©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transcriptional Regulation by p53
FUNCTIONAL INTERACTIONS AMONG MULTIPLE REGULATORY DOMAINS (*)

(Received for publication, November 9, 1994; and in revised form, January 5, 1995)

Yu-Shen Hsu (1) Fen-Mei Tang (1) Wei-Li Liu (2) Jing-Yuan Chuang (1) (3) Ming-Yang Lai (2) Young-Sun Lin (1)(§)

From the  (1)Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan and the (2)Graduate Institute of Clinical Medicine and the (3)Department of Pathology, College of Medicine, National Taiwan University, Taipei 11529, Taiwan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The tumor suppressor p53 protein possesses activities typical of eukaryotic transcriptional activators; p53 binds to specific DNA sequences and stimulates transcription of the target genes. By a series of deletion and domain-swapping studies, we report here that (i) p53 has two auxiliary domains, which have little effect on the DNA binding activity of its core domain but are capable of modulating its transactivation activity in a target site-dependent manner; (ii) p53 contains two cell-specific transcriptional inhibitory domains, I1 and I2, which are active in Saos-2 and HeLa cells but not in HepG2 and Hep3B cells; and (iii) I1 inhibits the activity of several structurally different activating regions. These results demonstrate that the apparent transcriptional activity of p53 is determined by collaborations among its regulatory domains, its target sites, and the cellular environment.


INTRODUCTION

The tumor suppressor p53 protein negatively regulates cell growth (1) . Transformation of primary cells by oncogenes is inhibited in the presence of wild type p53 protein(1) . Reintroducing the wild type p53 gene into transformed cells blocks cell proliferation (2) and causes these cells to accumulate in the late G(1) phase of the cell cycle(3) . Loss of p53 function results in genome instability(4, 5) and eliminates growth arrest at the G(1) phase in response to DNA damage(6) , indicating that p53 functions as a checkpoint that is important in arresting the cell cycle progression under inadequate or detrimental growth conditions.

DNA tumor viruses and many human cancers employ different mechanisms to overcome the negative effects of p53 on cell proliferation (for a review, see (7) ). Whereas the viruses encode oncoproteins that inactivate the braking effects of p53 on the cell cycle, the natural forms of cancers almost universally contain mutations in the p53 gene. Mostly, they are single missense point mutations located within the conserved regions 2-5 of p53 protein. Furthermore, many of these mutants act in a trans-dominant fashion to promote neoplastic processes by forming hetero-oligomers with wild type p53, therefore abrogating its function(8) .

Although the molecular mechanisms responsible for the biological activities of p53 are still the subject of intensive study, several lines of evidence indicate that p53 functions in the regulation of transcription. For example, p53 binds to specific DNA sequences termed p53-responsive elements (or PREs)(^1)(6, 9, 10, 11, 12, 13, 14) and stimulates transcription of the target genes in vivo and in vitro(9, 15, 16) . p53 mutants that fail to suppress cell growth also cannot perform specific DNA binding and transactivation(15) . Paradoxically, p53 also represses transcription of many viral and cellular genes that apparently do not have PREs (17, 18, 19) . How p53 down-regulates gene transcription is not well understood, although a number of studies have shown that p53 interacts with the TATA binding protein, and, depending on the promoter context (20) , may somehow repress transcription.

Like typical eukaryotic transcription factors, p53 is endowed with several distinct functional domains, including those for transactivation, for DNA binding, and for the responsiveness to regulatory signals (for a review, see (21) ). An acidic activating region is located at codons 20-42. The sequence-specific DNA binding domain (i.e. the so-called core domain) has been roughly mapped to the central portion, approximately corresponding to residues 100-300. In addition, oligomerization and nuclear localization activities have been assigned to the carboxyl terminus. Nonetheless, a systematic study of the structure and functions of p53 is still missing, as is a description of the collective interactions among its functional domains, which result in modulating the transcriptional activity of p53.

Deletion and domain-swapping approaches have been routinely and successfully employed to define various functional domains of proteins that have versatile biochemical activities. This testifies, in general, to the modular nature of proteins (for a review, see (22) ). Using these approaches to perform a systematic genetic analysis of p53, we report here the identification and characterization of several new transcriptional regulatory activities of p53. The functional communications among these domains as well as superimposed effects of the DNA target sites and the cellular environment profoundly influence how p53 performs its transcriptional activity.


MATERIALS AND METHODS

Plasmid Constructions

Plasmids pE1BCAT, pG(5)E1BCAT, pSG424, pSGVP, and pSGE1A have been previously described(23) . Plasmids p1PRE(c)CAT and p3PRE(c)CAT were cloned by inserting one and three copies, respectively, of a consensus p53 binding site oligomer (PRE(c)) 5`-AGCTAGGCATGTCTAGACATGCCT-3` (12) into the HindIII site of pE1BCAT. p1PRE(r)CAT and p3PRE(r)CAT were made similarly by replacing the above oligomer with the one (PRE(r)) that is found in a region upstream of the transcription start site for the human ribosomal gene cluster (10) and has the sequence 5`-AGCTTGCCTGGACTTGCCTGGCCTTGCCTTTTC-3`. For expression of p53 derivatives and GAL4-p53 fusion proteins in mammalian cells, polymerase chain reaction-generated p53 DNA fragments were cloned between the HindIII and BamHI sites of pCEP4 (Invitrogen) and pSG424, respectively. Plasmid pSGC67 was constructed by ligating a DNA fragment encoding the carboxyl-terminal 67 amino acids of p53 between the KpnI and XbaI sites of pSG424, resulting in an in-frame fusion between the GAL4 and p53 modules. Plasmids pSGVPC67, pSGE1AC67, pSGProC67, and pSGGlnC67 were cloned by inserting DNA fragments encoding the activating regions of HSV VP16 (residues 413-490)(24) , adenovirus E1A (residues 121-222)(23) , NF1/CTF (residues 401-480)(25) , and Sp1 (residues 339-500)(26) , respectively, in-frame between the EcoRI and BamHI sites of pSGC67. Plasmids pSGPro and pSGGln were constructed by deleting the 67 amino acids of p53 from plasmids pSGProC67 and pSGGlnC67. Finally, for the expression of p53 protein in bacteria, pHisp53 was constructed by inserting the p53 protein coding sequence between the NdeI and BamHI sites of plasmid 8His-pET11d(27) .

Transfection and CAT Assay

Saos-2, HepG2, Hep3B, and HeLa cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Calcium phosphate-mediated DNA transfection was performed as previously described(28) , except that 5 µg each of the CAT reporter and activator plasmids were used and 2 µg of a LacZ reporter plasmid were included to monitor the transfection efficiency. CAT activity was measured and quantified according to Carey et al.(29) .

Nuclear Extracts and DNA Binding Assays

Nuclear extracts were prepared from Saos-2 and HepG2 cells transiently transfected with the mammalian expression plasmids of interest. Cells from a 100-mm diameter plate were lysed by briefly mixing them in 500 µl of ice-cold buffer A (10 mM Hepes (pH 8.0), 1.5 mM MgCl(2), 10 mM KCl, 0.5 mM dithiothreitol, and 0.5% Nonidet P-40). Nuclei were pelleted at 3,000 times g for 3 min and then resuspended and extracted on ice in 100 µl of buffer C (20 mM Hepes (pH 8.0), 25% glycerol, 0.42 mM NaCl, 1.5 mM MgCl(2), 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol) for 30 min. Nuclear extracts were then collected by centrifugation at 15,000 times g for 10 min. For bandshift assays, the probe was the P end-labeled HindIII-EcoRI fragment of pMH100(23) . 1 µg of nuclear proteins was used per reaction. All other methods concerning bandshift assays have been previously described(30) . Assays for the DNA binding activity of p53 derivatives were performed as described(10) , except that probes were the P end-labeled XhoI-SalI fragment of p3PRE(c)CAT and p3PRE(r)CAT and that 400 µg of proteins of nuclear extracts from transiently transfected cells and 10 µl of the polyclonal anti-p53 anti-serum described below were used.

Nuclear-Cytoplasmic Fractionation

Nuclear/cytoplasmic fractionation was done as follows. Saos-2 cells from a transiently transfected 100-mm diameter plate were lysed on ice in 100 µl of hypotonic buffer (25 mM Tris-HCl (pH 7.4), 1 mM MgCl(2), 5 mM KCl, 1 mM phenylmethylsulfonyl fluoride, and a mixture of protease inhibitors containing 1 µg/ml each of leupeptin, pepstatin, antipain, and chymostatin) plus 0.5% Nonidet P-40 by pipetting up and down a few times. 2 M sucrose was immediately added to a final concentration of 0.25 M, and nuclei were pelleted at 1,000 times g in a microcentrifuge for 2 min at 4 °C. The supernatant (i.e. the cytosol fraction) was further spun at 12,000 times g for 2 min to remove contaminating nuclei. Nuclei were washed three times in hypotonic buffer plus 0.5% Nonidet P-40 and then lysed in 100 µl of hypotonic buffer plus 1% SDS. After addition of an equal volume of 2times protein sample buffer, the nuclear fraction was sonicated to reduce its viscosity. These nuclear fractions were used for the immunoblotting studies shown in Fig. 1C and Fig. 2B.


Figure 1: The amino terminus of p53 possesses a target site-dependent transcriptional modulator. A, transactivation of reporter constructs 1PRE(c)CAT (lanes1-6) and 1PRE(r)CAT (lanes7-12) by the ND mutants of p53 in Saos-2 cells. Experiments were repeated three times. Plasmid pCH110 (Pharmacia Biotech Inc.), which contains a functional LacZ gene, was used as an internal control to monitor transfection efficiency. An autoradiogram of a typical experiment is shown. Diagrams of the structure of the activators are shown below the autoradiogram. The activator and relative CAT activity (RCA) are indicated above each track of the autoradiogram. B, experiments were performed as in A, except that reporter plasmids are 3PRE(c)CAT (lanes1-6) and 3PRE(r)CAT (lanes7-12). The activators are the same as in A, and therefore their diagrams are omitted. C, protein levels of p53 derivatives. Proteins of the nuclear fraction of Saos-2 cells transiently transfected with the vector (lane1) or p53 derivatives (lanes2-5) were fractionated on a 10% SDS-polyacrylamide gel electrophoresis gel. p53 derivatives were detected by immunoblotting as described under ``Materials and Methods.'' The p53 derivative is indicated above each track of the immunoblot. The positions of molecular mass markers in kilodaltons are indicated on the left.




Figure 2: The carboxyl terminus of p53 also possesses a target site-dependent transcriptional modulator. A, the experiments were performed as described in Fig. 1A, except that activators are the CD mutants of p53 shown below the autoradiogram. B, protein levels of p53 derivatives. As in Fig. 1C, except that the CD mutants of p53 were used.



Antibody Preparation and Western Immunoblotting Analysis

Anti-GAL4 antibody was purchased from UBI (New York). The His-tagged p53 protein was expressed in and purified from bacteria JM109DE3 (Promega) by the published procedure(27) . The purified protein was used to raise polyclonal antibodies against human p53 protein in rabbits according to standard protocols. For immunoblotting, approximately 50 µg of nuclear proteins were separated on a 10% SDS-polyacrylamide gel. All other methods concerning Immunoprecipitation and Western immunoblotting assays were as previously described(23, 31) .


RESULTS

p53 Contains Two Auxiliary Regions of Target Site-dependent Transcriptional Modulation

Partly because p53 binds to several dissimilar DNA sequences(6, 9, 10, 11, 12, 13, 14) and partly because its DNA binding domain, located approximately between amino acids 100 and 300, is structurally distinct from other known DNA binding domains(32) , we asked whether any regions outside the central DNA binding domain influenced the transactivation activity of p53. Since the p53 activation domain has been mapped to its amino-terminal end, to facilitate studies on amino-terminal deletion mutants (or ND mutants) we decided to fuse the VP16 activation domain of herpes simplex virus to the carboxyl-terminal end of p53. The VP16 fusion apparently caused no detriment to the transactivation activity of p53 (Fig. 1A, compare lanes1 and 3). Mutants p53ND50-VP and p53ND100-VP, which have a deletion of 50 and 100 amino acids, respectively, from the amino-terminal end of the fusion protein, showed a 3-4-fold reduction in activity toward a reporter gene containing one copy of the consensus PRE (or PRE(c)) in the promoter. The reduction in activity was probably due to a difference in the level of protein being made (Fig. 1C, lanes2-5) and in the number of activation domains (see below). Interestingly, although the protein level of p53ND100-VP was about one-third of that of p53ND50-VP, both stimulated transcription almost equally well. It suggested that even the low level of p53ND100-VP being expressed was probably sufficient to saturate all of the PRE(c) sites. A further deletion of 25 amino acids completely eliminated the transactivation activity of the fusion protein, although the protein was made to a level about half of that of p53-VP (Fig. 1C, compare lanes2 and 5). Thus, our data demonstrate that the amino-terminal boundary of p53 required for specific DNA binding toward the PRE(c) is located between amino acids 100 and 125.

The yeast transcriptional activator HAP1 binds, as p53 does, to several dissimilar sequences ( (33) and references therein). Deletions of HAP1 have distinct effects on its transactivation activity via different target sites(33) . This prompted us to look into the influence of the amino-terminal deletions of p53 on other PREs. We chose the one found in a region upstream of the transcription start site of the human ribosomal gene cluster (PRE(r)) because it is well characterized. Fig. 1A shows that p53 and p53-VP did not distinguish PRE(r) from PRE(c) (compare lanes1 and 3 to lanes7 and 9). Surprisingly, both deletion mutants, p53ND50-VP and p53ND100-VP, failed to activate transcription via PRE(r) (Fig. 1A, lanes10 and 11).

The experiments above used reporters containing one copy of PRE. Since the amino-terminal end of p53 possesses a transactivation activity, the abrupt drop in activity toward PRE(r) observed from p53-VP to p53ND50-VP or to p53ND100-VP might merely reflect the difference in the number of activation domains (two in p53-VP but one in each of mutants p53ND50-VP and p53ND100-VP). If this were correct, then an increase in the number of p53 binding sites would compensate the loss via the mechanism of transcriptional synergism(29, 34) . As shown in Fig. 1B, whereas the activity of p53ND50-VP and p53ND100-VP approached that of the parental p53-VP16 when the number of PRE(c) increased from one to three, only a marginal increase in their activity was observed when the same assays were repeated with three copies of PRE(r). In other words, the data collected from the above study of transcription synergism suggest that the low transcriptional activity of the two ND mutants toward PRE(r) is unlikely due to a difference in the number or strength of their activation domains. These data clearly demonstrated that the p53 amino-terminal region containing the first 100 residues is not required for the core domain to activate transcription via PRE(c) but is indispensable for its transactivation via PRE(r). Thus, our experiments uncovered a novel function of the amino-terminal end of p53: a target site-dependent transcriptional modulator.

In a parallel study, the transactivation activity of a series of carboxyl-terminal deletion mutants (or CD mutants) of p53 was measured. As can be seen in Fig. 2B, mutants containing carboxyl-terminal deletions of up to 95 amino acids were expressed to a level about double that of wild type p53. Fig. 2A shows that wild type p53 and these mutants exhibited similar transcriptional activity toward PRE(c). We noted a discrepancy between the levels of proteins being made and their transcriptional activity. For instance, p53 and p53CD55 stimulated transcription approximately equally, though the latter was about twice as high in protein concentration as the former. We have no reasonable explanation for this, except for mutant p53CD30, whose high activity was probably due to the deletion of a repression domain ( (35) and see below). Note that p53CD55, p53CD75, and p53CD95 do not contain the oligomerization domain but still stimulated transcription quite well, indicating that the oligomerization domain is dispensable for the transactivation activity of p53 via PRE(c). p53CD115 was completely inactive, although the protein was expressed and localized to the nucleus (Fig. 2B and data not shown). By densitometer, the level of p53CD115 expressed was about 73% that of wild type p53, a reduction too small to account for the inaction of p53CD115. The experiment done with p53CD115 thus suggested that the carboxyl-terminal boundary of p53 required for specific DNA binding toward PRE(c) was located between amino acids 278 and 298. In addition, since activator p53CD95 lacks all three previously identified nuclear localization signals (NLSI, residues 316-322; NLSII, residues 369-375; and NLSIII, residues 379-384) ( (21) and references therein) but was still able to stimulate transcription, this implied that all three NLSs are not required for p53 derivatives to be localized to the nucleus.

When the same CD mutants were assayed with a reporter driven by PRE(r), a different result was obtained. Activators p53 and p53CD30 behaved similarly, regardless of which PRE they were assayed with (Fig. 2A, compare lanes2 and 3 with lanes9 and 10). Interestingly, p53CD55, p53CD75, and p53CD95 exhibited hardly any significant transactivation activity toward PRE(r). Thus, in analogy to its amino terminus, the p53 carboxyl-terminal region between residues 298 and 363 also contained a target site-dependent transcriptional modulator.

The Transcriptional Modulators Have No Effect on the DNA Binding Activity of p53 Core Domain

We imagined that the transcriptional modulators may influence the transactivation activity of p53 derivatives by modulating the DNA binding activity of the core domain. To test this idea, we compared the PRE binding activity of mutants p53CD30 and p53-VP with that of mutants p53CD95 and p53ND50-VP, respectively, because of their obvious difference in transactivation activity toward PRE(r). To quantitatively reflect the affinity of each protein to the PRE sites, the amount of nuclear extract was titrated so that the incubation was carried out within the linear range of the protein-DNA interaction. As shown in Fig. 3, all four p53 derivatives seemed to bind PRE(c) equally well (lanes4, 6, 8, and 10). Unexpectedly, they also exhibited similar activity in binding to PRE(r) (lanes3, 5, 7, and 9). The binding of p53 derivatives to PREs was specific as demonstrated by the result that a nuclear extract of Saos-2 cells transfected with vector alone showed only residual binding (lanes1 and 2). Thus, our results did not support the idea that the modulator regions may augment transcription by increasing the DNA binding activity of p53 core domain toward PRE(r).


Figure 3: The transcriptional modulators have no effect on the DNA binding activity of p53 core domain. Labeled probes of PRE(c) (lanes2, 4, 6, 8, 10) and PRE(r) (lanes1, 3, 5, 7, 9) were incubated with a nuclear extract of Saos-2 cells transfected with the activator plasmid indicated above each track of the autoradiogram. The DNA-protein complexes were precipitated with an anti-p53 antibody. The binding reaction was performed under an equilibrium condition (that is, an increase in the amount of nuclear extract proportionally brought down more probes). DNAs were purified and separated by electrophoresis on a denaturing 5% polyacrylamide gel. The positions of probes are indicated on the left. LaneM, P-labeled DNA size markers of MspI-digested pBR322 DNA.



p53 Has Two Cell-specific Transcriptional Inhibitory Domains

The existence of an inhibition domain in p53 protein has been proposed(35) . Our results (Fig. 2A) demonstrated that removal of 30 amino acids from the carboxyl terminus of p53 caused an increase in its transactivation activity. Partly because of the rapid degradation of p53 (Fig. 1C and Fig. 2B) and partly to search for other p53 inhibition domain(s), which may overlap with the DNA binding domain and therefore cannot be characterized in the p53 background, a new assay system is needed. Fortunately, the GAL4-p53 hybrid seems to be a useful system because it has been shown to be relatively stable(36) , and the GAL4 module can provide a DNA binding activity. Furthermore, two lines of evidence indicate that there is probably little change in the configuration and activity of p53 when fused to the GAL4 DNA binding domain. First, it is known that various p53 mutants and their corresponding GAL4-p53 hybrids behave similarly in terms of transcriptional activation(36) . Second, the temperature-sensitive feature of the Val-135 mutant of p53 is maintained in the background of GAL4-p53 hybrid, regardless of which site (either a PRE or GAL4 binding site) it is assayed with(36, 37) . Thus, it should be appropriate and relevant to transfer conclusions drawn from studies of the fusion protein to p53.

As shown in Fig. 4A, fusion protein GAL4-p53 barely stimulated transcription from the reporter pG(5)E1BCAT. A deletion of the carboxyl-terminal 30 amino acids increased the transactivation activity about 40-fold (compare lanes3 and 4). Since the fusion protein binds DNA via the GAL4 module, the low transcriptional activity of GAL4-p53 was incompatible with the argument that the region containing the carboxyl-terminal 30 amino acids inhibits transcription by reducing the DNA binding activity of p53(35) . Rather, our results suggested that a repression domain, I1, exists in the carboxyl terminus of p53. More support for this conclusion is provided below.


Figure 4: p53 protein has two cell-specific transcriptional inhibitory domains. A, transactivation of reporter constructs pG(5)E1BCAT by derivatives of the GAL4-p53 fusion protein in Saos-2 cells (otherwise as in Fig. 1A). B, as described in the legend to part A, except that experiments were performed in HepG2 cells, and only activators B, C, J, and K were assayed. C, immunoprecipitation of GAL4-p53 derivatives expressed in Saos-2 (lanes2-5) and HepG2 (lanes7-10). Precipitated proteins were separated by 10% SDS-polyacrylamide gel electrophoresis. The positions of GAL4-p53 derivatives are indicated on the right of the autoradiogram. The positions of proteins coprecipitated with GAL4-p53 from Saos-2 but not HepG2 cells are indicated on the left. The numbers on the left of the autoradiogram indicate molecular sizes in kilodaltons. D, as described in the legend to B, except that Hep3B (leftpanel) and HeLa (rightpanel) cells were used instead.



Derivatives of the fusion protein with a deletion of up to carboxyl-terminal 95 amino acids showed only a minor reduction in their transactivation activity (compare lanes4-7 in Fig. 4A). Another transcriptional inhibitory domain, however, was revealed when the carboxyl-terminal truncation proceeded beyond 95 amino acids. This second inhibitory domain, or I2, presumably resides quite close to the amino-terminal end of p53, because GAL4-p53CD223, a fusion protein with only the amino-terminal 170 amino acids of p53 left, still failed to enhance transcription. The carboxyl-terminal boundary of domain I2 was localized to between amino acids 145 and 170 of p53 since a further deletion of 25 amino acids produced a strong activator, GAL4-p53CD248 (compare lanes11 and 12 in Fig. 4A).

All of the above experiments were done with osteosarcoma Saos-2 cells. However, it has been demonstrated that transcriptional inhibitory domains can function in a cell-specific manner. For instance, the domain of c-jun is a cell-specific transcriptional inhibitor (38) . We therefore measured the activity of I1 and I2 in another cell line. Fig. 4B shows that, in contrast to the results obtained from Saos-2 cells, fusion proteins GAL4-p53 and GAL4-p53CD223 were able to activate transcription to a high level in HepG2 hepatoma cells (see lanes2 and 4). We therefore concluded that domains I1 and I2 did not act as transcriptional inhibitors in HepG2 cells. As a control, we performed an immunoprecipitation study to show that GAL4-p53, GAL4-p53CD30, GAL4-p53CD223, and GAL4-p53CD248 were all expressed and, as expected, quite stable in both Saos-2 and HepG2 cells (Fig. 4C), indicating that the observed cell-specific difference in the ability of GAL4-p53 and GAL4-p53CD223 to stimulate transcription was not simply a trivial result of differential protein degradation between the two cell lines. We noted that GAL4-p53 reproducibly brought down two proteins with molecular masses of about 56 and 80 kDa from Saos-2 but not from HepG2 cells. They probably bind to the carboxyl end of p53 because a 30-amino acid deletion from the carboxyl terminus of p53 completely eliminated the interaction (Fig. 4C, compare lanes2 and 3).

To further investigate their cell-type specificity, we tested the repression activity of I1 and I2 in two other cell lines. As shown in Fig. 4D, I1 and I2 retained their activity in HeLa but not in Hep3B cells. These results therefore provided additional supports for the conclusion that I1 and I2 function in a cell-specific manner.

To map the boundary of I1 more precisely, we performed the experiments shown in Fig. 5. Fusion of the p53 carboxyl-terminal 67 amino acids to activator GAL4-p53CD267 greatly reduced its activity, while fusion of a smaller region containing the p53 carboxyl-terminal 46 amino acids had little effect. Immunoprecipitation analysis showed that the two proteins were expressed to a similar level (data not shown). Thus, the p53 carboxyl-terminal 67 amino acids were required and sufficient for the activity of I1. Moreover, since I1 could be transferred from its original position to a completely new environment without compromising its activity, we further concluded that I1 was a context-independent inhibitory domain. In support of this conclusion, we found that in addition to the p53 activation domain, I1 inhibited many activation domains consisting of different protein motifs (see below).


Figure 5: The carboxyl-terminal 67 amino acids are required and sufficient for the function of I1. The experiments were done as described in the legend to Fig. 1A, except that the reporter plasmid is pG(5)E1BCAT.



I1 Is a General Inhibitor

The finding that I1 functioned as a movable inhibitory domain toward the p53 activation region compelled us to ask whether it could repress other transactivation domains of different protein motifs. For this purpose, we focused on those of VP16, E1A, Sp1, and NF-1/CTF, which represent four well characterized prototypes of eukaryotic transactivators: acidic, zinc-containing, glutamine-rich, and proline-rich(23, 39) , respectively. In the absence of I1, fusion proteins of GAL4 and various activation domains all stimulated transcription to certain extents (Fig. 6A). VP16 and E1a were strong activators, while Sp1 and NF-1/CTF were weak ones. However, a large reduction in activity was observed for all four activating regions when I1 was linked in cis to them (Fig. 6A), though all the proteins were expressed to a similar level (for an example, see Fig. 6B).


Figure 6: I1 is a general inhibitor. A, as described in the legend to Fig. 5. B, bandshift assay. A P end-labeled HindIII-EcoRI fragment of pMH100 (23) was incubated in a buffer alone (lane1) or a nuclear extract from Saos-2 cells transiently transfected with the vector (lane2), with activator GAL4VP (lane3) or with activator GAL4VPC67 (lane4), followed by electrophoresis on a nondenaturing 5% polyacrylamide gel. Nuclear extracts containing approximately equal amounts of GAL4VP and GAL4VPC67 as quantified by immunoprecipitation were used for the binding reaction. The positions of the free probe and DNA-protein complex are indicated on the right.



Since the most carboxyl-terminal part of p53 can inhibit its own DNA binding activity(35) , I1, by analogy, might inhibit transcription by blocking the DNA binding activity of the GAL4 fusion protein. Alternatively, since I1 is overlapping with the p53 oligomerization domain, it might inhibit transcription by forcing the fusion proteins to form oligomers, which therefore abrogated the activity of fusion proteins by steric hindrance. Fig. 6B shows that neither case was responsible for the repression activity of I1. Indeed, both GAL4VP and GAL4VPC67 had comparable affinity toward the cognate GAL4 site, and the DNA-protein complexes exhibited similar mobility in the bandshift assay. We therefore concluded that I1 was a general inhibitor of various eukaryotic transactivating regions, and its function was probably unrelated to either the proposed DNA binding inhibition activity or the oligomerization one of the p53 carboxyl terminus.


DISCUSSION

Previous studies have shown that p53 binds to specific DNA sequences(6, 9, 10, 11, 12, 13, 14) and activates transcription both in vivo and in vitro(9, 15, 16, 40) . Although the domains responsible for the inhibition(41) , transactivation, and DNA binding activities of p53 have been roughly mapped, information regarding the interactions among them in the determination of the apparent transcriptional activity of p53 is still missing. In this report, we identify several new functional domains in p53 and demonstrate that the apparent transcriptional activity of p53 is determined by functional communications among its multiple domains as well as by superimposed effects of the DNA target sites and the cellular environment.

The Transcriptional Modulators

In conjunction with the VP16 activating region, the p53 core domain containing residues 100-298 was sufficient to activate transcription via a consensus binding site, PRE(c). However, this core domain requires two additional regions, which we refer to as transcriptional modulators, to stimulate transcription efficiently via PRE(r). We do not know how the two modulators help the core domain in the process of transactivation of a reporter driven by PRE(r). The oligomerization state of p53 is not a viable explanation. First, all the ND mutants we tested have an intact oligomerization domain, which is composed of the most carboxyl-terminal 50-60 amino acids(42, 43, 44) , but they failed to activate transcription via PRE(r). Second, although the CD mutant, p53CD30, contains an oligomerization domain that is partially truncated, it functions better than the wild type protein.

Conceivably, the minimal domain of p53 required for efficient DNA binding is target site-dependent. This possibility is not without precedent. Genetic and biochemical evidence indicates that in transcription factor Oct-1, the region responsible for specific DNA binding varies, depending on the DNA site(45) . Nevertheless, this proposition apparently contradicts the result shown in Fig. 3and that of an in vitro study by Pavletich et al.(46) , who detected specific DNA binding to PRE(r) with the purified core domain.

Alternatively, PRE(r) may function as a composite regulatory element (47) and interacts with transcription factors other than p53. Accordingly, p53 would be required but not sufficient by itself to stimulate transcription from PRE(r). Transcriptional stimulation by PRE(r) would be an outcome of the concerted interaction between p53 and other factors. This model predicts that the p53 transcriptional modulators must be somehow involved in the interaction and that p53 mutants without the modulators either cannot interact or interact in a nonproductive way with those factors and, therefore, fail to activate transcription through PRE(r). The finding that PRE(r) binds, in addition to p53, several nuclear proteins with high affinity and that the p53 amino terminus is involved in interaction with transcription factor Sp1 (48) is in agreement with this hypothesis. In light of this, it is worthwhile to note that most of the known p53 binding sites could be classified into two groups. One group, represented by PRE(c), mainly exhibits a discernible palindromic motif of 20 base pairs(12) . The other group, typified by PRE(r), usually consists of multiple copies of TGCCT repeat, frequently with an Sp1-like GC-rich region adjacent to the TGCCT repeats(9, 10) .

Furthermore, a few cancer cells express mutant p53 proteins that have only the carboxyl-terminal transcriptional modulator deleted(49) . In other words, cancers arise probably because the truncated p53 no longer functions as a transcriptional activator. In evidence, most p53-responsive genes(6, 9, 13, 50, 51, 52, 53, 54, 55, 56) contain PREs in their promoters that do not match with the consensus p53 binding sequences and therefore may become unresponsive to the truncated p53. Such observations support that at least the carboxyl-terminal transcriptional modulator indeed plays an important role in determining the apparent transcriptional activity of p53.

The Inhibitory Domains

Either as part of p53 (Fig. 2A, compare lanes9 and 10) or as part of GAL4-p53 fusion (Fig. 4A, compare lanes3 and 4), domain I1 represses the activity of the p53-activating region. Thus, the most carboxyl-terminal part of p53 not only represses the DNA binding activity of p53 (35) but is itself also a true transcriptional inhibitor. The activity of domain I1 varies; it represses much better in the context of GAL4-p53 than in that of p53 (compare Fig. 2A and Fig. 4A), which probably reflects a difference in the degree of protein degradation between them. Wild type p53 is degraded very quickly (Fig. 1C and Fig. 2B), probably resulting in a large fraction of protein without an intact domain I1. Thus, the relatively high transactivation activity detected with the wild type p53 protein (see Fig. 1, A and B, and 2A) may be largely contributed by some carboxyl-terminal truncated products. In contrast, the GAL4-p53 fusion protein is quite stable in the cell lines we worked with (for an example, see Fig. 4C), indicating that domain I1 is left intact to fully perform its inhibition function. The basis for us to propose that heterogeneous pattern of p53 derivatives probably due to a difference in the degree of protein degradation, but not of phosphorylation, is from the observation that all of the p53 carboxyl-terminal deletion mutants end with a degraded product of about 32 kDa in molecular mass (Fig. 2B). Moreover, the degradation products probably reflect the state of p53 derivatives within the cell, because neither the addition of protease inhibitors to the extraction buffers (Fig. 2B) nor boiling transfected cells directly in the Laemmli SDS-containing sample buffer (data not shown) had much effect on the profile of p53 derivatives.

The repression activity of domain I2 can be demonstrated only in the context of GAL4-p53 fusion protein, for the overlapping of I2 with the p53 DNA binding domain makes the assay on p53 impossible. The activity of I2 seems to be regulated by the region with a carboxyl-terminal boundary around amino acids 279-298 of p53 because the repression activity of I2 appeared only when this region was removed (see Fig. 4A). The underlying mechanism of the regulation is unknown. Notably, carboxyl-terminal deletions of GAL4-p53 that terminate within the conformationally sensitive DNA binding domain of p53 mostly fail to transactivate. It is thus arguable that I2 might simply reflect some structural requirement for an intact p53 core domain. The cell-specific activity of I2, however, is incompatible with this argument concerning the conformational state of the p53 fragments. Rather, a cell-specific factor(s) may be involved in the modulation of I2 activity.

Both domains I1 and I2 function in a cell-specific manner. Although we do not know the molecular mechanism responsible for this, it is not simply a consequence of differential protein degradation in one cell line versus in another one (for an example, see Fig. 4C). More possibly, a cell-specific cofactor(s) may be responsible for the observed difference. The finding of specific proteins coimmunoprecipitated with GAL4-p53 from Saos-2 but not from HepG2 cells should provide a clue for investigating the mechanisms responsible for the cell-specific negative effect of I1 on transcription.


FOOTNOTES

*
This work was supported by grants from the Academia Sinica and National Science Council of Taiwan (to Y. S. L.) and by grants from the National Science Council and Dept. of Health of Taiwan (to M. Y. L.). 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.: 886-2-7899133; Fax: 886-2-7853569.

(^1)
The abbreviations used are: PRE, p53-responsive elements; RCA, relative CAT activity; NLS, nuclear localization signals; CAT, chloramphenicol acetyltransferase; ND and CD mutants, amino- and carboxyl-terminal deletion mutants, respectively.


ACKNOWLEDGEMENTS

We acknowledge Dr. C. Shih for the human p53 clone. We thank Drs. J. Y. Chen, C. Fletcher, J. Yen, and K. King for comments.


REFERENCES

  1. Finlay, C. A., Hinds, P. W., and Levine, A. J. (1989) Cell 57, 1083-1093 [Medline] [Order article via Infotrieve]
  2. Baker, S. J., Markowitz, S., Fearon, E. R., Willson, J. K., and Vogelstein, B. (1990) Science 249, 912-915 [Medline] [Order article via Infotrieve]
  3. Michalovitz, D., Halevy, O., and Oren, M. (1990) Cell 62, 671-680 [Medline] [Order article via Infotrieve]
  4. Yin, Y., Tainsky, M. A., Bischoff, F. Z., Strong, L. C., and Wahl, G. M. (1992) Cell 70, 937-948 [Medline] [Order article via Infotrieve]
  5. Livingstone, L. R., White, A., Sprouse, J., Livanos, E., Jacks, T., and Tlsty, T. D. (1992) Cell 70, 923-935 [Medline] [Order article via Infotrieve]
  6. Kastan, M. B., Zhan, Q., El-Deiry, W. S., Carrier, F., Jacks, T., Walsh, W. V., Plunkett, B. S., Vogelstein, B., and Fornace, A. J., Jr. (1992) Cell 71, 587-597 [Medline] [Order article via Infotrieve]
  7. Prives, C. (1994) Cell. 78, 543-546 [Medline] [Order article via Infotrieve]
  8. Milner, J., and Medcalf, E. A. (1991) Cell 65, 765-774 [Medline] [Order article via Infotrieve]
  9. Zambetti, G. P., Bargonetti, J., Walker, K., Prives, C., and Levine, A. J. (1992) Genes & Dev. 6, 1143-1152
  10. Kern, S. E., Kinzler, K. W., Bruskin, A., Jarosz, D., Friedman, P., Prives, C., and Vogelstein, B. (1991) Science 252, 1708-1711 [Medline] [Order article via Infotrieve]
  11. Bargonetti, J., Friedman, P. N., Kern, S. E., Vogelstein, B., and Prives, C. (1991) Cell 65, 1083-1091 [Medline] [Order article via Infotrieve]
  12. el-Deiry, W. S., Kern, S. E., Pietenpol, J. A., Kinzler, K. W., and Vogelstein, B. (1992) Nat. Genet. 1, 45-49 [Medline] [Order article via Infotrieve]
  13. Wu, X., Bayle, J. H., Olson, D., and Levine, A. J. (1993) Genes & Dev. 7, 1126-1132
  14. Funk, W. D., Pak, D. T., Karas, R. H., Wright, W. E., and Shay, J. W. (1992) Mol. Cell. Biol. 12, 2866-2871 [Abstract]
  15. Kern, S. E., Pietenpol, J. A., Thiagalingam, S., Seymour, A., Kinzler, K. W., and Vogelstein, B. (1992) Science 256, 827-830 [Medline] [Order article via Infotrieve]
  16. Farmer, G., Bargonetti, J., Zhu, H., Friedman, P., Prywes, R., and Prives, C. (1992) Nature 358, 83-86 [CrossRef][Medline] [Order article via Infotrieve]
  17. Ginsberg, D., Mechta, F., Yaniv, M., and Oren, M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9979-9983 [Abstract]
  18. Mercer, W. E., Shields, M. T., Lin, D., Appella, E., and Ullrich, S. J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1958-1962 [Abstract]
  19. Santhanam, U., Ray, A., and Sehgal, P. B. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7605-7609 [Abstract]
  20. Mack, D. H., Vartikar, J., Pipas, J. M., and Laimins, L. A. (1993) Nature 363, 281-283 [CrossRef][Medline] [Order article via Infotrieve]
  21. Prives, C., and Manfredi, J. J. (1993) Genes & Dev. 7, 529-534
  22. Ptashne, M. (1992) A Genetic Switch , Cell Press & Blackwell Scientific Publications, Cambridge, MA
  23. Lillie, J. W., and Green, M. R. (1989) Nature 338, 39-44 [CrossRef][Medline] [Order article via Infotrieve]
  24. Triezenberg, S. J., Kingsbury, R. C., and McKnight, S. L. (1988) Genes & Dev. 2, 718-729
  25. Mermod, N., O'Neill, E. A., Kelly, T. J., and Tjian, R. (1989) Cell 58, 741-753 [Medline] [Order article via Infotrieve]
  26. Courey, A. J., Holtzman, D. A., Jackson, S. P., and Tjian, R. (1989) Cell 59, 827-836 [Medline] [Order article via Infotrieve]
  27. Hoffmann, A., and Roeder, R. G. (1991) Nucleic Acids Res. 19, 6337-6338 [Medline] [Order article via Infotrieve]
  28. Lin, Y. S., and Green, M. R. (1989) Nature 340, 656-659 [CrossRef][Medline] [Order article via Infotrieve]
  29. Carey, M., Lin, Y. S., Green, M. R., and Ptashne, M. (1990) Nature 345, 361-364 [CrossRef][Medline] [Order article via Infotrieve]
  30. Lin, Y. S., and Green, M. R. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 109-113 [Abstract]
  31. Lin, Y. S., Ha, I., Maldonado, E., Reinberg, D., and Green, M. R. (1991) Nature 353, 569-571 [CrossRef][Medline] [Order article via Infotrieve]
  32. Cho, Y., Gorina, S., Jeffrey, P. D., and Pavletich, N. P. (1994) Science 265, 346-355 [Medline] [Order article via Infotrieve]
  33. Kim, K. S., Pfeifer, K., Powell, L., and Guarente, L. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4524-4528 [Abstract]
  34. Lin, Y. S., Carey, M., Ptashne, M., and Green, M. R. (1990) Nature 345, 359-361 [CrossRef][Medline] [Order article via Infotrieve]
  35. Hupp, T. R., Meek, D. W., Midgley, C. A., and Lane, D. P. (1992) Cell 71, 875-886 [Medline] [Order article via Infotrieve]
  36. Raycroft, L., Schmidt, J. R., Yoas, K., Hao, M. M., and Lozano, G. (1991) Mol. Cell. Biol. 11, 6067-6074 [Medline] [Order article via Infotrieve]
  37. Scharer, E., and Iggo, R. (1992) Nucleic Acids Res. 20, 1539-1545 [Abstract]
  38. Baichwal, V. R., and Tjian, R. (1990) Cell 63, 815-825 [Medline] [Order article via Infotrieve]
  39. Mitchell, P. J., and Tjian, R. (1989) Science 245, 371-378 [Medline] [Order article via Infotrieve]
  40. Seto, E., Usheva, A., Zambetti, G. P., Momand, J., Horikoshi, N., Weinmann, R., Levine, A. J., and Shenk, T. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 12028-12032 [Abstract]
  41. Liu, X., Miller, C. W., Koeffler, P. H., and Berk, A. J. (1993) Mol. Cell. Biol. 13, 3291-3300 [Abstract]
  42. Shaulian, E., Zauberman, A., Ginsberg, D., and Oren, M. (1992) Mol. Cell. Biol. 12, 5581-5592 [Abstract]
  43. Sturzbecher, H. W., Brain, R., Addison, C., Rudge, K., Remm, M., Grimaldi, M., Keenan, E., and Jenkins, J. R. (1992) Oncogene 7, 1513-1523 [Medline] [Order article via Infotrieve]
  44. Iwabuchi, K., Li, B., Bartel, P., and Fields, S. (1993) Oncogene 8, 1693-1696 [Medline] [Order article via Infotrieve]
  45. Verrijzer, C. P., Kal, A. J., and van der Vliet, P. C. (1990) Genes & Dev. 4, 1964-1974
  46. Pavletich, N. P., Chambers, K. A., and Pabo, C. O. (1993) Genes & Dev. 7, 2556-2564
  47. Diamond, M. I., Miner, J. N., Yoshinaga, S. K., and Yamamoto, K. R. (1990) Science 249, 1266-1272 [Medline] [Order article via Infotrieve]
  48. Borellini, F., and Glazer, R. I. (1993) J. Biol. Chem. 268, 7923-7928 [Abstract/Free Full Text]
  49. Levine, A. J., Momand, J., and Finlay, C. A. (1991) Nature 351, 453-456 [CrossRef][Medline] [Order article via Infotrieve]
  50. Weintraub, H., Hauschka, S., and Tapscott, S. J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4570-4571 [Abstract]
  51. Barak, Y., Juven, T., Haffner, R., and Oren, M. (1993) EMBO J. 12, 461-468 [Abstract]
  52. Deffie, A., Wu, H., Reinke, V., and Lozano, G. (1993) Mol. Cell. Biol. 13, 3415-3423 [Abstract]
  53. Juven, T., Barak, Y., Zauberman, A., George, D. L., and Oren, M. (1993) Oncogene 8, 3411-3416 [Medline] [Order article via Infotrieve]
  54. Aoyama, N., Nagase, T., Sawazaki, T., Mizuguchi, G., Nakagoshi, H., Fujisawa, J. I., Yoshida, M., and Ishii, S. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5403-5407 [Abstract]
  55. Makos, M., Nelkin, B. D., Reiter, R. E., Gnarra, J. R., Brooks, J., Isaacs, W., Linehan, M., and Baylin, S. B. (1993) Cancer Res. 53, 2719-2722 [Abstract]
  56. el-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Parsons, R., Trent, J. M., Lin, D., Mercer, W. E., Kinzler, K. W., and Vogelstein, B. (1993) Cell 75, 817-825 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.