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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Human CG is produced by the placenta and regulates steroid production from the corpus luteum (1). CG is a dimeric protein composed of an {alpha}- and a ß-subunit. The {alpha}-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 {alpha}-subunit or the CGß subunit in the placenta. Recent studies have shown that the trophoblast-specific element in the {alpha}-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 {alpha}-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 {alpha}- and ß-subunit gene expression (15).

AP-2{alpha} is present in the mouse placenta, as well as another family member, AP-2{gamma} (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{alpha} (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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo. 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.

 
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. 3Go). 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. 2Go and Fig. 3Go). Three protein complexes were detected in nuclear extracts of JEG-3 cells using the -309 to -288 fragment as a labeled probe (Fig. 2AGo). 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 3–5) or 5'Sp1-m3 (lanes 9–11), 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 6–8) as well as binding to the labeled 5'Sp1-m2 oligonucleotide (lane 13). Mutants 5'Sp1-m5 (lanes 6–8) and 5'Sp1-m7 (lanes 12–14) retain competition, whereas mutants 5'Sp1-m4 (lanes 3–5) and 5'Sp1-m6 (lanes 9–11) did not compete for Sp1/Sp3 binding (Fig. 2BGo). 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 1–11). Mutant oligonucleotides were also 32P labeled and incubated with nuclear extracts (5 µg) (lanes 12–14). B, Competition for binding to the labeled CGß -309 to -288 fragment with various oligonucleotides (lanes 1–14). 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. 2CGo.

 
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. 2CGo). 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. 2CGo, lanes 15–17). However, these same substitutions in 5'Sp1-m6 are necessary for Sp1 binding (Fig. 2BGo, lanes 6–8) to the wild-type -309 to -288 fragment. Similarly, a mutation (5'Sp1-m7) that weakly affects Sp1 binding (Fig. 2BGo, lanes 12–14) is necessary for AP-2 binding (5' AP-2-m2) to the wild-type -300- to -279-bp fragment (Fig. 2CGo, lanes 6–8). These experiments are summarized in Fig. 3Go 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. 4Go). 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 (1–3 µl) inhibited AP-2 binding and generated an Sp1 complex (lanes 3–5). 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 2–5). Lanes 6–9 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 7–9). AP-2- and Sp1-specific bands are indicated. Nonspecific bands seen with the unprogrammed lysate alone are also indicated.

 
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. 1BGo) 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. 5Go). 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 1–6).



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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 1–3), 32P-labeled CGß 5'-Sp1 (-309 to -288) oligonucleotides (lanes 4–6), or 32P-labeled CGß 3'-Sp1 (-205 to -188) oligonucleotides (lanes 7–9). Antibodies were added 60 min before electrophoresis (30 min before labeled oligonucleotide addition). Protein complexes corresponding to Sp1 and Sp3 are indicated by arrows.

 
A fragment containing both the AP-2 and Sp1 binding sites (see Fig. 1BGo) 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. 6Go). 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. 1BGo 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.

 
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. 7Go, inset). A mutation that eliminated Sp1 binding to the 5'Sp1 binding site (mut1: 5'Sp1 mut6; see Fig. 3Go) caused 84% reduction in basal activity (Fig. 7AGo). 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. 7BGo). 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.

 
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. 8Go). 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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {alpha}-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 {alpha}-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. 2BGo, 5Go'-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. 3Go), 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. 6Go). 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. 9Go). 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.

 

    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 (5–10 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 18–24 h before isolating extracts for luciferase assays (42).


    FOOTNOTES
 
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 15–709, 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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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