(Received for publication, June 9, 1995)
From the
Tumor necrosis factor (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.
Transcriptional activation of HIV-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-
B and
Sp1, have been studied intensively. NF-
B 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-
B
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.
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-,
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.
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.
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.
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.
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 to G
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.