©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Involvement of a Protein Distinct from Transcription Enhancer Factor-1 (TEF-1) in Mediating Human Chorionic Somatomammotropin Gene Enhancer Function through the GT-IIC Enhanson in Choriocarcinoma and COS Cells (*)

Shi-Wen Jiang (1), Norman L. Eberhardt (1)(§)

From the (1) Endocrine Research Unit, Departments of Medicine and Biochemistry/Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Previous studies suggested that transcription enhancer factor-1 (TEF-1) was involved in mediating the human chorionic somatomammotropin (hCS) gene enhancer (CSEn) function (Jiang, S.-W., and Eberhardt, N. L.(1994) J. Biol. Chem. 269, 10384-10392). We now show that an unrelated protein (CSEF-1) found in BeWo and COS-1 cells binds to the GT-IIC enhanson in CSEn and is correlated with CSEn activity in these cells. TEF-1 and CSEF-1 were distinguished by differential migration as GT-IIC complexes, thermal stability, molecular mass, and cross-reactivity with chicken TEF-1 antibodies. TEF-1 and CSEF-1 bound to the GT-IIC and Sph-I/Sph-II enhansons with identical binding properties, and in vitro generated TEF-1 competed with CSEF-1 binding to the GT-IIC motif, suggesting that their actions might be mutually exclusive. Up- and down-regulation of TEF-1 levels by expression systems and antisense oligonucleotides demonstrated that TEF-1 inhibited the hCS promoter in a manner independent of the enhancer or a known TEF-1 DNA binding site. The data suggest that TEF-1 may provide a counter-regulatory stimulus to the actions of CSEF-1, which may be involved in mediating enhancer stimulatory activity.


INTRODUCTION

Previous studies indicate that the human chorionic somatomammotropin enhancer (CSEn)() is involved in regulating the placental-specific expression of the human chorionic somatomammotropin genes (hCS-1 or hCS-B and hCS-2 or hCS-B; or placental lactogen, hPL) and the growth hormone variant gene (hGH-2 or hGH-V). CSEn was first identified by Saunders and co-workers (Rogers et al., 1986; Walker et al., 1990). Since the enhancer was localized nearest to the hCS-2 gene, which is expressed at high levels and is the 3`-most member in the hGH/hCS gene cluster on chromosome 17q22-24 (Miller and Eberhardt, 1983; Hirt et al., 1987; Chen et al., 1989), it was implicated in mediating hCS cell-specific expression (reviewed by Walker et al.(1991)). Three copies of the enhancer are present within the hGH/hCS chromosomal locus and are located downstream of the hCS-5 (putative pseudogene; Hirt et al., 1987), hCS-1, and hCS-2 genes; however, only the enhancer downstream of the hCS-2 gene appears to have significant enhancer activity (Jacquemin et al., 1994). Cell-specific expression of the hCS genes does not depend on the enhancer alone, since we recently demonstrated that an initiator element downstream of the TATA box contributes significantly to the basal- and enhancer-stimulated hCS promoter activity (Jiang et al., 1995).

The hCS enhancer is composed of multiple DNA elements that are homologous to several SV40 enhansons (Jiang and Eberhardt, 1994; Jacquemin et al., 1994). Both the SV40 GT-IIC and Sph-I/Sph-II enhanson motifs present in CSEn are protected by placental cell nuclear proteins in DNase I footprinting assays, and mutation of these elements results in marked loss of enhancer activity (Jiang and Eberhardt, 1994; Jacquemin et al., 1994). Transcription enhancer factor-1 (TEF-1) has been shown to bind to the GT-IIC and Sph-I/Sph-II enhansons (Davidson et al., 1988; Xiao et al., 1991) and a few variant elements with the consensus 5`-TGTGG(T/A)(T/A)(T/A)G-3` (Kariya et al., 1993). Accordingly, TEF-1 has been implicated in the mechanism of CSEn action. TEF-1 is a widely distributed transcription factor that appears to be involved in mediating the regulation of a diverse set of genes, including SV40 enhancer function (Davidson et al., 1988; Xiao et al., 1991; Hwang et al., 1993), human papillomavirus-16 E6 and E7 oncogene transcription (Ishiji et al., 1992), cardiac and skeletal muscle-specific gene expression (Stewart et al., 1994; Farrance et al., 1992; Kariya et al., 1993, 1994), early gene expression in mouse development (Melin et al., 1993), and hCS gene tissue-specific expression (Jacquemin et al., 1994; Jiang and Eberhardt, 1994; Lytras and Cattini, 1994).

The mechanism of TEF-1 action appears to be complex. Overexpression of TEF-1 in a variety of cells results in squelching of transcriptional activity (Xiao et al., 1991; Ishiji et al., 1992; Hwang et al., 1993), suggesting that other limiting transcription factors or adapters are required for TEF-1 function (Xiao et al., 1991). Additionally, co-repressors may be involved in repressing stimulation by TEF-1 (Chaudhary et al., 1994). Since such co-activators or co-repressors have not been isolated or cloned, we do not understand how TEF-1 mediates positive enhancer function. In the current studies we sought to establish whether TEF-1 was involved in CSEn function and provide information about its mechanism of action. We establish the presence of TEF-1 in the BeWo choriocarcinoma cell line and demonstrate that a 30-kDa protein (CSEF-1) competes for TEF-1 binding to the GT-IIC and Sph-I/Sph-II enhansons with identical binding specificity. CSEF-1 is distinguishable from TEF-1 on the basis of molecular mass, thermal stability and absence of cross-reactivity with antibodies to TEF-1. Overexpression of TEF-1 in BeWo cells resulted in marked inhibition of basal hCS promoter activity without affecting the relative enhancer-mediated stimulation of transcription. In contrast, down-regulation of TEF-1 expression in BeWo cells resulted in increased basal promoter activity and increased enhancer-mediated stimulation. Also, the enhancer was very active in COS-1 cells, a cell line that contained very low levels of TEF-1 but contained a CSEF-1-like protein. Thus CSEF-1 may be involved in the stimulatory properties of CSEn. The data suggest alternate models for TEF-1 action, including the possibility that it mediates negative enhancer functions.


MATERIALS AND METHODS

Cell Transfection and Luciferase Assays

COS-1 and HeLa cells (ATCC) were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.); BeWo cells (ATCC) were maintained in RPMI 1640 (Life Technologies, Inc.). All media were supplemented with 10% fetal bovine serum (Whittaker), 100 units/ml penicillin (Life Technologies, Inc.), 100 µg/ml streptomycin (Life Technologies, Inc.) and 2 mML-glutamine (Life Technologies, Inc.). Cells were maintained at 37 °C in an atmosphere containing 5% CO and 100% humidity.

Cell harvesting, transfection by electroporation, and luciferase assays have been described in detail (Jiang and Eberhardt, 1994). Sense and antisense TEF-1 phosphorothioate oligonucleotides (35 mM, Molecular Biology Core Facility, Mayo Clinic) () were introduced into BeWo cells via electroporation.

Data Analysis

Data were subjected to analysis of variance (ANOVA). For experimental groups satisfying the initial ANOVA criterion (p < 0.05), comparisons were performed using Student's t tests and a Bonferroni inequality to compensate for the multiple comparisons (Snedecor and Cochran, 1980). When the variances were not equivalent, logarithmically transformed (log) data were analyzed.

Plasmid Constructions

The plasmids pALUC (Maxwell et al., 1989); EnA_CSp.LUC, containing the CSEn and the hCS-1 5`-flanking region (493 bp); and EnA_EnAP_CSp.LUC, containing an additional copy of CSEn inserted in EnA_CSp.LUC at the PstI site (nucleotide -300 relative to the transcription initiation site) have been described previously (Jiang and Eberhardt, 1994).

Complementary oligonucleotides (Molecular Biology Core Facility, Mayo Clinic) GT-IICD(+) and GT-IICD(-) (), containing tandem copies of the SV40 GT-IIC enhanson (OGT2-56; Xiao et al. (1991)) were synthesized to construct reporter plasmids containing multiple GT-IIC binding sites. Single-stranded oligonucleotides were phosphorylated with T4 polynucleotide kinase (New England BioLabs), annealed and ligated with T4 DNA ligase (Boehringer Mannheim). The ligation products were resolved on a 2.5% agarose gel, and 4-mer oligonucleotides containing 8 copies of the GT-IIC site were selected by eluting the 120-bp DNA fragment. Following purification (GeneClean), BglII linkers were added and the DNA fragments were cloned into CSp.LUC at the BglII site, to yield (GT-IIC)CSp.LUC. Additionally, the 120-bp (GT-IIC) fragments were blunt-ended by T4 dNA Polymerase treatment, ligated to PstI linkers, and inserted in the PstI site of (GT-IIC)CSp.LUC to yield (GT-IIC)CSp.LUC and (GT-IIC)CSp.LUC.

Nuclear Extracts

Large scale nuclear extracts were isolated from cultured cells according to the method of Dignam et al. (1983). For microscale extracts, 1 10 transfected cells were plated onto 6-cm dishes. After 20 h, cells were harvested by scraping in 1.5 ml of phosphate-buffered saline and centrifugation (800 g for 1 min). After aspirating the phosphate-buffered saline, cells were resuspended in 3 packed cell volumes of 20 mM HEPES (pH 7.9), 20% glycerol, 0.5 M KCl, 0.2 mM EDTA, 0.5 mM PMSF and 0.5 mM DTT at room temperature. The cell suspension was rapidly frozen on dry ice and thawed in a 37 °C water bath, centrifuged (30 s at 14,000 g), and the supernatant was diluted with 3.5 volumes of 20 mM HEPES (pH 7.9), 20% glycerol, 0.2 mM EDTA, 0.5 mM PMSF, and 0.5 mM DTT. Protein concentration was determined by Coomassie binding, and nuclear extracts were stored at -70 °C.

Gel Shift Assays

The synthetic oligonucleotides used in gel shift assays () were labeled with [-P]ATP (Amersham Corp.), and the conditions for DNA-protein binding and gel shift analyses were performed as described in detail by Jiang et al.(1995).

In Vitro Translation and Escherichia coli Expression of TEF-1

The pXJ40-TEF-1A plasmid (Xiao et al., 1991) was generously provided by Dr. Pierre Chambon (Pasteur Institute, Paris) and was used to generate TEF-1 protein by in vitro translation (TNT, Promega). The reaction was performed per the manufacturer's protocol using 3.0 µg of double CsCl gradient-purified pXJ40-TEF-1A plasmid DNA and 50 µl of TNT reticulocyte lysate in a 100-µl reaction volume. Typically 1.5 µl of in vitro translational product was used in each gel shift reaction. Product from a mock translation with 3.0 µg of pBluescript was used as negative control.

TEF-1 was also expressed and purified from E. coli HB101. TEF-1 coding sequences were amplified from pXJ40-TEF-1A by PCR using primers GST-TEF_1 and GST-TEF_2 (). The DNA fragments were digested with EcoRI and SalI and ligated to pGEX-4T-3 vector (Pharmacia) digested by the same enzymes. Bacterial lysates were prepared by ultrasonic lysis and TEF-glutathione S-transferase fusion proteins were bound to glutathione-Sepharose 4B (Pharmacia) and cleaved by thrombin (Sigma). Cleavage products were immediately stored at -70 °C and used in gel shift assays without further purification.

Western and Southwestern Blotting and UV Cross-linking

The rabbit anti-chicken TEF-1 antibody was generously provided by Drs. Charles Ordahl and Iain Farrance (University of California, San Francisco). For blots, 10 µl of in vitro translation product and 80 µg of BeWo or HeLa cell nuclear extracts were resolved on a 9% SDS-polyacrylamide gel. Proteins were electro-transferred onto 0.2 µM nitrocellulose membrane (Trans-Blot, Bio-Rad) with 48 mM Tris (pH 9.2), 39 mM glycine, 20% methanol, and 1.3 mM SDS, using a semidry electrophoretic transfer cell (Trans-Blot SD, Bio-Rad). After 1 h of blocking in 3% gelatin in 20 mM Tris (pH 7.5), 500 mM NaCl, TEF-1 antibody diluted at 1:2500 in 20 mM Tris (pH 7.5), 500 mM NaCl, and 0.05% Tween 20 was applied. Following alkaline phosphatase-conjugated antibody binding, color development was carried out using the Immun-Blot kit (Bio-Rad) according to the manufacturer's specifications.

Southwestern blotting experiments were performed on proteins transferred onto membrane that were denatured/renatured with guanidine hydrochloride as described by Vinson et al.(1988). Nonspecific DNA binding activities were eliminated by incubation in 0.5 Dignam buffer D, 5% nonfat dried milk, 10 mM MgCl, 0.5 mM PMSF, and 0.5 mM DTT at 4 °C for 90 min. The filters were incubated with 5` end-labeled GT-IIC probe (), which was diluted to 2 10 cpm/ml in 0.5 Dignam buffer D, 0.2% nonfat dried milk, 10 mM MgCl, 0.5 mM PMSF, 0.5 mM DTT, and 50 µg/ml sonicated salmon sperm DNA at 4 °C for 180 min. The filters were then washed in the same buffer and exposed to Kodak x-ray film with intensifying screens at -70 °C for 48 h.

UV cross-linking was performed essentially as described in detail previously (Jiang et al., 1995). Authentic in vitro generated TEF-1 (5 µl) and 15 µg of BeWo nuclear extract were incubated with the GT-IIC probe (50,000 cpm), and the complexes were resolved on a 10% SDS-PAGE gel.

Heparin-Sepharose Chromatography

A 0.5-ml heparin-Sepharose (L-6B, Pharmacia, Sweden) column prepared in a 1.5-ml Pasteur pipette was washed with 5 ml of distilled water and equilibrated with 5 ml of Dignam buffer D containing 10 mM MgCl (Catala et al., 1989; Ristiniemi and Oikarinen, 1989; Hatamochi et al., 1993). Crude BeWo cell nuclear extract (200 µl, 5 µg/µl) was applied, and the column was washed with 5 ml of loading buffer. The bound proteins were then step-eluted with 1 ml of buffer D containing 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 M KCl. Four 250-µl fractions were collected for each elution and stored immediately at -70 °C. The protein concentration was measured by Coomassie dye binding, and GT-IIC DNA binding activities were detected by gel shift assay.


RESULTS

Proteins Present in BeWo and COS-1 Cells Other than TEF-1 Bind to the GT-IIC Motif

Previous footprint analyses of CSEn with nuclear extracts from a variety of cells were indistinguishable, suggesting that the factors that bound to CSEn might be ubiquitously distributed (Jiang et al., 1994). To determine if different proteins might bind to the various enhanson motifs in CSEn, we compared the binding of nuclear proteins from BeWo, COS-1, and HeLa cells along with in vitro generated TEF-1 to the GT-IIC enhanson. Three distinct complexes were formed with nuclear proteins from these cell types (Fig. 1). Nonspecific binding was distinguished with the GT-IICMUT, containing a mutated GT-IIC core consensus (). No differences were observed in the specific binding patterns using the SV40 (GT-IIC) or CS (GT-IIC) TEF-1 binding motifs (data not shown). The slowest migrating complex was tentatively identified as containing TEF-1, since it was the only complex observed in HeLa cell extracts and it co-migrated with authentic in vitro generated TEF-1. BeWo extracts appeared to contain significant quantities of a TEF-1-like binding activity as well as a protein, designated CSEF-1, which formed a more rapidly migrating complex with the GT-IIC enhanson. COS-1 cell extracts produce a complex that migrates identically to that of the CSEF-1 complex, produce a unique, more rapidly migrating complex and appear to contain only minor amounts of TEF-1. Since the CS enhancer is very active in COS-1 cells (discussed below), the data suggest that the protein forming the CSEF-1 complex might be involved in mediating CSEn function.


Figure 1: BeWo and COS-1 cells contain nuclear factors in addition to TEF-1 that form distinct, specific complexes with the GT-IIC enhanson. E. coli expressed TEF-1 (50 ng) or 3.0 µl of in vitro generated TEF-1 translation products (TNTTEF) were compared with 7.0 µg of BeWo, COS-1, and HeLa nuclear extracts in the gel shift assay as described under ``Materials and Methods.'' The various extracts were incubated with 15,000 cpm of P-labeled GT-IICWT or GT-IICMUT probes and 1.5 µg of poly(dI-dC) as a nonspecific competitor in each reaction. Probe GT-IICMUT contains mutations in the ``core consensus,'' but identical flanking sequences as the wild-type GT-IIC probe (Table I). No significant DNA binding activity was present in the mock translation products (TNT) using pBluescript plasmid as template to provide negative control. The TEF-1 complex appeared to be absent in COS-1 extracts, very abundant in BeWo extracts, and present in only minor amounts in GC cell extracts. The rapidly migrating complex that is abundantly present in BeWo and COS-1 cells is designated CSEF-1.



Both CSEF-1 and TEF-1 Recognize the GT-IIC and Sph-I/Sph-II Motifs

To further compare the properties of CSEF-1 and TEF-1, we examined their ability to bind the Sph-I/Sph-II enhanson. TEF-1 is known to bind to both the Sph-I/Sph-II and GT-IIC motifs (Davidson et al., 1988). Oligonucleotides containing the wild-type GT-IIC (Fig. 2A) and Sph-I/Sph-II (Fig. 2B) motifs compete for the binding of TEF-1 and CSEF-1 about equally, whereas neither the GT-IIC or Sph-I/Sph-II mutant oligonucleotides competed for TEF-1 or CSEF-1 binding to the GT-IIC wild-type DNA (Fig. 2, A and B). Both TEF-1 and CSEF-1 bind the GT-IIC enhanson with relatively higher affinity than the Sph-I/Sph-II enhanson. Thus TEF-1 and CSEF-1 have identical binding specificities and raise the question whether these two proteins might be related.


Figure 2: CSEF-1 and TEF-1 bind to the GT-IIC and Sph-1/Sph-II enhansons with nearly identical binding specificity. The DNA binding specificity of CSEF-1 was examined by a gel shift competition assay. Gel shift assays were performed under the same conditions as those of Fig. 1 using 15,000 cpm of P-labeled GT-IICWT oligonucleotide as the probe. Increasing amounts of GT-IICWT or GT-IICMUT (A) and SphWT or SphMUT oligonucleotides (B) were included in the reactions.



CSEF-1 and TEF-1 Are Distinguished by Differential Thermal Stability

To assess the relatedness of CSEF-1 and TEF-1, BeWo (Fig. 3A) and COS-1 (Fig. 3B) nuclear extracts were subjected to heat treatment at various temperatures for 2 min. The data demonstrate that the CSEF-1-containing complex in both BeWo and COS cells is very heat-stable, since it survived 100 °C treatment. In contrast, the TEF-1-containing complex in BeWo cells (Fig. 3A) was virtually absent after 2 min at 70 °C. This latter behavior was identical to authentic in vitro generated TEF-1 (data not shown). Thus CSEF-1 and TEF-1 may be unrelated proteins that independently recognize the same DNA elements. Additionally, the CSEF-1 binding activity in BeWo and COS cells appears to be due to a very similar or identical heat-stable protein.


Figure 3: CSEF-1 and TEF-1 exhibit differential thermal stabilities. BeWo (A) and COS-1 (B) cell nuclear extracts were heated at different temperatures, and the ability of the proteins to bind P-labeled GT-IICWT DNA was evaluated in gel shift assays (see ``Materials and Methods''). Incubation at 70 °C for 2 min destroyed almost all the TEF-1 binding activity. In contrast, CSEF-1 was resistant to heating at 100 °C for 2 min.



TEF and CSEF-1 Exhibit Partially Different Heparin-Sepharose Chromatography Profiles

To further characterize the potential relationship of CSEF-1 and TEF-1, heparin-Sepharose chromatography of BeWo nuclear extracts was performed. Crude BeWo nuclear extract was loaded onto a heparin-Sepharose column equilibrated with Dignam buffer D (0.1 M KCl). After extensive washing with the same buffer, DNA binding activities were stepwise eluted with buffer D containing 0.2-0.7 M KCl. The flow-through and salt-eluted fractions were collected and DNA binding activities characterized by gel shift assay (Fig. 4). Only very weak or nonspecific DNA binding activities were detected in the flow-through and 0.2 M eluate, which contained about 65% of the initial protein. CSEF-1 eluted in a sharp peak at 0.4 M KCl, whereas apparent TEF-1 binding activity was eluted broadly as two separate peaks of activity with 0.3 and 0.4 M KCl. The bulk of the TEF-1 binding activity eluted at 0.3 M KCl. Accordingly TEF-1 binding activity may be composed of different proteins, multiple isoforms, or differentially modified forms of TEF-1. Thus the profiles of the two proteins can be partially distinguished, supporting the concept that the two proteins may be distinct.


Figure 4: CSEF-1 can be separated from the bulk of TEF-1 binding activity with heparin-Sepharose chromatography. BeWo nuclear proteins were separated on a heparin-Sepharose column as described under ``Materials and Methods.'' The flow-through and salt-eluted fractions were analyzed by gel shift analysis to P-labeled GT-IICWT oligonucleotide. Most nonspecific DNA binding activity was eluted in the 0.2 M KCl eluate. The bulk of the TEF-1 binding activity eluted in the 0.3 M KCl eluate. CSEF-1 and a portion of the TEF-1 binding activity were co-eluted with 0.4 M KCl. The specific and nonspecific (GT-IICMUT) DNA binding activities from the original extract are shown to the right of the figure.



TEF-1 Is Present in BeWo Cells and Is Distinguished from CSEF-1 by Its Molecular Mass and Immunoreactivity

To further characterize TEF-1 and CSEF-1 binding activities in BeWo and COS-1 cells, Western and Southwestern analyses were performed. As demonstrated in Fig. 5A, an antibody to chicken TEF-1 (Kariya et al., 1993), which cross-reacts with human TEF-1, recognizes a major protein species with mass of 55 kDa and a minor species with mass of 57 kDa in both HeLa and BeWo nuclear extracts. TEF-1 was not detected by Western analysis in COS-1 cells (Fig. 5B). The finding of two immunoreactive TEF-1 species of differing molecular mass may help to explain the complexity of the heparin-Sepharose profile (Fig. 4). These two immunoreactive proteins exhibit identical electrophoretic properties as TEF-1 prepared by in vitro translation, demonstrating that BeWo cells contain TEF-1 (Fig. 5A). In addition, these data indicate that BeWo and HeLa cell nuclear extracts contain comparable levels of TEF-1, a finding supported by the previous gel shift analyses (Fig. 1). The antibody did not appear to recognize any other proteins in BeWo, HeLa, or COS cells with electrophoretic migration characteristics different from TEF-1, indicating that TEF-1 and CSEF-1 are unrelated proteins. In addition, although the chicken TEF-1 antibody was able to supershift TEF-1-DNA complexes containing two copies of the GT-IIC motif, no supershift of CSEF-1-containing complexes was observed with the same DNA in the presence of heat-treated BeWo cell nuclear extract (data not shown).


Figure 5: CSEF-1 is a 30-kDa protein that is immunologically distinct from TEF-1. 50 µg of HeLa or BeWo cell nuclear extracts and 5 µl of in vitro generated TEF-1 were resolved by SDS-PAGE, transferred to nitrocellulose membranes and detected by either antibody (A and B) or P-labeled GT-IICWT probe (C and D) as described under ``Materials and Methods.'' A and B, TEF-1 resolved in a band with an apparent molecular mass of 55 kDa on the basis of cross-reactivity with antibody raised against chicken TEF-1 (Kariya et al., 1993). The 55-kDa TEF-1 band was observed in BeWo and HeLa cells, but was not detected in COS cell extracts. C, a 55-kDa band was also observed in Southwestern blots of in vitro generated TEF-1 using the labeled GT-IIC probe. D, Southwestern analysis of BeWo, HeLa, and COS cell extracts using the GT-IIC probe revealed the presence of a 55-kDa band corresponding to TEF-1 in BeWo and HeLa cells, whereas a 30-kDa factor that bound the probe was detected in BeWo and COS cells but not HeLa cells. The 30-kDa factor was not recognized by the TEF-1 antibody (C). E, UV cross-linking analysis of the TEF-1 and CSEF-1-GT-IIC complexes present in BeWo nuclear extracts (Fig. 1).



To determine the relative molecular mass of CSEF-1, Southwestern analysis was done comparing the binding of in vitro generated TEF-1 (Fig. 5C) and BeWo, HeLa, and COS nuclear proteins (Fig. 5D) to the wild-type GT-IIC motif. The 55- and 57-kDa proteins present in BeWo and HeLa cells bind the GT-IIC probe (Fig. 5D) as does in vitro generated TEF-1 (Fig. 5C). In contrast, a protein with mass of 30 kDa present in BeWo and COS-1 nuclear extracts binds the GT-IIC motif (Fig. 5D). UV cross-linking indicated that the CSEF-1-GT-IIC complex migrates with a size of about 33 kDa (Fig. 5E), indicating that the 30-kDa protein (Fig. 5D) very likely corresponds to CSEF-1. Thus CSEF-1 appears to be present in BeWo and COS-1 cells and can be distinguished from TEF-1 on the basis of mass and the lack of common epitopes for binding the polyclonal antibody to chicken TEF-1.

CSEF-1 and TEF-1 Compete for Binding to the GT-IIC Motif

To establish whether TEF-1 and CSEF-1 compete for binding to the same site, we added in vitro generated TEF-1 to heat-treated BeWo nuclear extracts, which lack TEF-1 (Fig. 3), and measured TEF-1 and CSEF-1 binding to the GT-IIC enhanson. With varying amounts of TEF-1, increasing amounts of the heat-treated BeWo nuclear extract result in diminished TEF-1 binding and a corresponding increase in CSEF-1 binding to the GT-IIC enhanson (Fig. 6). Thus TEF-1 and CSEF-1 compete for the same binding site, suggesting that both proteins may be involved in enhancer function through mutually exclusive mechanisms.


Figure 6: TEF-1 and CSEF-1 compete for the same DNA motif in vitro. TEF-1 was produced by in vitro translation. Boiled BeWo cell nuclear extracts were used as source of CSEF-1 that lacked detectable TEF-1 binding as shown in Fig. 3. Varying amounts of in vitro generated TEF-1 and heat-treated BeWo cell nuclear extract were mixed and assayed by gel shift analysis using P-labeled GT-IICWT DNA as a probe. The increase in the relative amount of the TEF-1-GT-IIC complex with increasing amounts of in vitro generated TEF-1 was accompanied by a corresponding decrease in CSEF-1-GT-IIC complex formation.



The Stimulatory Effects of CSEn and Artificial Enhancers Containing GT-IIC Multimers Correlate with the Presence of CSEF-1, but Not TEF-1

The relative roles of TEF-1 and CSEF-1 in mediating CSEn activity were assessed by functional assays with constructs containing multimers of the GT-IIC enhanson to determine if the activity of enhancers could be correlated with the levels of CSEF-1 and/or TEF-1 in HeLa, COS-1, and BeWo cells. Initially, plasmids containing one or two copies of CSEn were transfected into the three cell lines (Fig. 7, A and B). As previously demonstrated (Jiang and Eberhardt, 1994), one or two copies of CSEn significantly increased CS promoter activity in BeWo cells, but failed to stimulate the CS promoter in HeLa cells. CSEn was as potent a stimulator of the CS promoter in COS-1 cells as it was in BeWo cells. This stimulation does not depend on the expression of the SV40 T antigen in COS-1 cells, since CSEn also stimulated CS promoter activity in the parent CV-1 cell line (data not shown). Thus CSEn function is not restricted to placental cells as had been previously thought. Since COS-1 cells contain only minor TEF-1 binding activity and contain a protein that produces similar GT-IIC complexes as CSEF-1, the presence of CSEF-1 is positively correlated with enhancer stimulatory activity.


Figure 7: The hCS enhancer and multiple copies of the GT-IIC enhanson functioned efficiently in BeWo and COS-1 cells, but was inactive in HeLa cells, providing a negative correlation of TEF-1 binding activity with enhancer function. Cells (5 10) were transfected by electroporation using 15 µg of luciferase reporter plasmids as described under ``Materials and Methods.'' Reporter activity was normalized to protein concentration and expressed as light units/µg of protein ± S.E. (A and C) or as -fold stimulation ± S.E. over the basal activity (B and D) to provide a measure of the relative enhancer activity. A, comparison of hCSp.LUC (openbars), EnA_CSp.LUC (stripedbars), and EnA_EnP_CSp.LUC (solidbars) gene activities in BeWo, COS-1, and HeLa cells. Separate ANOVA analyses for the effect of the enhancer in BeWo and COS-1 cells were significant at the p = 0.001 and 0.0004 levels, respectively. There was no effect of the enhancer in HeLa cells (ANOVA analysis, p > 0.05). B, -fold stimulation of CSp.LUC constructs containing one (stripedbars) or two (solidbars) copies of CSEn. ANOVA analyses for the effect of the enhancer in BeWo, COS-1, and HeLa cells were p = 0.0038, 0.0043, and >0.05, respectively. C, effect of inserting multiple copies of the SV40 GT-IIC enhanson upstream of the CSp.LUC gene. Eight (stripedand stippledbars), 16 (checkeredbars), or 24 (solidbars) copies of the GT-IIC enhanson were inserted upstream of the CSp.LUC gene in either the syn (A alignment, striped, checkered, and solidbars) and/or anti (B alignment, stippledbars) orientation with respect to the promoter and the relative activities were assessed in BeWo, COS-1, and HeLa cells. ANOVA analyses for the effect of the enhanson in all of these cells were significant at the p = 0.0001 level. D, -fold stimulation of the CSp.LUC gene containing 8, 16, or 24 copies of the GT-IIC enhanson in BeWo, COS-1, and HeLa cells. ANOVA analyses for the effect of the enhanson in BeWo, COS-1, and HeLa cells were p = 0.0307, 0.0062, and 0.017, respectively. Although constructs containing 16 or 24 copies of the enhanson were about 2-3 times higher than those containing 8 copies, none of the individual t test comparisons were significant when corrected for multiple comparisons using the Bonferroni correction factor.



To study more directly the function of GT-IIC binding factors in the absence of participation by other enhansons, artificial enhancers containing multiple GT-IIC enhansons were constructed and tested (Fig. 7, C and D). Significant enhancer activity was observed with GT-IIC 8-, 16-, and 24-mers when introduced into BeWo and COS-1 cells. Neither the trend of increased enhancer activity with an increasing number of GT-IIC enhansons nor the small increase in activity induced by the GT-IIC 24-mer in HeLa cells was statistically significant. Interestingly, in contrast to the wild-type enhancer (Fig. 7, A and B), the GT-IIC multimers were much more active in COS-1 cells than in BeWo cells (Fig. 7, C and D), suggesting that BeWo cells might contain a factor that down-regulates enhancer activity through the GT-IIC enhanson. These data are consistent with the concept that the GT-IIC site is a major contributor to enhancer stimulatory activity and suggest that TEF-1 might account for the reduced GT-IIC multimer activity in BeWo cells.

Modulation of TEF-1 Levels in BeWo Cells Resulted in Dramatic Alterations of Basal CS Promoter and Enhancer Function

To study the influence of TEF-1 on CSEn function directly, we analyzed the effects of up- and down-regulation of TEF-1 expression on basal and CSEn- or GT-IIC-stimulated CS promoter activity in BeWo cells. Up-regulation of TEF-1 expression was accomplished by transient transfection of a TEF-1 cDNA expression plasmid, and down-regulation was accomplished by co-transfection of TEF-1 antisense oligonucleotides. We first verified by gel shift and Western analysis that these manipulations modulated TEF-1 expression in the appropriate manner. Increasing amounts of co-transfected TEF-1 expression plasmid resulted in an increase in the amount of GT-IIC-TEF-1 complex and co-transfection of the TEF-1 antisense oligonucleotide decreased the levels of this complex (Fig. 8A). The levels of the CSEF-1-GT-IIC complex serves as an internal control. Similar changes in the levels of TEF-1 were observed in Western blots (Fig. 8B). Analysis of these data by scanning densitometry indicated that TEF-1 levels could be up- and down-regulated by at least 2-fold.


Figure 8: TEF-1 levels are up- and down-regulated respectively in BeWo cells transfected with a TEF-1 expression plasmid or antisense oligonucleotides. BeWo cells (1 10) were transfected with increasing amounts of the TEF-1A expression vector or 35 mM antisense TEF-1 oligonucleotide. After 20 h whole cell soluble proteins were extracted from the cells with the modified mini-prep protocol as described under ``Materials and Methods.'' In vitro translated TEF-1 was used as control. A, GT-IIC DNA binding activities were tested using 25 µg of whole cell extracts and 30,000 cpm of P-labeled GT-IIC probe. The TEF-1 levels, reflected by the intensities of the gel shift bends, were selectively up- or down-regulated, while the CSEF-1 concentration was relatively unchanged, serving as an internal control of protein concentration. B, nuclear extract samples (60 µg) were resolved on SDS-PAGE, transferred onto nitrocellulose membranes, and detected by the chicken TEF-1 antibody (Kariya et al., 1993) to provide an alternate measure of the modulation of TEF-1 levels in transfected BeWo cells.



We next examined the effects of co-transfecting the TEF-1 expression plasmid and antisense oligonucleotide on the basal and CSEn- and (GT-IIC)-stimulated CS promoter activity (Fig. 9, A and B). The plasmid pBluescript and the TEF-1 sense oligonucleotide served as negative controls. We utilized the EnA_EnP_CSp.LUC and (GT-IIC)CSp.LUC constructs for these studies, since these have the highest enhancer activities and should be the most sensitive toward sensing changes of TEF-1 concentrations on enhancer activity. Interestingly, up-regulation of TEF-1 resulted in a 7-fold decrease in basal CS promoter activity and down-regulation of TEF-1 produced a 3.1-fold increase in basal promoter activity (Fig. 9A). Virtually identical effects were observed with the EnA_EnP_CSp.LUC and (GT-IIC)CSp.LUC constructs (Fig. 9A). As shown in Fig. 9B, down-regulation of TEF-1 levels did result in a significant 2.7-fold increase in the relative enhancer activity, whereas up-regulation of TEF-1 levels had no effect. These data do not support the concept that TEF-1 mediates the positive stimulatory effects of CSEn in BeWo cells and suggest that TEF-1 is a negative modulator of CSEn and basal promoter function. The data provide additional evidence that factors other than TEF-1 that recognize the GT-IIC motif are required for positive CSEn function.


Figure 9: The modulated levels of TEF-1 in BeWo cells are inversely proportional to the activities of the CSp.LUC, EnA_EnP_CSp.LUC, and (GT-IIC)24_CSp.LUC genes in co-transfected BeWo cells in a GT-IIC site-independent manner. The reporter plasmids, CSp.LUC, EnA_EnP_CSp.LUC, and (GT-IIC)24_CSp.LUC, together with 5 µg each of pBluescript control DNA (openbars), the CMV promotor-driven expression plasmid TEF-1A (stripedbars), 35 mM TEF-1 antisense oligonucleotide (stippledbars), or 35 mM TEF-1 sense oligonucleotide (solidbars) were co-transfected into BeWo cells (see ``Materials and Methods''). Reporter activity was normalized to protein concentration and expressed as light units/µg of protein ± S.E. (A) or as -fold stimulation ± S.E. over the basal activity (B) to provide a measure of the relative effect of manipulating TEF-1 levels on the activities of the various reporter genes. ANOVA analyses for the different treatment groups with the CSp.LUC, EnA_EnP_CSp.LUC and (GT-IIC)24_CSp.LUC genes' basal activity (A) were significant at the p = 0.0001 level. ANOVA analyses for the -fold stimulation (B) of the EnA_EnP_CSp.LUC and (GT-IIC)24_CSp.LUC genes were significant at the p = 0.001 and 0.001 levels, respectively.




DISCUSSION

TEF-1 involvement in CSEn function has been considered likely, since mutation of the CSEn GT-IIC enhanson abolished enhancer activity (Jiang et al., 1994; Jacquemin et al., 1994). In the present studies we sought to establish whether TEF-1 operating through the GT-IIC and/or Sph-I/Sph-II enhansons was involved in mediating CSEn stimulatory function. Our finding of a rapidly migrating GT-IIC complex in BeWo nuclear extracts confirms the studies of Jacquemin et al.(1994), who reported a pattern of complexes with HeLa, JEG-3, and placental tissue extracts that is very similar to that observed with HeLa and BeWo cells (Fig. 1). We have demonstrated that the slower migrating complex, which appears to be present in all of these cell types, corresponds to TEF-1. The more rapidly migrating complexes CSEF-1 (Fig. 1) and f (Jacquemin et al., 1994) appear to be identical based on their relative migration and similar placental cell distribution. Since the GT-IIC enhanson is essential for CSEn function (Jiang et al., 1994; Jacquemin et al., 1994), the finding of multiple GT-IIC-binding proteins raises the question of the exact role of TEF-1 in mediating CSEn activity.

One important issue is whether CSEF-1 and TEF-1 are structurally related. The proteins have different molecular weights by Southwestern analysis (Fig. 5C) and exhibit differential heat stabilities (Fig. 3). CSEF-1 can be chromatographically separated from the bulk of TEF-1 binding activity (Fig. 4), although it does co-elute with at least one putative TEF-1 binding isoform. Introduction of TEF-1 antisense oligonucleotides into BeWo cells resulted in dramatic changes in the intracellular concentration of TEF-1 without affecting CSEF-1 levels (Fig. 8). Finally, a rabbit antibody raised against chicken TEF-1 (Kariya et al., 1993) only cross-reacted with TEF-1, but not CSEF-1 (Fig. 5A). Since mixed synthetic peptides corresponding to the entire chicken TEF-1 polypeptide were used to raise the polyclonal antibody and the antibodies cross-react with human TEF-1 (Kariya et al., 1993), which is 76% identical to chicken TEF-1, CSEF-1 does not appear to be closely related to TEF-1. Nevertheless, since their binding specificities to the GT-IIC and Sph-I/Sph-II enhansons are nearly identical (Fig. 2, A and B), it is possible that CSEF-1 is a member of the family of transcription factors containing the TEA DNA-binding domain (Bürglin, 1991). This domain has been identified in a diverse set of regulatory genes, including the scalloped gene, a neuronal-specific Drosophila gene that is 68% identical to TEF-1 (Campbell et al., 1992), the abaA gene of Aspergillus nidulans that regulates differentiation of asexual spores (Mirabito et al., 1989), and yeast TEC1 that regulates transposon Ty1 enhancer activity in Saccharomyces cerevisiae (Laloux et al., 1990).

Our finding that CSEn is a potent enhancer in COS-1 cells (Fig. 7, A and B) indicates that CSEn or a related activity may not be restricted to the placenta. Of interest, with respect to the potential role of TEF-1 in CSEn action, is the almost complete absence of this factor in COS-1 cells and the presence of a GT-IIC-protein complex, which co-migrates with CSEF-1 (Fig. 1). Thus in COS-1 cells CSEn stimulatory activity is correlated with CSEF-1, but not TEF-1 binding activity, suggesting that CSEF-1 mediates the positive enhancer function in these cells. This conclusion is strengthened by the absence of CSEn activity in HeLa cells (Fig. 7, A and B), which contain abundant TEF-1 binding activity but lack CSEF-1 binding activity (Fig. 1).

The finding that GT-IIC multimers introduced upstream of the CS promoter resulted in significant positive stimulation of promoter activity suggests that this enhanson plays a major role in mediating CSEn stimulatory activity (Fig. 7, C and D). Interestingly, these constructs are much more potent enhancers in COS-1 cells than in BeWo cells, suggesting that BeWo cells might contain factors that down-regulate enhancer responsiveness. This view is supported by studies with the TEF-1 antisense oligonucleotide ( Fig. 8and Fig. 9 ), which demonstrate that down-regulation of TEF-1 levels in BeWo cells results in a significant stimulation (3-fold) of either wild-type CSEn- or (GT-IIC)-mediated activity. Nevertheless, since modulation of TEF-1 levels results in significant effects on basal promoter activity in a manner independent from the GT-IIC enhanson (Fig. 9A), the data suggest that TEF-1 squelching does not require binding to the GT-IIC enhanson. Accordingly, the increased effectiveness of CSEn or the GT-IIC enhanson in COS-1 cells is correlated with the relative absence of TEF-1 in these cell types, supporting the concept that TEF-1 mediates negative but not positive effects on enhancer function.

Our data appear to parallel somewhat those found with the myosin heavy chain (MHC) promoter. Shimizu et al.(1993) have reported that both the ubiquitous mouse (m) TEF-1 (99% amino acid sequence homology to hTEF-1; Blatt et al.(1993)) and a distinct muscle-specific factor A1 bind to the GT-IIC variant motif, M-CAT, present in the MHC promoter. The binding specificities of A1 and mTEF-1 are indistinguishable. Over expression of mTEF-1 had no effect on MHC promoter activity, implicating a role for A1 in mediating MHC muscle-specific gene expression. Shimizu et al.(1993) suggested that stimulation might involve the heterodimerization of A1 and mTEF-1; however, our data do not support such a mechanism, since down-regulation of TEF-1 levels in BeWo cells resulted in an increase in CS promoter and enhancer activity (Fig. 9, A and B) and the enhancer functioned in COS-1 cells which lack high levels of TEF-1 (Fig. 7). In addition, no evidence for an interaction between TEF-1 and CSEF-1 was observed in the gel shift experiments where in vitro generated TEF-1 was added to a TEF-1-depleted cell extract containing CSEF-1 (Fig. 6). The simplest model to explain our data would be that down-regulation of TEF-1 levels results in increased occupancy of the GT-IIC domain by CSEF-1, which acts as a positive modulator of enhancer activity.

It is important to emphasize that our findings do not exclude the possibility that TEF-1 and a co-activator as proposed initially for the SV40 and HPV enhancers (Xiao et al., 1991; Ishiji et al., 1992; Hwang et al., 1993) account for CSEn function. For example, the endogenous TEF-1 levels in BeWo cells may exceed the available co-activator concentration such that ``endogenous squelching'' occurs. Accordingly, down-regulation of endogenous TEF-1 levels could result in the net liberation of a co-activator and concomitant activation. Since the modulation of TEF-1 levels in BeWo cells affects the hCS promoter (Fig. 9A), herpes simplex thymidine kinase and Rous sarcoma virus promoters as well as a minimal hCS promoter containing only a TATA box and initiator element,() the putative co-activator could be a component of the normal basal transcription apparatus.

Recently, Stewart et al.(1994) cloned a novel TEF-1 isoform, TEF-1B containing 13 extra amino acids immediately downstream of the TEA domain, that is probably derived from TEF-1 pre-mRNA by alternative splicing. Interestingly, the TEF-1B, but not the TEF-1A, transactivation domain was capable of stimulating transcription when fused to a GAL4 DNA binding domain in a GAL4-dependent reporter gene in muscle cells. Thus different TEF-1 isoforms may be involved in mediating positive enhancer functions. Given the apparent heterogeneity in TEF-1 binding isoforms in BeWo cells (Fig. 4), we cannot exclude the possibility that one of these TEF-1 isoforms plays a role in mediating positive CSEn functions. However, since the TEF-1 antisense oligonucleotide was directed toward the 5`-end of the mRNA, this should deplete all isoforms of TEF-1 except those containing a different translation start codon.

The mechanisms that control the cell-specific character of enhancer activity appear to depend on their multipartite structure. Single copies of individual enhansons usually lack or have very weak enhancer activity; however, when homogeneously multimerized, the individual enhansons can work independently and generate significant stimulatory activity. An interesting feature of these homogeneous enhancers is that they exhibit relatively restricted cell type dependence (Schirm et al., 1987; Ondek et al., 1987; Forsberg and Westin, 1991). Schirm et al.(1987) demonstrated that two related SV40 core consensus motifs (GT-IIC, GTGGAATGT; TC-II, GTGGAAAGT), displayed markedly different behavior in a variety of cells. These data could be explained by multiple TEF-1 isoforms that recognize the distinct enhansons as supported by the muscle cell expression studies of Stewart et al.(1994). Our data indicate that the GT-IIC multimer is sufficient in mimicking the wild-type CSEn activity in BeWo and COS-1 cells, emphasizing the important role that this enhanson plays in mediating positive enhancer function and suggest that CSEF-1 may play a role in mediating cell-specific enhancer function.

  
Table: Oligonucleotide use in studies



FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK41206 (to N. L. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: 4-407 Alfred, SMH, Mayo Clinic, Rochester, MN 55905. Tel: 507-255-6554; Fax: 507-255-4828; E-mail: eberhardt@mayo.edu.

The abbreviations used are: CSEn and En, placental-specific chorionic somatomammotropin gene enhancer associated with the hGH/hCS gene locus; hGH, human growth hormone; hCS, human chorionic somatomammotropin (also known as placental lactogen); TEF-1, transcription enhancer factor-1; CSEF-1, chorionic somatomammotropin enhancer factor-1; SV40, simian virus 40; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; LUC, luciferase; bp, base pair(s); PAGE, polyacrylamide gel electrophoresis; ANOVA, analysis of variance; GST, glutathione S-transferase.

S.-W. Jiang and N. L. Eberhardt, manuscript in preparation.


ACKNOWLEDGEMENTS

We express our appreciation to Drs. Pierre Chambon and Irwin Davidson for providing the pXJ40-TEF-1 expression plasmid, Drs. Charles Ordahl and Iain K. G. Farrance for the anti-chicken TEF-1 antibodies, Dr. Drew Arnold (Endocrine Research Unit, Mayo Clinic/Mayo Foundation) for advice with data analyses, Dr. Whyte Owen for helpful discussions of the studies involving characterization of the CSEF-1 protein, Dr. Miguel Trujillo for careful reading and criticism of the manuscript, and Nicole Henry for secretarial and editorial help.


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