). SV40 small t antigen enhances the transformation activity of limiting concentrations of SV40 large T antigen. Cell 48, 321–330.[CrossRef][Medline]
Blair, W. S., Bogerd, H. P., Madore, S. J. & Cullen, B. R. (1994). Mutational analysis of the transcription activation domain of RelA: identification of a highly synergistic minimal acidic activation module. Mol Cell Biol 14, 7226–7234.
Carbone, M., Lewis, A. M., Jr, Matthews, B. J., Levine, A. S. & Dixon, K. (1989). Characterization of hamster tumors induced by simian virus 40 small t deletion mutants as true histiocytic lymphomas. Cancer Res 49, 1565–1571.
Choi, Y. W., Lee, I. & Ross, S. R. (1988). Requirement for the simian virus 40 small tumor antigen in tumorigenesis in transgenic mice. Mol Cell Biol 8, 3382–3390.
Cicala, C., Pompetti, F. & Carbone, M. (1993). SV40 induces mesotheliomas in hamsters. Am J Pathol 142, 1524–1533.[Abstract]
Cladaras, C. & Wold, W. S. (1985). DNA sequence of the early E3 transcription unit of adenovirus 5. Virology 140, 28–43.[CrossRef][Medline]
Cole, N. C. (1996). Polyomavirinae: the viruses and their replication. In Fields Virology, 3rd edn, pp. 1997–2025. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: Lippincott – Raven.
Damania, B. & Alwine, J. C. (1996). TAF-like function of SV40 large T antigen. Genes Dev 10, 1369–1381.
Ferreira, V., Tarantino, N. & Körner, M. (1998). Discrimination between RelA and RelB transcriptional regulation by a dominant negative mutant of IB. J Biol Chem 273, 592–599.
Folgueira, L., McElhinny, J. A., Bren, G. D., MacMorran, W. S., Diaz-Meco, M. T., Moscat, J. & Paya, C. V. (1996). Protein kinase C- mediates NF-B activation in human immunodeficiency virus-infected monocytes. J Virol 70, 223–231.[Abstract]
Frost, J. A., Alberts, A. S., Sontag, E., Guan, K., Mumby, M. C. & Feramisco, J. R. (1994). Simian virus 40 small t antigen cooperates with mitogen-activated kinases to stimulate AP-1 activity. Mol Cell Biol 14, 6244–6252.
Furukawa, T. & Tanese, N. (2000). Assembly of partial TFIID complexes in mammalian cells reveals distinct activities associated with individual TATA box-binding protein-associated factors. J Biol Chem 275, 29847–29856.
Garcia, A., Cereghini, S. & Sontag, E. (2000). Protein phosphatase 2A and phosphatidylinositol 3-kinase regulate the activity of Sp1-responsive promoters. J Biol Chem 275, 9385–9389.
Glenn, G. M. & Eckhart, W. (1990). Transcriptional regulation of early-response genes during polyomavirus infection. J Virol 64, 2193–2201.
Green, M. R. (2001). TBP-associated factors (TAFIIs): multiple, selective transcriptional mediators in common complexes. Trends Biochem Sci 25, 59–63.
Guermah, M., Tao, Y. & Roeder, R. G. (2001). Positive and negative TAFII functions that suggest a dynamic TFIID structure and elicit synergy with traps in activator-induced transcription. Mol Cell Biol 21, 6882–6894.
Hahn, W. C., Dessain, S. K., Brooks, M. W., King, J. E., Elenbaas, B., Sabatini, D. M., DeCaprio, J. A. & Weinberg, R. A. (2002). Enumeration of the simian virus 40 early region elements necessary for human cell transformation. Mol Cell Biol 22, 2111–2123.
Herber, B., Truss, M., Beato, M. & Müller, R. (1994). Inducible regulatory elements in the human cyclin D1 promoter. Oncogene 9, 1295–1304.[Medline]
Hirano, F., Tanaka, H., Hirano, Y., Hiramoto, M., Handa, H., Makino, I. & Scheidereit, C. (1998). Functional interference of Sp1 and NF-B through the same DNA binding site. Mol Cell Biol 18, 1266–1274.
Howe, A. K., Gaillard, S., Bennett, J. S. & Rundell, K. (1998). Cell cycle progression in monkey cells expressing simian virus 40 small t antigen from adenovirus vectors. J Virol 72, 9637–9644.
Janssens, V. & Goris, J. (2001). Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem J 353, 417–439.[CrossRef][Medline]
Johnson, S. A. S., Mandavia, N., Wang, H.-D. & Johnson, D. L. (2000). Transcriptional regulation of the TATA-binding protein by Ras cellular signaling. Mol Cell Biol 20, 5000–5009.
Kolzau, T., Hansen, R. S., Zahra, D., Reddel, R. R. & Braithwaite, A. W. (1999). Inhibition of SV40 large T antigen induced apoptosis by small T antigen. Oncogene 18, 5598–5603.[CrossRef][Medline]
Lacroix, I., Lipcey, C., Imbert, J. & Kahn-Perlès, B. (2002). Sp1 transcriptional activity is up-regulated by phosphatase 2A in dividing T lymphocytes. J Biol Chem 277, 9598–9605.
Li, J.-M., Shen, X., Hu, P. P.-C. & Wang, X.-F. (1998). Transforming growth factor stimulates the human immunodeficiency virus 1 enhancer and requires NF-B activity. Mol Cell Biol 18, 110–121.
Li, X. Y., Bhaumik, S. R., Zhu, X., Li, L., Shen, W. C., Dixit, D. L. & Green, M. R. (2002). Selective recruitment of TAFs by yeast upstream activating sequences. Implications for eukaryotic promoter structure. Curr Biol 12, 1240–1244.[CrossRef][Medline]
Ma, W., Lim, W., Gee, K., Aucoin, S., Nandan, D., Kozlowski, M., Diaz-Mitoma, F. & Kumar, A. (2001). The p38 mitogen-activated kinase pathway regulates the human interleukin-10 promoter via the activation of Sp1 transcription factor in lipopolysaccharide-stimulated human macrophages. J Biol Chem 276, 13664–13674.
Martin, R. G., Setlow, V., Edwards, C. A. & Vembu, D. (1979). The roles of the simian virus 40 tumor antigens in transformation of Chinese hamster lung cells. Cell 17, 635–643.[CrossRef][Medline]
Moens, U., Seternes, O. M., Johansen, B. & Rekvig, O. P. (1997). Mechanisms of transcriptional regulation of cellular genes by SV40 large T- and small T-antigens. Virus Genes 15, 135–154.[CrossRef][Medline]
Moens, U., Van Ghelue, M., Kristoffersen, A. K., Johansen, B., Rekvig, O. P., Degré, M. & Rollag, H. (2001). Simian virus 40 large T antigen, but not small T antigen, trans-activates the human cytomegalovirus major immediate early promoter. Virus Genes 23, 215–226.[CrossRef][Medline]
Morike, M., Quaiser, A., Muller, D. & Montenarh, M. (1988). Early gene expression and cellular DNA synthesis after stimulation of quiescent NIH3T3 cells with serum or purified simian virus 40. Oncogene 3, 151–158.[Medline]
Mullane, K. P., Ratnofsky, M., Cullere, X. & Schaffhausen, B. (1998). Signaling from polyomavirus middle T and small T defines different roles for protein phosphatase 2A. Mol Cell Biol 18, 7556–7564.
Mungre, S., Enderle, K., Turk, B., Porrás, A., Wu, Y.-Q., Mumby, M. C. & Rundell, K. (1994). Mutations which affect the inhibition of protein phosphatase 2A by simian virus 40 small t antigen in vitro decrease viral transformation. J Virol 68, 1675–1681.
Näär, A. M., Lemon, B. D. & Tjian, R. (2001). Transcriptional coactivator complexes. Annu Rev Biochem 70, 475–501.[CrossRef][Medline]
Ogris, E., Mudrak, I. & Wintersberger, E. (1992). Polyomavirus large and small T antigens cooperate in induction of the S phase in serum-starved 3T3 mouse fibroblasts. J Virol 66, 53–61.
Olsen, H. S. & Rosen, C. A. (1992). Contribution of the TATA motif to Tat-mediated transcriptional activation of human immunodeficiency virus gene expression. J Virol 66, 5594–5597.
Patikoglou, G. A., Kim, J. L., Sun, L., Yang, S. H., Kodadek, T. & Burley, S. K. (1999). TATA element recognition by the TATA box-binding protein has been conserved throughout evolution. Genes Dev 13, 3217–3230.
Perera, L. P. (2000). The TATA motif specifies the differential activation of minimal promoters by varicella zoster virus immediate-early regulatory protein IE62. J Biol Chem 275, 487–496.
Périer, R. C., Praz, V., Junier, T., Bonnard, C. & Bucher, P. (2000). The eukaryotic promoter database (EPD). Nucleic Acids Res 28, 302–303.
Ploegh, H. L. (1998). Viral strategies of immune evasion. Science 280, 248–253.
Porrás, A., Bennett, J., Howe, A., Tokos, K., Bouck, N., Henglein, B., Sathyamangalam, S., Thimmapaya, B. & Rundell, K. (1996). A novel simian virus 40 early-region domain mediates transactivation of the cyclin A promoter by small-t antigen and is required for transformation in small-t antigen-dependent assays. J Virol 70, 6902–6908.
Porrás, A., Gaillard, S. & Rundell, K. (1999). The simian virus 40 small-t and large-T antigens jointly regulate cell cycle reentry in human fibroblasts. J Virol 73, 3102–3107.
Rundell, K. & Parakati, R. (2001). The role of the SV40 ST antigen in cell growth promotion and transformation. Semin Cancer Biol 11, 5–13.[CrossRef][Medline]
Saluja, D., Vassalo, M. F. & Tanese, N. (1998). Distinct subdomains of human TAFII130 are required for interactions with glutamine-rich transcriptional activators. Mol Cell Biol 18, 5734–5743.
Schüchner, S., Nemethova, M., Belisova, A., Klucky, B., Holnthoner, W. & Wintersberger, E. (2001). Transactivation of murine cyclin A by polyomavirus large and small T antigens. J Virol 75, 6498–6507.
Seternes, O. M., Johansen, B. & Moens, U. (1999). A dominant role of the Raf-MEK pathway in forskolin, 12-O-tetradecanoyl-phorbol acetate, and platelet-derived growth factor-induced CREB (cAMP-responsive element-binding protein) activation, uncoupled from serine 133 phosphorylation in NIH 3T3 cells. Mol Endocrinol 13, 1071–1083.
Seth, A., Alvarez, E., Gupta, S. & Davis, R. J. (1991). A phosphorylation site located in the NH2-terminal domain of c-Myc increases transactivation of gene expression. J Biol Chem 266, 23521–23524.
Shenk, T., Carbon, J. & Berg, P. (1976). Construction and analysis of viable deletion mutants of simian virus 40. J Virol 18, 664–671.
Sleigh, M. J., Topp, W. C., Hanich, R. & Sambrook, J. F. (1978). Mutants of SV40 with an altered small t protein are reduced in their ability to transform cells. Cell 14, 79–88.[CrossRef][Medline]
Sontag, E., Fedorov, S., Kamibayashi, C., Robbins, D., Cobb, M. & Mumby, M. (1993). The interaction of SV40 small tumor antigen with protein phosphatase 2A stimulates the MAP kinase pathway and induces cell proliferation. Cell 75, 887–897.[CrossRef][Medline]
Sontag, E., Sontag, J.-M. & Garcia, A. (1997). Protein phosphatase 2A is a critical regulator of protein kinase C signaling targeted by SV40 small t to promote cell growth and NF-B activation. EMBO J 16, 5662–5671.[CrossRef][Medline]
Tone, M., Powell, M. J., Tone, Y., Thompson, S. A. & Waldmann, H. (2000). IL-10 gene expression is controlled by the transcription factors Sp1 and Sp3. J Immunol 165, 286–291.
Travali, S., Ku, D.-H., Rizzo, M. G., Ottavio, L., Baserga, R. & Calabretta, B. (1989). Structure of the human gene for the proliferating cell nuclear antigen. J Biol Chem 264, 7466–7472.
Tsukihashi, Y., Miyake, T., Kawaichi, M. & Kokubo, T. (2000). Impaired core promoter recognition caused by novel yeast TAF145 mutations can be restored by creating a canonical TATA element within the promoter region of the TUB2 gene. Mol Cell Biol 20, 2385–2399.
van Straaten, F., Muller, R., Curran, T., Van Beveren, C. & Verma, I. M. (1983). Complete nucleotide sequence of a human c-onc gene: deduced amino acid sequence of the human c-fos protein. Proc Natl Acad Sci U S A 80, 3183–3187.
Watanabe, S., Ito, Y., Miyajima, A. & Arai, K. (1995). Granulocyte macrophage-colony stimulating factor-dependent replication of polyomavirus replicon in hematopoietic cells. Analyses of receptor signals for replication and transcription. J Biol Chem 270, 9615–9621.
Watanabe, G., Howe, A., Lee, R. J. & 7 other authors (1996). Induction of cyclin D1 by simian virus 40 small tumor antigen. Proc Natl Acad Sci U S A 93, 12861–12866.
Westphal, R. S., Anderson, K. A., Means, A. R. & Wadzinski, B. E. (1998). A signaling complex of Ca2+-calmodulin-dependent protein kinase IV and protein phosphatase 2A. Science 280, 1258–1261.
Whalen, B., Laffin, J., Friedrich, T. D. & Lehman, J. M. (1999). SV40 small T antigen enhances progression to >G2 during lytic infection. Exp Cell Res 251, 121–127.[CrossRef][Medline]
Wheat, W. H., Roesler, W. J. & Klemm, D. J. (1994). Simian virus 40 small tumor antigen inhibits dephosphorylation of protein kinase A-phosphorylated CREB and regulates CREB transcriptional stimulation. Mol Cell Biol 14, 5881–5890.
Yamashita, M., Dimayuga, P., Kaul, S., Shah, P. K., Regnstrom, J., Nilsson, J. & Cercek, B. (1999). Phosphatase activity in the arterial wall after balloon injury: effect of somatostatin analog octretide. Lab Invest 79, 935–944.[Medline]
Yamit-Hezi, A. & Dikstein, R. (1998). TAFII105 mediates activation of anti-apoptotic genes by NF-B. EMBO J 17, 5161–5169.[CrossRef][Medline]
Yang, J., Fan, G. H., Wadzinski, B. E., Sakurai, H. & Richmond, A. (2001). Protein phosphatase 2A interacts with and directly dephosphorylates RelA. J Biol Chem 276, 47828–47833.
Yuan, H., Veldman, T., Rundell, K. & Schlegel, R. (2002). Simian virus 40 small tumor antigen activates AKT and telomerase and induces anchorage-independent growth of human epithelial cells. J Virol 76, 10685–10691.
Zhang, Q., Didonato, J. A., Karin, M. & McKeithan, T. W. (1994). BCL3 encodes a nuclear protein which can alter the subcellular location of NF-B proteins. Mol Cell Biol 14, 3915–3926.
Received 20 December 2002;
accepted 21 February 2003.
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