From The Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, New York 10029
Received for publication, December 22, 2000
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ABSTRACT |
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An important regulator of the proapoptotic
BAX is the tumor suppressor protein p53. Unlike the
p21 gene, in which p53-dependent transcriptional activation is mediated by a response element containing two consensus p53 half-sites, it previously was reported that activation of the BAX element by p53 requires additional
sequences. Here, it is demonstrated that the minimal BAX
response element capable of mediating p53-dependent
transcriptional activation consists of two p53 half-sites plus an
adjacent 6 base pairs (5'-GGGCGT-3'). This GC-rich region constitutes a
"GC box" capable both of binding members of the Sp family of
transcription factors, including Sp1 in vitro, and of
conferring Sp1-dependent transcriptional activation on a
minimal promoter in cells. Mutations within this GC box abrogated the
ability of p53 to activate transcription without affecting the affinity
of p53 for its binding site, demonstrating that these 6 bases are
required for p53-dependent activation. In addition, a
positive correlation was observed between the ability of p53 to
activate transcription in cells and the ability of Sp1 to bind this
response element in vitro. Mutations that inhibited Sp1
binding also blocked the ability of p53 to activate transcription
through this element. Together, these results suggest a model in which p53 requires the cooperation of Sp1 or a Sp1-like factor to mediate transcriptional activation of the human BAX promoter.
The BCL-2 family of proteins are key mediators of the
apoptotic response. One member of this family is the proapoptotic BAX. Preceding apoptosis, cytosolic BAX translocates to the
mitochondria and homodimerizes. Homodimeric BAX then is thought to
cause the release of cytochrome c (1-3) which subsequently
functions as a coactivator of Apaf-1 in the cleavage of pro-caspase-9,
initiating programmed cell death (4). BAX exists in equilibrium with
two of its homologs, BCL-2 and BCL-XL. Unlike BAX, these
two homologs exert antiapoptotic effects by heterodimerizing with BAX
in the mitochondria, blocking its ability to release cytochrome
c (5, 6). Thus, an important determinant of the apoptotic
response of a cell is the balance between the levels of BAX and
BCL-2/BCL-XL. In this regard, regulation of the level of
expression of BAX protein is key.
An important regulator of BAX gene expression is the tumor
suppressor protein p53 (7, 8). The p53 protein has been implicated in
several growth-related pathways, including apoptosis and cell cycle
arrest (9, 10). The ability of p53 to function as a sequence-specific
DNA-binding protein appears to be central to its role as a tumor
suppressor (11, 12). At its amino terminus, the protein contains a
potent transcriptional activation domain (13) that is linked to a
central core domain that mediates sequence-specific DNA binding
(14-16). Both of these domains have been shown to be important for
p53-mediated growth suppression (17).
A DNA consensus sequence through which p53 binds and activates
transcription has been identified. This sequence consists of two
palindromic decamers of 5'-RRRCWWGYYY-3' (where R is a purine; Y is a
pyrimidine; and W is an adenine or thymine) separated by 0-13 base
pairs, forming four repeats of the pentamer 5'-RRRCW-3' alternating
between the top and bottom strands of the DNA duplex (18, 19). Through
sequences similar to this consensus, p53 has been shown to activate the
transcription of many genes, including BAX, p21,
mdm2, gadd45, IGF-BP3, and
cyclin G (8, 20-26). When compared with alternate p53
targets, studies demonstrate that the BAX gene is
differentially regulated by wild-type p53 in a cell type-specific
manner (7, 27, 28). In the mouse, p53-dependent regulation
of BAX expression following ionizing radiation is seen in
the prostate, thymus, spleen, small intestine, and lung, as well as
sympathetic, Purkinje, and olfactory cortical neurons. In the kidney,
heart, liver, and brain, however, no p53-dependent regulation of BAX is observed (7, 27). Furthermore, the
myeloid leukemia ML-1, Burkitt's lymphoma WMN and AG876, and
lymphoblastoid NL2 and FWL cell lines induce BAX following
ionizing radiation, whereas the fibroblast AG1522 and WI38, colorectal
carcinoma RKO, and osteosarcoma U2-OS cell lines fail to do so (28). In
addition, several tumor-derived p53 mutants have been identified that
are capable of activating transcription through the promoter of the p21 gene but not through the BAX promoter
(29-32). This correlates with an inability of these mutants to trigger
apoptosis (29, 31, 32), suggesting that a failure in the ability of p53
to transactivate the BAX gene may play an important role in
tumor formation and progression. Supporting this, Yin et al.
(33) demonstrated that BAX is an obligatory downstream effector for the
p53-mediated apoptosis that attenuates choroid plexus tumor growth in
the TgT121 mouse model. Thus, a complete understanding of the
transcriptional regulation of the BAX promoter by p53 may yield important information relevant to our understanding of tumorigenesis.
Here is presented a detailed analysis of the p53 response element
located in the promoter of the human BAX gene. The minimal BAX response element capable of mediating
p53-dependent transcriptional activation is found to
consist of two p53 half-sites plus an adjacent 6 base pairs
(5'-GGGCGT-3') that demonstrate sequence-specific binding to the
transcription factor Sp1. Mutational analysis of this "GC box"
shows it to be required for p53-dependent activation, and a
positive correlation between the ability of p53 to activate transcription in cells and the ability of Sp1 to bind this response element in vitro is observed. These results are consistent
with a model in which p53 requires the cooperation of Sp1 or a Sp1-like factor to mediate transcriptional activation of the human
BAX gene. This presents the intriguing possibility that
regulation of this cofactor may represent a novel basis for the cell
type-specific control of the proapoptotic BAX by wild-type p53.
Cells--
The osteosarcoma Saos-2 cell line was maintained in a
humidified tissue culture incubator at 37 °C with 5%
CO2. Cells were grown in Dulbecco's modified Eagle's
medium, containing 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Drosophila SL2 cells were cultured at 25 °C in
Schneider's Drosophila medium, containing 10%
heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin.
Oligonucleotides--
For use in electrophoretic mobility shift
assays and for subsequent cloning into luciferase reporter plasmids,
complementary single-stranded oligonucleotides were annealed to produce
double-stranded oligonucleotides with the following indicated
sequences: BAX Plasmids--
The following synthetic double-stranded
oligonucleotides were digested with KpnI and NheI
and cloned into pGL3-E1bTATA (34), which also had been double-digested
with KpnI and NheI to produce pTATA vectors with
corresponding names: BAX Transfections--
Saos-2 cells were transfected using
LipofectAMINE Plus Reagent (Life Technologies, Inc.). 2 × 105 cells were seeded into 35-mm plates. Cells were
transfected 24 h later according to the manufacturer's
instructions. Cellular lysates were prepared 24 h
post-transfection, and total protein concentration was determined by
protein assay (Bio-Rad), and luciferase assays were quantitated using a
commercially available kit (Promega) and a TD-20e Luminometer (Turner).
Drosophila SL2 cells were transfected using Cellfectin (Life
Technologies, Inc.). 60-mm dishes were seeded with 2 × 106 cells in Schneider's Drosophila media
containing 10% heat-inactivated fetal bovine serum but no penicillin
or streptomycin. The DNA to be transfected was added to 500 µl of
serum-free media containing 8 µl of Cellfectin reagent, mixed gently,
and incubated at room temperature for 20 min. This mixture then was
added directly to the cells. 48 h post-transfection cells were
lysed by sonication (6 × 20 s pulse). Total protein and
luciferase activity was determined as above.
HeLa Cell Nuclear Extraction--
Unless otherwise stated, all
procedures were conducted at 4 °C. HeLa S3 cells were obtained as a
packed cell pellet from the National Cell Culture Center (Minneapolis,
MN). Cell pellets were resuspended in 5 volumes of Buffer A (10 mM HEPES, pH 7.6, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT) and incubated on ice for 10 min. Cells then were centrifuged at 500 × g for 12 min. The supernatant was removed, and the pellet was resuspended in two
packed cell volumes of Buffer A. Cells were homogenized 10 times in a
Dounce homogenizer with pestle A (tight). The resulting solution was centrifuged at 430 × g for 10 min to pellet the
nuclei. The supernatant was decanted, and the pellet was recentrifuged
at 24,000 × g for 20 min. The supernatant again was
removed. The pellet was resuspended in 3 ml of Buffer C (20 mM HEPES, pH 7.6, 25% glycerol, 420 mM NaCl,
1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT) per 109 cells. The solution was
homogenized 10 times with pestle B (loose). The resulting solution was
transferred to a beaker and stirred for 30 min on ice. The solution
then was centrifuged at 24,000 × g for 30 min. The
resulting nuclear extract was dialyzed against Buffer D (20 mM HEPES, pH 7.6, 20% glycerol, 0.1 M
KCl, 0.2 mM EDTA, 1.5 mM MgCl2, 0.5 mM DTT) for 5 h. The extract was clarified by
centrifugation at 24,000 × g for 20 min. Nuclear
extracts were aliquoted, frozen in a dry ice/ethanol bath, and stored
at Electrophoretic Mobility Shift Assay--
Production of
baculovirus-infected Sf9 cell extracts and purification of
recombinant human p53 protein were done as described previously (34).
Purified p53 protein, extract from Sf9 cells expressing
recombinant human Sp1 protein, or HeLa cell nuclear extract was
incubated with 3 ng of radiolabeled double-stranded oligonucleotide and
antibody (Sp1 PEP-2X, p300 N-15X, and CBP 451X, Santa Cruz
Biotechnology), where appropriate, in a total volume of 30 µl of DNA
binding buffer (20 mM HEPES, pH 7.5, 83 mM
NaCl, 0.1 mM EDTA, 12% glycerol, 2 mM
MgCl2, 2 mM spermidine, 0.7 mM DTT,
and 17 µg/ml poly(dI-dC)) for 20 min at room temperature. Samples
were loaded on a native 4% acrylamide gel in 0.5× TBE and
electrophoresed at 4 °C at 225 V for 2 h. The gel was dried and
exposed to Kodak XAR film using an intensifying screen at All Three Potential p53 Half-sites Are Required for the
p53-dependent Transcriptional Activation of the Human BAX
Promoter--
Previously it was demonstrated that in isolation the p53
response element from the human BAX promoter required
sequences from three adjacent half-sites to confer
p53-dependent transcriptional activation on a minimal
promoter (37). To confirm the requirement of all three half-sites in
the context of the BAX promoter, luciferase reporter
plasmids with various deletions in the BAX promoter, both in
and around the p53 response element, were cotransfected with either
pCMV or a wild-type p53 expression vector into the p53-negative
osteosarcoma Saos-2 cell line (Fig. 1).
The previously characterized p53 response element of the BAX
promoter is contained within the sequence from
The region from The First Two Potential p53 Half-sites Constitute a Bona Fide p53
Response Element--
Each of the three potential p53 half-sites
located in the BAX promoter from Sp1 Binds with Sequence Specificity to and Activates Transcription
through the p53 Response Element from the Human BAX Promoter--
We
previously reported the identification of a nuclear factor, termed
Binder of BAX 1 (BoB1), that interacts with sequence specificity with
the same region of the human BAX promoter that is required
for p53-dependent transcriptional activation (37). These
previous studies demonstrated that this factor binds to sequences
within the region of
To delineate further the sequences important for Sp1 binding,
oligonucleotides were synthesized that replaced portions of the
BAX sequence with corresponding sequence from the
p21 5' p53 response element. The sequence from
To determine whether or not Sp1 can interact with this element in
cells, a pTATA luciferase reporter plasmid containing The Ability of Sp1 to Bind the p53 Response Element of the BAX
Promoter in Vitro Correlates with the Ability of p53 to Activate
Transcription through This Element in Cells--
To explore the
significance of the Sp1-binding site to the ability of p53 to activate
transcription through the BAX promoter, nucleotide
substitutions were identified that differentially affected the ability
of p53 to activate transcription through its response element in the
BAX promoter (
Both of these mutant sequences were assayed for their ability to bind
purified p53 in an EMSA. An oligonucleotide corresponding to
The results with the GG
To confirm that the bases from The data presented in this report demonstrate that the minimum p53
response element in the BAX promoter consists of the
sequence from Previous studies have suggested a connection between p53 and Sp1. The
two proteins physically interact under certain circumstances (40-42),
and, transcriptionally, p53 and Sp1 have been shown to function in a
cooperative manner in some settings and an antagonistic manner in
others (41, 43, 44). In addition to p53, Sp1 has been found to
synergize with other transcription factors, including YY1 and SREBP
(45-47). Studies with the Sp family of transcription factors, however,
are complicated by the fact that there are at least 16 mammalian
members of this family. Due to marked conservation in the DNA-binding
domain, many of these family members have similar if not identical
in vitro DNA binding characteristics (48, 49). Originally,
this led to the misclassification of many GC boxes solely as
Sp1-binding sites because of the ubiquitous nature of Sp1 and the fact
that it was the first family member cloned. Given this, the possibility
exists that the true in vivo cofactor required for the
p53-dependent transactivation of the BAX
promoter is an Sp1-related family member that is obscured in in
vitro assays by the sheer abundance of Sp1 in nuclear extracts
from tissue culture cells. Consistent with this, antibodies used in a
supershift EMSA identified other Sp family members as minor components
of the Sp1-DNA complex.2
Furthermore, cotransfection assays in the Sp1-deficient
Drosophila SL2 cell line failed to demonstrate cooperation
between Sp1 and p53 in transcriptionally activating the p53 response
element of the BAX promoter.2 The
Drosophila assays, however, are difficult to interpret as the ability of p53 alone to activate transcription through a control plasmid was significantly impaired in the SL2 cell line. Complicating interpretation of the results in the Drosophila system is
the recent identification of a Drosophila p53 homolog (50,
51) that may affect the ability of transiently expressed human p53 to
function properly in this system.
Regardless of whether the cofactor required for the
p53-dependent transactivation of the BAX
promoter is Sp1 or a related family member, the requirement of this
cooperating protein suggests a model for the observed cell type- and
tumor type-specific regulation of the BAX gene by wild-type
p53 (Fig. 9). In this model, cells that
are permissive to p53-dependent up-regulation of the
BAX gene express both p53 and the cofactor, and these
proteins function together to activate transcriptionally the gene. In
those cells that fail to show p53-dependent BAX
expression, one can propose three possible mechanisms to explain the
apparent failure of wild-type p53 to activate the BAX gene
(Fig. 9). First, the required cofactor may be absent, either due to
mutation or due to cell type-specific limitations on its expression.
Second, this factor may be inactivated by post-translational
modification. Finally, another factor that cannot cooperate with p53
may compete with the cofactor for binding to its site in the
BAX promoter. Data with the Sp family of transcription factors support each of these possibilities. Although several of the Sp
family members, like Sp1, are ubiquitously expressed, other members of
the family display high degrees of tissue specificity (48, 49). Even
the ubiquitously expressed family members fluctuate in levels under
particular cellular conditions (52-55). Sp1 mRNA, for example,
varies up to 100-fold depending on the cell type and developmental
stage of the mouse (56). Consistent with a model of post-translational
modification, certain Sp family members, including Sp1 and EKLF, are
phosphorylated, glycosylated, and acetylated (57-59). Finally, given
the high level of conservation in the DNA-binding domain of the Sp
family of transcription factors, it is not surprising that DNA binding
competition can be observed between various members of this family. In
certain cases, including Sp1/Sp3, BTEB1/AP-2rep, and BKLF/EKLF, this
competition has ramifications on gene expression (60-62). In each
case, transcriptional activation by one family member is repressed by
the other member by competing for the same DNA-binding site. The
data in this report, in combination with the previous studies of the Sp
family of transcription factors, support a model in which the
regulation of a required cofactor controls cell type-specific
p53-dependent expression of the BAX gene.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
113/
77, AATTCGGTACCTCACAAGTTAGAGACAAGCCTGGGCGTGGGCTATATTGCTAGCGAATT; BAX
113/
83, AATTCGGTACCTCACAAGTTAGAGACAAGCCTGGGCGTGGGCGCTAGCGAATT; BAX
113/
92, AATTCGGTACCTCACAAGTTAGAGACAAGCCTGCTAGCGAATT; BAX
102/
83, AATTCGGTACCAGACAAGCCTGGGCGTGGGCGCTAGCGAATT; BAX-113/
83(sc
102/
93), AATTCGGTACCTCACAAGTTAGCTCACCTAAGGGGCGTGGGCGCTAGCGAATT; BAX(
113/
93)3,
AATTCGGTACCTCACAAGTTAGAGACAAGCCTCACTGGTCACAAGTTAGAGACAAGCCTCACTGGTCACAAGTTAGAGACAAGCCTGCTAGCGAATT; BAX
92/
83, AATTCGGTACCGGGCGTGGGCGCTAGCGAATT; p21 5',
AATTCGGTACCGAACATGTCCCAACATGTTGGCTAGCGAATT; BAX/p21 5' hybrid,
AATTCGGTACCAGACAAGCCTCAACATGTTGGCTAGCGAATT; p21 5'/BAX hybrid,
AATTCGGTACCGAACATGTCCGGGCGTGGGCGCTAGCGAATT; Sp1 consensus,
ATTCGATCGGGGCGGGGCGAGC; BAXGG
92/
91AA,
AATTCGGTACCTCACAAGTTAGAGACAAGCCTAAGCGTGGGCGCTAGCGAATT; BAXGG
85/
84AA,
AATTCGGTACCTCACAAGTTAGAGACAAGCCTGGGCGTGAACGCTAGCGAATT; BAXG
92A,
AATTCGGTACCTCACAAGTTAGAGACAAGCCTAGGCGTGGGCGCTAGCGAATT; BAXG
91A,
AATTCGGTACCTCACAAGTTAGAGACAAGCCTGAGCGTGGGCGCTAGCGAATT; BAXG
90A,
AATTCGGTACCTCACAAGTTAGAGACAAGCCTGGACGTGGGCGCTAGCGAATT; BAXC
89A,
AATTCGGTACCTCACAAGTTAGAGACAAGCCTGGGAGTGGGCGCTAGCGAATT; BAXG
88A,
AATTCGGTACCTCACAAGTTAGAGACAAGCCTGGGCATGGGCGCTAGCGAATT; BAXT
87G,
AATTCGGTACCTCACAAGTTAGAGACAAGCCTGGGCGGGGGCGCTAGCGAATT; BAXG
86T,
AATTCGGTACCTCACAAGTTAGAGACAAGCCTGGGCGTTGGCGCTAGCGAATT; BAXG
85T,
AATTCGGTACCTCACAAGTTAGAGACAAGCCTGGGCGTGTGCGCTAGCGAATT; BAXG
84T,
AATTCGGTACCTCACAAGTTAGAGACAAGCCTGGGCGTGGTCGCTAGCGAATT; BAXC
83A,
AATTCGGTACCTCACAAGTTAGAGACAAGCCTGGGCGTGGGAGCTAGCGAATT; BAX sc
92/
83, AATTCGGTACCTCACAAGTTAGAGACAAGCCTTTTATGTTTAGCTAGCGAATT; BAX
sc
86/
83, AATTCGGTACCTCACAAGTTAGAGACAAGCCTGGGCGTTTTAGCTAGCGAATT.
113/
83, BAX
113/
93, BAX
102/
83,
BAX
113/
83(sc
102/
93), BAX(
113/
93)3, BAX
92/
83, BAX
133/
77, p21 5', BAXGG
92/
91AA, BAXGG
85/
84AA, BAXG
92A, BAXG
91A, BAXG
90A, BAXC
89A, BAXG
88A, BAXT
87G, BAXG
86T,
BAXG
85T, BAXG
84T, BAXC
83A, BAX sc
92/
83, BAX sc
86/
83.
pBAX
315/+51, pBAX
127/+51, and pBAX
76/+51 were generated by
PCR1 amplification of the
appropriate fragments from the original pBAX luciferase reporter
plasmid (8). Upstream primers were engineered with the NheI
restriction site. Downstream primers contained the HindIII
restriction site. Following PCR, products were digested with both
NheI and HindIII and cloned into pGL3-E1bTATA which was also double-digested with NheI and
HindIII, removing the adenovirus E1b minimal
promoter. To construct pBAX
126/
77, PCR amplification of the
original pBAX was used to generate two fragments corresponding to
315
to
127 and to
76 to +51 from the start site of transcription. The
315 to
127 fragment was engineered to contain the NheI
restriction site on the upstream side and the SacI
restriction site on the downstream side. The
76 to +51 fragment was
engineered to contain the SacI site upstream and the
HindIII site downstream. Following PCR amplification each fragment was double-digested with the appropriate restriction enzymes
(NheI and SacI or SacI and
HindIII). A three-way ligation with the two PCR-generated
fragments and pGL3-E1bTATA, double-digested with NheI and
HindIII, then was performed, replacing the BAX
sequence from
126 to
77 with the SacI restriction site.
To construct pBAX
113/
104, PCR amplification of the original pBAX
was used to generate two fragments corresponding to
315 to
114 and
to
103 to +51 from the start site of transcription. The
315 to
114 fragment was engineered to contain the NheI
restriction site on the upstream side and the NcoI
restriction site on the downstream side. The
103 to +51 fragment was
engineered to contain the NcoI site upstream and the
HindIII site downstream. Following PCR amplification each
fragment was double-digested with the appropriate restriction enzymes
(NheI and NcoI or NcoI and
HindIII). A three-way ligation with the two PCR-generated
fragments and pGL3-E1bTATA, double-digested with NheI and
HindIII, then was performed, replacing the BAX
sequence from
113 to
104 with the NcoI restriction site.
The generation of pBAX
103/
93, pBAX
92/
83, and
pBAX
113/
93 was accomplished as above with pBAX
113/
104 but
using PCR-generated fragments corresponding to
315 to
104 and
92
to +51,
315 to
93 and
82 to +51, and
315 to
114 and
92 to
+51, respectively. The expression plasmid pCMV-p53wt,
originally referred to as pC53-SN3 (35), encodes the wild-type human
p53 protein under the control of the cytomegalovirus promoter. The
expression plasmid pPacSp1 contains the 2.1-kilobase pair XhoI restriction fragment of Sp1 cloned downstream of the
Actin 5C promoter (36). pPacU was generated by removing the
2.1-kilobase pair XhoI fragment from pPacSp1.
70 °C.
70 °C.
Phosphorimaging and densitometry data were collected with a Personal
Molecular Imager FX and a GS-710 Calibrated Imaging Densitometer
(Bio-Rad), and analyzed with Quantity One software (Bio-Rad).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
113 to
83 from the
start site of transcription. There was no significant difference
between the p53-dependent transactivation of either a
reporter construct lacking sequences 5' to the p53 response element
(pBAX
127/+51) or the full-length promoter construct (pBAX
315/+51)(Fig. 1A). Deletion of a larger fragment,
including the p53 response element (pBAX
76/+51), produced a reporter
construct that was unresponsive to wild-type p53 (Fig. 1A).
Furthermore, targeted deletion of the promoter region containing the
p53 response element (pBAX
126/
77) also produced a reporter plasmid that was unresponsive to wild-type p53 (Fig. 1A).
These results show that
113 to
83 is the only region, within the
366-base pair promoter fragment investigated, that affects the ability of p53 to activate transcription.
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Fig. 1.
All three potential p53 half-sites are
required for the p53-dependent transcriptional activation
of the human BAX promoter. A and
B, Saos-2 cells were transfected as described under
"Experimental Procedures" with 1 µg of the indicated pBAX
reporter constructs in the presence of either 10 ng of pCMV
(white bars) or 10 ng of pCMV-p53wt (gray
bars). 24 h post-transfection cells were lysed and assayed
for total protein and luciferase activity as described under
"Experimental Procedures." The indicated values are the average of
three independent experiments each performed in duplicate. Error
bars correspond to one S.D. The numbers above each bar
indicate the fold activation for each reporter construct observed with
pCMV-p53wt as compared with pCMV. A, the
previously identified p53 response element is indicated by the
dark gray box at 113 to
83. B, the three
potential p53 half-sites are represented by the light gray
(
113 to
104), white (
102 to
93), and dark
gray (
92 to
83) boxes.
113 to
83 contains three potential p53 half-sites
(represented in Fig. 1B as the light gray, white,
and dark gray boxes). The role of each of these half-sites
in the p53-dependent activation of the BAX
promoter was examined. Removal of the first half-site from
113 to
104 (pBAX
113/
104) significantly reduced the ability of p53 to
activate transcription through this promoter (Fig. 1B,
compare 63-fold with pBAX
315/+51 to 7-fold with pBAX
113/
104),
whereas removal of the second (pBAX
103/
93) or the third
half-site (pBAX
92/
83) completely abolished the ability of p53 to
activate transcriptionally the promoter (Fig. 1B).
Consistent with the above results, removal of the first and second
half-sites in combination (pBAX
113/
93) also abolished the
ability of p53 to activate transcriptionally the promoter (Fig.
1B). These results demonstrate that, as was observed with the isolated response element (37), p53 requires sequences from all
three potential half-sites to mediate transcriptional activation of the
BAX promoter.
113 to
83 closely
resembles the consensus sequence of 5'-RRRCWWGYYY-3' (represented in
Fig. 2 by the light gray, white, and dark gray boxes). The first, located at
113 to
104, deviates from the consensus at 2 bases (
113 and
104). The second half-site matches the consensus sequence at all 20 base pairs and is located at
102 to
93. The third half-site is
located at
92 to
83 and deviates from the consensus at three bases
(
84,
85, and
88)(see Fig. 2). These three half-sites can combine in different ways to produce a total of three possible p53 complete binding sites (half-sites 1 and 2, 2 and 3, and 1 and 3). Previous studies demonstrated that in electrophoretic mobility shift assays (EMSA), double-stranded oligonucleotides representing both
113 to
93 (half-sites 1 and 2) and
102 to
83 (half-sites 2 and 3) are
capable of binding p53 in a sequence-specific manner with similar
affinities (37). When cloned upstream of the adenovirus E1b
minimal promoter in the pTATA luciferase reporter plasmid, however, the
combination of the first and second half-sites (
113 to
93) is
unable to mediate p53-dependent transcriptional activation (37). To examine further the ability of p53 to interact with this
sequence in cells, the
113 to
93 sequence was multimerized (as
three copies) and cloned into the pTATA luciferase reporter plasmid.
This reporter plasmid was cotransfected with either pCMV or a wild-type
p53 expression vector in the Saos-2 cell line (Fig. 2). These three
copies of this p53-binding site were capable of mediating a significant
degree of activation in response to p53 (Fig. 2, compare 4-fold with
pTATA-113/
93 to 142-fold with pTATA(
113/
93)3), demonstrating that the sequence from
113 to
93 is indeed a
bona fide p53 response element capable of both binding p53
in a sequence-specific manner in vitro and mediating
p53-dependent transcriptional activation in cells.
Confirming previous results, p53 was able to activate transcription
through the second and third half-sites (
102 to
83), but this
activation was significantly reduced as compared with that mediated by
all three half-sites combined (Fig. 2, compare 44-fold with
pTATA-102/
83 and 153-fold with pTATA-113/
83). To test the ability
of half-sites one and three to mediate p53-dependent transcriptional activation, a synthetic oligonucleotide corresponding to
113 to
83 of the BAX promoter, with
102 to
93
scrambled to remove any contribution of the second half-site, was
cloned into the pTATA reporter plasmid. This construct failed to be
activated by p53 (Fig. 2, pTATA-113/
83(sc
102/
93)). The third
half-site in isolation (
92 to
83) also failed to mediate
p53-dependent transcriptional activation (Fig. 2,
pTATA-92/
83).
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Fig. 2.
The first two potential p53 half-sites
constitute a bona fide p53 response element.
Saos-2 cells were transfected as described under "Experimental
Procedures" with 1 µg of the indicated pTATA reporter constructs in
the presence of either 10 ng of pCMV (white bars) or 10 ng
of pCMV-p53wt (gray bars). 24 h
post-transfection cells were lysed and assayed for total protein and
luciferase activity as described under "Experimental Procedures."
The indicated values are the average of three independent experiments
each performed in duplicate. Error bars correspond to 1 S.D.
The numbers above each bar indicate the fold activation for
each reporter construct observed with pCMV-p53wt as
compared with pCMV. The sequence of the BAX promoter from
113 to
83 is given at the top of the figure. Potential
p53 quarter-sites are indicated by the solid bars above and
below the sequence. Bases that deviate from the p53
DNA-binding consensus sequence are indicated by asterisks.
The three potential half-sites are indicated by the brackets
labeled 1-3, respectively, and are represented graphically as the
light gray, white, and dark gray boxes,
respectively. The vertical arrow above the BAX
sequence indicates the 1-base pair insert between the first and second
half-sites.
102 to
83. Analysis of this region using a
MatInspector search of the TRANSFAC data base (38, 39) showed that it
contains a sequence that potentially could bind the transcription
factor Sp1. To test this, a synthetic oligonucleotide corresponding to
102 to
83 of the BAX promoter was used as a radiolabeled probe in an EMSA with HeLa cell nuclear extract
(Fig. 3). As reported previously for
Saos-2 (37), HeLa cell nuclear extract contains a factor that
demonstrated marked sequence specificity for the labeled BAX
probe. This factor was successfully competed by increasing amounts of
unlabeled probe (Fig. 3, lanes 7-9) as well as by
increasing amounts of oligonucleotide corresponding to the DNA-binding
consensus sequence of Sp1 (Fig. 3, lanes 13-15). This
binding was specific, as an oligonucleotide corresponding to the 5' p53
response element from the human p21 promoter failed to
compete for binding (Fig. 3, lanes 10-12). In addition,
this factor was successfully bound by an anti-Sp1 antibody, as
demonstrated by a "supershifted" complex (Fig. 3, lanes
2 and 3), whereas a control anti-p300 antibody failed
to bind the factor (Fig. 3, lanes 4 and 5).
Together, these data demonstrate that Sp1 can bind a portion of the p53
response element from the human BAX promoter in a
sequence-specific manner.
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Fig. 3.
Sp1 binds with sequence specificity to the
p53 response element from the human BAX promoter.
An electrophoretic mobility shift assay was performed using an
oligonucleotide corresponding to the 102/
83 sequence from the human
BAX promoter as radiolabeled probe. 8 µg of HeLa cell
nuclear extract was incubated with 3 ng of the probe alone (lanes
1 and 6), in the presence of 2 or 4 µl of anti-Sp1
antibody (Ab) (lanes 2 and 3, respectively), 2 or 4 µl of anti-p300 antibody (lanes 4 and 5, respectively), a 100-, 200-, or 300-fold molar excess
of either the unlabeled BAX
102/
83 oligonucleotide (lanes
7-9) or p21 5' oligonucleotide (lanes 10-12), or a
10-, 20-, or 30-fold molar excess of the unlabeled Sp1 consensus
oligonucleotide (lanes 13-15). The arrows
indicate the positions of the Sp1-DNA complex and the supershifted
complex containing antibody, Sp1, and DNA.
102 to
83
in the BAX promoter contains two p53 half-sites (
102 to
93 and
92 to
83), and the p21 5' element also consists of two p53
half-sites. Hybrid oligonucleotides were synthesized in which the first
of the two half-sites in the BAX element was combined with
the second half-site of the p21 5' element and vice versa.
The oligonucleotide corresponding to
102 to
83 of the
BAX promoter again was used as a radiolabeled probe with
HeLa nuclear extract in an EMSA (Fig. 4).
Competitions, using unlabeled probe as well as the oligonucleotides
corresponding to the p21 5' element and the two hybrid
elements, were conducted. Sp1 bound the radiolabeled probe (Fig. 4,
lane 1) and was recognized by an anti-Sp1 antibody (Fig. 4,
lane 2) but not by a control anti-CBP antibody (Fig. 4,
lane 3). Both unlabeled probe and the Sp1 DNA-binding
consensus site oligonucleotide effectively competed for Sp1 binding
(Fig. 4, lanes 4-5 and 12-13, respectively),
whereas the p21 5' element did not (Fig. 4, lanes
10-11). Consistent with the notion that Sp1 binds DNA through GC
box regions, the hybrid oligonucleotide in which the first half-site is
derived from the p21 sequence and the second half-site from
the BAX sequence (
92 to
83, 5'-GGGCGTGGGC-3')
effectively competed for Sp1 binding (Fig. 4, lanes 8-9),
whereas the other hybrid oligonucleotide that replaces this GC-rich
region with sequence from the p21 5' element demonstrated a
significantly reduced affinity for Sp1 binding (Fig. 4, lanes
6-7). These data indicate that Sp1 binds to sequence within
92 to
83 of the BAX promoter.
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Fig. 4.
The Sp1-binding site is localized to a
GC-rich region of the p53 response element. A, an
electrophoretic mobility shift assay was performed using an
oligonucleotide corresponding to the 102/
83 sequence from the human
BAX promoter as radiolabeled probe. 8 µg of HeLa cell
nuclear extract was incubated with 3 ng of the probe alone (lane
1), in the presence of 4 µl of anti-Sp1 antibody (Ab)
(lane 2), 4 µl of anti-CBP antibody (lane 3), a
100- or 200-fold molar excess of either the unlabeled BAX
102/
83
oligonucleotide (lanes 4 and 5), p21 5'
oligonucleotide (lanes 10 and 11), BAX/p21 5'
hybrid oligonucleotide (lanes 6 and 7) or p21
5'/BAX hybrid oligonucleotide (lanes 8 and 9), or
a 10- or 20-fold molar excess of the unlabeled Sp1 consensus
oligonucleotide (lanes 12 and 13). The
arrows indicate the positions of the Sp1-DNA and the
supershifted antibody-Sp1-DNA complexes. B, the sequences of
the human BAX promoter from
102 to
83 from the start
site of transcription (gray boxes) and the human
p21 promoter from
2281 to
2262 from the start site of
transcription (white boxes; corresponding to the p21 5'
oligonucleotide) are shown. Each sequence is divided into two with the
first half indicated as A and the second half indicated as
B. Oligonucleotides in A are represented
graphically according to this color and letter scheme. For example, the
BAX/p21 5' hybrid oligonucleotide that corresponds to the first half of
the BAX sequence followed by the second half of the
p21 sequence is indicated by a gray box labeled
A followed by a white box labeled
B.
113 to
77 of
the human BAX promoter was cotransfected with increasing amounts of an Sp1 expression vector into the Sp1-deficient
Drosophila SL2 cell line (Fig.
5). Expression of Sp1 successfully
activated transcription of this reporter and yet failed to activate
transcription of a control plasmid containing the 5' p53 response
element of the p21 promoter (Fig. 5). Consistent with the
in vitro EMSA results, this confirms that Sp1 is capable of
activating transcription through the p53 response element of the human
BAX promoter.
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Fig. 5.
Sp1 can activate transcription through the
p53 response element of the human BAX promoter.
Drosophila SL2 cells were transfected as described under
"Experimental Procedures" with 2 µg of the indicated pTATA
reporter constructs in the presence of 0, 300, 600, or 900 ng of
pPacSp1. Appropriate amounts of the vector pPacU were added to each
transfection mixture to maintain a constant level of total plasmid DNA
of 2.9 µg/sample. 48 h post-transfection cells were lysed and
assayed for total protein and luciferase activity as described under
"Experimental Procedures." The indicated values are the average of
four independent experiments expressed as the fold activation for each
reporter plasmid with pPacSp1 as compared with pPacU. Error
bars correspond to 1 S.D.
113 to
83). Two mutated forms of the p53
response element from the BAX promoter, in which the
indicated guanine bases were replaced with adenines (Fig.
6A, GG
92/
91AA and GG
85/
84AA), were cloned into the pTATA luciferase
reporter plasmid. In cotransfection assays with a wild-type p53
expression vector in the Saos-2 cell line, substitution of bases
92
and
91 completely abolished the ability of p53 to activate
transcription through this element (Fig. 6A, compare
113/
83 to GG
92/
91AA), whereas substitution of
bases
85 and
84 did not (Fig. 6A). As observed in Figs.
1 and 2, removal of the third potential half-site (
92 to
83)
inhibited the ability of p53 to mediate transcriptional activation
through this element (Fig. 6A, compare
113/
83
and
113/
93), demonstrating the requirement for this
Sp1-binding sequence in the p53-dependent transcriptional
activation of this element.
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Fig. 6.
A mutant element that fails to bind Sp1
in vitro also fails to confer
p53-dependent transcriptional activation in cells.
A, Saos-2 cells were transfected as described under
"Experimental Procedures" with 1 µg of the indicated pTATA
reporter constructs in the presence of either 10 ng of pCMV
(white bars) or 10 ng of pCMV-p53wt (gray
bars). 24 h post-transfection cells were lysed and assayed
for total protein and luciferase activity as described under
"Experimental Procedures." The indicated values are the average of
five independent experiments each performed in duplicate. Error
bars correspond to 1 S.D. The GC-rich region that binds Sp1 is
shown by the boxed sequence. Bases in the wild-type sequence
that were mutated are shown in gray with the corresponding
mutations indicated above. B, an electrophoretic
mobility shift assay was performed using an oligonucleotide
corresponding to the 113/
77 sequence from the human BAX
promoter as radiolabeled probe. 50 ng of purified p53 was incubated
with 3 ng of the probe alone (lane 1) or in the presence of
a 500-, 1000-, or 1500-fold molar excess of either the unlabeled BAX
113/
83 oligonucleotide (lanes 2-4), the GG
92/
91AA
oligonucleotide (lanes 5-7), or the GG
85/
84AA
oligonucleotide (lanes 8-10). The arrow
indicates the position of the p53-DNA complex. The vertical
bar between lanes 4 and 5 represents the
removal of irrelevant lanes from the gel. C, bands were
quantitated by densitometry. D, an electrophoretic mobility
shift assay was performed using an oligonucleotide corresponding to the
113/
77 sequence from the human BAX promoter as
radiolabeled probe. Extract from Sf9 cells expressing human
recombinant Sp1 protein was incubated with 3 ng of the probe alone
(lane 2), in the presence of anti-Sp1 antibody (lane
1), or in the presence of a 50-, 100-, or 200-fold molar excess of
either the unlabeled BAX
113/
83 oligonucleotide (lanes
3-5), the GG
92/
91AA oligonucleotide (lanes 6-8),
or the GG
85/
84AA oligonucleotide (lanes 9-11). The
arrow indicates the position of the Sp1-DNA complex, and the
asterisk indicates the position of the supershifted
anti-Sp1-DNA complex. The vertical bar between lanes
5 and 6 represents the removal of irrelevant lanes from
the gel. E, bands were quantitated by densitometry.
113 to
77 of the BAX promoter was used as a radiolabeled probe
with purified p53 in an EMSA (Fig. 6B). Competitions were performed with increasing amounts of an oligonucleotide corresponding to
113 to
83 of the BAX promoter and the two mutant
oligonucleotides. When compared with the wild-type oligonucleotide,
both mutant oligonucleotides displayed a slightly decreased affinity
for p53 (Fig. 6B, compare lanes 2-4 with
lanes 5-7 and 8-10; Fig. 6C). Compared with one another, however, both mutant oligonucleotides demonstrated a comparable affinity for p53 (Fig. 6, A and
B), suggesting that the differences in
p53-dependent transcriptional activation observed in Fig.
6A are not due to differences in the affinity of p53 for the
two sequences. In contrast, the abilities of the two mutant sequences
to bind Sp1 differed (Fig. 6, D and E). An
oligonucleotide corresponding to
113 to
77 of the BAX promoter was used as radiolabeled probe with extract from Sf9 cells expressing recombinant human Sp1 protein in an EMSA (Fig. 6D). Sp1 bound the probe and was recognized by an anti-Sp1
antibody (Fig. 6D, lanes 1-2). Sp1 binding was successfully
competed by unlabeled BAX
113/
83 oligonucleotide as well as by the
GG
85/
84AA mutated oligonucleotide (Fig. 6D, lanes 3-4
and 9-11, respectively; Fig. 6E). The
GG
92/
91AA mutant, however, demonstrated a significant decrease in
affinity for Sp1 (Fig. 6D, compare lanes 3-5 and
9-11 to lanes 6-8; Fig. 6E).
85/
84AA mutant presented in Fig. 6 suggest
that not all of the bases contained within the third potential half-site of the p53 response element are required for
p53-dependent transcriptional activation. To identify the
minimal sequence elements required to mediate p53-dependent
transactivation, a series of oligonucleotides was synthesized in which
each of the 10 bases of the third potential half-site (
92 to
83) was individually replaced. These mutant oligonucleotides then
were cloned into the pTATA luciferase reporter plasmid and tested for
their responsiveness to p53 in a cotransfection assay in the Saos-2
cell line (Fig. 7). Consistent with the
results in Fig. 6A, substitution of the bases at either
85
or
84 did not inhibit the ability of p53 to activate transcription
through this element (Fig. 7, G
85T and G
84T).
Furthermore, substitution of
86 and
83 also failed to affect
significantly the ability of p53 to activate transcription (Fig. 7,
compare
113/
83 to G
86T and
C
83A). Substitution of the base at
87, however,
significantly reduced the ability of p53 to activate transcription
through this element (Fig. 7, compare
113/
83 to
T
87G). Together, these results suggest that the minimal response element consists of sequence from
113 to
87, with
86 to
83 being dispensable for p53-dependent
transactivation.
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Fig. 7.
Mutational analysis shows that the
BAX promoter sequence from 86 to
83 is not
required for p53-dependent transcriptional activation.
Saos-2 cells were transfected as described under "Experimental
Procedures" with 1 µg of the indicated pTATA reporter constructs in
the presence of either 10 ng of pCMV (white bars) or 10 ng
of pCMV-p53wt (gray bars). 24 h
post-transfection cells were lysed and assayed for total protein and
luciferase activity as described under "Experimental Procedures."
The indicated values are the average of three independent experiments
each performed in duplicate. Error bars correspond to 1 S.D.
The GC-rich region that binds Sp1 is shown by the boxed
sequence. Bases in the wild-type sequence that were mutated are
shown in gray with the corresponding mutations indicated
above.
86 to
83 are not required for
p53-dependent transcriptional activation, two additional
mutant oligonucleotides were synthesized. The first mutant was
generated by replacing all 10 nucleotides from
92 to
83 (Fig.
8A, sc
92/
83). The 4 bases
from
86 to
83 were substituted as indicated to generate the second
mutant oligonucleotide (Fig. 8A, sc
86/
83). Each
oligonucleotide was cloned into the pTATA vector and tested for its
responsiveness to p53 in a cotransfection assay (Fig. 8A).
As observed with the reporter plasmid in which the sequence from
92
to
83 is removed entirely (pTATA
113/
93), the first mutant, in
which all 10 bases of the third potential half-site (
92 to
83) are
replaced, showed little to no response to p53 (Fig. 8A,
compare pTATA
113/
93 to pTATA
113/
83 and
pTATAsc
92/
83). In contrast, the second mutant, in which
only the last 4 bases of the element (
86 to
83) are replaced, was
efficiently activated by p53 (Fig. 8A, compare 312-fold with
pTATA
113/
83 to 323-fold with
pTATAsc
86/
83). This result demonstrates that the minimal
p53 response element in the BAX promoter consists of
sequence from
113 to
87. In an EMSA both mutants displayed a
decreased affinity for p53 as compared with the wild-type sequence
(Fig. 8B, compare lanes 2-4 to lanes
8-10 and 11-13; Fig. 8C). When compared
with each other, there was no significant difference in the affinity of
p53 for the two mutant sequences (Fig. 8B, compare
lanes 8-10 to 11-13; Fig. 8C). This suggests that the differences in transcriptional activation observed in
Fig. 8A cannot be explained by differences in p53
affinities. Furthermore, the oligonucleotide corresponding to
113 to
93 displayed a similar affinity for p53 as the two mutant
oligonucleotides (Fig. 8B, compare lanes 5-7 to
lanes 8-10 and 11-13; Fig. 8C) consistent with the idea that, in the case of the two mutants, p53 is
interacting with the first and the second half-sites only. The sc
86/
83 mutant oligonucleotide efficiently competed for Sp1 binding
in an EMSA (Fig. 8D, compare lanes 2-4 to
lanes 8-10; Fig. 8E), whereas the ability of the
sc
92/
83 mutant to bind Sp1 was significantly reduced compared with
the wild-type sequence (Fig. 8D, compare lanes
2-4 to lanes 5-7; Fig. 8E), further
strengthening the correlation between Sp1 binding in vitro
and p53 activation in cells.
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Fig. 8.
The minimal element from the BAX
promoter that confers p53-dependent transcriptional
activation consists of a single p53-binding site and an adjacent
Sp1-binding site. A, Saos-2 cells were transfected as
described under "Experimental Procedures" with 1 µg of the
indicated pTATA reporter constructs in the presence of either 10 ng of
pCMV (white bars) or 10 ng of pCMV-p53wt
(gray bars). 24 h post-transfection cells were lysed
and assayed for total protein and luciferase activity as described
under "Experimental Procedures." The indicated values are the
average of three independent experiments each performed in duplicate.
Error bars correspond to 1 S.D. The numbers above each
bar indicate the fold activation for each reporter construct
observed with pCMV-p53wt as compared with pCMV. The GC-rich
region that binds Sp1 is shown by the boxed sequence. Bases
in the wild-type sequence that were mutated are shown in
gray with the corresponding mutations indicated
above. B, an electrophoretic mobility shift assay
was performed using an oligonucleotide corresponding to the 113/
83
sequence from the human BAX promoter as radiolabeled probe.
50 ng of purified p53 was incubated with 3 ng of the probe alone
(lane 1) or in the presence of a 500-, 1000-, or 1500-fold
molar excess of either the unlabeled BAX
113/
83 oligonucleotide
(lanes 2-4), the BAX
113/
93 oligonucleotide, the BAX sc
92/
83 oligonucleotide (lanes 5-7), or the BAX sc
86/
83 oligonucleotide (lanes 8-10). The
arrow indicates the position of the p53-DNA complex.
C, bands were quantitated by phosphorimaging. D,
an electrophoretic mobility shift assay was performed using an
oligonucleotide corresponding to the
113/
83 sequence from the human
BAX promoter as radiolabeled probe. Extract from Sf9
cells expressing human recombinant Sp1 protein was incubated with 3 ng
of the probe alone (lane 1) or in the presence of a 10-, 50-, or 100-fold molar excess of either the unlabeled BAX
113/
83
oligonucleotide (lanes 2-4), the BAX sc
92/
83
oligonucleotide (lanes 5-7), or the BAX sc
86/
83
oligonucleotide (lanes 8-10). The arrow
indicates the position of the Sp1-DNA complex. E, bands were
quantitated by phosphorimaging.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
113 to
87 from the start site of transcription. This
sequence contains a p53-binding site (
113 to
93) that can function
as a bona fide response element as demonstrated by its
ability when multimerized to confer p53-dependent
transcriptional activation on a minimal promoter (Fig. 2). Immediately
adjacent to this p53-binding site are 6 base pairs that are GC-rich in
nature (
92 to
87: 5'-GGGCGT-3'). These 6 bases are required for
p53-dependent transcriptional activation as deletion or
mutation of this region in the context of either the promoter or the
isolated response element completely abrogates the ability of p53 to
activate transcription through this sequence (Figs. 1B, 2, 6A, and 8A). The addition of these bases to the
113/
93 sequence appears to have little effect on the affinity of
p53 for this sequence (Fig. 8, B and C),
consistent with a model in which these 6 bases function to recruit a
co-activator as opposed to simply enhancing p53 binding. Furthermore,
these 6 base pairs mediate sequence-specific binding to the Sp1
transcription factor (Figs. 3, 4, 6D, and 8D),
and a positive correlation is seen between the ability of Sp1 to bind
this element in vitro and the ability of p53 to mediate
transcriptional activation through its response element in cells (Figs.
6 and 8). In addition, the results with electrophoretic mobility shift
assays with the GG
92/
91AA mutant oligonucleotide (Fig.
6B) are not consistent with the published p53 DNA-binding
consensus sequence of (RRRCWWGYYY)2 (18, 19). This
consensus allows for a purine in the first three positions of each
half-site. The GG
92/
91AA mutant contains a conservative substitution of purines (adenines) for purines (guanines) and, as such,
does not represent a substantive change in terms of the p53 DNA-binding
consensus sequence. This substitution, however, did produce a
significant decrease in the ability of p53 to bind to this
oligonucleotide in vitro (Fig. 6B), suggesting
that, in these limited circumstances, the p53 DNA-binding sequence
involves greater specificity than implied by the consensus.
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Fig. 9.
Model for the cell type-specific regulation
of the BAX promoter by the tumor suppressor protein
p53. A, in cells that are permissive to
p53-dependent transcriptional activation of the
BAX gene, p53 and the required cofactor cooperate to mediate
activation. In cells that do not support the p53-BAX pathway, three
possible mechanisms may explain the apparent failure of wild-type p53
to activate the BAX gene. B, the cofactor may be
absent due to mutation or to cell type-specific limitations on its
expression. C, the cofactor may be inactivated by
post-translational modification as follows: P,
phosphorylation; G, glycosylation; or A,
acetylation. D, another factor that cannot cooperate with
p53 may compete with the required cofactor for binding to its site in
the BAX promoter. The p53-binding site ( 113 to
93) is
represented by the black box. The Sp1-binding site (
93 to
87) is represented by the white box. p53, the required
cofactor, and the inhibitory factor are represented by the gray
circle, the dotted oval, and the cross-hatched
triangle, respectively.
The ability of the proapoptotic BAX to function as a tumor suppressor protein has been substantiated by several studies. In certain mouse models, BAX has been shown to be an important mediator of p53-dependent apoptosis and a suppressor of oncogenic transformation, with loss of BAX leading to accelerated rates of tumor growth, increased tumor numbers, larger tumor mass, and decreased survival rates (63, 64). A significant correlation between decreased BAX expression and both a corresponding resistance to apoptotic stimuli, as well as a shorter survival period also have been observed in a number of human tumor types, including breast, ovarian, pancreatic, colorectal, and non-Hodgkin's lymphoma (65-69). In addition, in colon and gastric cancers of the microsatellite mutator phenotype mutational inactivation of the BAX gene has been shown to confer a strong survival advantage during tumor clonal evolution (70). Complimenting these data are observations showing that overexpression of the BAX protein in certain tumor cell lines both sensitizes these cells to chemotherapy- and radiation-induced apoptosis and reduces their ability to form tumors in SCID mice (71-73). Together, these results strongly support a tumor suppressor role for the BAX protein.
An important regulator of the BAX gene is the tumor
suppressor protein p53. Several reports have demonstrated the
significance of the p53-BAX pathway in tumor suppression. Both the
identification of tumor-derived p53 mutants that selectively fail to
activate transcription through the BAX promoter and
subsequently fail to induce apoptosis (29-32) as well as the TgT121
transgenic studies that demonstrate that BAX is an
obligatory downstream effector of p53 in the suppression of choroid
plexus tumor growth (33) suggest that the ability of p53 to activate
transcription through the BAX promoter is important to the
tumor suppressor function of p53. Furthermore, the resistance of
certain tumor cell lines to radiation therapy is associated with a
failure of wild-type p53 to induce BAX expression (28, 74),
and certain human tumors have been identified that are genetically
wild-type for both p53 and BAX and yet fail to
express significant levels of BAX protein (75). Thus, a complete
understanding of the transcriptional regulation of the BAX
gene by the tumor suppressor p53 may provide important information
concerning both the molecular origins of cancer as well as the
development of tumor resistance to certain cancer treatments.
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ACKNOWLEDGEMENTS |
---|
We thank Bert Volgelstein (The Johns Hopkins University) for the wild-type p53 expression plasmid; John Reed (Burnham Institute) for the original pBAX reporter construct; Robert Tjian and Andres Naar (University of California at Berkeley) for the pPacSp1 expression plasmid and the recombinant baculovirus expressing human Sp1; and Ze'ev Ronai (Mount Sinai) for the recombinant baculovirus expressing His-tagged human p53. We thank Ron Magnusson for help with the recombinant baculovirus infections. We also thank Lois Resnick-Silverman of the Manfredi laboratory for help and support. The technical assistance of Andrea DaCosta is gratefully acknowledged.
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FOOTNOTES |
---|
* This work was supported by NCI Grant CA69161 from the National Institutes of Health and the Breast Cancer Program of the United States Army Medical Research and Materiel Command Grants DAMD-17-97-1-7336, DAMD-17-97-1-7337, and DAMD-17-99-1-9308).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Cancer Center, Box
1130, Mount Sinai School of Medicine, New York, NY 10029. Tel.: 212-659-5495; Fax: 212-849-2446.
Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc. M011643200
2 E. C. Thornborrow and J. J. Manfredi, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PCR, polymerase chain reaction; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assays.
![]() |
REFERENCES |
---|
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