SP1/SP3-Binding Sites and Adjacent Elements Contribute to Basal and Cyclic Adenosine 3',5'-Monophosphate-Stimulated Transcriptional Activation of the Rhesus Growth Hormone-Variant Gene in Trophoblasts

Judith T. Schanke1, Maureen Durning, Kimberly J. Johnson2, Lindsey K. Bennett and Thaddeus G. Golos

Wisconsin Regional Primate Research Center and Department of Obstetrics and Gynecology University of Wisconsin Medical School University of Wisconsin Madison, Wisconsin 53715-1299


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transcriptional activation of the rhesus monkey GH-variant gene in syncytiotrophoblasts is developmentally regulated by trophoblast-specific and cAMP-responsive mechanisms. Progressive deletions of 5'-flanking DNA defined the most proximal 140 bp as the minimal region retaining full cAMP-stimulated mGH-V transcription. To identify the regions of this promoter critical for transcription, transient transfections of reporter plasmids containing systematic 10 base mutations throughout this proximal region were performed. Mutation of the region from -140/-131 decreased transcription in syncytiotrophoblasts by 50%, and gel mobility-shift analyses demonstrated that Sp1 and Sp3 bound to a region containing a GGGAGG motif at -136/-131. Mutation of the -62/-53 region decreased transcriptional activation by 66–99%, and Sp1 and Sp3 bound to a GGTGGG motif overlapping this region (at -65/-60). Selective mutation of this Sp1/Sp3 site decreased basal transcription by approximately 80%, and cAMP-stimulated transcription by up to 75% (with the greatest effect in primary syncytiotrophoblast cultures), indicating that the Sp1/Sp3 site is critical for transcriptional activation. Mutations in the regions adjacent to the Sp1/Sp3 sites (-130/-111 and -52/-43) also dramatically reduced (by 75%) transcriptional activation in trophoblasts. We conclude that two Sp1/Sp3 sites as well as additional elements directly adjacent to these sites contribute to trophoblast-specific cAMP-responsiveness of the mGH-V proximal promoter.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Gene expression is regulated by the interplay of both ubiquitous and cell-specific transcription factors. The varied expression and activation of these factors during cell differentiation and development control the pattern of cellular gene transcription and ultimately determine cell phenotype. The placental trophoblast is an excellent model for investigating the developmental and cell-specific regulation of gene transcription. In primates, the trophoblast is the cellular compartment responsible for the endocrine activity of the placenta and secretes a vast array of hormones and regulatory factors, including steroids, peptides, cytokines, growth factors, and placenta-specific protein hormones (1). The primate placenta is unique in that it expresses placenta-specific members of the GH gene locus, the chorionic somatomammotropins (CS) and placental GH-variant (GH-V) (2, 3, 4). Roles for both CS and GH-V in the regulation of maternal/fetal energy balance and metabolism have been proposed (5, 6). The expression of these genes is related to the state of differentiation of trophoblasts and is predominantly localized to the terminally differentiated syncytiotrophoblast (STB) (7, 8, 9). The expression of GH receptors in the placenta (10, 11), as well as the observation that GH-V is the primary somatotropin in maternal blood during the second half of pregnancy (5), underscores the importance of characterizing the regulation of GH-V expression throughout primate pregnancy. However, the mechanisms controlling the tissue specificity and developmental regulation of GH-V expression are not well understood.

We have previously described the transcriptional regulation of the placental GH-V gene in primary cultures of rhesus monkey STBs (12) and have demonstrated that transcriptional activation by cAMP is developmentally regulated as well as trophoblast specific (12). The gene is not responsive to cAMP in cells from the first trimester of pregnancy, when endogenous gene expression is low, but is strongly up-regulated by cAMP in cells from placentas obtained during the second or third trimester, when expression is maximal in vivo (12, 13). Thus, the activation by cAMP in vitro coincides with developmental activation of this gene during normal pregnancy.

In the current study we have examined the locus of cAMP activation of monkey GH-V gene transcription in both trophoblast and nontrophoblast cell lines to begin to identify the transcription factors responsible for this activation. Our results define the promoter of the mGH-V gene and demonstrate a critical role for multiple regions in cAMP-activated transcription. Two elements were identified that bind the zinc finger transcription factors Sp1 and Sp3. Mutagenesis studies demonstrated that the distal and proximal Sp1/Sp3-binding elements contribute to transcriptional activation of the mGH-V gene. Additionally, elements immediately adjacent to each Sp1/Sp3 site also are critical for both basal and cAMP-stimulated transcription in placental cells.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Majority of the cAMP-Responsive mGH-V Promoter Activity Resides within the Most Proximal 140 Bases of 5'-Flanking DNA
To identify the specific regulatory regions responsible for mGH-V transcriptional activation by cAMP (12), deletions were introduced throughout the 492-bp 5'-flanking region of mGH-V. These constructs were transiently transfected into the choriocarcinoma cell line JEG-3 and primary cultures of rhesus monkey cytotrophoblasts that differentiate in culture to STBs (14). Figure 1Go, A and B, illustrates that the fold-activation by 8-Br-cAMP of luciferase constructs, containing as little as 140 bp of 5'-flanking DNA, was equivalent to activation of the parent 492 bp construct when transfected into JEG-3 cells or second and third trimester STBs, respectively. However, responsiveness to cAMP decreased dramatically upon further deletion. Deletions from -140 to -107 and -107 to -66 resulted in up to 50% and 90% losses in transcriptional activity, respectively, in STBs (Fig. 1BGo), and 30% and 50% losses in activity in JEG-3 cells (Fig. 1AGo). Transcription is equivalent to a promoterless reporter in the absence of the TATA element (deletion to -19).



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Figure 1. Localization of Regions Conferring Transcriptional Activation by Deletion of the 5'-Flanking DNA of the Rhesus Monkey GH Variant Gene

Luciferase constructs containing 19–492 bp of 5'-flanking mGH-V DNA were transiently transfected into JEG-3 choriocarcinoma cells (A and C), primary cultures of rhesus STBs during the second or third trimesters of pregnancy (B and D), or nonplacental COS-7 cells (E and F). Panels A, B, and E present the fold induction of each construct by 8-Br-cAMP. Panels C, D, and F present the basal luciferase activity of each construct, relative to the parental 492-bp construct. The results presented are the means of five to seven individual experiments, with each treatment group represented by triplicate dishes in each experiment.

 
Deletions down to 140 bp did not decrease basal transcription; in fact, the 140-bp construct had higher basal activity than the parent 492-bp construct in JEG-3 cells (Fig. 1CGo). A reproducible increase in all cell types was seen upon deletion from -295 to -214 (discussed further below). However, deletions from -140 to -107, and further deletion to -66, decreased basal transcription by 73% and 88%, respectively, in STBs, compared with the full-length construct (Fig. 1DGo). Similar results were seen with -140, -107, and -66 constructs in JEG-3 cells (Fig. 1CGo). Thus, basal as well as cAMP-stimulated transcriptional elements are located within 140 bp of mGH-V 5'-flanking DNA.

To examine the differences in transcriptional activation between placental and nonplacental cells, we transfected COS-7 monkey kidney cells with the same deletion constructs. Although there is essentially no responsiveness to cAMP treatment (Fig. 1EGo), basal transactivation in nonplacental cells is localized to the same region as described for cAMP-responsive transactivation in trophoblasts (Fig. 1FGo).

A notable decrease in cAMP-stimulated transcriptional activity in JEG-3 cells and STBs was seen upon deletion from -338 to -295 (Fig. 1Go, A and B). Basal as well as cAMP-stimulated transcriptional activation typically rebounded, however, upon subsequent deletion to -214 (Fig. 1Go, C, D, and F), suggesting the presence of a negative regulatory element between -295 and -214. Since this rebound in GH-V transcription was found in all cells, this is not a cell-specific phenomenon and may be the result of the previously noted negative regulatory element in the rat GH gene upstream silencer-1 region (15).

Scanning Mutagenesis of the 140-bp mGH-V Promoter Identifies Multiple Functional Regions
To more precisely define the functional elements within the 140-bp mGH-V promoter that confer transcriptional activation, 10-bp block mutations were introduced that scan the entire region from -140 to -33. Reporter gene constructs containing these mutated promoters were transfected into primary STBs and JEG-3 cells, as well as nonplacental COS-7 cells. Elements critical for cAMP-stimulated and basal transcriptional activity were identified by comparison of mutated promoters with the wild-type 140-bp promoter construct. Mutation of bases -130/-121 resulted in 91% and 84% losses in cAMP-activated transcription in primary trophoblasts and JEG-3 cells, respectively (Fig. 2Go, A and B), whereas mutation of bases -62/-53 resulted in a 66% (JEG-3 cells) and 99% (STBs) loss in cAMP-stimulated transcriptional activation, in comparison to the wild-type construct. Although the borders of the regions varied somewhat among cell types, the bases consistently most critical for transcriptional activation were -130/-121 and -62/-53 in both JEG-3 cells and primary STBs, confirming the appropriate use of JEG-3 cells to model mGH-V transcriptional control in STBs.



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Figure 2. Localization of Transcriptional Regulatory Elements by Block Mutagenesis of the 5'-Flanking DNA of the Rhesus Monkey GH Variant Gene

Luciferase constructs containing 140 bp of 5'-flanking mGH-V DNA were transiently transfected into primary cultures of rhesus STBs (A), JEG-3 choriocarcinoma cells (B and C), or nonplacental COS-7 cells (D). Ten-base pair mutations within the 140-bp promoter were created scanning the region from -140 to -33 as listed in Table 1Go. Panels A and B indicate the fold induction by 8-Br-cAMP for each mutant construct, over its untreated control in primary STBs and JEG-3 cells, respectively. Panels C and D indicate the control levels of luciferase activity of all mutant constructs transfected into JEG-3 and COS-7 cells, respectively, relative to the wild-type 140 construct. Activity of the promoterless vector (pGL2) is also shown. The results presented are the means of three to eight individual experiments, with each treatment group represented by triplicate dishes in each experiment.

 
Mutations in regions adjacent to these elements also have significant effects in either STBs or JEG-3 cells. Mutation in the -140/-131 region decreased activity by 50% in primary STB cultures (Fig. 2AGo), while cAMP-stimulated transcription was decreased by approximately 25% in JEG-3 cells (Fig. 2BGo). In JEG-3 cell experiments, regions -120/-111 and -72/-63 appeared to contribute significantly to basal and cAMP-responsive transcription (Fig. 2Go, B and C). However, these mutations appeared to have wild-type levels of activity in primary STB cultures (Fig. 2AGo). In STB experiments, mutation of the region -52/-43 essentially eliminates cAMP-stimulated transcriptional activity from the mGH-V promoter (Fig. 2AGo) and reduced induction by 8-Br-cAMP by approximately 40% in JEG-3 cells (Fig. 2BGo).

An examination of basal transcriptional activation of these scanning mutant constructs in JEG cells, as presented in Fig. 2CGo, demonstrated the importance of regions -130/-121, -72/-63, and -62/-53. Basal activity in STBs was also consistent with an important role for the -130/-121 and -62/-53 regions in basal transcription (not shown).

Transfection assays also assessed the effects of these mutations on basal promoter activity in nonplacental COS-7 cells. The mGH-V promoter construct has detectable basal activity in COS-7 cells, but these cells do not significantly respond to cAMP stimulation (Fig. 1EGo). Transfection of COS-7 cells with the scanning mutagenesis constructs (Fig. 2DGo) indicates that the regions -130/-121 and -72/-53 are important for basal transcription in COS-7 cells, as they were for basal and cAMP-stimulated transcription in trophoblasts.

There Is Significant Sequence Homology to Recognition Elements for a Number of Known Transcription Factors within the mGH-V Promoter
Examination of the sequence of the 140-bp mGH-V promoter region revealed a number of potential recognition elements for DNA-binding proteins, including Sp1-, ets-, and GATA-binding sites, and a putative thyroid hormone response element (TRE) (16) (Fig. 3Go). There are also elements homologous to the proximal and distal Pit-1/GHF-1 binding sites and the Z box of the pituitary GH gene (17). These elements, with the exception of the Z box and proximal Pit-1 site, are localized within the two regions containing functional activity, as defined by the scanning mutagenesis study (-140/-111 and -72/-43). Oligonucleotide probes for electrophoretic mobility shift assays were designed to investigate binding of nuclear proteins to these regions of the mGH-V promoter.



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Figure 3. Sequence Homology of the Rhesus Monkey GH-V Gene Promoter Region with Previously Described Elements

Elements homologous to consensus sequences are indicated by boxes (labeled above) or arrows (labeled below).

 
Sp1 Family Members Bind to Two Elements within the 140-bp mGH-V Promoter
A radiolabeled oligonucleotide probe spanning -140 to -119 formed three specific DNA-protein complexes when incubated with JEG-3, COS-7, HeLa, or GH3 cell nuclear extracts (Fig. 4AGo). This region contains an Sp1-like binding site (GGGAGG; GA box) at -136/-131 (Fig. 3Go). All three complexes were specifically competed by excess unlabeled -140/-119 oligonucleotide as well as by a consensus Sp1 oligonucleotide, but not by an oligonucleotide containing the overlapping region -132/-105, which does not contain the entire GA box (Fig. 4Go B). The GA box region consensus site also has some homology to an AP-2 motif, but competition with a consensus AP-2 oligonucleotide had no effect on the bound complexes (Fig. 4BGo).



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Figure 4. Mobility Shift Analysis of Factors Binding to the -140/-119 Region within the mGH-V Promoter

A, Mobility shift assays with the -140/-119 probe and a range of nuclear extracts including the choriocarcinoma cell line JEG-3, with or without 48 h stimulation with 8-Br-cAMP, and the nonplacental lines COS-7, HeLa, and GH3. One microgram per reaction of sheared salmon sperm DNA was included as a nonspecific competitor. Arrows on the left indicate specific complexes. B, Mobility shift assays with labeled -140/-119 mGH-V oligonucleotide incubated with JEG-3 choriocarcinoma cell line nuclear extract and the indicated fold-excess of nonradiolabeled competitor oligonucleotides. C, Supershift analysis of the JEG-3 -140/-119 complex by preincubation with 2 µg (2 µl) of each indicated antibody. The lanes with 4 µg (4 µl) of AP-2 antiserum serve as a nonspecific control for the lanes containing 2 µg anti-Sp1 plus 2 µg anti-Sp3. Extracts from cells untreated or exposed to 1.5 mM 8-Br-cAMP were used as indicated. Oligonucleotides used in competition analysis are given in Table 1Go.

 
The identity of the bands was investigated with antisera to the zinc finger transcription factors Sp1, Sp3, and Sp4, which all bind Sp1 recognition sites (18, 19). We also explored whether the relative amounts of these factors bound to the -140/-119 probe was changed in cells that had been treated for 48 h with 8-Br-cAMP. Supershift analysis (Fig. 4CGo) showed that while no bands were shifted in response to anti-AP2 or anti-Sp4 serum, the top-most complex was completely supershifted by antiserum to Sp1. Incubation with antiserum to Sp3 eliminated the lower two bands and in the presence of both antisera, negligible amounts of the three original DNA-protein complexes were observed (Fig. 4CGo). Thus, Sp1 and Sp3 account for essentially all detectable DNA-protein interactions with this probe. The indistinct smear seen below the supershifted Sp1 band in extracts incubated with antibodies to both Sp1 and Sp3 is likely to represent a supershifted Sp3 DNA-protein complex, since this appears only in the presence of anti-Sp3 serum. There was no apparent effect of 8-Br-cAMP treatment on the amount or relative proportions of these factors bound to the -140/-119 probe.

Mutation of the region from -62/-53 also dramatically reduced transcriptional activity in all cell types, and gel mobility-shift assays with a probe spanning -75/-35 demonstrated the presence of two specific bands in both JEG-3 extracts and COS-7 cell extracts (Fig. 5AGo). Competition with overlapping unlabeled competitor oligonucleotides localized the DNA-protein interactions within the -75/-54 region. We performed competitions with sterol response element-binding protein-1 (SREBP-1) (20) and Sp1 consensus binding site oligonucleotides since the mGH-V motif AGGTGGGG (-66/-59) is highly homologous to both of these elements (Fig. 5CGo), and with a consensus GATA-binding site oligonucleotide, since a GATAA motif is seen at -45/-40 (Fig. 5CGo). Fig 5AGo shows that only the Sp1 consensus oligonucleotide competed effectively for binding. To determine the identity of the factors that presumably bind to the GGTGGG element (GT box), we performed supershift assays with antisera to Sp1 family transcription factors as in Fig. 4Go. Figure 5BGo demonstrates that while antisera to Sp4 or AP-2 failed to shift the electrophoretic mobility of any band, formation of the specific complex of fastest mobility was completely blocked by antiserum to Sp3, and the intensity of the upper band was diminished. Incubation with an antiserum to Sp1 did not affect the lower band, but the upper band was partially shifted by anti-Sp1 serum. As with the -140/-119 probe in Fig. 4Go, incubation with both Sp1 and Sp3 antisera supershifted or essentially eliminated all specific DNA-protein complexes. Short exposure times (not shown) clearly demonstrated that the upper portion of the complex of slowest electrophoretic mobility contained Sp1, while the lower portion contained Sp3. As with the -140/-119 probe, there was no detectable effect of 8-Br-cAMP on complexes formed at the -75/-34 probe. Supershift analysis with COS-7 extracts and both the -140/-119 and -75/-35 probes demonstrated essentially identical binding of Sp1 and Sp3 (not shown).



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Figure 5. Mobility Shift Analysis of Factors Binding to the -75/-35 Region within the mGH-V Promoter

A, Mobility shift assays with labeled -75/-35 mGH-V oligonucleotide incubated with JEG-3 choriocarcinoma cell nuclear extracts and the indicated fold-excess of nonradiolabeled competitor oligonucleotides. SRE-1 is an oligonucleotide from the sterol-regulatory element of the human low-density lipoprotein receptor gene (20). GATA is a consensus GATA-binding site oligonucleotide, and Sp1 is a consensus Sp1-binding site oligonucleotide, both from Promega (Madison, WI). One microgram per reaction of sheared salmon sperm DNA was included as a nonspecific competitor. Ar-

 
The GT Box at -65/-60 Binds Sp1/Sp3 and Has Significant Transcriptional Activity
We examined the effects of the scanning mutations at -72/-63 and -62/-53 as well as specific mutation of the GT box at -65/-60, on binding of Sp1/Sp3 to the -75/-35 mGH-V probe (Fig. 6Go). Although wild-type unlabeled oligonucleotides competed effectively, none of the mutant oligonucleotide competed for Sp1 or Sp3 binding (Fig. 6AGo), demonstrating that Sp1/Sp3 could not bind to the promoters with mutations at -72/-63, -62/-53, or -65/-60.



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Figure 6. Competitive Mobility Shift Analysis of Sp1/Sp3 Binding to the -75/-35 Region of the mGH-V Promoter

A, Two micrograms of JEG-3 extract were incubated in the absence or presence of various competitors at the indicated fold-excess. Arrows on the left indicate complexes containing Sp1 or Sp3 as determined by supershift analysis in Fig. 5Go. B, Sequences of the -75/-35 region of the mGH-V promoter and mutated oligonucleotide competitors. The putative Sp1/Sp3 site is highlighted; m72/63 and m62/53 oligonucleotides are identical to the mutations introduced into the promoter for scanning mutagenesis analysis of transcriptional activity rows on the left indicate specific and nonspecific complexes. B, Supershift analysis of the JEG-3 -75/-35 complex by preincubation with 2 µg (2 µl) of the indicated antibody. Anti-AP-2 served as a nonspecific control as in Fig. 4Go. Extracts from untreated JEG-3 cells or cells treated for 48 h with 8-Br-cAMP were used. C, Sequences of the -75/-35 region of the mGH-V promoter and oligonucleotides used in competition analysis. Consensus binding sites for the relevant transcription factors in competitor oligonucleotides are underlined.

 
To directly examine the importance of the GT box at -65/-60 Sp1/Sp3 site in transcription, we prepared a reporter construct with the 140- bp mGH-V promoter containing the -65/-60 mutation shown to disrupt Sp1 and Sp3 binding and compared its activity to the wild-type promoter as well as the overlapping scanning mutations. Figure 7Go demonstrates that the -72/-63, -65/-60, and -62/-53 mutations had similar effects on basal or cAMP-stimulated transcriptional activation within each cell type. All mutations resulted in up to a 90% loss of basal transcriptional activity (Fig. 7BGo). As previously shown in Fig. 1Go, transcriptional activity of the promoter constructs in COS-7 cells was unaffected by treatment with 8-Br-cAMP (Fig. 7DGo). Mutation of the GT box at -65/-60 decreased cAMP-stimulated transcription to levels not significantly different from the -72/-63 and -62/-53 mutations in both primary STB cultures and JEG-3 cells (~50–90% decrease, respectively; Fig. 7Go, C and E). These results also indicate that this downstream GT box may play a more prominent role in transcriptional activation than does the distal GA box disrupted by the -140/-131 mutation (Fig. 2Go), since the effects of the -65/-60 mutation were more dramatic than the effects of the -140/-131 mutation in either JEG-3 cells or primary STBs (see Fig. 2Go, B and D, respectively).



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Figure 7. Mutational Analysis of the Proximal GT Box of the mGH-V Promoter

Luciferase constructs containing 140 bp of 5'-flanking mGH-V DNA were transiently transfected into JEG-3 choriocarcinoma cells (A and C), primary cultures of rhesus STBs (E), or nonplacental COS-7 cells (C). The constructs included either the wild-type (wt) promoter or promoters mutated at nucleotides -72/-63, -65/-60, or -62/-53. The -65/-60 construct selectively eliminates the GT box shown in Fig. 3Go; the other mutants are as listed in Table 1Go. The pGL2 basic plasmid serves as a promoterless control. Panels A and B present the basal luciferase activity of each construct, relative to the WT140 construct, in JEG and COS cells. Panels C and D present the fold-induction of each construct by 8-Br-cAMP. Because of substantial variation in the basal activity between experiments with primary STB cultures, we have presented cAMP-stimulated luciferase activity in panel E relative to the activity with the wild-type promoter. In Panels C and E, cAMP-stimulated luciferase activity of the wild-type promoter was significantly greater than the mutated promoters (P < 0.05), which were not significantly different from each other. The results presented are the means of two individual experiments for COS cells and three individual experiments with trophoblasts, with each treatment group represented by triplicate dishes in each experiment. Individual means for each experiment are indicated by + signs in panels B and D. All dishes were cotransfected with 1–2 µg of a cytomegalovirus-lacZ plasmid, and luciferase activity was normalized to ß-galactosidase activity.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
cAMP-regulated pathways have been identified and implicated in the control of the expression of a diverse range of endocrine genes in the placenta, including CG{alpha} and -ß (21, 22), CS (23, 24, 25), mouse placental lactogen-I, proliferin and proliferin-related protein (26), CRH (27), and enzymes of the steroid synthesis pathway (28, 29). We have previously demonstrated developmentally regulated, trophoblast-specific expression of the rhesus GH-V gene and have performed a detailed analysis of the rhesus monkey GH-V promoter to identify the DNA elements involved in its cell-specific function. We have defined the most proximal 140 bases of the 5'-flanking region to be sufficient for cAMP-responsive transcription in both choriocarcinoma cells and primary trophoblast cultures. These are the first studies to define cAMP-responsive elements controlling transcription of this member of the GH gene family in the primate placenta. We demonstrated that Sp1 and Sp3 bind to two elements that contribute to (-136/-131) or are critical for (-65/-60) mGH-V promoter activation. Interestingly, elements adjacent to both of these Sp1/Sp3 sites (at -130/-121 and -52/-43) also are important for basal and cAMP-stimulated transcription; however, the identity of protein(s) that may bind in this region remains unknown.

cAMP has long been known to activate GH expression in the pituitary gland. Responsiveness to cAMP in the human and rat GH genes is localized to within 145 bp of the start site of transcription (30, 31, 32). It has recently been reported that cAMP-response element binding protein (CREB) regulates human pituitary GH (hGH-N) transcription via a variant recognition element at -98/-95 (33). However, this CREB site is not conserved within the homologous region of the mGH-V promoter, and mutation of the region analogous to this hGH-N CREB site in the mGH-V promoter did not identify a significant involvement in cAMP-stimulated transcription.

The GH-V, GH, and CS genes contain Sp1-binding sites located approximately 136 bp from the start site of transcription. Sp1 sites are important in the placental transcription of the genes for the steroidogenic enzymes cytochrome P450 cholesterol side-chain cleavage enzyme (34, 35, 36), cytochrome P450 aromatase (37), and cytochrome P450 11ß-hydroxysteroid dehydrogenase (38). In addition to activity as an element of the hCS proximal promoter, an Sp1-binding site at -136/-131 is important for functional cooperation of the hCS promoter with a 3'-enhancer located downstream of the CS-B gene (39). In our studies, mutation of the mGH-V region including the -136/-131 Sp1 motif resulted in a 50% reduction in transcription in STBs, similar to the 2- to 3-fold decreases reported for the hCS and hGH genes (16, 39, 40). Supershift and binding site competition analysis demonstrated that both Sp1 and Sp3 are bound to this element. However, mutations in other regions of the promoter (e.g. -62/-53) drastically decreased transcription, despite the presence of an intact Sp1 site at -136/-131. This result indicates that while this Sp1 site contributes to transcriptional activity, it is not sufficient to sustain transcription after disruption of other DNA-protein interactions.

One of these critical regions for transcription is centered at -62/-53. Examination of the promoter sequence revealed an Sp1-like site (GGTGGG) at -65/-60, and subsequent supershift and competition analysis demonstrated Sp1 and Sp3 binding at this element. With both the upstream and downstream Sp1 sites, bands of two distinctly different mobilities contained Sp3, while a single band contained Sp1. Previous studies of the platelet-derived growth factor-B promoter have shown that Sp3 variants of different molecular weights account for a mobility shift pattern remarkably similar to that seen with our elements (41). Whether the two Sp3-containing bands seen with trophoblast extracts represent splice variants or Sp3 complexed with additional proteins remains to be determined.

It is interesting to note that although very similar complexes are formed at the two Sp1 sites, mutations in the regions containing these sites have different effects on transcription. One explanation for the distinct differences in functional importance may be that the site at -65/-60 is in close proximity to the TATAA element and is thus more critically positioned to interact with the basal transcriptional complex (42, 43, 44). In its absence, transcription is severely limited. Interaction of factors bound to the more distal Sp1 site with the basal transcriptional initiation complex may be of relatively less importance. Alternatively, Sp1/Sp3 bound to the proximal but not distal binding site may facilitate other DNA-protein interactions as yet unidentified. For example, the adjacent -52/-43 region was critical for transcription in primary STB cultures, and factors bound at this region may have critical interactions with Sp1/Sp3 bound at -65/-60.

We identified additional elements not previously reported to be involved in GH-V expression that appear to be critical for transcriptional activation. Mutations at -130/-121 dramatically reduced transcriptional activation, despite the presence of intact upstream and downstream Sp1 sites. Mutations in the adjacent region, -120/-111, also significantly reduced transcriptional activation in JEG-3 cells. This -120/-111 region contains a potential Pit-1-binding site. Pit-1 plays an important role in the cAMP-stimulated transcription of GH and Pit-1 promoters (45, 46, 47). We and others have recently detected the expression of Pit-1 in human and monkey trophoblasts (48, 49); however, competition and supershift analyses with JEG-3 cell extracts could not detect Pit-1 binding to this region (J. T. Schanke and T. G. Golos, unpublished). Additionally, scanning mutations including either the distal or proximal Pit-1 sites did not reveal a role in mGH-V transcription in rhesus STBs, as has previously been noted in studies of the hCS promoter by Jiang et al. (39). Further studies are needed to identify the factor(s) acting at the -130/-121 region.

Finally, although Sp1 and Sp3 bind to regions important for trophoblast-specific transcriptional activation by cAMP, they are not trophoblast-specific factors; likewise, scanning mutagenesis demonstrated that the cis-elements important for basal and cAMP-stimulated transcription in STBs also contribute to basal transcription in COS-7 cells. Sp1 effects transcriptional activation through interactions with multiple components of the basal transcriptional complex, including Drosphila TATA-binding protein-associated factorII110 (dTAFII 110) (42), human (h)TAFII55 (43), and TATA-binding protein (44). We do not yet know how cAMP responsiveness involving Sp1/Sp3 is restricted to trophoblasts. It is possible that placenta-specific coactivators may interact with Sp1/Sp3 bound to the mGH-V promoter in the placenta to confer transcriptional regulation. For example, a B cell-specific coactivator for Oct-1 (50) and a B cell-enriched TAF, hTAFII105 (51), have recently been identified, and it seems likely that cell-specific mechanisms will be identified in other tissues for fine tuning the effects of widely distributed transcription factors.

Alternatively, the effects of cAMP may include modulating protein-protein interactions or transcriptional complex assembly rather than recruitment of new or trophoblast-specific proteins to DNA-binding sites. Posttranslational modifications of trans-acting protein activity by the protein kinase A pathway has been widely recognized (52, 53, 54, 55, 56, 57), and extensive phosphorylation previously noted for Sp1 (58) provides incentive to investigate the role of this mechanism in mGH-V transcriptional activation by cAMP in future studies.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Placental Cell Isolation, Culture, and Transfection
Chorionic villi obtained from placentas at cesarean section were dispersed with trypsin and DNAse as previously described (14). All procedures involving the use of animals were approved by the University of Wisconsin Graduate School Animal Care and Use Committee and were conducted in accordance with the NIH Guide for the Care and Use of Experimental Animals. A highly purified fraction of cytotrophoblasts was obtained by Percoll gradient centrifugation, and cells were plated for transient transfection experiments. Typically, freshly isolated cytotrophoblasts were plated in 60-mm dishes at 2 x 106 cells per dish in serum-free medium. Plasmid-liposome complexes formed using Lipofectamine (Life Technologies, Inc., Gaithersburg, MD) or freshly prepared liposomes [1 µg/ml each of dioleylphosphatidyl-ethanolamine and dimethyldioctadecylammonium bromide (59)] and 10 µg plasmid DNA were immediately added. Four hours after plasmid addition, FCS was added to a final concentration of 10%. Cells were scraped from dishes approximately 48 h posttransfection, harvested by centrifugation, and lysed with Luciferase Assay Buffer (Promega, Madison, WI). Cell debris was pelleted and supernatants were assayed for luciferase activity. Relative light units were determined as the integral of 8 sec of light output after a 2-sec stabilization period, in a EG&G model 9505 tube luminometer (EG&G, Natua, NH). The data are expressed in most figures as means with SE relative light units (RLU)/µg protein in cell extracts, normalized to luciferase activity in untreated controls or a wild-type or full-length promoter, as is appropriate for the experiment at hand. Protein concentrations in cell lysates were determined with Coomassie protein assay reagent from Pierce (Rockford, IL). Some cultures were treated with 1.5 mM 8-bromo-cAMP (Sigma, St. Louis, MO) for 48 h. For the experiments in Fig. 7Go, all luciferase constructs were cotransfected with a cytomegalovirus (CMV) immediate early enhancer/promoter-ß-galactosidase reporter (60), ß-galactosidase activity was determined with the Galacto-light assay kit (Tropix, Bedford, MA), and luciferase activity was normalized to ß-galactosidase activity in each dish. The conclusions reached with normalized data were consistent with studies done without cotransfection presented in Figs. 1Go and 2Go.

Luciferase Reporter Gene Constructs
The mGH-V 5'-flanking DNA used in deletion constructs was subcloned into the vector pZLUC (12) (kindly provided by Dr. Paul Deutsch, Cornell University Medical Center, New York, NY). 5'-Deletion fragments, containing the denoted number of base pairs including the transcriptional start site, were subcloned, using convenient restriction sites or PCR amplification, just upstream of the luciferase gene.

A modified version of the three-step PCR mutagenesis strategy described by Li and Shapiro (61) was used to create six to 10 base block mutations scanning the 140-bp mGH-V promoter fragment, which spans -140 to the BamHI site at +1 (the start site of transcription). The fragment was PCR-amplified with primers that added BglII and HindIII sites to the 5' and 3' ends, respectively, and was subcloned into the BglII/HindIII site of the Luciferase reporter vector pGL2 (Promega, Madison, WI) and was used as the template for PCR mutagenesis. Primers used for PCR mutagenesis are depicted in Table 1Go. Typically 50-µl reactions contained 200 ng mGH-V wt140-pGL2 template and 1.5 U Tfl DNA polymerase (Epicentre Technologies, Madison, WI). The first PCR was carried out with 20 pmol of each mutation primer paired with 20 pmol of the pGL2 downstream primer: 5' CCT TTC TTT ATG TTT TTG GCG TCT 3'. The reactions were carried out at 95 C for 50 sec, 45 C for 60 sec, and 72 C for 60 sec for 40 cycles. The products from the first round of amplifications were used directly in the second-round asymmetric amplification with 20 pmol pGL2 downstream primer. The second-round amplification conditions were 95 C for 50 sec, 55 C for 60 sec, and 72 C for 60 sec for 30 cycles. The products of the second PCR were gel purified and used as template in a third round of amplifications using 20 pmol of the pGL2 upstream primer: 5' GGT ACT GTA ACT GAG CTA ACA TAA CC 3' and the original wild-type mGH-V 140 template. After 40 cycles with the first-round amplification conditions, the products were purified by gel electrophoresis and were restricted with BglII and HindIII and ligated into the luciferase reporter plasmid pGL2. All constructions and mutations were verified by sequencing.


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Table 1. Oligonucleotides are listed in 5' to 3' Orientation

 
Nuclear Extracts and Electrophoretic Mobility Shift Analysis
Nuclear extracts were prepared essentially by the method of Dignam et al. (62) in the presence of 1 µM leupeptin and 0.5 mM 4-(2-aminoethyl) benzenesulfonly-flouride, hydrochloride (Boehringer Mannheim Corp., Indianapolis, IN). Nuclear extracts were prepared from the human JEG-3 choriocarcinoma cell line, the monkey kidney line COS-7, and the rat pituitary line GH3. HeLa cell nuclear extracts were purchased from Promega Corp. (Madison, WI).

Mobility shift binding reactions typically contained 5–10 µg nuclear extract, 1–3 µg nonspecific competitor (indicated in figure legends), and 20,000 cpm (0.2–1.0 ng) end-labeled double-stranded oligonucleotide probe in a final reaction volume of 20 µl [final buffer composition: 10 mM HEPES (pH 7.9), 50 mM KCl, 2.5 mM MgCl2, and 10% glycerol]. Reactions were incubated at room temperature for 15 min. Oligonucleotides for competitive binding studies were preincubated with nuclear extract reaction mix for 5 min at room temperature before the addition of the radiolabeled probe. Oligonucleotide sequences are depicted in Table 1Go. The reactions were then loaded onto a 5% polyacrylamide gel with 5% glycerol in 0.5 x Tris-borate-EDTA and were run for 2 h at 150 V, dried, and exposed to x-ray film. For supershifts, extracts were preincubated with 1–3 µg of antibody (Sp1, Sp3, Sp4, and AP-2 polyclonal antibodies, Santa Cruz Biotechnology, Santa Cruz, CA) or 1–3 µl normal rabbit serum for 1 h on ice before the addition of radiolabeled probe.


    ACKNOWLEDGMENTS
 
We thank Dr. W. D. Houser, Dr. C. O’Rourke, and Dr. Jan Ramer for assistance with animal surgery and S. G. Eisele for coordinating timed matings. We thank Dr. William Fahl for Sp3 antiserum for preliminary experiments and Peter Schams for assistance with luciferase and ß-galactosidase assays.


    FOOTNOTES
 
Address requests for reprints to: Dr. Thaddeus G. Golos, Wisconsin Regional Primate Research Center, 1223 Capitol Court, University of Wisconsin, Madison, Wisconsin 53715-1299.

This work was supported by NIH Grants HD-26458 to Dr. Golos, HD-08069 to Dr. Schanke, and RR-00167 to the Wisconsin Regional Primate Research Center.

Preliminary reports of portions of this work were presented at the 77th Annual Meeting of The Endocrine Society, Washington D.C., June, 1995, and at the 10th International Congress on Endocrinology, San Francisco, CA, June, 1996.

This is Wisconsin Regional Primate Research Center Publication 37–028.

1 Current address: Epicentre Technologies, Inc., Madison, WI. Back

2 Current address: Pharmacopeia, Cranbury, NJ. Back

Received for publication August 12, 1997. Revision received February 11, 1997. Accepted for publication November 26, 1997.


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 RESULTS
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