AP-2 (Activating Protein 2) and Sp1 (Selective Promoter Factor 1) Regulatory Elements Play Distinct Roles in the Control of Basal Activity and Cyclic Adenosine 3',5'-Monophosphate Responsiveness of the Human Chorionic Gonadotropin-ß Promoter
Wade Johnson and
J. Larry Jameson
Division of Endocrinology, Metabolism and Molecular Medicine
Northwestern University Medical School Chicago, Illinois 60611
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ABSTRACT
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The CG ß-subunit gene (CGß) arose
evolutionarily from the LH ß-subunit gene (LHß) through gene
duplication. Although the promoter sequences of the CGß and human (h)
hLHß genes are greater than 90% homologous, their expression
patterns are distinct. LHß is expressed in pituitary gonadotrope
cells and CGß is expressed in placental trophoblast cells. The
placental specific and cAMP-inducible region within the CGß promoter
has been mapped to a complex enhancer element spanning 118 bp (-318 to
-200). Transcription factor-binding sites within this enhancer have
been partially characterized and include multiple binding sites for
AP-2 (activating protein 2) and Sp1 (selective promoter factor 1),
which activate basal and cAMP-induced expression. In this study, we
performed a detailed analysis of the recognition sites for these
transcription factors and examined the functional roles of these
elements in the control of CGß expression. An upstream Sp1/AP-2
binding site (-318 to -279) preferentially binds Sp1, which occludes
AP-2 binding to an adjacent site. In contrast, both Sp1 and AP-2 bind
concurrently to a downstream composite Sp1/AP-2 element (-220 to
-188). Functionally, mutations in any of the Sp1 or AP-2 binding sites
cause a progressive decrease in basal CGß expression. However, cAMP
stimulation of the CGß promoter is reduced by AP-2 mutations, whereas
Sp1 mutations enhance cAMP activation. We conclude that multiple AP-2
and Sp1 elements are required to maintain basal CGß promoter
activity, but these factors have opposing effects on cAMP regulation,
which is mediated primarily by AP-2.
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INTRODUCTION
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Human CG is produced by the placenta and regulates steroid
production from the corpus luteum (1). CG is a dimeric protein composed
of an
- and a ß-subunit. The
-subunit is shared among the
glycoprotein hormone family (FSH, TSH, and LH), whereas the ß-subunit
is specific for each family member. The CGß gene shows a high degree
of homology with the LHß gene and is proposed to have arisen
evolutionarily from an ancestral LHß gene through gene duplication
(2). The human LHß and CGß genes are located on a 56-kb region of
chromosome 19 and comprise a cluster that includes six independent
CGß genes (CGß 5, 8, 7, 3, 2, and 1) and a single copy of the LHß
gene (2, 3, 4).
The CGß and LHß promoters share greater than 90% homology at the
nucleotide level, reflecting their relatively recent evolutionary
divergence. Despite this sequence similarity, the patterns of
expression and regulation of the CGß and LHß genes are quite
different. The LHß gene is expressed exclusively in the gonadotrope
cells of the pituitary gland whereas the CGß genes are expressed in
the trophoblast cells of the placenta and occasionally in certain
neoplasms where it can serve as a tumor marker (1). One potential clue
regarding the unique patterns of expression of the CGß and LHß
genes is that the architecture of their promoters has diverged. The
LHß gene contains a consensus TATA box and transcribes a very short
(9 bp) 5'-untranslated sequence (5). In contrast, the CGß genes are
transcribed from an upstream site that does not contain a canonical
TATA box. As a consequence, the CGß transcript contains an extended
(356 bp) 5'-untranslated sequence that encompasses the homologous
region in the LHß promoter.
Initially, it was believed that sequence comparisons, and the
generation of chimeric promoter constructs that exchanged regions of
the CGß and LHß promoters, might reveal the regulatory elements
involved in their unique transcriptional initiation sites and patterns
of expression (6). However, these strategies have yielded limited
information and have given way to more traditional studies of
transcription factor binding and promoter mutagenesis. In the case of
the LHß gene, binding sites for steroidogenic factor-1 (SF-1) and
early-growth-response-1 gene (Egr-1) appear to play a critical role in
gonadotrope-specific expression (7). Targeted mutagenesis of the murine
Ftz-f1 gene (which encodes SF-1) impairs LH production, and
mutagenesis of the Egr-1 gene eliminates LH expression
(8, 9, 10). The homologous binding sites for these factors reside within
the 5'-untranslated sequence of the CGß transcript, and experiments
have shown that these factors are not involved in CGß expression in
JEG-3 cells (W. Johnson and J. L. Jameson, unpublished data).
In the case of the CGß genes, chimeric exchanges between active and
inactive members of the CGß gene cluster, or between the CGß and
LHß genes, indicate that multiple divergent sequences contribute to
CGß expression in placental cells (6). These studies, and others
(11, 12, 13, 14), suggest that CGß promoter activity requires a relatively
extended sequence between -315 and -188 bp for maximal activity. This
region is a very rich in GC-nucleotide content and deoxyribonuclease I
footprinting studies suggest the presence of multiple protein binding
sites (11, 12). Experiments using heterologous promoter constructs
reveal that a short fragment between -311 to -279 bp maintains basal
expression, whereas cAMP stimulation requires a more extended sequence
spanning -311 to -202 bp (11).
No placental-restricted factors have been isolated to date that might
account for the specific expression of either the
-subunit or the
CGß subunit in the placenta. Recent studies have shown that the
trophoblast-specific element in the
-subunit promoter binds
the transcription factor activating protein-2 (AP-2) (15). This
element, in combination with a GATA-binding site and the cAMP-response
element, appears to be important for
-subunit expression in the
placenta, even though these factors are also expressed in many other
tissues (16, 17, 18). The basal element in the CGß promoter (-315 to
-279) also binds AP-2, along with selective promoter factor-1 (Sp1)
(15). An additional AP-2 binding site is located between -220 and
-200 bp of the CGß promoter, a region that is involved in cAMP
regulation. Levels of AP-2 increase during placental cell
differentiation from cytotrophoblasts to syncytiotrophoblasts, which is
also associated with a marked increase in CG
- and ß-subunit gene
expression (15).
AP-2
is present in the mouse placenta, as well as another family
member, AP-2
(19). Sp1 levels are shown to be concentrated in the
trophectoderm of mouse blastocysts, as compared with the inner cell
mass (20, 21). However, mice generated with a targeted disruption of
AP-2
(22, 23, 24), AP-2ß (25), or Sp1 (26) show no placental defects.
These mice indicate that neither of these transcription factors is
essential for mouse placental development.
Both AP-2 and Sp1 are responsive to the protein kinase A (PKA) pathway.
Phosphorylation of Sp1 by the PKA catalytic subunit increased Sp1
transactivation of the SV40 promoter (27). However, in aged liver,
phosphorylation of Sp1 is associated with decreased DNA binding and
decreased transactivation (28). AP-2 is associated with and
phosphorylated by the PKA catalytic subunit; however, phosphorylation
did not correlate with increased binding to DNA (29).
Sp1 and AP-2 have similar consensus binding sites, composed of short
GC-rich elements. Reflecting the similarities in binding sites, it has
been observed that Sp1 and AP-2 bind to the same, or overlapping
regions within a variety of promoters (30, 31, 32, 33). However, depending on
promoter context and relative ratios of Sp1 to AP-2, these factors can
function additively or antagonistically (31, 32).
In this study, we performed a detailed analysis of the Sp1 and AP-2
binding sites within the basal and cAMP-responsive region of the CGß
promoter. These studies identify a complex array of adjacent and
overlapping Sp1 and AP-2 binding sites and demonstrate divergent
effects of these factors in cAMP regulation.
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RESULTS
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Sequence Determinants for Sp1 Binding to the -302- and -294-bp
Elements of the CGß Promoter
Some of the transcription factor binding sites in the CGß
promoter are summarized in Fig. 1A
. An
enhancer element between -318 and -187 bp is involved in
placental-specific and cAMP-inducible expression (11). Transcription
factor binding sites within this region include two upstream AP-2
binding sites on either side of an Sp1 site, an OCT3 binding site, a
binding site for JUN family members, and downstream AP-2 and Sp1
binding sites (13, 14, 15).

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Figure 1. Identification of Proteins That Bind to the CGß
Promoter
A, Graphic representation of known binding sites for AP-2, Sp1, OCT3,
and c-JUN in the the CGß promoter. B, DNA sequence of the composite
CGß -220 to -188 bp element. The locations of the 3' AP-2 (-220 to
-200) and 3' Sp1 (-205 to -188) oligonucleotides are indicated by
solid lines under the sequence. The AP-2-specific
binding site is underlined and the Sp1-specific binding
site is double underlined.
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Because the nucleotide sequence of this enhancer is very GC rich,
additional studies were performed to define sequences involved in Sp1
and AP-2 binding. Initially, the region between -318 to -279 was
separated into three overlapping oligonucleotides that are known to
bind to Sp1 (-309 to -288) or AP-2 (-318 to -296 and -300 to
-279) (15) (Fig. 3
). Competition experiments using mutated
oligonucleotides were used in electrophoretic gel mobility shift assays
(EMSAs) to determine which nucleotides were necessary for
transcription factor binding to these sequences (Fig. 2
and Fig. 3
). Three protein complexes were detected
in nuclear extracts of JEG-3 cells using the -309 to -288 fragment as
a labeled probe (Fig. 2A
). The uppermost band corresponds to
Sp1, and the lower two bands contain Sp3 complexes, as shown
below. Competition with a 50-fold excess of unlabeled wild-type
oligonucleotide eliminates all three bands (lane 2). In contrast, there
was no competition by mutants 5'Sp1-m1 (lanes 35) or 5'Sp1-m3 (lanes
911), indicating that these nucleotide changes eliminate binding to
Sp1 and Sp3. Mutant 5'Sp1-m2 retains partial binding to Sp1/Sp3
complexes, as revealed by competition by the unlabeled oligonucleotide
(lanes 68) as well as binding to the labeled 5'Sp1-m2 oligonucleotide
(lane 13). Mutants 5'Sp1-m5 (lanes 68) and 5'Sp1-m7 (lanes 1214)
retain competition, whereas mutants 5'Sp1-m4 (lanes 35) and 5'Sp1-m6
(lanes 911) did not compete for Sp1/Sp3 binding (Fig. 2B
). Consistent
with previous studies (34, 35), none of the nucleotide substitutions
distinguished between Sp1 or Sp3 binding or eliminated Sp3 binding but
preserved Sp1 binding. This series of mutant oligonucleotides
demonstrates that the Sp1/Sp3 binding site is localized to the sequence
GGACACACC.

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Figure 3. Sequences of Wild-Type and Mutant Oligonucleotides
Wild-type sequences are indicated for each of the three overlapping
fragments of -318 to -279 hCGß oligonucleotide. +/- Indicates
whether the mutant oligonucleotides were able to bind and compete for
specific protein complexes with the wild-type oligonucleotide.
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Figure 2. Identification of Sp1 and AP-2 Binding Sites in
-318 to -279 Fragment
In each panel, the labeled CGß oligonucleotide is indicated by a
bold line and the transcription factor is
highlighted with white lettering. JEG-3 nuclear extracts
(5 µg) were incubated with the 32P-labeled
oligonucleotides and the indicated competitor oligonucleotides (50x,
100x, or 200x excess) for 30 min before electrophoresis. A,
Competition for binding to the labeled CGß -309 to -288 fragment
with various mutant oligonucleotides (lanes 111). Mutant
oligonucleotides were also 32P labeled and incubated with
nuclear extracts (5 µg) (lanes 1214). B, Competition for binding to
the labeled CGß -309 to -288 fragment with various oligonucleotides
(lanes 114). The bands representing Sp1 and Sp3 complexes are shown
at the left side of the gels. C, Competition for binding
to the labeled AP-2 binding element (-300 to -279 bp) with various
mutant oligonucleotides. The band corresponding to AP-2-specific
complex is indicated at the left of Fig. 2C .
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AP-2 Binds to Two Sites within the -318- to -279-bp Element of
the CGß Promoter
A similar experimental design was used to determine the nucleotide
sequence determinants for AP-2 binding to the -318- to -279-bp
element. AP-2 bound to CCCGTGGGC between -318 to -296 of the CGß
promoter (data not shown). AP-2 also bound to a second site (CCTGCGGG)
between -300 to -279 (Fig. 2C
). When EMSA was performed with the
complete composite element (-318 to -279), AP-2 binding was only
observed in the presence of excess unlabeled Sp1 oligonucleotide (15).
Mutational analysis reveals that the AP-2 and Sp1 binding sites within
this element do not overlap. For example, nucleotide substitutions in
mutant 5'AP2-m4 are not involved in AP-2 binding (Fig. 2C
, lanes
1517). However, these same substitutions in 5'Sp1-m6 are necessary
for Sp1 binding (Fig. 2B
, lanes 68) to the wild-type -309 to -288
fragment. Similarly, a mutation (5'Sp1-m7) that weakly affects
Sp1 binding (Fig. 2B
, lanes 1214) is necessary for AP-2 binding (5'
AP-2-m2) to the wild-type -300- to -279-bp fragment (Fig. 2C
, lanes
68). These experiments are summarized in Fig. 3
and indicate that the
core sequences required for AP-2 binding do not overlap with those
involved in Sp1 binding. However, there is some dependency of Sp1
binding on the nucleotides outside of the core sequence, as shown by
5'Sp1-m7 mutation. Therefore, the competition between Sp1 and
AP-2 may involve steric hindrance, as well as competition for shared
nucleotide sequences. Individual mutations in both the Sp1 and AP-2
binding sites, when combined in a full-length oligonucleotide,
eliminated all protein complexes in EMSA (data not shown).
Sp1 Binds Preferentially to AP-2 in the Composite -318- to
-279-bp Element
Because Sp1 is known to interact with other proteins
(36, 37, 38, 39), it is possible that its ability to inhibit AP-2 binding
involves complexes with other nuclear factors. Additional experiments
using in vitro translated proteins were therefore used to
assess whether Sp1 alone is sufficient to inhibit AP-2 binding (Fig. 4
). Using unprogrammed reticulocyte
lysate as a control, two nonspecific bands were seen with the -318 to
-279 oligonucleotide (lane 1). The upper nonspecific band migrates
near the Sp1 in vitro translated band and may be native Sp1
protein found in the in vitro lysate system. The lower
nonspecific band migrates at a faster mobility than either AP-2- or
Sp1-specific bands. Experiments to define these two bands are being
pursued within the laboratory. The addition of AP-2 programmed lysate
(1 µl), in the presence of a constant amount of total lysate (4
µl), generates an AP-2 complex (lane 2). The addition of increasing
amounts of Sp1 programmed lysate (13 µl) inhibited AP-2 binding and
generated an Sp1 complex (lanes 35). The reverse experiment was also
performed in which 1 µl of programmed Sp1 lysate was used with
increasing amounts of AP-2 programmed lysate. Again, competition was
observed between AP-2 and Sp1, although AP-2 appears to compete less
effectively than Sp1 (compare lanes 5 and 9). No slower migrating band
was observed in the in vitro EMSA, indicating that AP-2 and
Sp1 do not bind simultaneously to the -318 to -279
oligonucleotide.

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Figure 4. Binding of in Vitro Translated AP-2
and Sp1 to the CGß -318 to -279 Element
The 32P-labeled -318 to -279 hCGß oligonucleotide was
incubated with 4 µl of unprogrammed lysate (lane 1). Increasing
amounts of Sp1 programmed lysate (1, 2, or 3 µl) were added to 1 µl
of AP-2 programmed lysate, and the total amount of lysate was kept
constant (4 µl) by the addition of unprogrammed lysate (lanes 25).
Lanes 69 contain 1 µl of Sp1 programmed lysate incubated with 3
µl of unprogrammed lysate (lane 6) or 1, 2, or 3 µl of AP-2
programmed lysate (lanes 79). AP-2- and Sp1-specific bands are
indicated. Nonspecific bands seen with the unprogrammed lysate alone
are also indicated.
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Identification of a New Sp1 Binding Site between -200 and -188 bp
of the CGß Promoter
An Sp1 binding site was proposed between -200 and -188 (Fig. 1B
)
of the CGß promoter, based on inspection of the nucleotide sequence
(11). Due to the close proximity of an AP-2 binding site and the
putative Sp1 binding site, EMSA were performed to determine whether Sp1
binds to the -200 and -188 element. A DNA fragment corresponding to
the 3'-Sp1 site was used in EMSA, and antibodies directed against Sp1
and Sp3 were used to characterize the proteins in the complexes (Fig. 5
). Antibodies to Sp1 caused a supershift
of the uppermost band, whereas antibodies against Sp3 eliminated the
two lower bands (lanes 8 and 9). A consensus Sp1 site and the CGß
5'-Sp1 binding site were used as controls and confirmed that the Sp1
antibody supershifts the complex, and the Sp3 antibody ablates DNA
binding (lanes 16).

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Figure 5. Identification of Sp1 and Sp3 Complexes
Gel mobility shift assays were performed with 5 µg of JEG-3 nuclear
extracts incubated with the 32P-labeled consensus Sp1
binding site (lanes 13), 32P-labeled CGß 5'-Sp1 (-309
to -288) oligonucleotides (lanes 46), or 32P-labeled
CGß 3'-Sp1 (-205 to -188) oligonucleotides (lanes 79). Antibodies
were added 60 min before electrophoresis (30 min before labeled
oligonucleotide addition). Protein complexes corresponding to Sp1 and
Sp3 are indicated by arrows.
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A fragment containing both the AP-2 and Sp1 binding sites (see Fig. 1B
)
was used to assess protein binding when both sites are present. Unlike
the 5'-Sp1/AP-2 binding site, which showed preferential Sp1 binding,
the composite element showed equal binding of Sp1 and AP-2 (Fig. 6
). Antibodies to Sp1 and AP-2
supershifted the respective binding proteins. Antibodies to Sp3
indicate that little or no Sp3 protein binds to the 3'-composite
element (lane 3). Competition with the single binding sites for AP-2 or
Sp1 eliminated their specific protein complexes, suggesting that
binding to this composite element is not cooperative. A faint slower
mobility band was also observed and may correspond to DNA complexes
with both AP-2 and Sp1. AP-2 antibody supershifts this band, and
competition with single binding sites for either AP-2 or Sp1 eliminates
this band. These experiments indicate that in contrast to the distal
composite AP-2/Sp1 element, Sp1 and AP-2 bind to the proximal composite
element independently and with similar affinity.

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Figure 6. Identification of Protein Complexes That Bind to
the Proximal AP-2 and Sp1 Element
Gel mobility shift assays were performed with 5 µg of JEG-3 nuclear
extract incubated with the 32P-labeled CGß -220 to -188
oligonucleotide (see Fig. 1B and graphic representation above
figure). Antibodies to Sp1 (lane 2), Sp3 (lane 3), or AP-2
(lane 4) were added 60 min before electrophoresis. The indicated
unlabeled competitor oligonucleotides were added at 100-fold excess.
Arrows indicate protein complexes corresponding to Sp1
and AP-2. A slower mobility Sp1/AP-2 complex is also indicated.
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Functional Significance of the Sp1 and AP2 Binding Sites for Basal
Activity and cAMP Regulation
Based on the results of the binding experiments, five mutant
luciferase reporter constructs were created to specifically alter
individual AP-2 or Sp1 binding sites in the context of -345 to +114
CGß promoter (Fig. 7
, inset). A mutation that eliminated Sp1 binding to the 5'Sp1
binding site (mut1: 5'Sp1 mut6; see Fig. 3
) caused 84% reduction in
basal activity (Fig. 7A
). However, cAMP induction was 3-fold greater
with this mutant in comparison to the wild-type reporter construct
(59-fold compared with 22-fold) (Fig. 7B
). Mutation of the 3'-Sp1
binding site (mut2) caused a similar reduction in basal activity (83%)
and an increase in cAMP stimulation (51-fold). Mutation of the two AP-2
binding sites that reside between -318 to -279 (mut3) decreased basal
activity 86% but had little or no effect on cAMP induction (19-fold).
A mutation in the proximal AP-2 binding site resulted in the most
marked reduction in basal activity (93%). When all three AP-2 binding
sites were mutated (mut5), basal activity was reduced by 88%, and
there was also a significant reduction in cAMP responsiveness
(11-fold). These mutations indicate that each of the Sp1 and AP-2 sites
are involved in the maintenance of basal expression. However, the AP-2
sites appear to be more important for stimulation by cAMP.

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Figure 7. Functional Effects of AP-2- and Sp1-Specific
Mutations within the CGß Promoter
JEG-3 cells were transiently transfected with 500 ng of reporter
constructs. A schematic representation of the mutations is shown in the
inset. A, Basal activity of CGß mutants 48 h
after transient transfection. The numbers above each bar
represent the percentage of repression relative to wild-type (CGß
-345+114) activity. B, Transfected JEG-3 cells were treated with 0.5
M 8-Br-cAMP for 24 h. The numbers above each
bar represent fold stimulation relative to basal activity for
each construct. Results represent mean ± SE of six
individual transfections. A significant difference,
P < 0.05, is indicated by an asterisk above
the bar; the Tukey test of one-way analysis of variance was
used to determine statistical significance.
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Cotransfection experiments were performed with increasing amounts of
Sp1 or Sp3 expression vectors with either the PKA catalytic subunit or
8-bromo-cAMP (8-Br-cAMP) treatment. Neither Sp1 nor Sp3 overexpression
was able to inhibit cAMP activation of the hCGß promoter (data not
shown).
cAMP Treatment of JEG-3 Nuclear Extracts Increases AP-2 Binding to
Response Elements
Nuclear extracts were prepared from JEG-3 cells treated for 6 or
24 h with 0.5 mM 8-Br-cAMP, and EMSA was performed
using the -220 to -188 probe (Fig. 8
).
cAMP treatment for 24, but not 6 h, increased AP-2 binding,
whereas Sp1 binding was constant (upper band). The AP-2
consensus site from human metallothionein II A promoter (hMTIIA) also
showed an increase in AP-2 binding (data not shown).

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Figure 8. cAMP Treatment Increases AP-2 Binding Activity
Nuclear extracts were prepared from JEG-3 cells treated with 0.5
M 8-Br-cAMP for 6 or 24 h. Nuclear extracts (5 µg)
were incubated with the 32P-labeled CGß -220 to -188
oligonucleotide. Nuclear extracts were prepared from untreated,
normally growing cells (lane 1), 6 h cAMP treatment (lane 2), and
24 h cAMP treatment (lane 3). AP-2- and Sp1-specific complexes are
indicated at the left of the gel.
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DISCUSSION
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The regulatory elements in the CGß promoter have been
difficult to identify. Its level of activity in transient expression
assays is relatively low, particularly in comparison to the
-subunit
promoter, which contains a very strong promoter (11, 13, 16, 40). The
absence of a TATA box, and other canonical sequences, has also made it
difficult to identify candidate protein binding sites in the CGß
promoter. Finally, the sequence similarities between the CGß and
LHß genes have not provided the anticipated insights about conserved
regulatory regions, as the functional regions of these promoters are
distinct. For these reasons, we have taken an empirical approach to
characterize the critical CGß promoter sequences that confer basal
activity in placental cells and responsiveness to cAMP.
In previous studies, it was shown that multiple regions of the CGß
promoter function in a combinatorial manner to maintain basal
expression (11, 12, 13). For example, when different regions of the CGß
5'-flanking region were linked to a minimal heterologous promoter,
basal activity and cAMP inducibility were fully restored only when a
region between -315 and -279 was combined with additional
3'-sequences that extend to -202 bp (11). Consistent with this
finding, a variety of different point mutations that convert CGß
sequences to those found in LHß markedly reduce basal activity (6).
These observations suggested that a unique combination of factors in
the CGß promoter act to regulate its expression, as opposed to a
discrete, strong enhancer element that might have evolved during the
divergence of the CGß and LHß genes.
Recently, we demonstrated that two broadly expressed transcription
factors, AP-2 and Sp1, bind to several different regions within the
proximal CGß promoter (15). AP-2 was also found to bind to the
-subunit promoter as part of a composite enhancer element that
confers placental expression and cAMP regulation (12, 15, 18). These
findings raise the possibility that, despite their broad expression,
AP-2 and Sp1 might play an important role in placental expression of
the CGß promoter. In this study, we have defined the complex
relationship of these redundant binding sites. In the upstream region,
-315 to -279, Sp1 binding predominates and competes with AP-2. More
proximally, AP-2 and Sp1 bind concomitantly to adjacent sites.
Mutational studies reveal that the Sp1 sites play a critical role in
basal expression, whereas the AP-2 sites sustain basal activity but
also enhance cAMP responsiveness.
Identification of these binding sites required detailed
mutagenesis because several of the elements deviate considerably from
consensus sequences. For example, an Sp1/Sp3 site was localized between
-309 and -288 bp. EMSA experiments using various mutations within
this region further localized the Sp1/Sp3 binding site to the sequence,
GGACACACCTCC. Unexpectedly, mutation of the 5'-GG pair or the 3'-CC
only slightly reduced binding (see Fig. 2B
, 5
'-Sp1 mut2 and mut7),
leaving a core binding site, CACACC. This sequence deviates from the
consensus Sp1 binding site (GGGGCGGGGC) (41) used as a competitor.
However, it is similar to other Sp1 sites that are GT rich rather than
GC rich. The observation that antibodies supershift these Sp1
complexes, and that in vitro translated Sp1 binds to this
sequence, confirms that the complexes contain Sp1, or a highly related
protein. AP-2 binding in this region was also increased in the presence
of excess unlabeled Sp1 oligonucleotides. Using shorter overlapping
oligonucleotides, including one between -318 and -296 and another
between -300 and -279, localized two AP-2 binding sites. Although
neither of the adjacent binding sites for AP-2 overlap with the minimal
Sp1/Sp3 binding site (see Fig. 3
), Sp1 effectively competes away AP-2
binding to these sites. The ability of Sp1 to compete for AP-2 may
reflect its relatively strong binding to this Sp1 sequence. The
mechanism for competition appears to involve steric hindrance or
occlusion, since these binding sites appear to be separable in
mutagenesis studies. Whether such competition occurs in vivo
is unknown. The fact that mutations in either the Sp1 site (Mut 1), or
the AP-2 sites (Mut 3) reduce basal activity suggests that these
elements may interact in the context of the native promoter. On the
other hand, the Sp1 mutation enhances cAMP responsiveness, perhaps
because AP-2 binding is increased in the absence of Sp1 or Sp3.
Given the high GC content of the proximal CGß promoter (-220 to
-188), it was not unexpected to find additional Sp1 binding sites
within this region. However, it is notable to compare the relationship
of the AP-2 and Sp1 sites within this region to the interactions in the
distal enhancer described above. Unlike the 5'-element, which exhibits
preferential binding of Sp1, the hCGß -220 to -188 fragment binds
AP-2 and Sp1 with similar affinities. It is also notable that although
Sp3 binds to the isolated Sp1 site, Sp3 binding is no longer detectable
in the context of the extended -220 to -188 fragment, even when
unlabeled AP-2 binding site is used as a competitor (see Fig. 6
). This
suggests that additional sequences can distinguish Sp1 and Sp3
binding.
Treatment of JEG-3 cells with cAMP dramatically increases the
binding of AP-2 to its respective binding sites. This effect on AP-2
binding is seen most clearly in the presence of a mutation in the Sp1
binding site such that competition by Sp1 is eliminated. Consistent
with this finding, cAMP induction is 3-fold greater in reporter
constructs with mutations in the 5'-Sp1/Sp3 binding site.
Interestingly, mutation of the proximal Sp1 binding site produces a
similar enhancement of cAMP stimulation, even though Sp1 and AP2 do not
appear to compete for binding to this region. Thus, it appears that Sp1
may abrogate AP-2 action by a second mechanism. Because the Sp1 site in
the proximal enhancer is downstream of the AP-2 site, it might impair
its access to the basal transcription complex. Alternatively, the Sp1
sites may inhibit cooperative interactions of AP-2 sites. This type of
mechanism might also account, in part, for the ability of c-JUN and
OCT3, which bind between the upstream and downstream enhancers,
to inhibit CGß expression (13, 14).
Based on these studies, we propose a model for control of the CGß
promoter by Sp1, Sp3, and AP-2 (Fig. 9
).
Sp1 is capable of contributing to basal promoter activity, whereas
competition with Sp3 inhibits basal activity. However, differentiation
of placental cells (15), or agonists that raise cAMP levels, increases
the amount of AP-2. Greater levels of AP-2 further activate the CGß
promoter. An analogous mechanism for modulating promoter activity has
been proposed for the keratin 3 (K3) promoter (32). In this case, Sp1
and AP-2 binding sites overlap, and binding is mutually exclusive. In
undifferentiated keratinocytes, when levels of AP-2 and Sp1 are
similar, AP-2 inhibits Sp1 binding and activation of the K3 promoter.
As keratinocytes differentiate and levels of AP-2 decrease, the
relative binding of Sp1 increases, thereby stimulating activity of the
K3 promoter. Although this model may explain the relationship at the
hCGß 5'-composite element, there is a different relationship at the
3'-composite element. At this site, Sp1 and AP-2 bind together
inhibiting Sp3 binding. Although the model is different, the function
of the specific proteins stays the same; Sp1 and AP-2 are necessary for
basal activities with Sp3 acting as an inhibitor. The models (hCGß
and K3 promoters) suggest that the relative amounts of AP-2, Sp-1, and
Sp3, which can vary during cellular differentiation, are capable of
modulating the responses of target genes.

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Figure 9. Model of CGß Activation by Various Transcription
Factors
Basal expression is controlled by Sp1 and AP-2 binding to specific
regions within the minimal CGß promoter. cAMP agonists increase AP-2
binding to the promoter. The increase in AP-2 binding stimulates CGß
expression. Inhibitors of CGß expression (Jun and Oct3) bind between
the two composite binding sites and block CGß activation. Sp3
inhibits Sp1 binding and acts as a repressor of basal and
cAMP-inducible transcription.
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MATERIALS AND METHODS
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EMSA Conditions
Nuclear extracts were prepared by scraping a single plate of
JEG-3 choriocarcinoma cells in PBS+ [PBS, 1 mM EDTA, 1
mM dithiothreitol (DTT), 1 mM
phenylmethylsulfonylfluoride (PMSF)]. Scraped cells were pelleted in
microcentrifuge tubes and resuspended in 400 µl buffer A (10
mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM
EDTA, 0.1 mM EGTA, 1.0 mM DTT, 1.0
mM PMSF, 0.15 mM spermine, 0.75 mM
spermidine) plus a cocktail of protease inhibitors (1 µg/ml
aprotinin, 1 µg/ml pepstatin, 1 µg/ml leupeptin, and 1 µg/ml
p-aminobenzimide), all purchased from Sigma
(St. Louis, MO). The resuspended cells were incubated on ice for 15
min, 25 µl of 10% IGEPAL (Sigma) were added, and tubes
were vortexed vigorously for 10 sec. After the cells were pelleted in a
microfuge, the supernatant was removed and the pellet was resuspended
in 50 µl of buffer C (20 mM HEPES, pH 7.9, 0.4
M KCl, 0.1 mM EDTA, 0.1 mM EGTA,
1.0 mM DTT, 1.0 mM PMSF, 20% glycerol, and the
above protease cocktail). The resuspended pellet was shaken at 4 C for
15 min and pelleted by centrifugation. The supernatant was removed and
used in EMSAs. Nuclear extracts (510 µg protein) were added to a 20
µl reaction containing 2 µl 10x buffer (20 mM HEPES,
pH 7.9, 40 mM KCl, 1 mM MgCl2, and
0.1% NP-40), 500 ng poly dI-dC, with the addition of AP-2, Sp1, or Sp3
antibodies as indicated in individual experiments. After incubation on
ice for 30 min, 32P-labeled oligonucleotides (30 fmol) were
added with or without unlabeled competitor sequences. Reactions were
incubated at room temperature for another 30 min before electrophoresis
through a nondenaturing 5% polyacrylamide gel at 180 V for 3 h.
EMSAs using in vitro transcribed and translated
transcription factors were performed under similar conditions except
that poly dI-dC was not added to the reaction buffer. In
vitro translated reactions for Sp1 and AP-2 were performed
according to the instructions of the manufacturer (Promega Corp., Madison, WI). Unprogrammed lysate refers to in
vitro translated reactions that were performed simultaneously with
the Sp1 and AP-2 reactions, but without expression vectors.
Reporter Genes and Transient Expression Assays
The CGß promoter-luciferase constructs in the pA3LUC plasmid
were described previously (11). The method of generating site-directed
mutations in the CGß promoter has also been described (15). All
plasmids were purified using Qiagen columns (Santa
Clarita, CA). Transient transfections of JEG-3 cells were performed in
12-well plates using the calcium phosphate method. Typical reactions
consisted of 500 ng of reporter plasmid, 5 µl of 2 M
CaCl2, 150 µl of HBS (137 mM NaCl, 5
mM KCl, 0.7 mM Na2PO4,
6 mM dextrose, 21 mM HEPES with a final pH of
7.05). Expression vectors for transcription factors (20 ng) were
included in some experiments, and an equivalent amount of empty plasmid
was added to control reactions. Cells were incubated with the
transfection mix at 37 C for 4 to 6 h. After removing the media,
cells were washed in PBS and fresh media were added with or without 0.5
mM 8-Br-cAMP for 1824 h before isolating extracts for
luciferase assays (42).
 |
FOOTNOTES
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Address requests for reprints to: J. Larry Jameson, M.D., Ph.D., Endocrinology, Metabolism and Molecular Medicine, Northwestern University Medical School, 303 East Chicago Avenue, Tarry Building 15709, Chicago, Illinois 60611.
This work was supported in part by NIH Grant HD-23519.
Received for publication March 15, 1999.
Revision received August 10, 1999.
Accepted for publication August 17, 1999.
 |
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