©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
p53 and Sp1 Interact and Cooperate in the Tumor Necrosis Factor-induced Transcriptional Activation of the HIV-1 Long Terminal Repeat (*)

(Received for publication, June 9, 1995)

Antonio Gualberto Albert S. Baldwin , Jr. (1)(§)

From the Lineberger Comprehensive Cancer Center and Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Tumor necrosis factor alpha (TNF) is a potent activator of transcription directed by the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). We have recently reported that the p53 tumor suppressor gene product binds to a site within the Sp1 binding region of the HIV-1 LTR and contributes to the TNF induction of this promoter. In this study we show that the transcription factor Sp1 cooperates with p53 in the transcriptional activation directed by the HIV-1 LTR. The presence of Sp1 increased p53 binding to its recognition sequence in the HIV-1 LTR, and experiments in Drosophila cells show that Sp1 is necessary for full transactivation by mutant p53. Importantly, TNF induced the association between p53 and Sp1 in Jurkat T cells. These data demonstrate a synergistic role for these proteins in the mechanism of TNF induction of HIV-1 LTR-mediated transcription and suggest that Sp1 may play an important role in modulating certain functions of p53.


INTRODUCTION

Transcriptional activation of HIV-1 (^1)gene expression is controlled in part by the interaction of sequence-specific transcription factors with the long terminal repeat (LTR) region(1, 2) . Two of these factors, NF-kappaB and Sp1, have been studied intensively. NF-kappaB is an inducible transcription that binds two described DNA elements in the HIV-1 LTR. Sp1 is an approximate 100-kDa protein that is ubiquitously expressed in mammalian cells, and three Sp1 sites have been described in the LTR that are downstream of the NF-kappaB sites(1, 2, 3) . Although a number of cytokines have been shown to modulate the expression of the human immunodeficiency virus 1 (HIV-1), only TNF is known so far to activate viral replication in both T cells and mononuclear phagocytes(4) . This cytokine is a potent activator of transcription factors that have been shown to regulate HIV-1 LTR-mediated expression(5, 6) .

The p53 tumor suppressor gene product is a sequence-specific DNA-binding protein, which acts as a transcriptional activator or repressor of a number of genes(7, 8, 9, 10, 11, 12, 13, 14, 15) . The frequent appearance of tumors in the p53 null mice suggests that p53 functions as a tumor suppressor agent(16) . However, experiments with antisense p53(17) , microinjection of anti-p53 antibodies(18) , and transcriptional assays utilizing transforming mutant p53 forms (6, 13, 14, 19) indicate that p53 presents additional functions that are independent of growth suppression. It has been demonstrated recently that overexpression of mutant p53 in cells that completely lack endogenous p53 led to a dramatic increase in HIV-1 replication in vivo(20) . In addition, it has been shown that mutant and wild type p53 transactivate and repress, respectively, HIV-1 LTR-directed transcriptional activity (6, 19) and that the p53, Sp1, and TATA box sites are involved in the p53-induced effects on this promoter(6, 19, 20) . Additionally, we identified an inducible form of p53 in Jurkat T cells that directly interacts with a sequence element positioned immediately 3` to the most 5` Sp1 binding site(6) .

Here we report that TNF induces a physical interaction between p53 and Sp1 and that this interaction is important for p53 DNA sequence recognition and transactivation of the HIV-1 LTR. These data indicate a significant functional role for the interaction of both proteins in the mechanism of TNF induction of HIV-1 transcription and suggest a role for Sp1 in modulating specific biological functions of p53.


EXPERIMENTAL PROCEDURES

Electrophoretic Mobility Shift Assays

The LTR A and B sequences were as indicated in Fig. 1(duplexes). Other oligonucleotides used were (duplexes): p53m ( CAGGGAGGCGTAAACTGGGCGGGACTGGGG) and Sp1m (CAGGGAGGCGTGGCCTGGGTTTTACTGGGG). DNA oligonucleotides and gel shift mobility assays were prepared as described previously(6) , but 0.5 µg/µl bovine serum albumin was added when recombinant proteins were assayed. Purified Sp1 from HeLa cells was from Promega. Purified wild type p53 was a gift from P. Tegtmeyer. Purified proteins GST-YB-1, GST-Z (BZLF1), E2F, CEBPbeta, and Egr-1 were a gift from G. MacDonald and J. Ting, S. Kenney, N. Sung, S. McKnight, and V. Sukhatme, respectively. YY1 was expressed and purified as described(21) .


Figure 1: A, EMSA showing a comparison of the TNF-induced complexes with the LTR A and LTR B probes. Jurkat cells were incubated in the absence (-TNF) or presence (+TNF) of rTNF-alpha, processed for the preparation of nuclear extracts assayed by EMSA. B, EMSA-antibody blocking experiment characterizing TNF-induced protein-LTR B complexes. C, EMSA-Western blotting identifying the presence of Sp1 and p53 in TIC3 using an LTR B probe. D, EMSA competition experiment with TNF-induced extracts and the LTR B probe. 500-fold excess of the oligonucleotide competitors were used as indicated in the figure. E, Western blot of biotin-LTR B affinity-purified p53 form TNF-induced Jurkat nuclear extracts. Biotin-LTR B (53m) and biotin-LTR B (Sp1m) are biotinylated LTR B oligonucleotides with mutations in the p53 and Sp1 sites, respectively.



UV Cross-linking

Several templates for UV cross-linking were prepared. LTR A-UV is an LTR A probe with a BrdUrd for T substitution at position 25. LTR B-UV is a LTR B probe with BrdUrd for T substitutions at positions 11 and 24. 10-fold scale-up EMSA reactions were prepared and irradiated for 20 min at room temperature with 312 nm UV light. Reactions were loaded onto 3.8% polyacrylamide gels and run as indicated. DNA-protein complexes were identified by autoradiography, excised, and loaded on a 10% SDS-PAGE gel (Bio-Rad).

Biotin-Oligonucleotide Affinity Precipitation of p53

100 µg of nuclear extracts from TNF-induced Jurkat cells were incubated with 100 ng of biotinylated probes and 100 µg of poly(dI-dC)bullet(dI-dC) for 20 min at room temperature. Streptavidin Magnesphere beads (150 µl, Promega) were added and the mix incubated for an additional 15 min, followed by precipitation through a magnetic field. The pellet was resuspended in 250 µl of phosphate-buffered saline with 10 µg of poly(dI-dC)bullet(dI-dC), reprecipitated, and the presence of p53 determined by Western analysis.

Antibodies, Immunoprecipitations, and Western Blotting

Mouse monoclonal anti-p53 PAb 240, PAb 421, and PAb 1801 and rabbit polyclonal anti-Sp1 antibodies were purchased from Santa Cruz. Mouse monoclonal anti-p53 1620 and anti-MDM2 were from Oncogene Sciences. Immunoprecipitations and Western blotting were carried out as recommended by the suppliers. EMSA-Western blotting experiments were performed essentially as described(22) . EMSA reactions were carried out with probe LTR B and the position of the TIC3 band identified by autoradiography. Gel slices containing this band were isolated, and proteins were transferred to Immobilon-P membranes (Millipore). The membranes were probed with anti-Sp1 antibody (1:2000) and anti-p53 antibody PAb 1801 (1:500).

Plasmids

HIV-1 LTR CAT reporter plasmid constructs were a gift from B. Stein. The cytomegalovirus (CMV) enhancer-promoterdriven expression vectors containing wild type p53 and the tumor-promoting p53ala form were a gift from B. Vogelstein. Sp1 mammalian expression vectors were as described in (23) . The pPAC and pPAC-Sp1 expression plasmids were kindly provided by R. Tjian.

Cell Culture, Cell Extracts, Transfections, and CAT Assays

Jurkat T leukemia and Akata cells from ATCC were cultured and transfected as described(6) . Nuclear cell extracts were prepared as described(24) , but a final concentration of 2 mM phenylmethylsulfonyl fluoride was used. Extracts were aliquoted and stored at -80 °C. Drosophila melanogaster SL2 cells were cultured in Schneider's media (LCCC Tissue Culture Facility) and supplemented with 10% fetal bovine serum. Cells were transfected by electroporation (300 V, 960 microfarads) at 5 10^6 cells/0.5 ml of phosphate-buffered saline and resuspended in 10 ml of media. Incubations were carried out for 48 h (Jurkat and Akata cells) or 72 h (SL2 cells). CAT activity was assayed as described in (6) .


RESULTS AND DISCUSSION

We have shown previously that a binding site interacting with a TNF-inducible form of p53 is present in the HIV-1 LTR (Fig. 1)(6) . This binding site is situated between the most 5` (Sp1 III) and the middle (Sp1 II) HIV-1 LTR Sp1 sites(3, 6) . The presence of the p53 site overlapping the 5` flank of the Sp1 II site prompted as to study a possible interaction between both transcription factors on the HIV-1 LTR sequences. For this purpose, two oligonucleotides, LTR A and LTR B, were synthesized. Oligonucleotide A contains the most 5` Sp1 site (site III) along with the recently reported p53 binding site. Oligonucleotide probe LTR B (Fig. 1) contains the p53 site and the complete Sp1 site II. Jurkat T cells were incubated for 1 h in the presence or absence of 10 ng/ml TNF, after which nuclear extracts were prepared and assayed by EMSA using the LTR A and B probes. Two TNF-inducible complexes (TIC1 and TIC2) were detected with the LTR A probe (Fig. 1A). It has been previously reported that these complexes contain p53 and that p53 binding to this sequence is blocked by incubation of the extracts with antibodies that recognize p53 in a proliferative (mutant-like) conformation(6) . Another two inducible complexes of slower mobility (TIC3 and TIC4) were detected using LTR B (Fig. 1A).

The slower mobility of TIC3 suggested that, aside from p53, an additional factor was present in this complex. To determine whether p53 and Sp1 were part of TIC3 and TIC4, the same extracts were incubated with antibodies that recognize either Sp1 or p53 epitopes and then analyzed by EMSA with the LTR B probe (Fig. 1B). The anti-Sp1 antibody supershifted the upper TIC complex (TIC3) indicating the presence of this transcription factor. Three anti-p53 monoclonal antibodies were used: PAb 421, PAb 1620, and PAb 1801. PAb 421 is a monoclonal antibody against wild type and mutant p53 C-terminal domain. PAb 1620 is specific for a nonproliferative conformational epitope of wild type p53. PAb 1801 recognizes wild type and mutant p53 N-terminal domain. Only PAb 421 was able to detect p53 in the EMSA. Addition of this antibody completely blocked TIC formation (Fig. 1B). The effect of PAb 421 was not proteolytic since this antibody was able to supershift p53 bound to the consensus RGC p53 site (not shown). Interestingly, the p53 PAb 421 epitope has been shown to be expressed when lymphocytes are induced to proliferate by mitogens(25) , and expression of this epitope in the absence of PAb 1620 reactivity is considered to identify a p53 conformational state associated with cell proliferation(26) . In order to provide further evidence for the presence of Sp1 and p53 in the TICs, we transferred the major inducible complex, TIC3, from the EMSA to a membrane and probed with antibodies specific for Sp1 or for p53. Rabbit anti-Sp1 and PAb 1801 identified the presence of both proteins in TIC3 (Fig. 1C). In conclusion, the EMSA-supershift and EMSA-Western experiments indicated that p53 is present in both TIC3 and TIC4 and that p53 and Sp1 constitute the TIC3.

The specificity of the TICs was demonstrated by EMSA oligonucleotide competition experiments. Nuclear extracts from TNF-induced Jurkat cells were preincubated with a 500-fold molar excess of oligonucleotides containing native LTR B sequences or LTR B sequences with mutations in the p53 (LTR B p53m) or Sp1 (LTR B Sp1m) sites, or with the p53 consensus RGC sequence. EMSA analysis is shown in Fig. 1D. The TIC bands were completely competed by the LTR B and LTR B Sp1m sequences, indicating that the p53 site is necessary for the TIC complexes to bind. Competition with LTR B p53m only partially competed TIC formation and the RGC oligonucleotide had no effect. As demonstrated previously(6) , TNF-inducible p53 does not bind the RGC consensus p53 site (Fig. 1D).

In order to determine if the presence of the Sp1 binding site substantially modified the affinity of p53 for the HIV-1 LTR, experiments were designed to test the ability of biotinylated oligonucleotides containing LTR B native or mutant sequences to affinity precipitate p53 from nuclear extracts. TNF-induced Jurkat nuclear extracts were incubated with oligonucleotides biotin-LTR B, biotin-LTR B p53 mutant, or biotin-LTR B Sp1 mutant. Protein-DNA complexes were precipitated with streptavidin magnetic beads and were analyzed by immunoblotting. A typical result of these experiments, shown in Fig. 1E, indicated that Sp1 binding to the DNA probe enhanced the affinity of p53 for its LTR binding site. Mutation of the p53 site completely blocked p53 binding (Fig. 1E, lane2). Although mutation of the Sp1 site did not block p53 binding, it affected p53 binding in a significant manner, ranging from 50% to 25% of p53 bound to the Sp1 mutant probe relative to the native probe (Fig. 1E, lane3). These data suggested that an interaction between Sp1 and p53 alters the binding affinity of p53 for its target on the HIV-1 LTR or that Sp1 binding to its site enhances p53 binding through alteration of DNA conformation.

TNF Induces the Association of p53 and Sp1 in Jurkat Cells

To determine if p53 and Sp1 associate in Jurkat cells following TNF induction, we performed coimmunoprecipitation studies. Sp1 and associated proteins present in the cell extracts were immunoprecipitated with anti-Sp1 and analyzed by PAGE. The proteins were transferred to a membrane and the potential presence of p53 was measured by probing with antibody PAb 1801. The results shown in Fig. 2A demonstrate that TNF induced the association of p53 and Sp1. In the experiment shown in Fig. 2A, the amount of p53 bound to Sp1 in uninduced cells was practically undetectable. In similar experiments, some p53 was coimmunoprecipitated with Sp1 in untreated cells but always the amount of p53 in uninduced cells was significantly less that the amount coimmunoprecipitated with Sp1 in TNF-induced cells. The overall levels of p53, as determined by Western analysis, did not change during the incubation with TNF (not shown). Interestingly, it has been shown previously that Sp1 and p53 are able to interact in an erythroleukemia cell line induced with granulocyte/macrophage colony-stimulating factor(27) .


Figure 2: A, effect of TNF on the coimmunoprecipitation of p53 with Sp1 in Jurkat cells. Cell lysates were prepared and Sp1 immunoprecipitated. Immunoprecipitates were assayed by Western blotting for the presence of p53. B, effect of TNF on the coimmunoprecipitation of Sp1 and MDM2 with p53 in Jurkat cells. Cells were assayed as in A and p53 immunoprecipitated with PAb 240. The presence of Sp1 and MDM2 was detected Western blotting.



In reciprocal experiments, cells were TNF induced, lysed and p53 was precipitated with PAb 240. Extracts were analyzed by PAGE and probed with anti-Sp1 antibody (Fig. 2B). The amount of Sp1 interacting with p53 was increased by the treatment of the cells with TNF. Sp1 was also coimmunoprecipitated with p53 when anti-p53 PAb 421 was used (not shown). As a control, the membrane was probed for the presence of MDM2, a protein that has been shown to interact with p53 (28) . Intriguingly, a decrease in the amount of MDM2 interacting with p53 was found in the TNF induced cells. It is known that human MDM2 inhibits the ability of p53 to stimulate transcription by binding to the acidic activation domain of p53(28, 29) . We speculate that MDM2 may also be inhibiting p53 transcriptional activity by blocking the ability of p53 to interact with other transcription factors. This would also suggest that the regulation of MDM2-p53 interaction could be involved in the mechanism of TNF function. It has been previously shown that the p53 PAb 240 epitope is induced by the incubation of lymphocytes with TNF(6) . The present data suggest that posttranslational modifications induced by TNF allow p53 to interact with other transcriptional activators, such as Sp1. As previously stated, it has been shown (27) that granulocyte/macrophage colony-stimulating factor induced the interaction of p53 with Sp1 in erythroleukemia cells. It is presumable that the cytokine-induced modifications to p53 and the subsequent interaction with Sp1 play an important role in the ability of p53 to bind and transactivate certain of its DNA targets.

Sp1 Interaction Is Necessary for Wild Type p53 to Bind the HIV LTR p53 Site in Vitro

In order to further analyze the interaction between Sp1 and p53, we assayed the ability of purified proteins to bind different probes in vitro. As expected, baculovirus-expressed wild type p53, but not purified Sp1 from HeLa cells, was able to bind the RGC probe (Fig. 3B). As indicated previously, our conditions of assay do not favor Sp1 binding. Sp1-LTR A complexes were identified when the reaction conditions were modified (lower stringency) for that purpose (not shown). p53 and Sp1 were not able to independently bind the LTR A probe (Fig. 3A). As shown in Fig. 1, the LTR A probe does not contain the high affinity GGGCGG consensus Sp1 binding site. Addition of Sp1 dramatically increased p53 binding to this probe. The fact that p53 is the protein that binds the LTR A site in these gels was demonstrated by a cross-linking experiment using an LTR A probe with a BrdUrd substitution at position 25 (probe LTR A-UV). Sp1 and p53 were incubated with LTR A-UV for 1 h and irradiated for 15 min with 312 nm UV light, and complex formation was determined by EMSA. The unique band present in the EMSA was excised and analyzed by PAGE (Fig. 3C, LTRA), and only p53 was detected. This result shows that p53 is able to bind the LTR A probe, and, although p53 and Sp1 may be able to interact in solution, the strength of this interaction may not be sufficient to resist the conditions of the EMSA. Several experiments were done to demonstrate the specificity of the activation effect of Sp1 on p53 binding. First, Sp1 did not increase the binding of recombinant p53 to the RGC or other unrelated sequences (data not shown). No binding was obtained when p53 was incubated with purified YB-1, C/EBP-beta, the Epstein-Barr virus transactivator Z protein, Erg1, YY1, or heat-inactivated Sp1 (not shown). A minor activation of p53 binding was observed with Egr-1. Interestingly, Egr1 is a zinc finger protein with a high homology to Sp1 in the zinc finger domains. Erg1 has been shown to bind not only its specific binding sites, but also Sp1 consensus sequences(30) . Interestingly, another zinc finger protein, WT1, has been shown to interact with p53(31) .


Figure 3: A, EMSA of p53 and Sp1 using the LTR A probe. 100 ng of purified Sp1 and p53 were utilized. Gel shift assay conditions were as indicated under ``Experimental Procedures.'' B, EMSA of p53 and Sp1 using the RGC and LTR B probes. Conditions were as in A. C, cross-linking of purified Sp1 and p53 proteins to LTR A-UV (LTRA) and LTR B-UV (LTRB) probes.



Further information was obtained using the LTR B sequence (Fig. 3B). As indicated (see Fig. 1A), this probe contains the p53 site and the flanking consensus Sp1 binding site. Direct binding of Sp1, but not recombinant p53, was detected. When both proteins were assayed together, the presence of a slower mobility band was detected. The addition of p53 also decreased the intensity of the Sp1 band, suggesting that the upper band may contain both proteins. When this slow mobility complex was assayed by UV cross-linking, using an LTR B probe with BrdUrd for T substitutions at position 11 and 24 (probe LTR B-UV), both Sp1 and p53 were identified. This experiment demonstrated that both proteins can bind simultaneously to the DNA probe (Fig. 3C, LTRB). These experiments suggested that the interaction with Sp1 plays a role in the sequence recognition properties of p53. A possible interpretation is that interaction with Sp1 allows p53 to adopt the adequate conformation for binding to the LTR DNA sequence. However, it is also possible that in these assays Sp1 is selecting p53 protein molecules in the preparation of wild type p53 that exhibit an appropriate conformation for interaction. The latter hypothesis would imply growth factors or cell cycle regulators induce the necessary posttranslational modifications in p53 and Sp1 that allow these proteins to interact and bind Sp1-p53 composite sites.

Synergistic Transactivation between Sp1 and p53 in Vivo

It has been previously shown that site-directed mutagenesis of the LTR p53 site decreased the transactivation of the HIV-1 LTR by mutant p53 forms (6) and also that mutation of the Sp1 sites did not abolish, but significantly diminished, mutant p53 transactivation(19) . These results indicated that multiple sites mediate the ability of p53 to transactivate the HIV-1 LTR and that Sp1 is not necessary but may be synergistic with p53 transactivation. To characterize the functional role of the physical interaction between Sp1 and p53, we sought to analyze the role of these transcription factors in a D. melanogaster cell line, SL2, that lacks proteins that are related to the mammalian Sp1 transcription factor(32) . SL2 cells were transfected with expression vectors encoding p53 143ala and Sp1 together with the -121,+232 HIV-1 LTR CAT reporter plasmid. Transfection of p53 143ala or Sp1 alone produced a minimal activation of HIV-1 LTR CAT activity. However, cotransfection of both transcription factors resulted in a significant increase of transcription (Fig. 4). This synergistic effect was dependent in the presence of the p53 and Sp1 composite site as both factors failed to synergize when a -65,+232 LTR deletion mutant promoter was used (Fig. 4). At high levels of input Sp1 expression vector the synergy is lost. Possibly at these levels the 5` Sp1 site may be saturated for binding, thus preventing p53 from binding to its target site. The finding of this synergistic activation supports the in vitro data presented in this paper indicating the cooperativity of Sp1 and P53 for DNA binding. In addition, the juxtaposition of the p53 and Sp1 responsive elements in the HIV-1 LTR suggests that this close proximity may be necessary for the stabilization of the interaction between these transcription factors.


Figure 4: Transient-transfection assay showing the Sp1 and p53 synergy on the HIV-1 LTR transcription. SL2 cells were transfected with 10 µg of -121,+232 HIV-1 LTR CAT or -65,+232 HIV LTR CAT reporter plasmids, 3 µg of a CMV-p53 143ala (p53), and increasing concentration of pPAC-Sp1 (Sp1). Transfection samples were adjusted using CMV and pPAC empty vectors. Cells were incubated for 72 h and CAT activity measured as indicated under ``Experimental Procedures.'' CAT activity was normalized to basal expression of the HIV-1 LTR -121,+232 (5% CAT conversion). The figure represents three independent experiments.



It may be important to consider that the role of the p53-Sp1 interaction could be extended to the regulation of the expression of other promoters that contain Sp1 sites. Recent studies suggest that phosphorylation of Sp1 during the G(0) to G(1) transition may regulate its ability to transactivate gene expression (33) . Sp1 binding sites, called GC boxes, are present in the promoters of many growth-regulated genes(33) . It is possible that some of these promoters may contain Sp1-p53 composite sites. This hypothesis is most interesting when promoters that have been shown to be transactivated by transforming mutant forms of p53 but do not contain known p53 binding sites are considered.

Some examples of these genes are the PCNA(14) and MDR1 genes(13) . The identification of these putative p53-Sp1 composite sites may help to understand the functions of the proliferative p53 conformational state (34) and the gain-of-function effect (26, 34) attributed to p53 mutant forms.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants CA52515 and AI35098 and by Grant DAMD17-94-J-4053 from the Department of the Army. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 919-966-3652; Fax: 919-966-3015.

(^1)
The abbreviations used are: HIV-1, human immunodeficiency virus type 1; LTR, long terminal repeat; TNF, tumor necrosis factor; BrdUrd, bromodeoxyuridine; PAGE, polyacrylamide gel electrophoresis; EMSA, electrophoretic mobility shift assay; CMV, cytomegalovirus; TIC, TNF-inducible complex.


ACKNOWLEDGEMENTS

We thank R. Tjian, B. Stein, P. Tegtmeyer, G. McDonald, J. Ting, S. K. Kenney, N. Sung, S. McKnight, and V. Sukhatme for reagents and suggestions. We especially thank T. S. Finco and G. L. Youngblood for helpful comments on the manuscript.


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