From the Department of Biochemistry and Molecular Biology, Program in Genes and Development, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
Received for publication, November 13, 2000, and in revised form, February 9, 2001
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ABSTRACT |
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We previously identified transcription factor
AP-2 as the nuclear factor that interacts with the tissue-specific
repressor element in the rat serum amyloid A1 (SAA1)
promoter. In this report, we provide evidence for a second AP-2-binding
site and show that both AP-2 sites participate in mediating the
transcription repression of SAA1 promoter. This proximal
AP-2 site overlaps with the NF Expression of cell type-specific genes is tightly controlled by
the combined actions of positive and negative transcription regulators
that interact with specific DNA sequences (1-3). In addition, the
tissue-specific expression of transcription factors themselves can
contribute significantly to their functional role in regulating
cell-type determination or differentiation (4, 5). To achieve accurate
tissue-specific transcription, a complex array of signals must be
integrated through the actions of transcription activators and
repressors, culminating at the gene promoter to regulate initiation of
RNA synthesis (6-8). For instance, expression of MyoD is restricted to
the skeletal muscle cells and contributes to myogenesis by interacting
with other positive transcription factors to activate the transcription
of muscle-specific genes (9). In contrast, neuron-restrictive silencer
factor is expressed in most nonneuronal tissues and in undifferentiated
neuronal progenitors (10, 11). Because of its tissue distribution and
its repressive effects on neuronal-specific genes, this silencer factor
may function as a master negative regulator for neurogenesis (10,
12).
A growing list of transcription factors has been shown to function as
either transcription activators or transcription repressors, depending
on the promoter and cellular context of their target genes (13, 14).
Examples of this group of transcription factors include the
Drosophila transcription factor Kruppel, which
converts from an activator to a repressor in a
concentration-dependent manner (15, 16); human thyroid hormone
receptor Serum amyloid A (SAA), one of the major
acute-phase proteins (32, 33), represents an excellent model system to
study cytokine-induced regulation and liver-specific expression. Its
serum concentration increases 1,000-fold after acute inflammation,
being regulated primarily by the 200-300-fold increase in
SAA gene transcription (32, 34, 35). In mice, the
SAA gene family consists of four genes, SAA1,
SAA2, SAA3, and SAA5, and a pseudogene
(36, 37). Whereas expression of SAA1, SAA2, and
SAA3 is dramatically induced after inflammation and each
contributes equally to the increased SAA mRNA levels in the liver,
SAA5 expression is induced to a much lower level and with
different induction kinetics (38, 39). In addition to being highly
regulated by the inflammatory cytokines, expression of SAA1
and SAA2 are also highly cell type-specific, restricted to
the liver hepatocytes (32, 34, 35).
Studies of the rat SAA1 promoter demonstrate that 304 bp of
its 5'-flanking sequence are sufficient not only for its liver cell-specific expression but also for its cytokine-induced expression in response to inflammatory mediators (40). Further deletion analyses
identified a 66-bp DNA fragment spanning bp We have recently purified this DNA-binding protein from HeLa nuclear
extracts and shown by protein sequencing and biochemical and
immunological analyses that it is identical to the transcription factor
AP-2 (43). Here, we report on the repressive effects of AP-2 on the
expression of transfected and endogenous liver genes and suggest that
it may function as a negative regulator to repress the expression of
some liver genes in nonhepatic cells.
Cell Culture and Conditioned Medium--
HeLa, HepG2, and Hep3B
cells were grown in basal medium that contained minimum essential
medium (Life Technologies, Inc.) supplemented with 10% fetal calf
serum, 0.1 mM nonessential amino acids, 1 mM
pyruvate, 100 units/ml penicillin, and 100 µg/ml streptomycin. HepG2
and Hep3B cells were used for our transient transfection and stable
cell line studies because they have been demonstrated to confer proper
regulation of many endogenous and transfected genes, including
SAA (44-47). Conditioned medium (CM) known to contain a
mixture of inflammatory mediators capable of inducing acute-phase gene
expression was prepared from activated mixed lymphocyte cultures as
described (48) and used as a mixture with an equal volume of basal medium.
Oligonucleotides and Plasmids--
The wild-type AP-2
consensus sequence (AP-2) (5'-GGAACTGACCGCCCGCGGCCGTGGTCAG-3') and its
mutant (mAP-2)
(5'-GGAACTGACCGaCCGCtGCCGTGGTCAG-3') are from the
human metallothionein IIA promoter (bp
pTK/SAA1( Transient Transfection Assay--
HepG2 cells (1.5-2 × 105) were seeded into 60-mm culture dishes 16 h before
they were transfected with plasmid DNAs using the FuGENETM6
transfection kit (Roche Molecular Biochemicals). Cells were incubated
for 16-20 h before being stimulated with basal medium or with 50% CM
to induce the reporter gene activities. Sixteen to 20 h after
treatment, cells were harvested for cell extract preparation and CAT or
luciferase assays.
CAT and Luciferase Assays--
CAT assays were performed
essentially as described (48). The conversion of acetylated forms of
chloramphenicol was calculated by measuring the radioactivity with a
PhosphorImager (Molecular Dynamics). Luciferase activity was measured
with luciferase assay kit (Tropix, Inc.) using a Lumat LB 9507 luminometer (EG & G Berthold). The averages and S.E. from 3 or 4 independent experiments were calculated relative to the activities for
the control samples, to which a value of 1.0 was assigned.
EMSA--
For EMSA, 2 × 104 cpm (~1 ng) of
32P-labeled DNA fragments were mixed with indicated amounts
of protein samples in reaction buffer (48). After incubation for 30 min
at room temperature, the samples were loaded on a 5% polyacrylamide
gel and subjected to electrophoresis at 200 V for 100 min. The gel was
then dried and autoradiographed. The intensities of shifted protein-DNA
complexes were quantified using a PhosphorImager (Molecular Dynamics).
In oligonucleotide competition experiments, the protein was incubated
simultaneously with the labeled probe and an excess amount of unlabeled
competitor oligonucleotides. Recombinant AP-2 and the p50 subunit of
NF Establishment of AP-2-expressing Stable Hep3B Cell
Lines--
Hep3B cells were plated at 70-80% confluence in 100-mm
culture dishes 16-24 h before transfection with pIND/AP-2 or empty vector (3 µg/dish). Cells were trypsinized 48 h after
transfection, washed with basal medium, and split into four 100-mm
culture dishes in selection medium containing 400 µg/ml G418. Cells
were fed with fresh selection medium every 3-4 days. After culturing
for 2-3 weeks, individual resistant clones were picked and transferred to 24-well plates. Clones that express AP-2 were screened by EMSA using
the AP-2d sequence as a probe and further verified by RT-PCR. Three
AP-2-expressing clones, designated H3B.A1, H3B.A2, and H3B.A6, were
selected for further analysis. For comparison, three control clones
that contain the empty pIND vector were selected similarly and were
designated H3B.V1, H3B.V2, and H3B.V3.
RT-PCR--
Total cellular RNAs were isolated from the cloned
Hep3B cell lines using the High Pure RNA Isolation kit (Roche Molecular Biochemicals). TitanTM One Tube RT-PCR system (Roche
Molecular Biochemicals) was used to optimize conditions for PCR
(55 °C for 30 min, 94 °C for 30 s, 64 °C for 1 min, and
68 °C for 1 min) and to quantify mRNA levels. The amplification
reactions contained 1 µCi of [ The Proximal Region of SAA1 Promoter Contains Two AP-2-binding
Sites--
We had previously identified a repressor element in the rat
SAA1 promoter that could inhibit its promoter activity in
HeLa cells (42). Furthermore, we showed this repressor element
contained a high affinity binding site for AP-2 (43). In this study, we further investigated the mechanisms for the repressive effects of AP-2
by searching for additional AP-2-binding sites in the SAA1
promoter. In addition to the AP-2-binding site (bp
Interestingly, the position of the AP-2p site overlaps that of NF Binding of AP-2 to the SAA1 Promoter Is Required for Its Inhibitory
Effects--
Site-specific mutation of NF Mutation of AP-2-binding Sites Results in Derepression of SAA1
Promoter in HeLa Cells--
Because liver and liver-derived cell lines
such as HepG2 do not normally express AP-2, a study of the effects of
AP-2 in these cells requires cotransfection with AP-2 expression
vectors. In contrast, HeLa cells express abundant amounts of AP-2, thus
making cotransfection unnecessary as well as providing an alternative approach to examine the specificity of AP-2 repression effects. To
investigate whether the lack of SAA1 promoter activities in HeLa cells is, at least in part, due to repression by the endogenous AP-2 through its binding at AP-2d and AP-2p sites, we generated a
double AP-2-site mutant construct,
pTK/SAA1( Other Liver Gene Promoters Contain Potential AP-2-binding
Sequences--
Two AP-2-binding sites were identified in the
SAA1 promoter region, a high affinity AP-2d site and a
weaker AP-2p site. To determine whether AP-2 may also regulate the
transcription of other liver genes, we searched for potential
AP-2-binding sites using the AP-2d sequence 5'-ATACCTCAGGCAGC-3' and
the AP-2 consensus sequence 5'-GGCN3GCC-3' (25, 53). As
shown in Table I, at least 12 genes that
are expressed mainly in the liver were found to contain putative
AP-2-binding sites. To determine whether these sequences in the liver
gene promoters could interact with AP-2 and therefore compete for its
binding, oligonucleotides corresponding to six of the genes, chosen for
their acute-phase response or liver-enriched expression, were
synthesized and used as competitors in EMSA. HeLa cell nuclear extracts
were used as the source of AP-2 binding activity, and radiolabeled
AP-2d sequence was used as a probe. As shown in Fig.
5, all six oligonucleotides showed dose-dependent competition for AP-2 binding. Although they
contained highly homologous core AP-2 binding sequences, they
nevertheless exhibited considerable differences in their abilities to
compete for AP-2 binding. For example, although the sequence from the human complement C3 promoter was 20 times more effective in competing for AP-2 binding than the SAA1 AP-2d site, sequences from
the AP-2 Inhibits Cytokine-induced Human Complement C3 Promoter
Activity in HepG2 Cells--
The fact that human complement C3
promoter contains such a high affinity AP-2-binding site suggests AP-2
may play a functional role in C3 gene regulation as well. To determine
whether AP-2 can similarly repress C3 promoter activity, we transfected
HepG2 cells with the cytokine-inducible pC3/Luc( Stable Expression of AP-2 in Hep3B Cells Reduced the Expression of
Endogenous SAA, Albumin, and
To determine whether AP-2 expressed in Hep3B cells could inhibit the
expression of endogenous liver genes, we first examined its effects on
the expression of endogenous SAA genes. To induce the
endogenous SAA genes, control (H3B.V1, V2, and V3) and
AP-2-expressing (H3B.A1, A2, and A6) cells were treated with basal
medium or with 50% CM for 12 h. Total RNA was isolated, and the
levels of SAA mRNA were determined by RT-PCR and quantified. In
nonstimulated cells, SAA mRNA was undetectable in both control and
AP-2-expressing cells (Fig. 7C). Upon cytokine stimulation,
the expression of endogenous SAA mRNA in control cells was
dramatically induced. In contrast, in AP-2-expressing cell lines, an
increase in SAA mRNA was also detected after stimulation, albeit
the magnitude of induction was much lower than that in control cells
(Fig. 7, C and D).
To determine whether AP-2 could also exert inhibitory effects on other
liver genes, we chose to examine the expression of albumin and
AFP because they are two prototypic liver genes that are
constitutively expressed in Hep3B cells and because putative AP-2-binding sites were also found in their promoters. When compared with control cells, albumin and AFP expression levels were consistently 2-fold lower in the AP-2-expressing cells (Fig. 7D). This
reduced albumin and AFP expression in H3B.A1, A2, and A6 cells was not due to nonspecific inhibition since the expression of control genes,
GAPDH, and Transcription factor AP-2 joins a growing list of transcription
regulators that are able to function as either transcription activators
or transcription repressors depending on the cellular and promoter
context of their target genes (13, 14, 54). Initially characterized as
an activating protein in SV40 gene transcription (21, 22), AP-2 has
since been shown to participate in the transcription activation of many
other genes, including those related to neuronal and epidermal
development (23-25, 55, 56). More recently, AP-2 was also shown to
function as a transcription repressor in repressing the expression of
cell type-specific genes (29, 31).
Expression of SAA1 is highly inducible by the
proinflammatory cytokines and is highly tissue-specific, being
expressed primarily in the liver. Our earlier studies identified a
66-bp CRU in the SAA1 promoter that is responsible for its
cytokine-induced regulation (41, 50). Within the CRU, two
cis-regulatory elements, the NF This study together with our earlier studies leads us to
formulate a model for the regulatory mechanisms that control the inflammation-induced and tissue-specific expression characteristics of
SAA1 (Fig. 8). In
nonstimulated hepatic cells such as HepG2, YY1 functions as a
constitutively expressed transcription repressor that occupies its site
in the CRU and accounts for the low basal expression of
SAA1. In response to stimulation, inflammatory mediators dramatically increase the activities of the positive transcription factors NFB-binding site known to be essential
for SAA1 promoter activity. Protein binding competition
experiments demonstrated that AP-2 and NF
B binding to these
overlapping sites were mutually exclusive. Furthermore, the addition of
AP-2 easily displaced prebound NF
B, whereas NF
B could not
displace AP-2. These results thus suggest that one mechanism by which
AP-2 negatively regulates SAA1 promoter activity may be by
antagonizing the function of NF
B. Consistent with a repression function, transient expression of AP-2 in HepG2 cells inhibited conditioned medium-induced SAA1 promoter activation. This
inhibition was dependent on functional AP-2-binding sites, since
mutation of AP-2-binding sites abolished inhibitory effects of AP-2 in HepG2 cells as well as resulted in derepression of the SAA1
promoter in HeLa cells. In addition to SAA1, we found that
several other liver gene promoters also contain putative AP-2-binding
sites. Some of these sequences could specifically inhibit AP-2·DNA
complex formation, and for the human complement C3 promoter,
overexpression of AP-2 also could repress its cytokine-mediated
activation. Finally, stable expression of AP-2 in hepatoma cells
significantly reduced the expression of endogenous SAA,
albumin, and
-fetoprotein genes. Taken together, our results suggest
that AP-2 may function as a transcription repressor to inhibit the
expression of not only SAA1 gene but also other liver genes
in nonhepatic cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, which converts from a repressor to an activator upon
ligand binding (17, 18); and YY1, which exerts positive or negative
effects on transcription depending on the presence or absence of the
E1A protein (19, 20). Transcription factor AP-2 is yet another
transcription regulator with dual functions. First identified in HeLa
cell nuclear extracts (21, 22), AP-2 was named for its transcription
activation function. It acts as an activator in regulating many genes,
including those involved in the morphogenesis of peripheral nervous
system, face, limbs, skin, and nephric tissues (23-25). Recently, AP-2 has been shown to negatively regulate the transcription of stellate cell Type I collagen, K3 keratin, acetylcholinesterase, prothymosin, ornithine decarboxylase, retina fatty acid-binding protein, and CCAAT/enhancer binding protein
(C/EBP
)1
(26-31). Exactly when a particular transcription factor with dual functions acts as an activator or as a repressor depends not only on
its intrinsic features and concentration and on the presence of other
transcription factors but perhaps also on the structure, relative
position, and orientation of the promoter sequence itself (13, 14).
138 to
73 that could
confer its cytokine responsiveness. Within this cytokine response unit
(CRU) reside binding sites for transcription factors NF
B, C/EBP, and
YY-1 (41). Whereas NF
B and C/EBP function cooperatively to induce
SAA1 promoter activity in response to cytokine stimulation,
YY-1 functions as a repressor in opposing NF
B-mediated transcription
activation and contributes to the low basal expression of
SAA1 and perhaps the transience of its expression after
inflammation (41). Subsequent transient transfection analyses revealed
a tissue-specific repressor element distal to the CRU that conferred
repression on the SAA1 promoter in HeLa cells but had no
such inhibitory activity in liver cells (42). This repressor element
formed an intense DNA-protein complex with nuclear extracts prepared
from HeLa cells but not from liver cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
188 to
161) (49). The
wild-type (5'-CCTTCACTCTATACCTCAGGCAGCTAAGGAA-3') and mutant
(5'-CCTTCACTCTATACCTtAaaCAGCTAAGGAA-3') AP-2d and
wild-type (5'-CATGGTGGGACTTTCCCCAGGGACCAA-3') and mutant (5'-CATGGTGGGACTTTCCCCAaaGACCAA-3') AP-2p
oligonucleotides correspond to the distal (bp
285 to
255) and
proximal (bp
97 to
70) AP-2-binding sites, respectively, in the
SAA1 promoter. These oligonucleotides were used as primers
in polymerase chain reactions (PCR) or as probes or competitors in
electrophoretic mobility shift assay (EMSA).
285/
70) was constructed by inserting a DNA fragment from
the rat SAA1 promoter (bp
285 to
70) into the
SmaI site of pBLCAT vector, which contains the thymidine
kinase (TK) minimal promoter and the chloramphenicol acetyltransferase
(CAT) reporter gene. This SAA1 promoter fragment was
synthesized by PCR using AP-2d and the complement of AP-2p
oligonucleotides. The constructs pTK/SAA1(
285/
70)mAP-2d/p is identical to
pTK/SAA1(
285/
70) except the distal and the proximal AP-2 sites have
been mutated. pSAA1/CAT(
120) contains the intact SAA1
promoter (bp
120 to +18) in front of the CAT reporter (50), and the
pSAA1/CAT(
120)mAP-2p construct has the proximal AP-2 site
mutated. The AP-2 expression construct, pIND/AP-2, was made by
inserting a 1.9-kilobase fragment containing the AP-2 cDNA and
-globin intronic sequences into the
HindIII/XbaI site of pIND expression vector
(Invitrogen). Expression constructs for the wild-type AP-2 (pSAP2), the
frameshift AP-2 mutant (pSAP2/FS21), and the dominant-negative AP-2
mutant (pSAP2
166-277) were kindly provided by Dr. Michael Tainsky
(49). The reporter construct pC3/Luc(
199) containing 199 bp of
the human C3 promoter in a luciferase reporter was kindly provided by
Dr. Gretchen Darlington (51).
B were purchased from Promega.
-32P]dCTP for the
detection of reaction products and were carried out to ensure that they
are within the linear range of the amplification. The reaction products
were resolved on 5% native polyacrylamide gels and quantified using a
PhosphorImager (Molecular Dynamics).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
272 to
265)
identified initially, one additional sequence (5'-CCCCAGGG-3') located
at bp
85 to
78 was found to be a potential AP-2-binding site (Fig.
1A). These two sites were
designated, relative to their positions in the SAA1
promoter, the distal AP-2-binding site (AP-2d) and the proximal
AP-2-binding site (AP-2p) (Fig. 1A). It is interesting to
note that the sequences at the AP-2p site are highly conserved between
rat, human, and mouse SAA1 genes (40, 52). To determine whether the AP-2p site could indeed interact with AP-2, oligonucleotide competition experiments were carried out using radiolabeled AP-2d sequence and purified AP-2 in EMSA. In the absence of any competitors, a strong AP-2·DNA complex was detected (Fig. 1B). As
expected, both the consensus AP-2 binding sequence from the
metallothionein IIA promoter and the AP-2d site very effectively
competed for AP-2·DNA complex formation, whereas the mutated
AP-2d-site oligonucleotides could not compete (Fig. 1B).
When the wild-type and mutated AP-2p oligonucleotides were used as
competitors, the wild-type AP-2p oligonucleotides showed
dose-dependent competition of the complex formation,
whereas the mutant AP-2p site did not. This result indicates that the
AP-2p site functioned as a binding site for AP-2, although it had lower
binding affinity than the AP-2d site.
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Fig. 1.
SAA1 promoter contains two
AP-2-binding sites. A, schematic diagram of the
5'-flanking region of the rat SAA1 gene. The positions of
NF B-, YY1-, C/EBP-, and AP-2-binding sites as well as the TATA motif
are indicated. DNA sequences corresponding to the distal AP-2-binding
site (AP-2d), the proximal AP-2-binding site
(AP-2p), and the overlapping NF
B-binding site are shown.
The arrow represents the direction of transcription.
B, competition with AP-2d and AP-2p oligonucleotides. The
AP-2d sequence was radiolabeled and used as probe in EMSA. Purified
AP-2 was incubated with labeled probe in the presence of 5, 25, or 125 ng of oligonucleotides corresponding to the consensus AP-2 binding
sequence from the metallothionein IIA promoter, AP-2d, mAP-2d, AP-2p,
and mAP-2p.
B,
which is known to be essential for SAA1 promoter function (40, 50). The overlapping nature of these two transcription factor-binding sites raises an intriguing question, that is, whether binding of one factor to the DNA would affect the binding of the other
factor. To address this question, gel shift assays were performed using
the AP-2p probe, which contains both AP-2- and NF
B-binding sites,
and recombinant AP-2 and NF
B. The reactions were carried out under
probe-limiting conditions to allow AP-2 and NF
B to compete for
limited amounts of target DNA. When added individually to the
reactions, both AP-2 and NF
B formed specific DNA-protein complexes
(Fig. 2). However, when AP-2 and NF
B
were added together, no DNA-protein complexes with slower mobilities were observed, arguing against simultaneous binding of these two transcription factors to the same DNA molecule. To examine their relative binding affinities, radiolabeled AP-2p probe was first incubated with a constant amount of AP-2 or NF
B. After a brief incubation, increasing amounts of NF
B or AP-2 were added to the reaction mixture. As shown in Fig. 2, the addition of AP-2 readily displaced the prebound NF
B from this overlapping region (lanes 8-12). At a 2.3-fold molar excess of AP-2, no NF
B remained
bound to the probe. In sharp contrast, NF
B had little effect on the prebound AP-2, even at a 28-fold molar excess of NF
B (lanes
2-6). Taken together, our results indicate that AP-2 and NF
B
bind to this overlapping region in a mutually exclusive manner.
Furthermore, AP-2 has much higher DNA binding affinity than NF
B. It
is therefore tempting to speculate that one mechanism by which AP-2 may
inhibit cytokine-induced SAA1 promoter function is by
antagonizing the ability of activated NF
B to bind to its cognate
binding site.
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Fig. 2.
Mutually exclusive binding of AP-2 and
NF B to the overlapping region. A limited
amount of labeled DNA probe (2 × 103 cpm, ~0.1 ng)
containing the overlapping AP-2- and NF
B-binding sites was incubated
with 0.6 ng of recombinant AP-2 (lane 1) or 2.2 ng of
recombinant NF
B (lane 7). AP-2 and NF
B were
preincubated with the probe for 15 min before an increasing amount of
NF
B (lanes 2-6) or AP-2 (lanes 8-12) was
added to the reaction mixtures. The AP-2·DNA and the NF
B·DNA
complexes were resolved on polyacrylamide gels. Positions of specific
protein-DNA complexes are indicated by the solid
arrows.
B- or C/EBP-binding sites
in the CRU completely abolished SAA1 promoter cytokine
responsiveness (40, 50), indicating the importance of these two
transcription factors in SAA1 gene transcription. Since we
showed that the NF
B-binding site overlaps with that of AP-2 and the
presence of AP-2 effectively prevents NF
B from binding to its
cognate site, we sought to determine whether AP-2 binding to the AP-2p
site is required for its repressive effects on the SAA1
promoter. Two reporter constructs, pSAA1/CAT(
120) (wild-type AP-2p
site) and pSAA1/CAT(
120)mAP-2p (mutant AP-2p site), were
generated. As determined by EMSA, the 2-bp mutation at the AP-2p site
completely abolished AP-2 binding but still retained full NF
B
binding activities (data not shown). These two reporter constructs were
cotransfected into HepG2 cells with the AP-2-expression vector, pSAP2,
to assess the effects of AP-2p site mutation on the ability of AP-2 to
repress the reporter gene expression. As shown in Fig.
3, the CAT activities for both the wild-type and the mutant constructs could be induced 5-6-fold by CM.
However, cotransfection with AP-2 expression vector completely inhibited CM-induced expression of the wild-type pSAA1/CAT(
120) reporter. In contrast, expression of AP-2 had no significant effects on
the cytokine responsiveness of the mutant
pSAA1/CAT(
120)mAP-2p reporter. As controls,
cotransfection with the frameshift AP-2 mutant construct had no
inhibitory effects on either reporter construct. These results indicate
that AP-2-mediated repression on a pSAA1/CAT(
120) reporter requires a
functional AP-2p site.
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Fig. 3.
Mutation of AP-2-binding site abolishes the
inhibitory effects of AP-2 on the SAA1 promoter.
Wild-type pSAA1/CAT( 120) (WT) and mutant
pSAA1/CAT(
120)mAP-2p (mt) reporter plasmids
were cotransfected into HepG2 cells with empty vector
(Vector), wild-type AP-2-expression vector pSAP2
(AP-2), or frameshift AP-2 mutant pSAP2/FS21
(fsAP-2). Approximately 20 h after transfection, cells
were treated with basal medium (
) or 50% CM (+). Cells were
harvested 20 h later for CAT assays.
285/
70)mAP-2d/p, in which both the distal and
the proximal AP-2 sites were mutated. As expected, the construct
containing the wild-type AP-2 sites, pTK/SAA1(
285/
70), was
nonresponsive to CM stimulation when transfected into HeLa cells,
presumably due to inhibition by the endogenous AP-2. Mutation of
AP-2-binding sites, however, resulted in derepression of
SAA1 promoter activity (Fig.
4). This result indicates that endogenous
AP-2 in HeLa cells can indeed inhibit the cytokine-induced SAA1 activation and that binding of AP-2 is required for its
repressive effects on the SAA1 promoter.
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Fig. 4.
Mutation of AP-2-binding sites derepresses
the SAA1 promoter in HeLa cells. Schematic
diagrams of wild-type pTK/SAA1( 285/
70) construct (WT) and the
double AP-2-binding site mutant
pTK/SAA1(
285/
70)mAP-2d/p (mt) are shown at
the top. HeLa cells were transfected with 8 µg of wild-type or mutant
CAT reporter plasmids and treated with (+) or without (
) CM. After
20 h of treatment, cells were harvested for CAT assays.
-fibrinogen and apolipoprotein E promoters were 4 and 20 times, respectively, less effective. The fact that these oligonucleotides could inhibit AP-2·DNA complex formation suggests that AP-2 may bind
to these sequences and perhaps regulate their expression in
vivo.
Putative AP-2-binding sequences in liver gene promoters
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Fig. 5.
Competition of various oligonucleotides from
liver gene promoters for AP-2 binding. The distal AP-2-binding
sequence from the SAA1 promoter was radiolabeled and used as
probe in EMSA. HeLa cell nuclear extracts were incubated with labeled
probe in the presence of increasing amounts of double-stranded
oligonucleotide competitors. The competing oligonucleotides were from
the promoters of rat SAA1 (AP-2d site), human C3, human
apolipoprotein B (ApoB), rat phosphoenolpyruvate
carboxykinase (PEPCK), rat 1-acid
glycoprotein (
1-AGP),
-fibrinogen
(
-FG), and apolipoprotein E (ApoE). The
efficiency of competition was calculated and presented as the amount of
oligonucleotides required for 50% inhibition of AP-2·DNA complex
formation.
199) reporter with or
without the AP-2-expression vector. When cotransfected with the empty
vector, the luciferase activity was dramatically induced (25-fold) in
response to CM stimulation. However, cotransfection with AP-2
expression vector pSAP2 resulted in more than 90% inhibition of the
pC3/Luc(
199) reporter gene activity (Fig.
6A), whereas cotransfection
with the frameshift AP-2 mutant pSAP2/FS21 had no inhibitory effects.
To further demonstrate that the inhibitory effects were specific for
AP-2, HepG2 cells were also transfected with the dominant-negative AP-2
mutant pSAP
166-277 (53). As shown in Fig. 6B,
coexpression of this mutant effectively neutralized the inhibitory
effects of AP-2 on human C3 promoter.
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Fig. 6.
AP-2 inhibits CM-mediated activation of human
C3 promoter in HepG2 cells. A, HepG2 cells were
cotransfected with pC3/Luc( 199) reporter construct and 0.5 µg of
wild-type AP-2-expression vector pSAP2 (AP-2) or frameshift
AP-2 mutant pSAP2/FS21 (fsAP-2). Approximately 20 h
after transfection, cells were treated with (+) or without (
) CM.
Cells were harvested 20 h after stimulation. Extracts were
prepared and assayed for luciferase activities. The fold induction of
luciferase activities was calculated relative to the nonstimulated
control sample, to which a value of 1.0 was assigned. B,
pC3/Luc(
199) DNA was cotransfected into HepG2 cells with 1 µg of
AP-2-expressing vector pSAP2 alone or with 1 or 2 µg of
dominant-negative AP-2 mutant pSAP2
166-277 construct
(AP2
166-277). Twenty hours after transfection, cells were treated
with (+) or without (
) CM. Relative luciferase activities were
calculated as above.
-Fetoprotein Genes--
Our transient
transfection experiments demonstrated that expression of AP-2 in HepG2
cells can inhibit the expression of transiently transfected reporter
genes driven by either SAA1 or C3 promoter. Together with
the oligonucleotide competition results that suggest many other liver
gene promoters contain AP-2-binding sites, we sought to examine whether
AP-2 may have similar effects on the expression of endogenous liver
genes in their normal chromosomal context. Stable Hep3B cell lines that
constitutively express moderate levels of AP-2 were generated by
transfecting AP-2 expression vector, pIND/AP-2, into cells followed by
selection with G418. Among the 11 stable cell lines isolated, three
clones, H3B.A1, H3B.A2, and H3B.A6, were chosen for further studies. As
controls, three Hep3B cell lines, H3B.V1, H3B.V2, and H3B.V3, that had
the vector DNA stably integrated were selected in a similar manner. The
AP-2 mRNA levels in these cell lines were determined by RT-PCR. As
with the parental Hep3B cells, no detectable AP-2 expression was
observed in H3B.V1, H3B.V2, and H3B.V3 cells (Fig.
7A). In contrast, AP-2
mRNA was easily detected in H3B.A1, H3B.A2, and H3B.A6 cells.
Likewise, AP-2 protein can be detected by Western blot in stably
transfected AP-2-expressing cells but not in the cells transfected with
vector only (data not shown). To determine AP-2 DNA binding activities
in these stable cells, EMSAs were performed using radiolabeled AP-2d
probe. Significant AP-2-binding activities were detected in H3B.A1,
H3B.A2, and H3B.A6 cells, whereas no detectable AP-2·DNA complexes
were observed in H3B.V1, H3B.V2, and H3B.V3 cells (Fig. 7B).
For comparison, HeLa cell extracts were also incubated with the AP-2d
probe. A strong AP-2·DNA complex with the same electrophoretic
mobility as those with the stable cell lines was detected. This
protein-DNA complex could be specifically competed by wild-type but not
mutant AP-2 oligonucleotides. When compared with HeLa cell extracts,
the levels of AP-2 in the stable cell lines were 2-3-fold lower (Fig.
7B).
View larger version (38K):
[in a new window]
Fig. 7.
Inhibition of SAA, albumin, and AFP
expression in AP-2-expressing Hep3B cells. A, AP-2
mRNA levels in stable AP-2-expressing Hep3B cells. Total RNAs were
isolated from G418-resistant Hep3B cells that contain the empty vector
(H3B.V1, H3B.V2, and H3B.V3) or the AP-2-expression vector (H3B.A1,
H3B.A2, and H3B.A6), and the levels of AP-2 and GAPDH mRNAs were
determined by RT-PCR. B, AP-2 binding activities in stable
AP-2-expressing Hep3B cells. AP-2d sequence was 32P-labeled
and used as probe in EMSA. Nuclear extracts from control and stable
Hep3B cell lines were incubated with labeled AP-2d probe. For
comparison, HeLa nuclear extracts were also incubated with the probe in
the absence ( ) or presence of wild-type (WT) or mutant
(mt) competitors. The position of the AP-2·DNA complex is
indicated by the solid arrow. C, inhibition of
CM-induced endogenous SAA gene expression. Control and
AP-2-expressing cell lines were treated with (+) or without (
) 50%
CM for 12 h to induce endogenous SAA gene expression.
After stimulation, total RNAs were isolated, and the levels of SAA and
GAPDH mRNAs were determined by RT-PCR. D, expression of
SAA, albumin, AFP, GAPDH, and
-actin in AP-2-expressing and control
cells. Total RNAs were isolated from control and AP-2-expressing Hep3B
cell lines, and mRNA levels for SAA, albumin, AFP, GAPDH, and
-actin were measured by RT-PCR. For SAA mRNA, comparisons were
made between CM-stimulated control and AP-2-expressing cells. The
expression levels of these genes in AP-2-expressing cells were
calculated relative to those in the vector control cells, to which a
value of 100 was assigned.
-actin were not altered. Taken together, our results
indicate that AP-2 when expressed in a liver-cell background is still
capable of conferring repression on inducible- and constitutively expressed liver-specific genes but has no effect on the expression of genes that are also expressed in nonliver cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B- and C/EBP-binding
sites, were essential for its promoter responsiveness to cytokine
stimulation (40, 50). In addition to the CRU, we identified two DNA
regulatory elements that have the characteristics of transcription
silencers and can serve as binding sites for the transcription factor
AP-2. Interestingly, AP-2 is expressed abundantly in HeLa and many
other nonliver tissues but is absent in liver or liver-derived hepatoma
cells (57, 58). This inverse correlation between AP-2 and
SAA1 expression is consistent with the repressive function
of AP-2 on SAA1 gene transcription and may contribute to
liver-specific expression pattern of SAA1 by diminishing or
abrogating its expression in nonliver cells.
B and C/EBP in the nucleus. In the absence of AP-2, as in
liver cells, these two potent transactivators could displace YY1 and
bind to their respective binding sites in the SAA1 promoter and function cooperatively to activate SAA1 gene
transcription (Fig. 8). In HeLa cells, however, in addition to YY1,
AP-2 is also constitutively expressed and could bind to its binding
site in the CRU. Because of its higher binding affinity, AP-2 prevents activated NF
B from binding to the overlapping binding site.
Consequently, without NF
B binding, the SAA1 promoter
cannot respond to cytokine induction despite the presence of activated
NF
B and C/EBP in the nucleus. Thus, in this case, the
tissue-specific expression of AP-2 itself plays an important role in
determining the expression pattern of SAA1. It is noteworthy
that removal of the repressive effects of AP-2 in HeLa cells by
mutating the two AP-2 sites did not lead to full activation of the
SAA1 promoter. This may reflect the nature of the cellular
milieu in HeLa cells, which is not optimum for SAA1 gene
transcription. For example, compared with HepG2 cells, HeLa has
relatively low levels of C/EBP, a critical transcription factor for
SAA1 gene activation. In addition, results from
AP-2-expressing cell lines suggest that AP-2 is likely not the only
repressor that inhibits SAA1 promoter activity in nonhepatic cells.
View larger version (16K):
[in a new window]
Fig. 8.
A model for inflammation-induced and
tissue-specific regulation of SAA1 gene
expression. A, schematic diagram of four known
transcription factors binding at the proximal region of the
SAA1 promoter with NF B-binding site flanked by two
repressors, YY1 and AP-2. B, in HepG2 cells, YY1 normally
binds and represses SAA1 gene transcription, accounting for
its low basal expression. Upon stimulation, activated NF
B and C/EBP
displace YY1 from the promoter and confer synergistic transcription
activation. In HeLa cells, however, the presence of AP-2 binding to its
high affinity site prevents activated NF
B from binding to the
overlapping site. Consequently, SAA1 gene transcription
remains in the repressed state after induction despite the presence of
NF
B and C/EBP in the nucleus.
Five lines of evidence support the notion that AP-2 plays a central
role in repressing SAA1 expression. First, two high affinity AP-2-binding sites were localized to within 300 bp of the
SAA1 gene transcription initiation site. Moreover, protein
binding studies indicated that the proximal AP-2 binding sequence that overlaps with that of NFB binds to AP-2 with significantly higher affinity than NF
B at these overlapping sites. The fact that
AP-2 binds with higher affinity and the constitutive presence of AP-2 in the nucleus plus the normally inactive state of NF
B in
nonstimulated cells gives AP-2 a distinct advantage over NF
B in
competing for the overlapping binding sites. Consequently, in cells
that express AP-2, i.e. many nonliver cells, this
overlapping site would normally be occupied by AP-2 and prevent
subsequent NF
B binding. Thus, AP-2 may mediate repression on the
SAA1 promoter by antagonizing the function of NF
B.
Second, a 20-bp region spanning the NF
B site to the AP-2p site is
100% conserved between rat and human SAA1 genes and 80%
with that of mouse SAA1 gene (40, 52). Such high sequence
conservation suggests functional importance of this region. Third,
forced expression of AP-2 in HepG2 cells efficiently inhibited
CM-mediated induction of the SAA1 promoter. This inhibition
was highly specific because mutation of the AP-2-binding sites or
expression of a dominant-negative mutant of AP-2 relieved AP-2-mediated
inhibitory effects. Fourth, whereas the wild-type SAA1
promoter is nonresponsive to cytokine stimulation when transfected into
HeLa cells, mutation of AP-2-binding sites led to derepression of the
promoter and responsiveness to cytokine induction. Finally, stable
expression of AP-2 in Hep3B cells significantly reduced endogenous
SAA gene expression in response to inflammatory cytokines.
Transcription repression may be achieved by various mechanisms. In
principle, negative regulators could inhibit transcription by
interfering with any of the several steps in the transcription initiation pathway (60, 61). A repressor might interfere with the
activity of a DNA-bound activator by quenching or masking its
activation potential (62). Alternatively, it may block nuclear localization of an activator (63). In addition, negative regulators might interfere with the function of general transcription machinery (6, 64, 65). Finally, repressors might block transcription activation
by binding to DNA at a region that overlaps that of transcription
activators, thus excluding the binding of activators (26, 41). In the
rat SAA1 promoter, one possible mechanism by which AP-2
represses the SAA1 promoter in HeLa cells is by blocking the
activity of the transactivator NFB. In this regard, it is also
interesting to note that although the 3' region of NF
B-binding site
overlaps with the AP-2p site, its 5' region overlaps with the binding
site of another transcription repressor YY1 (41). However, unlike AP-2,
YY1 is expressed in all tissues examined, including the liver, and
modulates the cytokine responsiveness of the SAA1 promoter
rather than controls its cell-specific expression. Another
distinguishing feature between these two repressors is that whereas
NF
B is unable to displace prebound AP-2 at the overlapping region,
it can readily displace prebound YY1 (41). Because the binding between
AP-2 and NF
B and between YY1 and NF
B at their respective
overlapping sites is mutually exclusive, the function of NF
B is
therefore opposed by two repressors, with relative binding affinities
at these overlapping sites of AP-2 > NF
B > YY1.
Transcription repressors that can function as general negative regulators and contribute to cell differentiation have been reported (10, 12, 66-68). One such negative regulator is the neuron-restrictive silencer factor, which could potentially repress a large number of neuron-specific genes in nonneuronal cells (10, 12). In this regard, the function of AP-2 in repressing liver gene expression in nonliver cells may resemble that of neuron-restrictive silencer factor. In addition to the SAA1 and C3 promoters, we identified potential AP-2-binding sites in at least 10 other liver gene promoters. Several of these sequences could effectively compete for AP-2 binding in EMSA, implying AP-2 may also bind to these promoters and perhaps regulate their expression. Thus, AP-2 may, by restricting its expression in AP-2-expressing nonliver cells, help regulate the expression of many liver genes in a cell-specific manner.
Our results indicate that AP-2, when expressed in a liver cell
background, is capable of conferring repression not only on cytokine-inducible genes but also on constitutively expressed liver-specific genes. The reduced expression of SAA, albumin, and AFP
was not due to nonspecific effects caused by the manipulation of cells
during G418 selection because (a) the comparisons were made
with control Hep3B cell lines that were transfected with an empty
vector and were selected in a similar manner, (b) consistent repression was observed in three independent AP-2-expressing Hep3B cell
lines, and (c) the expression of two housekeeping genes, GAPDH and -actin, in these cell lines was not altered. Therefore, together with our transient transfection studies, these results further
support the notion that AP-2 may have broad functional role as a
general negative regulator in repressing the expression of some liver
genes in nonhepatic cells. Although AP-2 may possess the ability to
repress the expression of certain liver genes, it is unlikely to be the
only regulatory factor responsible for the tissue-specific repression
of these liver genes. An indication is that although the levels of SAA,
albumin, and AFP were reduced in AP-2-expressing Hep3B cells, they were
nevertheless significantly higher than that in HeLa or other nonliver
cells. Therefore, repression of liver genes in nonliver cells is likely
to be determined by multiple factors, including other repressors and
chromatin structures. Together with AP-2, these determinants contribute
to the silencing of SAA1 and other liver genes in nonliver
cells. It should be noted that although overexpression of AP-2 in
hepatic cells could repress the expression of a number of liver genes,
our results could not conclusively prove the converse would be true.
That is, removal or reduction of AP-2 in nonhepatic cells will lead to
enhanced expression of SAA or other liver genes.
In summary, we report that transient expression of AP-2 in HepG2 cells
effectively inhibited cytokine-induced expression of SAA1
and human C3 promoter activities. This inhibition was dependent on
functional AP-2-binding sites and could be neutralized by a dominant-negative mutant of AP-2. In addition to SAA1 and
C3, 10 other liver gene promoters were found to contain potential AP-2-binding sites, some of which are very effective competitors for
AP-2 binding. Finally, stable expression of AP-2 in a liver cell
background significantly reduced the expression of several endogenous
liver genes. Although it is tempting to speculate that the observed
inhibition in human SAA1 expression in AP-2-expressing Hep3B
cells was due to the repressive effects of the conserved AP-2p site,
additional studies are required to specifically address this issue.
Taken together, our studies suggest that AP-2 may function as a general
transcription repressor that not only inhibits SAA1 gene
transcription in nonhepatic cells but also as a contributor to the
cell-specific expression of other liver genes.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. M. Tainsky and P. Kannan for providing the AP-2 expression vectors and Dr. G. Darlington for the human C3 promoter construct. We also thank Karen Hensley and Rama Grenda for preparing the graphics.
![]() |
FOOTNOTES |
---|
* This research was supported in part by National Institutes of Health Public Health Service Grant AR 38858 (to W. S.-L. L.).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: Dept. of Biochemistry
and Molecular Biology, Box 117, Program in Genes and Development, The
University of Texas M. D. Anderson Cancer Center, Houston, Texas
77030. E-mail: liao@ijm.jussieu.fr or
wliao@odin.mdacc.tmc.edu.
Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M010307200
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ABBREVIATIONS |
---|
The abbreviations used are:
C/EBP, CCAAT/enhancer binding protein
;
SAA1, serum amyloid A1;
bp, base pair(s);
CRU, cytokine response unit;
CM, conditioned medium;
RT-PCR, reverse transcription-polymerase chain reaction;
CAT, chloramphenicol
acetyltransferase;
TK, thymidine kinase;
AFP,
-fetoprotein;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
EMSA, electrophoretic
mobility shift assay;
mAP-2, mutant AP-2.
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