The Albert Einstein Cancer Center, Departments of Medicine and
Developmental and Molecular Biology Albert Einstein College of
Medicine Bronx, New York 10461
Department of Biology
(A.D., T.W.) Yale University New Haven, Connecticut 06520
Division of Endocrinology, Metabolism, and Molecular
Medicine (I-W.S.) Northwestern University Medical School
Chicago, Illinois 60611
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The CYP11A1 gene promoter regions of a number of different species contain conserved GC-rich DNA sequences governing basal and regulated expression (11, 12, 13, 14, 15). The ovine CYP11A1 promoter GC-rich sequences are referred to as ovine footprint 5 (OF5) and OF3 because nuclear proteins from human trophoblastic JEG-3 cells and ovine primary placental and adrenal cells bound these sequences in deoxyribonuclease 1 (DNAse 1) footprinting experiments (11). These GC-rich sequences were shown to be well conserved between species (11, 16). The porcine CYP11A1 promoter contains GC-rich sequences involved in IGF-responsive gene expression (10). One of the human CYP11A1 promoter GC-rich sequences binds Sp1-like proteins and conveys basal level regulatory function (17). The ovine and bovine CYP11A1 promoter GC-rich sequences control basal level and cAMP-responsive function (7, 16). The sequences governing cAMP-regulated expression of the human CYP11A1 gene in JEG-3 cells resembles an activator protein-2 (AP-2) binding site (13, 14, 15). Thus, GC-rich sequences, conserved between the CYP11A1 promoters of several species, play a role in basal and/or cAMP-regulated expression, do not resemble the canonical cAMP response element (CRE) known to bind CREB (18, 19), and may involve AP-2 and/or Sp1 transcription factors.
AP-2 was originally isolated as a protein binding to GC rich basal regulatory sequence of the SV40 and human metallothionein IIA (hMT IIA) promoters (20, 21). AP-2 is expressed in the trophoblast (22) and widely during development (23, 24). Activation of the cAMP pathway has been shown to increase AP-2 activity in Hela cells (21, 25), and AP-2 responsive elements have been identified in the regulatory regions of the genes encoding the murine major histocompatability complex H-2 Kb, collagenase, human GH, proenkephalin, ornithine decarboxylase, keratin 14, CG ß-subunit, and vascular endothelial growth factor (VEGF) (20, 21, 22, 25, 26, 27, 28). AP-2 can also form heterodimeric complexes binding Myc to regulate gene transcription through a Myc-binding site (27), and the AP-2 response element of the VEGF promoter is a GC-rich enhancer that bound Sp1 proteins (28), suggesting that AP-2 may regulate gene transcription through combinatorial interactions. Sp1 is a widely expressed transcription factor of approximately 100 kDa that binds GC-rich sequences through a DNA-binding domain consisting of three C-terminal zinc fingers (29). The activity of Sp1 is generally constitutive but can be regulated (30, 31). Sp1 activity can be induced directly by O-glycosylation (32, 33) or indirectly by other proteins such as the retinoblastoma protein pRB, which can induce Sp1 transactivation function (34).
Because AP-2 can bind to GC-rich sequences (GCC NNN GGC) and has been implicated in basal and cAMP-regulated gene expression and the conserved GC-rich sequences of the CYP11A1 promoters convey basal and/or cAMP-regulatory function, we evaluated a role for AP-2 in regulating the ovine CYP11A1 gene promoter. AP-2 induced the CYP11A1 promoter through the GC-rich conserved DNA sequences OF5 and OF3 in JEG-3 cells. Deletion within the basic helix-span-helix (bH-S-H) region of AP-2 abolished induction of the native CYP11A1 promoter. The AP-2 responsive elements bound Sp1 and Sp3 in JEG-3 cell nuclear extracts but did not bind AP-2. AP-2, however, directly associated with Sp1 in both native JEG-3 cells and in vitro binding assays. Deletion of the bH-S-H domain of AP-2 or the carboxy terminus of Sp-1 abolished binding. When Sp1 and Sp3 were linked to a GAL4 DNA-binding domain and assessed using a heterologous reporter system dependent upon GAL4 DNA binding only, AP-2 induced Sp1 and Sp3 activity. Thus, induction of Sp1/Sp3 transactivation by AP-2 can occur independently of AP-2 binding to DNA. These studies suggest AP-2 may regulate CYP11A1 promoter activity through direct association with Sp1.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
To identify AP-2-responsive sequences within the
CYP11A1 promoter, several CYP11A1 5'-promoter
deletion constructs were examined in the presence of the AP-2
expression vector (Fig. 2). The reporter
-2700 CYPLUC was induced 4.5-fold, the -117 fragment was induced
7-fold, and the -92 CYPLUC reporter was induced only 2-fold,
suggesting elements located between -117 and -92 are also required
for optimal induction by AP-2 (Fig. 2A
). Further deletion to -77
resulted in a fragment that was induced 5-fold by AP-2, and deletion to
-55 abolished AP-2 activation of CYP11A1. PCR-directed
point mutation of the OF3 sequence within the -77 bp fragment was
performed to produce the mutant reporter -77 CYPOF3mutLUC. The OF3
sequence was mutated in the context of the AP-2-responsive -77 bp
fragment from CCG CCC TGT to CaG aaC TGT (Fig. 2B
, inset).
The effect of AP-2 on this mutant reporter was examined in JEG-3 cells
(Fig. 2A
). Unlike the wild type -77 CYP reporter, the -77CYPOF3mtLUC
reporter was not induced by the overexpression of AP-2 (Fig. 2A
). These
results suggested that AP-2-responsive sequences were located between
-117 and -92 and between -77 and -55.
|
OF3 Conveys Basal Level Activity in JEG-3 Cells
Previous studies had shown that the CYP11A1 promoter
OF5 region contained basal level regulatory elements that could also
function as a cAMP-responsive element in JEG-3 cells (7). The
homologous sequences of the bovine CYP11A1 promoter and a
related GC-rich sequence homologous to OF3 also functioned as a basal
regulatory element and cAMP-responsive sequences in murine Y1 adrenal
cells (16). We examined the basal level activity of the OF3 sequence in
JEG-3 cells (Fig. 3). To examine the
basal level function of OF3 in the context of the native promoter,
comparison was made between the -77 CYPLUC and the -55 CYPLUC
constructions. Basal level activity was reduced approximately 50% by
deletion from -77 to -55 (Fig. 3A
). To determine whether OF3 conveyed
basal activity in JEG-3 cells in the context of an heterologous
promoter, the basal activity of the OF3 reporter was compared with the
parental TKLUC reporter. The basal activity of (OF3)2TKLUC
was 6-fold higher than TKLUC, and the basal activity of
(AP2)2TKLUC was 10-fold higher than TKLUC (Fig. 3B
). These
findings suggest that OF3 conveys basal level activity in JEG-3
cells.
|
|
To examine further the role of the AP-2 bH-S-H domain in
regulation of the CYP11A1 promoter, the -77
CYP11A1 promoter fragment was assessed with the AP-2 mutant
(AP-2 N278). The -77 CYP11A1 promoter fragment was
examined because it contained the proximal 5'-promoter sequences
induced by AP-2. The induction of -77 CYPLUC reporter activity by AP-2
was reduced 80% by deletion of the amino terminus (Fig. 4B
). The
heterologous reporter containing the OF3 sequence was also examined
with either the wild-type or the AP-2 mutant expression vectors to
determine the role of the amino terminus that includes the bH-S-H
domain (Fig. 4C
). In these experiments the OF3 reporter was induced
2.7-fold by AP-2 (Fig. 4C
). The induction by AP-2 was sustained for the
AP-2
N165 mutant; however, further deletion to AP-2
N278
abolished induction of the promoter. The loss of induction with
deletion of the bH-S-H domain, rather than the amino-terminal
activation domain, distinguishes the induction of the
CYP11A1 promoter mechanistically from the induction of the
hMT IIA promoter. These studies provide further support for a role of
the AP-2 bH-S-H domain in CYP11A1 activation through
OF3.
The Basal Regulatory Sequences of OF and OF3 Bind Sp1 and Sp3 in
JEG-3 Cells
Electrophoretic mobility shift assays (EMSA) were performed
using JEG-3 cell nuclear extracts to characterize nuclear protein
interactions with OF5 and OF3 (Fig. 5).
The mobility of the complex binding the CYP11A1 OF3 and OF5
site was similar (Fig. 5
, A and B). The OF3-binding complex consisted
of three bands (AC), which were competed by 100-fold excess of either
self-competitor or Sp1 sequences (Fig. 5A
, lanes 23) but not by an
equimolar excess of cold AP-2 oligodeoxynucleotides (not shown).
Addition of Sp1 antibody supershifted the band labeled A (Fig. 5A
, lane
6), and the Sp3 antibody supershifted complexes B and C (Fig. 5A
, lane
8). The addition of Sp2 antibody (Fig. 5A
, lane 7), preimmune serum
(lane 5) or AP-2 antibody (lane 10) was without effect. In similar
experiments with the OF5 sequence (Fig. 5B
), the Sp1 antibody
supershifted complex A (lane 6), and the Sp3 antibody shifted complexes
B and C (lane 8). The OF5 complex was not shifted with antibodies to
Sp2, Sp4, or AP-2 (lanes 7, 9, and 10) and was not shifted with
preimmune serum (lane 4). An additional faster migrating band was
observed with both OF5 and OF3; however, the naure of this complex is
currently unknown.
|
Endogenous AP-2 and Sp1 Associates in JEG-3 Cells
Our studies indicated that OF5 and OF3 bound Sp1 and Sp3 in JEG-3
cells. We hypothesized that AP-2 may induce CYP11A1 through
these sequences by one or more different mechanisms. AP-2 may enhance
the transactivation function of Sp1/Sp3. Alternatively, AP-2 may
interact with Sp1 or alter the abundance of Sp1. Finally, it was
possible that AP-2 may affect one or more of the many proteins that
have been shown to interact with Sp1 and thereby indirectly affect Sp1
activity. For example AP-2 might recruit coactivator proteins known to
regulate Sp1 activity. We first sought to determine whether AP-2 could
directly interact with Sp1. We examined the immunospecificity of the
Sp1 and AP-2 antibodies, by introducing expression plasmids encoding
the cDNAs for AP-2 and Sp1 into 293 cells. The 293 cell derivative BOSC
23 was chosen because of its high transfection efficiency. Cells were
harvested 48 h after transfection with expression vectors for AP-2
and Sp1. Western blotting was performed using specific antibodies for
Sp1 and AP-2. Western blotting with the Sp1-specific antibody
identified an immunoreactive band at 97 kDa (Fig. 6A, upper panel, lane 4). The
relative abundance of this band was increased in the cells transfected
with the Sp1 expression vector (Fig. 6A
, upper panel,
compare lane 1 and lane 3). This immunoreactive band was of identical
mobility to that observed with in vitro translated Sp1 (not
shown). Western blotting of the same extracts was also performed using
an AP-2-specific antibody. An immunoreactive band was identified at
approximately 46 kDa (Fig. 6A
, middle panel, lanes 2, and
4), which was identical in mobility to in vitro translated
AP-2 (Fig. 7
). Reprobing of the
Western blot with anti-
-tubulin antibody demonstrated equal amounts
of protein in each lane (Fig. 6A
, lower panel).
|
|
It was important to determine whether the association observed in 293
cells through overexpression occurred normally in JEG-3 cells. To
determine whether Sp1-immunoreactive material was associated with
endogenous AP-2 in JEG-3 cells, IP was performed with either an Sp1 or
an AP-2 antibody. (Unlike the 293 cell experiments, the JEG-3 cells
were not transfected with expression plasmids encoding either Sp1 or
AP-2). The immunoprecipitates were electrophoresed on an 10% SDS-PAGE
and subjected to Western blotting with an Sp1 antibody. An
Sp1-immunoreactive band was identified at 97 kDa in cell extracts
immunoprecipitated with either the Sp1 or the AP-2 antibody (Fig. 6C, lanes 2 and 3). The specificity of the IP was confirmed by the finding
that equal amounts of a nonspecific antibody (Fig. 6C
, lanes 1) or IgG
(not shown) did not precipitate any Sp1 immunoreactive material.
In Vitro Protein-Binding Assays
Together, these studies indicate that an AP-2-immunoreactive
antibody is capable of coprecipitating Sp1 in JEG-3 cells but do not
demonstrate whether a direct interaction occurs between these two
proteins. To extend these observations, a
glutathione-S-transferase (GST) fusion protein for Sp1 was
incubated with in vitro synthesized AP-2, and IP was
performed with an AP-2-specific antibody (Fig. 7A, lane 1).
35S-labeled AP-2 was immunoprecipitated by the AP-2
antibody as expected (Fig. 7
, lane 1). When GST-AP-2 was coincubated
with in vitro translated Sp1 and immunoprecipitated with the
AP-2 antibody (C-18, Santa Cruz Biotechnology, Inc., Santa
Cruz, CA), the band corresponding to 35S-labeled Sp1 was
observed at 97 kDa (Fig. 7A
, lane 2). This interaction was not observed
when GST-AP-2 was incubated with an unrelated in vitro
translated protein (Fig. 7A
, lane 3). To confirm the presence of AP-2
in the GST-AP-2 used to immunoprecipitate the in vitro
translated Sp1, Western blotting was performed on a component of the IP
using the AP-2-specific antibody 5E4 (24), and the AP-2 immunoreactive
band was identified in Fig. 7A
, lane 4.
The reciprocal coimmunoprecipitation also provided further evidence for
an interaction between Sp1 and AP-2 (Fig. 7B, lane 1). GST-Sp1 was
coincubated with 35S-labeled AP-2 and immunoprecipitated
with the Sp1 antibody. The immune complexes were resolved on a
denaturing gel. An 35S-labeled band corresponding to AP-2
was identified in the Sp1 immunoprecipitate (Fig. 7B
, lane 1). In
vitro translated Sp1 was immunoprecipitated with the Sp1 antibody
(Fig. 7B
lane 3), and immunoreactive AP-2 was identified in the Sp1
immunoprecipitate by Western blotting (Fig. 7B
, lane 4). Western
blotting with the AP-2 antibody 5E4 confirmed the presence of GST-AP-2
associated with in vitro translated Sp1 in the IP performed
with the Sp1 antibody (lane 4), but not with an unrelated control
in vitro translate (lane 5). Together, these studies provide
evidence for an association between AP-2 and Sp1 using in
vitro binding assays.
To characterize this interaction further, in vitro binding
assays were performed using GST-Sp1 and in vitro translated
AP-2. Comparison was made with equal amounts of either GST protein or
[35S]methionine-labeled control proteins. The full-length
GST-Sp1 bound in vitro translated AP-2 (Fig. 7C, lane 2).
The specificity of this interaction was indicated by the undetectable
binding of in vitro translated AP-2 to equal amounts of GST
protein (Fig. 7C
, lane 1). In vitro translated Sp1 also
bound the full-length GST-AP-2 (Fig. 7C
, lane 3), and the specificity
of this interaction was supported by the finding that no binding was
observed between in vitro translated Sp1 and GST protein
(Fig. 7C
, lane 4). Western blotting of the GST pull-down product of
GST-AP-2 and in vitro translated Sp1 using the AP-2-
specific antibody 5E4 demonstrated an AP-2-immunoreactive band in lane
5.
To identify the domains of AP-2 and Sp-1 required for this
protein-protein interaction, experiments were conducted with either
wild-type or mutant expression plasmids shown schematically in Fig. 7D.
The programmed lysate product for AP-2 (PL) (lane 1) and AP-2
N278
(lane 4) were incubated with GST-Sp1 proteins. In vitro
translated wild-type AP-2 bound to wild-type GST-Sp1 as shown in Fig. 7D
(lane 3). To identify the region of AP-2 required for binding to
Sp1, the amino-terminal deletion mutant AP-2
N278 was produced by
in vitro translation (IVT AP-2
, lane 4) and its abundance
assessed on SDS-PAGE, and the protein was coincubated with GST-Sp1
protein. In contrast with the binding of wild-type AP-2 to Sp1, no
binding was observed between Sp1 and the AP-2
N278 deletion mutant
(Fig. 7D
, lane 6). A 5-fold longer exposure did not show any evidence
of specific binding (data not shown).
The carboxy-terminal domain of Sp1 had previously been implicated in
protein-protein interactions with GATA-1, YY1, and E2F-1. To determine
whether the Sp1 carboxy-terminus was required for AP-2 binding, an Sp1
deletion mutant that lacks the carboxy-terminal zinc finger domain
(GST-Sp1AB) was used (37) (Fig. 7D). Equal amounts of wild-type or
mutant Sp1 GST fusion protein (GST-Sp1AB) was incubated with equal
amounts of in vitro translated AP-2 protein. We confirmed
that the amount of GST-Sp1AB protein was equal to the amount of wild
type Sp1 protein through GST Western blotting of the protein
transferred from an SDS-PAGE (data not shown). The binding of AP-2 to
Sp1 was reduced approximately 90% by deletion of the Sp1 carboxy
terminus (Fig. 7D
, lane 8 vs. 9). Together, these findings
demonstrate that the carboxy terminus of Sp1 is required for optimal
binding to AP-2.
The Domains of AP-2 Required for Induction of Sp1 Transactivation
Function
Because AP-2 did not bind the AP-2 response element of the
CYP11A1 promoter, the current studies supported a model in
which AP-2 activated CYP11A1 independently of binding to
DNA. To examine the possibility that AP-2 could induce Sp1/Sp3 activity
independently of AP-2 binding to DNA, we used an heterologous reporter
system (UAS)5E1BTATALUC (38), which consists of multimeric
GAL4 DNA-binding sites linked to a luciferase reporter gene. The Sp1
and Sp3 transactivation domains were linked to the GAL4 DNA-binding
domain (Fig. 8A), and the basal and
AP-2-regulated activity of Sp1 and Sp3 were examined in conjunction
with the heterologous reporter construction. Sp1 and Sp3 conveyed basal
enhancer function, and overexpression of the wild-type AP-2 expression
vector enhanced Sp1 and Sp3 activity approximately 3-fold
(P < 0.05) (Fig. 8C
). AP-2 did not induce the
(UAS)5E1BTATALUC reporter in the absence of the GAL4-Sp
constructions (as shown in Fig. 1A
). Because the
(UAS)5E1BTATALUC reporter does not contain AP-2 binding
sequences, these studies demonstrate that AP-2 can activate Sp1/Sp3
transactivation function without binding to DNA.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
AP-2 can activate gene expression through binding a DNA sequence
identified in the promoter region of many different genes (35). The DNA
sequences implicated in signaling by AP-2, however, do not always
resemble an AP-2 site (27), and the proline-rich activation domain of
AP-2 is a poor transactivator when positioned in a distal position
(39), suggesting AP-2 may function through combinatorial mechanisms.
Our findings that AP-2 formed a protein-protein complex with Sp1 to
regulate CYP11A1 promoter activity are consistent with
recent studies showing that AP-2 forms a heteromeric protein-protein
interaction with c-Myc (27) and E1A (40) to regulate gene
transcription. AP-2 heterodimerized with c-Myc to activate the
ornithine decarboxylase gene through a Myc-binding site (27). As with
our studies in which induction of the CYP11A1 promoter
required the AP-2 basic helix-span-helix (bH-S-H) domain and extreme
carboxy terminus, the AP-2 bH-S-H domain was also required for the
interaction with c-Myc (27) and the adenovirus E1A protein in
vitro (40). AP-2 homodimerizes in solution or when bound to DNA
via a H-S-H motif (35). Heterodimerization with AP-2 can occur either
with other AP-2 isoforms or with the products of the related AP-2ß
and AP-2 genes (41, 42). The bH-S-H motif of AP-2 may form a surface
capable of interacting with several different proteins as this region
was also necessary for binding to the amino terminus of the adenovirus
E1A protein (40). The carboxy terminus of AP-2 was required for
regulation of c-Myc transactivation function (27) and Sp1 activity when
linked to a GAL4 DNA-binding domain. These studies demonstrate that
AP-2 is capable of regulating gene transcription indirectly through
forming heteromeric protein-protein interactions and suggest a common
surface of AP-2 may be involved.
When Sp1 and Sp3 were linked to a GAL4 DNA-binding domain and their transactivation function assayed using a heterologous reporter containing only GAL4 DNA-binding sequences, AP-2 induced their activity 3- to 4-fold. Previous studies had demonstrated that Sp1 transactivation function can be induced by several different proteins including the retroviral oncoprotein v-Rel (43) and the retinoblastoma protein pRB (34). Induction of Sp1 transactivation function in our studies required the AP-2 bH-S-H domain and the extreme carboxy terminus. The physical interaction between Sp1 and AP-2 that we observed in native JEG-3 cell extracts required the Sp1 carboxy terminus when examined using in vitro pull-down assays. The carboxy-terminal zinc finger DNA-binding domain of Sp1 was also involved in binding to GATA-1 (44), YY1 (45), E1A (46), and E2F-1 (47). It has been proposed that the interactions between Sp1 and these additional transcription factors facilitate activity of specific target genes. It remains to be determined whether the AP-2/Sp1 complex functions as a molecular bridge to coactivator proteins and the basal transcription apparatus.
AP-2 is capable of inducing promoter activity through sequences that are responsive to cAMP (21, 22, 48, 49). The site of cAMP-induced expression of the acetyl-CoA carboxylase gene, for example, binds AP-2 (50). The human CG ß (hCGß) subunit gene promoter also contains an important basal level enhancer and cAMP-responsive region (CRE) that bind AP-2 (22, 50). The GC-rich OF5 sequence was previously shown to convey basal level and cAMP responsiveness in JEG-3 cells (7). The ovine CYP11A1 promoter is induced by cAMP in murine adrenal Y1 cells (11, 51), and the homologous regions corresponding to OF5 and OF3 of the bovine CYP11A1 promoter bound Sp1 from Y1 cell extracts and conveyed cAMP responsiveness (16). The bovine CYP17 gene CRE bound Sp1 in thecal and luteal cells; however, the binding affinity to these sites was unchanged by forskolin treatment (52), and the binding of Sp1 and Sp3 to OF5 and OF3 was unchanged after 24 h of cAMP treatment of JEG-3 cells (P. Pena and R.G. Pestell, unpublished). Although the expression of AP-2 is increased by cAMP in primary astrocytes (41) and in JEG-3 cells (P. Pena, R.G. Pestell, unpublished), consistent with a role for AP-2 as an intermediary protein in cAMP-induced gene expression, the role of AP-2 in cAMP-mediated activation of the ovine CYP11A1 gene remains to be determined. We are currently investigating whether cAMP regulates AP-2 binding to Sp1 in JEG-3 cells or whether cAMP recruits the coactivator PC4 to AP-2. In this regard it is of interest that the mutants of AP-2 that were defective in binding PC4 (53) were also defective in activating the -2700CYPLUC reporter. The promoter regions of a number of steroidogenic genes including the CYP21, CYP17, CYP19, CYP11A1, and ferrodoxin gene (7, 52, 54, 55) contain GC-rich sequences that function as important basal level regulatory elements. In some circumstances these GC-rich sequences convey cAMP-mediated enhancer activity (7, 52, 54, 55). As many of the steroidogenic CYP genes are coordinately induced by hormonal stimuli (1, 56), it will be of interest to determine the role of AP-2 in regulating the other CYP genes.
The current studies suggest a model in which the Sp1/Sp3 binding sites
of the CYP11A1 promoter are required for induction by AP-2
(Fig. 9). Adrenal and ovarian
steroidogenic cells contain SF-1, which can both bind DNA sequences in
the CYP11A1 promoters of several species (57, 58, 59) and bind
to Sp1 in two-hybrid assays (57). In steroidogenic cells containing
SF-1, the coactivator p300 is brought to the basal apparatus through
SF-1 (60). Placental cells, including JEG-3 and RCHO-1, are deficient
in SF-1 (4, 61). In the current studies, AP-2 activated the
CYP11A1 promoter through OF3, which bound Sp1, but not AP-2,
in EMSA using JEG-3 cell extracts. Because there is flexibility in the
requirement for G or C sequences in the consensus AP-2 site (GCC NNN
GGC), the core of OF3 (CGC CCT GTC) differs primarily by the eighth
nucleotide from the AP-2 site, being T rather than G/C. Mutation of OF3
(from 5'-CGC CCT GTC-3' to 5'-aGa aCT GTC-3') abolished induction by
AP-2. Endogenous AP-2 in JEG-3 cells bound Sp1 in IP-Western blot
analysis. AP-2 activated Sp1 when it was linked to a GAL4 DNA-binding
site in a reporter system that contained only GAL4 DNA-binding
sequences. These studies indicate that AP-2 is capable of inducing Sp1
activity independently of an AP-2 binding site. These studies suggest
induction of CYP11A1 by AP-2 can occur independently of
binding DNA, but do not formally exclude the possibility that AP-2 can
bind to other sequences in the CYP11A1 promoter. We propose
that AP-2 may form a bridge to Sp1 in JEG-3 cells, which contain AP-2
but lack SF-1, and thereby recruit coactivator (s) such as PC4 to the
basal apparatus (Fig. 9
). In addition to placental JEG-3 cells, AP-2
may also regulate CYP11A1 in other SF-1-deficient tissues,
including the CNS and primitive gut, that express CYP11A1
(referenced in Refs. 3, 61, 62). In recent studies AP-2 was shown
to directly bind the coactivator PC4 (53). PC4 stimulates
transcriptional activity of several different activation surfaces,
including the glutamine-rich activation surface of Sp1 (63). A recent
model suggests that several PC4 molecules stabilize interactions
between multiple proteins in the preinitiation complex through pairwise
interaction (64), raising the possibility that through binding Sp1,
AP-2 may recruit PC4 to the CYP11A1 promoter preinitiation
complex (Fig. 9
).
|
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The wild-type and mutant RSV-driven mammalian AP-2 expression vectors
were previously described (35) (Fig. 4). The vector AP-2
C390 has a
deletion of the carboxy terminus between 390 and 437, which abolishes
DNA binding; AP-2
C413 contains a 24-amino acid carboxy-terminal
deletion. AP-2 Int 97/165 has an internal deletion of the acid-rich
activation domain. AP-2 INT/31/77 contains a deletion of the
proline-rich activation domain. AP-2
N51 and AP-2
N278 encode
sequential amino-terminal deletions of AP-2. In previous studies
nuclear and cytoplasmic extracts were prepared from transfected HepG2
cells, and the presence of AP-2 derivatives was determined by
immunoblotting (35). The data indicated that the wild-type and mutant
constructs containing an intact DNA-binding domain were all expressed
and translocated to the nucleus (35). CMV-Sp1 encodes the full-length
Sp1 protein and was a gift from Dr. G. Gill and Dr. R. Tjian. The Sp1
and Sp3 activation domains were linked to the GAL4 DNA-binding domain
to form GAL4-Sp1 (GAL4-Sp1(83621) (29, 43) (a gift from Dr. G. Gill),
and GAL4-Sp3(1382) (a gift from Dr. J. Horwitz). The wild-type AP-2
cDNA (35) was cloned into either pT7ßSal to form pT7ßSal AP-2 or
the GST expression vector pGEX2TK to form pGEX2TKAP-2 wt. The
amino-terminal deletion fragment of AP-2 N278 was cloned in frame into
pT7ßSal to form pT7ßSal AP-2
N278. GST-Sp1 and the C-terminal
deletion fragment of Sp-1 (Sp-1 AB) (37) were a gift from Dr. J.
Horwitz, and the in vitro expression plasmid T3-FL-Sp1
plasmid was a gift from Dr. R. Tjian. The luciferase T7 control DNA and
luciferase T3 control DNA were from Promega Corp.
(Madison, WI).
Cell Culture, DNA Transfection, and Luciferase Assays
Cell culture, DNA transfection, and luciferase assays were
performed as previously described (7, 65). JEG-3 choriocarcinoma cells
(American Type Culture Collection, Manassas, VA) and CV-1
cells (a green monkey kidney cell line) were cultured in DMEM with 10%
FCS, 1% penicillin, and 1% streptomycin. The 293 cell line
derivative, BOSC 23, was a gift from Dr. D. Baltimore (66). Cells were
transfected by calcium phosphate precipitation, the medium was changed
after 6 h, and luciferase activity was determined after 48 h.
At least two different plasmid preparations of each construct were
used. In cotransfection experiments, a dose response was determined in
each experiment with 300 ng and 600 ng of expression vector and the
CYP11A1 promoter reporter plasmids (4.8 µg). Luciferase
assays were performed at room temperature using an Autolumat LB 953
(EG&G Berthold, Gaithersburg, MD). Luciferase content was
measured by calculating the light emitted during the initial 10 sec of
the reaction, and the values are expressed in arbitrary light units
(ALU). Background activity from cell extracts was typically less than
150 ALU/10 sec. The -fold effect was determined for 300600 ng of AP-2
expression vector with comparison made to the effect of the empty
expression vector cassette (RSV) or the mutant vector C390, and
statistical analyses were performed using the Mann-Whitney U
test. Significant differences were established as P <
0.05.
Western Blots
For the detection of AP-2, Sp1, CYP11A1, GST, and -tubulin
protein, cell extracts were prepared as previously described (67).
Western blotting was performed using antibodies to the rat Cyp11a1 (51, 68), the AP-2 monoclonal antibody 5E4 (24), an
-tubulin monoclonal
IgM antibody (5H1) (69), and several antibodies from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) including antibodies to
AP-2 (C-18), GST (B-14), Sp1 (SC-1C6), and E2F-1 (C-20). Reactive
proteins were visualized using an antirabbit horseradish peroxidase
second antibody for CYP11A1 and AP-2 and an antimouse antibody for
-tubulin and AP-2 (5E4) and the enhanced chemiluminescence system
(Kirkegaard & Perry Laboratories, Gaithersburg, MD). The
abundance of immunoreactive protein was quantified by phosphoimaging
using a Computing Densitometer (Image Quant version 1.11,
Molecular Dynamics, Inc., Sunnyvale, CA).
EMSA and DNAase 1 Footprinting
The oligodeoxyribonucleotides used in EMSA correspond to the
CYP11A1 promoter OF3 region from -80 to -57 (5'-TGG AGG
AAG CTG ACC GCC CTG TCA-3'), the OF5 region (-112 5' GTT TGG GAG GAG
CTG TGT GGG CTG-3'), the wild-type AP-2 site (Promega Corp.), 5'-AGC TGA CCG CCC GCG GCC CGT-3'), and the wild-type
Sp1 site (Promega Corp.), (5'-ATT CGA TCG GGG CGG GGC
GAG-C3'). Nuclear extracts from JEG-3 cells were prepared as previously
described (70). Five micrograms of nuclear extract protein were mixed
with binding buffer (20 mM HEPES, pH 7.9, 80 mM
KCl, 5 mM MgCl2, 2% Ficoll, 5% glycerol, 0.1
mM EDTA), 25 ng/µl single-strand (ss)DNA, and
competitor or antibodies, on ice for 1 h. For competition assay,
10 pmol of unlabeled competitor oligonucleotide were used. In the case
of antibody supershift experiment, the incubation conditions were
identical, and 1 µl of anti-Sp1 (1C6), anti-Sp2 (K-20), anti-Sp3
(D-20), anti-Sp4 (V-20), or anti-AP-2 (C-18) (from Santa Cruz Biotechnology, Inc.) was used in each reaction. The
32P-labeled probe (10,000 cpm) was added after 1 h and
incubated at room temperature for 30 min. The protein-DNA complexes
were analyzed by electrophoresis through a 5% polyacrylamide gel in a
0.5 x TBE buffer (0.045 M Tris-borate, 0.001
M EDTA) and 2.5% glycerol. Autoradiography was performed
at -70 C using XAR5 film (Eastman Kodak Co., Rochester,
NY) with an intensifying screen.
Interactions between AP-2 and Sp1 in Cells
BOSC 23 cells were transfected with RSV-AP-2, CMV-Sp1 expression
plasmid, or RSV control vector. After 36 h, cells were rinsed with
PBS and harvested by scraping, and cell pellets were lysed for 10 min
on ice in RIPA buffer [150 mM NaCl, 1% Nonidet P-40,
0.5% deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 7.5, with
1 mM sodium orthovanadate (Sigma Chemical Co.,
St. Louis, MO), 1 mg/ml leupeptin, 0.1 mM
phenylmethylsulfonyl fluoride (PMSF)]. Extracts were cleared by
centrifugation and immunoprecipitated with rabbit polyclonal antibody
(1 µg of IgG) specific for AP-2 (Santa Cruz Biotechnology, Inc.) using protein A-Sepharose as directed in the Santa Cruz
product bulletin. Beads were washed four times with RIPA buffer and
boiled in SDS sample buffer, and released proteins were resolved by
10% SDS-PAGE. The gel was transferred to nitrocellulose and Western
blotting was performed using the AP-2 or Sp1 antibody.
In Vitro Protein-Protein Interaction
Coupled transcription-translation reactions were performed as
previously described (7) using either T7 polymerase [pT7ßSal AP-2
(35)] or T3 polymerase (T3-FL-Sp1) and either T7 or T3 from the
plasmid pGEMLUC as a control reaction. AP-2 and Sp1 proteins were
labeled with [35S]methionine by coupled
transcription-translation with a Promega Corp. TNT
reticulocyte lysate kit using 1.5 µg of plasmid DNA in a total of 50
µl (7).
GST fusion proteins were prepared as previously described (71). GST
fusion plasmids were used to transform Escherichia coli
DH5, and cells were grown to an absorbance of 0.50.7 at 600 nm.
Fusion proteins were induced for 3 h with 0.2 M
isopropyl-ß-D-thiogalactopyranoside, and crude
lysates were prepared at 4 C. Cell pellets were spun down, resuspended
in 20 mM Tris-HCL, pH 8.0, 1 mM EDTA, 1
mM PMSF, 1 mM leupeptin. Lysozyme (1 mg/ml) was
added and incubation was performed on ice for 1530 min. After the
addition of 1/6 volume of 5 M NaCl and 1% Tween 20, and a
10-min incubation on ice, the cells were spun at 35,000 rpm for 15 min
at 4 C. To the supernatant, 0.5 ml bed volume of GST beads
(Pharmacia Biotech, Piscataway, NJ) was added, and the
mixture was rotated for 612 h at 4 C, after which time centrifugation
was performed at 1500 rpm for 5 min. The supernatant was discarded, and
the pellet was resuspended in NETN buffer (0.5% NP-40, 20
mM Tris-HCL, pH 8, 100 mM NaCl, 1
mM EDTA). Glutathione elution buffer (0.5 ml of 10
mM glutathione in 50 mM Tris-HCl, pH 8) was
added, and incubation was performed at 4 C for 14 h. The beads were
separated through centrifugation, and the amount of protein was
determined by Bio-Rad Laboratories, Inc. (Richmond, CA)
assay and Coomassie Blue staining of an SDS-polyacrylamide gel. In
addition, to ensure equal amounts of wild-type and mutant GST fusion
proteins were used, Western blotting was performed using the GST
antibody on serial dilutions of the fusion protein electrophoresed on
an SDS-polyacrylamide gel.
In vitro protein-protein interactions were performed as previously described (72). The in vitro translated protein (6 µl of AP-2 or Sp1) was added to either 2 µg of GST, GST-AP-2, or GST-Sp1 in 200 µl of binding buffer (20 mM HEPES, pH 7.9, 1 mM MgCl2, 40 mM KCl, 0.1 mM EDTA, 0.1% NP-40, 1 mM PMSF, 1 mM leupeptin) and rotated for 1 h at 4 C. GST bead slurry (50 µl) was added for GST pull-down experiments. Protein A sepharose (50 µl) and 1 µg of AP-2 or Sp1 antibody (Sp1, PEP2-G, Santa Cruz Biotechnology, Inc.) was added in IP experiments. Samples were rotated for 612 h at 4 C and pelleted, and the beads were washed four times with binding buffer. Twenty five microliters of 2 x loading buffer were added, and the sample was electrophoresed on an SDS-polyacrylamide gel (810%). The electrophoresed samples were then transferred to nitrocellulose for 1 h and probed with antibodies to AP-2 (5E4 or C-18) and Sp1 (SC-1C6) as directed by the supplier. The gel was then fixed with 25% isopropanol and 10% acetic acid for 15 min, washed with Amplify (Amersham Pharmacia Biotech, Arlington Heights, IL) for 30 min to enhance the 35S signal, and dried for 1 h at 80 C, after which autoradiography was performed at -70 C using XAR5 film (Eastman Kodak Co.) with an intensifying screen.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
This work was supported by NIH Grants R29CA-70897, RO1CA-75503, and RO1CA-7755201; The Irma T. Hirschl Charitable Trust; and The Monique Weill-Caulier Charitable Trust (to R.G.P.). Work at the Albert Einstein College of Medicine was supported by Cancer Center Core NIH Grant 5-P30-CA-1333026. G.W. was supported in part by a Travel Fellowship from the Aichi Health Promotion Foundation, Owari Kenyu Committee, and the Takasu Foundation. R.J.L. was supported by NIH Training Grant 5 T32 GM-085210. A.T.R. was supported by a P.F. Sabotka Postgraduate Scholarship from the University of Western Australia, and M.D. was supported by NIH Training Grant CA-0947512. I-W.S. was supported by a David Shemin Undergraduate Fellowship at Northwestern University. P.P. was supported by a Fullbright Fellowship. T.W. is a Pew Scholar in the Biomedical Sciences and was also supported by American Cancer Society Grant RPG-98096-01-MG.
1 These authors contributed equally to the manuscript.
Received for publication January 5, 1999. Revision received May 19, 1999. Accepted for publication May 20, 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|