(Received for publication, November 9, 1994; and in revised form, January 5, 1995)
From the
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.
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 phase of the cell cycle(3) . Loss of p53
function results in genome instability(4, 5) and
eliminates growth arrest at the G
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)()(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.
Figure 1:
The
amino terminus of p53 possesses a target site-dependent transcriptional
modulator. A, transactivation of reporter constructs
1PRECAT (lanes1-6) and
1PRE
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
CAT (lanes1-6) and 3PRE
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.
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) because it is well characterized. Fig. 1A shows that p53 and p53-VP did not distinguish PRE
from
PRE
(compare lanes1 and 3 to lanes7 and 9). Surprisingly, both deletion
mutants, p53ND50-VP and p53ND100-VP, failed to activate transcription
via PRE
(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 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
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
. 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
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
but
is indispensable for its transactivation via PRE
. 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. 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
. 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
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, 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
. 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.
Figure 3:
The transcriptional modulators have no
effect on the DNA binding activity of p53 core domain. Labeled probes
of PRE (lanes2, 4, 6, 8, 10) and PRE
(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.
As shown in Fig. 4A, fusion protein
GAL4-p53 barely stimulated transcription from the reporter
pGE1BCAT. 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 pGE1BCAT 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
pGE1BCAT.
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.
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.
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 with the purified core domain.
Alternatively, PRE 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
. Transcriptional stimulation by
PRE
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
. The finding that
PRE
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
, mainly exhibits a
discernible palindromic motif of 20 base pairs(12) . The other
group, typified by PRE
, 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 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.