FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2043550216> ?p ?o ?g. }

• W2043550216 endingPage "553" @default.
• W2043550216 startingPage "544" @default.
• W2043550216 abstract "Article15 January 1998free access p53 binds and represses the HBV enhancer: an adjacent enhancer element can reverse the transcription effect of p53 Assaf Ori Assaf Ori Departments of Molecular Genetics and The Weizmann Institute of Science, Rehovot, 76100 Israel Search for more papers by this author Arie Zauberman Arie Zauberman Molecular Cell Biology, The Weizmann Institute of Science, Rehovot, 76100 Israel Search for more papers by this author Gilad Doitsh Gilad Doitsh Departments of Molecular Genetics and The Weizmann Institute of Science, Rehovot, 76100 Israel Search for more papers by this author Nir Paran Nir Paran Departments of Molecular Genetics and The Weizmann Institute of Science, Rehovot, 76100 Israel Search for more papers by this author Moshe Oren Moshe Oren Molecular Cell Biology, The Weizmann Institute of Science, Rehovot, 76100 Israel Search for more papers by this author Yosef Shaul Corresponding Author Yosef Shaul Departments of Molecular Genetics and The Weizmann Institute of Science, Rehovot, 76100 Israel Search for more papers by this author Assaf Ori Assaf Ori Departments of Molecular Genetics and The Weizmann Institute of Science, Rehovot, 76100 Israel Search for more papers by this author Arie Zauberman Arie Zauberman Molecular Cell Biology, The Weizmann Institute of Science, Rehovot, 76100 Israel Search for more papers by this author Gilad Doitsh Gilad Doitsh Departments of Molecular Genetics and The Weizmann Institute of Science, Rehovot, 76100 Israel Search for more papers by this author Nir Paran Nir Paran Departments of Molecular Genetics and The Weizmann Institute of Science, Rehovot, 76100 Israel Search for more papers by this author Moshe Oren Moshe Oren Molecular Cell Biology, The Weizmann Institute of Science, Rehovot, 76100 Israel Search for more papers by this author Yosef Shaul Corresponding Author Yosef Shaul Departments of Molecular Genetics and The Weizmann Institute of Science, Rehovot, 76100 Israel Search for more papers by this author Author Information Assaf Ori1, Arie Zauberman2, Gilad Doitsh1, Nir Paran1, Moshe Oren2 and Yosef Shaul 1 1Departments of Molecular Genetics and The Weizmann Institute of Science, Rehovot, 76100 Israel 2Molecular Cell Biology, The Weizmann Institute of Science, Rehovot, 76100 Israel *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:544-553https://doi.org/10.1093/emboj/17.2.544 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The transcription program of the hepatitis B virus (HBV) genome is regulated by an enhancer element that binds multiple ubiquitous and liver-enriched transcription activators. HBV transcription and replication are repressed in the presence of p53. Here we describe a novel molecular mechanism that is responsible for this repression. The p53 protein binds to a defined region within the HBV enhancer in a sequence-specific manner, and this, surprisingly, results in p53-dependent transcriptional repression in the context of the whole HBV enhancer. This unusual behavior of the HBV enhancer can be reconstituted by replacing its p53-binding region with the p53-binding domain of the mdm2 promoter. Remarkably, mutation of the EP element of the enhancer reversed the effect of p53 from repression to transcriptional stimulation. Furthermore, EP-dependent modulation of p53 activity can be demonstrated in the context of the mdm2 promoter, suggesting that EP is not only required but is also sufficient to convert p53 activity from positive to negative. Our results imply that the transcriptional effect of DNA-bound p53 can be dramatically modulated by the DNA context and by adjacent DNA–protein interactions. Introduction Hepatitis B virus (HBV) is the prototype of the hepadnaviruses. It is a primarily hepatotropic, enveloped DNA virus with a very small genome that is replicated by reverse transcription. It has a unique mode of gene expression: the synthesis of all viral transcripts is regulated at the level of transcription, not by RNA processing. The viral genome contains an enhancer element that regulates viral gene expression in liver cells (Shaul et al., 1985; Jameel and Siddiqui, 1986; Honigwachs et al., 1989). Since RNA synthesis precedes viral replication, this enhancer has a key role in viral replication and life cycle. Hepadnaviruses are tightly associated with development of hepatocellular carcinoma (Beasley et al., 1981) and at least in animals the homologous enhancer has a role in tumor formation (Wei et al., 1992; Etiemble et al., 1994). The HBV enhancer is divided into distinct functional elements (Dikstein et al., 1990a). The E element has intrinsic enhancer activity which works in isolation in a number of different cell lines (Faktor et al., 1990). This element binds multiple factors of the basic leucine zipper family, including C/EBP (Dikstein et al., 1990b), the AP-1 complex (Faktor et al., 1990) and ATFs (Maguire et al., 1991). A second important enhancer element, termed EF-C (Ostapchuk et al., 1989) or EP (Ben-Levy et al., 1989), has no intrinsic enhancer activity and in isolation is not functional as a positive element (Dikstein et al., 1990a). Its exact role in the context of the enhancer is not yet known, but it appears to cooperate with either the E element (Dikstein et al., 1990a) or the RXR/HNF4-binding element (Garcia et al., 1993). Two distinct proteins bind the EP element: RFX1 (Siegrist et al., 1993) and the proto-oncoprotein c-Abl, which has tyrosine kinase activity (Dikstein et al., 1992). EP-associated c-Abl has strong kinase activity and EP-bound proteins are heavily tyrosine phosphorylated (Dikstein et al., 1996). An interesting and open question is whether the ability of the EP element to cooperate in transcription activation is due to the kinase activity of c-Abl. NF1 is another ubiquitous cellular factor that binds the HBV enhancer at three sites (Ben-Levy et al., 1989). One of these NF1 sites has an important role in enhancer activity, and mutation of this site decreases the production of the 3.5 kb viral pregenomic transcript (Ori and Shaul, 1995). The p53 tumor suppressor gene product has a variety of transcription-regulatory activities (Haffner and Oren, 1995; Gottlieb and Oren, 1996; Ko and Prives, 1996). In a large body of independent studies, p53 has been shown to repress the activity of numerous viral and cellular promoters (Ginsberg et al., 1991; Santhanam et al., 1991; Kley et al., 1992; Moberg et al., 1992; Shiio et al., 1992; Subler et al., 1992). The emerging notion is that p53, a strong activator of transcription, has a general repressive effect on promoters that lack a specific p53-binding site, probably by sequestering components of the basal transcription machinery through protein–protein interactions (Seto et al., 1992; Agoff et al., 1993; Liu et al., 1993; Martin et al., 1993; Ragimov et al., 1993; Lu and Levine, 1995; Thut et al., 1995). It has been reported that p53 binds specifically viral DNA elements such as the GC boxes of the SV40 early promoter (Bargonetti et al., 1991) and the Tax-responsive element of the HTLV I enhancer (Aoyama et al., 1992). In the latter case, this interaction leads to activation of the viral promoter. The Tax-responsive element is similar in sequence to the E element of the HBV enhancer and these two elements respond to the Tax and X proteins in a similar manner (Faktor and Shaul, 1990). Therefore, we have investigated the effect of p53 on the HBV enhancer. We show here that p53 binds, sequence-specifically, to the 5′ portion of the HBV enhancer and represses its activity. This unexpected p53-mediated repression activity is an intrinsic property of the HBV enhancer and depends on an intact enhancer EP element. Furthermore, the EP element is sufficient to block the p53-mediated activation of the mdm2 promoter. Collectively, our results demonstrate a previously unknown transcription regulatory activity of p53 and raise the interesting possibility that the transcriptional effect of p53 may be modulated through interaction with an adjacent enhancer element. Results p53 binds specifically to the HBV enhancer To study whether there was any interaction between p53 and the HBV enhancer, an immune selection assay was performed (Zauberman et al., 1993). A plasmid containing the complete HBV enhancer was digested with appropriate restriction enzymes, radiolabeled and incubated with a nuclear extract containing wild-type p53 activity. The extract was prepared from Clone 6 cells overexpressing a temperature-sensitive p53 mutant (Michalovitz et al., 1990). The cells were maintained at 32°C before preparing the extracts, to induce the accumulation of p53 in a conformation like that of the wild-type. Resultant p53–DNA complexes were immunoprecipitated with the p53-specific monoclonal antibody PAb421 (Figure 1A). The HBV enhancer DNA fragment, but not the vector DNA, was specifically immunoprecpitated in complex with p53 (lane 2). A similar experiment was done with purified recombinant baculovirus-expressed p53 and a shorter HBV DNA fragment. Only the fragment containing the HBV enhancer sequence was immunoprecipitated (lane 6). No precipitated DNA fragment was obtained in the absence of recombinant baculovirus-expressed p53, suggesting that the antibody itself has no DNA-binding activity. Thus, the enhancer binds p53 in a sequence-specific manner. Interestingly, the sequence near the 5′ end of the HBV enhancer, between the EcoRV and StuI sites (Figure 1B), has a similar structure to the p53-binding region of the muscle creatine kinase promoter (Weintraub et al., 1991), as it contains stretches of partial homology to the 10 bp half-consensus (El-Deiry et al., 1992), as well as adjacent repeats of the TGCCT sequence (Kern et al., 1991). Figure 1.p53 binds the HBV enhancer. (A) Autoradiograms of the 32P-labeled DNA fragments separated on a polyacrylamide gel. The total end-labeled plasmid restriction digest, containing the vector DNA and the HBV insert, is shown in lanes 3 and 4. The DNA recovered after incubation with p53-containing nuclear extracts (lane 2) or recombinant p53 (lane 6) immune selection with the p53-specific antibody PAb421. M, DNA size marker (MspI-digested pBR322 DNA). (B) Schematic representation of the HBV enhancer region with the indicated functional motifs, and the sequence of EcoRV–StuI fragment. The TGCCT repeats found in certain p53-binding sites (Kern et al., 1991) are indicated by horizontal bars. Stretches with homology to the 10 bp half-consensus (RRRCWWGYYY; El-Deiry et al., 1992) for p53 binding are also indicated. Download figure Download PowerPoint p53 binds the 5′ portion of the HBV enhancer An EMSA was used to map the p53-binding site within the enhancer. Binding was performed with recombinant baculovirus-expressed p53 in the presence of the p53-specific monoclonal antibody PAb421, which is known to improve and to supershift the binding of p53 to its cognate sites (Hupp et al., 1993). The results (Figure 2) reveal that the complete HBV enhancer (nt 1043–1235) and the 5′ portion of it (nt 1043–1115) bind p53 specifically, as confirmed by inclusion of specific and nonspecific competitors in the reactions (Figure 2A). The antibody itself has no DNA-binding activity (lane 9). The 3′ StuI–SphI fragment (nt 1115–1235) did not generate a specific complex with p53 whereas the EcoRV–StuI fragment (nt 1043–1115) bound efficiently (2B). Thus, the HBV enhancer binds p53 specifically within the EcoRV–StuI region. Figure 2.p53 specifically binds the 5′ portion of the HBV enhancer. (A) The complete enhancer and the EcoRV–StuI fragments were 32P-end-labeled and used as probes for EMSA with 50 ng recombinant baculovirus-expressed p53. Reactions were supplemented with the monoclonal antibody PAb421 to improve binding.. When indicated, the following oligonucleotide competitors (‘comp DNA’) were included: TGT3, which binds the hepatocyte nuclear factor 3 protein; EP, which binds the RFX1–c-Abl complex; GLN LTR, a high affinity p53 binding element derived from an endogenous retrovirus-like element (Zauberman et al., 1993). (B) Fragments of HBV enhancer were subjected to EMSA with or without the addition of PAb421 monoclonal antibody, to show that only the EcoRV–StuI fragment binds p53. The antibody that was used failed to supershift the complex effectively, because cognate peptide epitope was present in the recombinant p53 protein samples (Zauberman et al., 1993). The probes used are shown in (C). Download figure Download PowerPoint To delineate the site of interaction of p53 with the enhancer, DNase I footprinting either with extracts enriched for wild-type-like p53 DNA-binding activity or with recombinant baculovirus-expressed p53 was performed. A conspicuous protected region was visible in each case (Figure 3). The footprint is extended and is centered around the second stretch of homology to the 10 bp half-consensus (AAACAGGCTT; Figure 1B) which exhibits the highest match (9/10) to the half-consensus (El-Deiry et al., 1992). The region at the 5′ end containing the TGCCT repeats was only poorly protected (lanes 4 and 5, and data not shown). Figure 3.Definition of the p53-binding site within the HBV enhancer by DNase I footprinting. An end-labeled EcoRV–HpaI fragment from the HBV enhancer (nt 1043–1196; the HpaI site was artificially inserted) was incubated with either cellular extract of Clone 6 cells (32°C) or recombinant p53 and then subjected to DNase I footprinting followed by polyacrylamide–urea gel electrophoresis. The amount of protein per reaction is indicated. The protected sequence is indicated; two Maxam–Gilbert reactions are shown in lanes 1 and 2. Download figure Download PowerPoint p53 represses the transcriptional activity of the HBV enhancer/X promoter To test whether the specific binding of p53 to the HBV enhancer has functional significance, the effect of p53 on the HBV enhancer and the X promoter was examined by cotransfection into p53-null cells. Since the HBV enhancer is active mostly in liver cells, the hepatoma cell line Hep3B was used. Unexpectedly, wild-type p53 significantly reduced the luciferase activity of the reporter containing HBV enhancer and X promoter (Figure 4A, columns 1–3 and 5–7). A mutant p53 had only a marginal effect (columns 4 and 8). A very similar dose-dependent repression pattern was observed in another p53-null cell line, Saos2 (columns 5–8). In contrast, the mdm2 promoter (Juven et al., 1993; Wu et al., 1993; Barak et al., 1994) was strongly activated by wild-type but not mutant p53 (Figure 4B). The activation of the mdm2 reporter indicates that p53 was efficiently produced in the transfected cells. As often observed at high p53 concentrations, the activation of the mdm2 reporter plasmid was reduced. Previously, p53 has been shown to repress an array of different promoters (Ginsberg et al., 1991; Santhanam et al., 1991; Kley et al., 1992; Moberg et al., 1992; Shiio et al., 1992; Subler et al., 1992). To check whether in the HBV context the negative effect of p53 is mediated by the X promoter, a reporter plasmid containing only that promoter (XpLuc; reporter II) was used. p53 had no significant effect on this promoter under these experimental conditions (Figure 4A, columns 9–12). The HBV enhancer had a similar effect in the context of a heterologous thymidine kinase (TK) promoter, in both enhancer orientations (Figure 4C), suggesting that the X promoter is not essential for the transcriptional repression activity of p53. Interestingly, no repression by p53 was seen in the presence of a fragment of the enhancer retaining functional enhancer activity but lacking the EcoRV–StuI fragment (columns 13–16). This implies that p53 represses the HBV enhancer activity through the EcoRV–StuI fragment, presumably by a mechanism requiring specific binding of p53 to a cognate site within this fragment. This is an unexpected finding, since sequence-specific DNA binding by p53 has so far always been reported to activate transcription (Kastan et al, 1991; Weintraub et al., 1991; El-Deiry et al., 1992; Zambetti et al., 1992; Juven et al., 1993; Wu et al., 1993; Okamoto and Beach, 1994; Miyashita and Reed, 1995; Zauberman et al., 1995). Figure 4.Wild-type p53 specifically represses HBV enhancer activity in p53-null cell lines. Various combinations of luciferase reporters and p53 expression plasmids were transiently transfected into Hep3B or Saos2 cells. (A) The HBV enhancer in context of the X promoter. (B) Effect of p53 on the mdm2 promoter. (C) Effect of p53 on the HBV enhancer activity in context of the TK promoter. In construct III the HBV enhancer was cloned in the opposite orientation. In all cases luciferase activity was determined 24 h later. The histogram represents the relative luciferase activity, where 100 is the basal activity without p53. Reporter plasmids (represented by roman numerals) and amounts of the wild-type and mutant p53 effector plasmids are indicated below. A schematic representation of each reporter plasmid is shown at the bottom. ‘R-S’ indicates the EcoRV–StuI fragment of the extended enhancer, containing the p53-binding site. ‘En’ indicates the StuI–SphI fragment containing the minimal HBV enhancer, lacking the p53-binding region. ‘Xp’, ‘Md2p’ and ‘TKp’ refer to the X (MscI–NcoI fragment) the mouse mdm2 promoter and HSV TK gene promoters, respectively. In Hep3B cells deletion of the EcoRV–StuI fragment reduced the enhancer activity by 20%. The Xp reporter shows 10-fold lower activity than the enhancer-containing reporter. Download figure Download PowerPoint The p53-binding regions of the HBV enhancer can confer transcriptional activation when uncoupled from the rest of the enhancer To test whether the unexpected repression of the HBV enhancer by p53 resulted from an intrinsic inhibitory activity of the p53-binding EcoRV–StuI fragment, four tandem repeats of this fragment were cloned upstream of the X promoter to yield 4(R-S)XpLuc (Figure 5B, reporter I). Surprisingly, luciferase activity was now induced rather than repressed by the cotransfected wild-type p53 (Figure 5A). The X promoter itself was refractory to the effect of p53 (columns 9–12). Since by itself the HBV EcoRV–StuI fragment conferred transcriptional activation upon interaction with wild-type p53, the repression activity must be mediated by the minimal HBV enhancer (nt 1115–1235). To examine this possibility, a construct containing four EcoRV–StuI (R-S) repeats upstream of the minimal enhancer [4(R-S)EXpLuc] was used (Figure 5B, construct II). The behavior of this reporter was exactly opposite that of 4(R-S)XpLuc: it was repressed dose-dependently by cotransfected wild-type p53 (Figure 5A, columns 5–7). Thus, the minimal HBV enhancer region can convert p53 from a positive regulator of transcription into a specific negative one. Figure 5.The HBV enhancer p53-binding element (R-S) responds to p53 in a context-dependent manner. Hep3B cells were transiently transfected by the combinations of indicated luciferase reporter and p53 expression plasmids. (A) Relative luciferase activity, where 100 is the basal activity without p53. The reporter plasmids (represented by roman numerals) and amounts of p53 effector plasmids are indicated below. (B) Schematic representation of the different reporters. Download figure Download PowerPoint The p53-binding region of the mdm2 gene represses the HBV enhancer in a p53-dependent manner If the observed negative effect of p53 is indeed an intrinsic property of the HBV minimal enhancer, the enhancer may be expected to act similarly in the context of other p53-binding sites. To test this possibility, the well characterized p53 response element of the mouse mdm2 gene (MPRE) was used. When inserted directly upstream of the X promoter, MPRE conferred a strong positive response to cotransfected wild-type p53 (Figure 6, columns 5–8), in line with its behavior in other contexts (Juven et al., 1993; Wu et al., 1993; Friedlander et al., 1996). However, exactly the opposite behavior was observed in the presence of the HBV enhancer, where MPRE now mediated strong transcriptional repression (columns 1–4). Repression was not seen in the context of the X promoter, either alone (columns 9–12) or together with the HBV minimal enhancer (column 13–16). These data strongly suggest that the HBV minimal enhancer converts the outcome of sequence-specific p53 binding from positive transactivation into transcriptional repression. Figure 6.The p53 responsive element of the mdm2 gene (MPRE) confers p53-dependent repression in the context of the HBV enhancer. (A) Various combinations of luciferase reporters and p53 expression plasmids were transiently transfected into Hep3B cells. Basal activity in the absence of p53 was taken as 100. Reporter plasmids (represented by roman numerals) and amounts of p53 effector plasmids are indicated below. (B) Schematic representation of the different reporters. Download figure Download PowerPoint The EP element of the HBV enhancer is required to convert p53 into a transcriptional repressor The HBV enhancer binds multiple cellular activators and has been divided into distinct functional elements (Shaul and Ben-Levy, 1987; Ben-Levy et al., 1989; Patel et al., 1989; Dikstein et al., 1990a; Trujillo et al., 1991; Ori and Shaul, 1995). To investigate which of these might be responsible for converting p53 into a transcriptional repressor, reporter plasmids carrying enhancer mutants under the regulation of the MPRE were used. Since this effect of the HBV enhancer is not cell-type-specific (Figure 4A, columns 1–8) the responsible element must interact with ubiquitous factors. The HBV enhancer contains two known elements which bind ubiquitous factors and have an important role in enhancer activity. One element binds the NF1 activator and the other generates a large DNA–protein complex termed EP (Ben-Levy et al., 1989) or EF-C (Ostapchuk et al., 1989). Two proteins are known to associate with the EP elements: RFX1, an evolutionarily conserved DNA-binding protein (Reith et al., 1994), and c-Abl, a tyrosine kinase proto-oncoprotein (Dikstein et al., 1992). Enhancer mutants that do not bind the corresponding EP and NF1 proteins were assayed for their p53 response. Remarkably, mutation of the EP site converted p53 into a positive activator, whereas mutation of the NF1 site had no effect on the p53-mediated, HBV enhancer-dependent transcriptional repression (Figure 7A, compare columns 9–12 and 13–16). These results indicate that the intact EP element of the HBV enhancer is required for converting p53 into a sequence-specific transcriptional repressor. Figure 7.Transcriptional repression by wild-type p53 is dependent on an intact enhancer EP element. Details are as in Figure 5. The altered sequences of the EPmEn and NF1bmEn, bearing mutations at the EP and the NF1b sites, respectively, are boxed. Download figure Download PowerPoint The EP element is sufficient to block p53-mediated transcriptional activation To test whether the EP element by itself can block transcriptional activation by p53, we introduced four tandem copies of the EP element into the mouse mdm2 promoter between the MPRE and the core promoter region (Juven et al., 1993). Remarkably, in the presence of the EP element, p53 almost completely lost its capacity to stimulate the mdm2 promoter (Figure 8, columns 1–4). This effect is specific, as a mutated EP sequence and an unrelated 5×Gal4 element did not behave in this way (Figure 8, columns 5–8 and 9–12 respectively). These data suggest that the HBV-enhancer EP element can antagonize the transcriptional activation function of p53, and is sufficient to render a p53-inducible promoter unresponsive to p53. In this particular context the presence of the four EP repeats did not turn p53 into a specific transcriptional repressor. This observation is addressed in the Discussion. Figure 8.The EP element blocks activation of the mouse mdm2 promoter by p53. Details as in Figure 5. The black bar in (B) represents the core region of the mouse mdm2 p53-dependent promoter. EP and EPm are as defined in Figure 7. 'Gal4′ is UASG, conferring specific binding of the yeast Gal4 transactivator. Download figure Download PowerPoint Repression of HBV gene expression by a p53 inducer DNA-damaging agent The HBV enhancer regulates the production of all the viral transcripts. The finding that p53 represses enhancer activity suggests that HBV gene expression and, hence, replication (since the pregenomic viral mRNA is the template for replication) should be repressed by overproduction of p53. HepG2 cells transfected with HBV DNA were treated with cis-platinum, a DNA-damaging agent that induces p53 overproduction (Fritsche et al., 1993), and the viral transcripts were analyzed. HepG2 cells were used because they contain wild-type p53 and support HBV gene expression. A significant reduction in the level of the viral transcripts was obtained mainly in the treated cells (Figure 9). The effect of cis-platinum was even greater when the X open reading frame was knocked out (lanes 7–12). This observation is in agreement with a previous report that pX can block p53 activity (Lee et al., 1995). Western analysis confirmed that p53 but not β-tubulin was overproduced by cis-platinum. These data suggest that physiological levels of p53 repress HBV gene expression. Figure 9.HBV gene expression is reduced in cells treated with cis-platinum. HepG2 cells were transfected with a plasmid that contains two tandem copies of HBV full-length DNA, either wild-type or a mutant with a stop codon at position 27 of the X gene open reading frame (X−HBV, lanes 7–12). Cells were treated with 2 μg/ml cis-platinum for the indicated time (in hours) before harvesting. RNA and proteins were extracted and analyzed. A 32P-labeled HBV DNA probe was used to detect the known viral transcripts and GAPDH probe to quantify the RNA in each lane. For Western analysis, anti human p53 (1801+DO-1) and anti-β-tubulin (clone no. TUB2.1, Sigma) antibodies were used. Download figure Download PowerPoint Figure 10.A model to explain the role of the EP element in p53-mediated transcriptional repression by the HBV enhancer. The distinct enhancer elements are shown. R-S is the p53-binding region, and GB, EP and E are the HNF4/RXR, RFX1/c-Abl and basic lucine zipper binding sites, respectively. (A) In the absence of p53 the EP element cooperates with the E and GB elements to mediate transcriptional activation (Dikstein et al., 1990a; Garcia et al., 1993). (B) In the presence of p53, an interfering interaction is generated between p53 and the EP-binding proteins. This interaction blocks the positive activity of p53, and simultaneously eliminates the cooperation between EP and other enhancer elements. Download figure Download PowerPoint Discussion In this study we report that the p53 tumor suppressor protein binds the HBV enhancer and modulates its activity. The strategic position of the p53-binding region between the previously reported UE1 site (Shaul and Ben-Levy, 1987) and the minimal enhancer region, and its sequence conservation at the homologous position in other hepadnaviruses that infect mammals (data not shown), suggest a potential function in modulating the enhancer activity. The role of p53 was tested in two different human p53-null cell lines, the Hep3B hepatoma (Ponchel et al., 1994) and the Saos2 osteosarcoma (Masuda et al., 1987; Chen et al., 1990). In both lines the p53 site repressed the enhancer activity and this repression depended on wild-type p53. It is well established that p53, a strong activator of transcription, has a general repressive effect on promoters that lack a corresponding binding site, probably by sequestering basal transcription factors through protein–protein interactions. To our knowledge this is the first example of p53 acting as a sequence-specific transcriptional repressor. It is unlikely that this is due to non-specific sequestration of transcription factors, for several reasons. First, we used very low amounts of transfected p53 plasmid to reduce the probability of such sequestration. Indeed, under these conditions the X and TK promoters were only marginally affected. Second, we showed that the repression activity is fully dependent on the p53-binding site; upon its removal, no p53 effect was retained despite the fact that the basal enhancer activity remained the same. Third, we demonstrated that this behavior can be reproduced with the heterologous p53-binding DNA element of the mdm2 gene. Finally, and perhaps most significantly, the positive transcriptional effect of DNA- bound p53 could be restored by mutating the EP element of the HBV enhancer. In the context of the HBV enhancer, p53 represses transcription in an EP-element-dependent manner. However, in the context of the mdm2 promoter, this element blocks the positive action of p53 but does not repress promoter activity. A likely explanation is that, in the former case, p53 and EP block each other's activity (Figure 9). Under these conditions the major positive role that the EP-binding proteins play in cooperation with the E and the GB element-binding proteins is eliminated as a result of p53 binding. Consequently, the contribution of the EP element to the rate of transcription is totally abolished, allowing p53 binding to re" @default.
• W2043550216 created "2016-06-24" @default.
• W2043550216 creator A5014467884 @default.
• W2043550216 creator A5023108013 @default.
• W2043550216 creator A5036387444 @default.
• W2043550216 creator A5046027770 @default.
• W2043550216 creator A5071233555 @default.
• W2043550216 creator A5081926768 @default.
• W2043550216 date "1998-01-15" @default.
• W2043550216 modified "2023-10-05" @default.
• W2043550216 title "p53 binds and represses the HBV enhancer: an adjacent enhancer element can reverse the transcription effect of p53" @default.
• W2043550216 cites W1480719064 @default.
• W2043550216 cites W1487724525 @default.
• W2043550216 cites W1494359007 @default.
• W2043550216 cites W1502887285 @default.
• W2043550216 cites W1525669528 @default.
• W2043550216 cites W1546139579 @default.
• W2043550216 cites W1581523186 @default.
• W2043550216 cites W1607419610 @default.
• W2043550216 cites W1608926015 @default.
• W2043550216 cites W1627175747 @default.
• W2043550216 cites W1661434665 @default.
• W2043550216 cites W1751026265 @default.
• W2043550216 cites W1823168351 @default.
• W2043550216 cites W1847864430 @default.
• W2043550216 cites W1854431161 @default.
• W2043550216 cites W1860047937 @default.
• W2043550216 cites W1874132424 @default.
• W2043550216 cites W1912385731 @default.
• W2043550216 cites W1962459157 @default.
• W2043550216 cites W1964224010 @default.
• W2043550216 cites W1972773643 @default.
• W2043550216 cites W1974355172 @default.
• W2043550216 cites W1974640412 @default.
• W2043550216 cites W1975122645 @default.
• W2043550216 cites W1979520405 @default.
• W2043550216 cites W1983171715 @default.
• W2043550216 cites W1991917274 @default.
• W2043550216 cites W1995856480 @default.
• W2043550216 cites W1998979789 @default.
• W2043550216 cites W2005546492 @default.
• W2043550216 cites W2015026645 @default.
• W2043550216 cites W2027157544 @default.
• W2043550216 cites W2029529223 @default.
• W2043550216 cites W2038182463 @default.
• W2043550216 cites W2041606694 @default.
• W2043550216 cites W2045086385 @default.
• W2043550216 cites W2046922918 @default.
• W2043550216 cites W2047697085 @default.
• W2043550216 cites W2052210851 @default.
• W2043550216 cites W2058737236 @default.
• W2043550216 cites W2059110341 @default.
• W2043550216 cites W2061154169 @default.
• W2043550216 cites W2064194464 @default.
• W2043550216 cites W2072763798 @default.
• W2043550216 cites W2074222004 @default.
• W2043550216 cites W2076059928 @default.
• W2043550216 cites W2081472291 @default.
• W2043550216 cites W2093089286 @default.
• W2043550216 cites W2096808350 @default.
• W2043550216 cites W2102930268 @default.
• W2043550216 cites W2109384031 @default.
• W2043550216 cites W2124380422 @default.
• W2043550216 cites W2125545372 @default.
• W2043550216 cites W2126537212 @default.
• W2043550216 cites W2127952872 @default.
• W2043550216 cites W2128060680 @default.
• W2043550216 cites W2133629762 @default.
• W2043550216 cites W2150710841 @default.
• W2043550216 cites W2151085515 @default.
• W2043550216 cites W2167983057 @default.
• W2043550216 cites W2168498502 @default.
• W2043550216 cites W2169321817 @default.
• W2043550216 cites W2170494099 @default.
• W2043550216 cites W2172270130 @default.
• W2043550216 cites W2234554087 @default.
• W2043550216 cites W2337944356 @default.
• W2043550216 cites W2411184464 @default.
• W2043550216 cites W2417046404 @default.
• W2043550216 doi "https://doi.org/10.1093/emboj/17.2.544" @default.
• W2043550216 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/1170404" @default.
• W2043550216 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9430645" @default.
• W2043550216 hasPublicationYear "1998" @default.
• W2043550216 type Work @default.
• W2043550216 sameAs 2043550216 @default.
• W2043550216 citedByCount "92" @default.
• W2043550216 countsByYear W20435502162012 @default.
• W2043550216 countsByYear W20435502162014 @default.
• W2043550216 countsByYear W20435502162015 @default.
• W2043550216 countsByYear W20435502162016 @default.
• W2043550216 countsByYear W20435502162017 @default.
• W2043550216 countsByYear W20435502162018 @default.
• W2043550216 countsByYear W20435502162019 @default.
• W2043550216 countsByYear W20435502162020 @default.
• W2043550216 countsByYear W20435502162021 @default.
• W2043550216 countsByYear W20435502162022 @default.
• W2043550216 countsByYear W20435502162023 @default.
• W2043550216 crossrefType "journal-article" @default.

• next