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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 7 3305-3311
Copyright © 2003 by The Endocrine Society

Identification of an Enhancer of the Human Activating Protein-2{alpha} Gene That Contains a Critical Ets1 Binding Site

You-Hong Cheng and Stuart Handwerger

Cincinnati Children’s Hospital Medical Center and Department of Pediatrics, Division of Endocrinology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45229-3039

Address all correspondence and requests for reprints to: You-Hong Cheng, Ph.D., Division of Endocrinology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229-3039. E-mail: cheny0{at}cchmc.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Deletion analysis studies of the 5'-flanking region of the activating protein-2{alpha} (AP-2{alpha}) gene indicates that the proximal 152 bp are essential for minimal promoter activity and that a 140-bp fragment from nucleotides -1279 to -1139 acts as an enhancer of basal transcriptional activity. Ligation of the 140-bp fragment to a minimal AP-2{alpha} promoter or a heterologous simian virus 40 promoter luciferase reporter plasmid conferred enhancer activity in trophoblast cells. In deoxyribonuclease I footprint studies, nuclear extracts from trophoblast cells protected two regions of the 140-bp fragment, E2 and E3. Site-directed mutagenesis of an Ets1-binding site in E2 significantly inhibited AP-2{alpha} enhancer activity, whereas mutagenesis of two putative polyomavirus enhancer activator-3-binding sites on E2 and an insulin upstream factor 1-binding site in E3 had no effect on enhancer activity. Gel shift and supershift assays indicated that Ets1 binds to the Ets site in E2, and overexpression of Ets1 in transfection studies induced AP-2{alpha} promoter activity. As the transcription factor Ets1 is abundant in trophoblast cells, these investigations strongly suggest that AP-2{alpha} gene expression in the placenta is enhanced by a cis-acting element at nucleotides -1279 to -1139 that contains a critical Ets1-binding site.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE TRANSCRIPTION FACTOR activating protein-2 (AP-2) was originally identified as a nuclear factor regulating the expression of the human metallothionein IIA gene (1). AP-2 is now known to be a family of transcription factors consisting of four different isoforms, AP-2{alpha}, AP-2ß, AP-2{gamma}, and AP-2{delta}, that are derived from separate genes. All four isoforms regulate the expression of genes involved in cell proliferation and differentiation (2, 3, 4, 5). Analysis of the patterns of AP-2 expression in mouse embryos indicates roles for the transcription factor family in development, including the hindbrain, spinal cord, face, and limbs (2, 3, 4). AP-2{alpha} knockout mice die at birth and exhibit a range of congenital abnormalities, including exencephaly, craniofacial defects, a failure of ventral body wall closure, and axial skeleton contortion and shrinkage (2, 3, 4). Knockout of AP-2{gamma} mice leads to a reduction in cell proliferation in the extraembryonic ectoderm and the ectoplacental cone as well as a reduced number of trophoblast giant cells (5). In addition, there is a reduction in the trophoblast-specific expression of eomesodermin and Cdx2, both of which are implicated in fibroblast growth factor-responsive trophoblast stem cell maintenance (6).

Several lines of evidence strongly suggest that AP-2{alpha} and AP-2{gamma}, the two AP-2 isoforms present in human placenta, play pivotal roles in the regulation of trophoblast differentiation (7, 8, 9, 10, 11). AP-2{alpha} expression increases markedly as human mononuclear cytotrophoblast cells differentiate to form multinucleated syncytiotrophoblast cells (7). In addition, AP-2{alpha} induces the expression of syncytiotrophoblast-specific genes during the trophoblast differentiation, including human chorionic gonadotropin (hCRH) (7), human placental lactogen (8), human CRH (9), and ovine P450 side-chain cleavage (CYP11A1) (10). Furthermore, many syncytiotrophoblast-specific genes have promoters with consensus AP-2 binding sites and are induced by overexpression of AP-2{alpha} in transfection studies. The hCRH gene lacks an AP-2-binding site, but AP-2{alpha} interacts with CREB to potentiate markedly the effect of cAMP on hCRH gene expression in trophoblast cells (9). AP-2{gamma} also regulates murine adenosine deaminase gene expression through a consensus AP-2 binding site (11).

Although AP-2{alpha} plays a pivotal role in the differentiation of cytotrophoblast and other cell types, relatively little is known about the transcriptional regulatory elements and their associated factors that regulate the expression of the gene. Recent studies analyzing the 5'-flanking sequence of the human AP-2{alpha} gene provide initial insights into the transcriptional regulation of AP-2{alpha} gene expression. The proximal promoter contains binding sites for the transcription factors AP-2, nuclear factor-1, and octamer protein, but lacks a TATA box motif. Initiation for AP-2{alpha} mRNA occurs upstream of an internal repeat-3-like repetitive element (12). The conserved octamer-binding site at nucleotide (nt) -50 of the proximal AP-2{alpha} promoter is critical for basal expression of AP-2{alpha} promoter activity. In addition, an AP-2 binding site located nt -335 confers positive autoregulation to the promoter (13). Imhof et al. (14) identified a zinc finger silence factor, called AP-2rep, which is involved in mediating positive and negative regulation of AP-2{alpha} gene expression through interaction with the AP-2 binding site located at nt -335. Creaser et al. (12), however, demonstrated by deletion analysis studies that this site is not critical for basal expression of the AP-2{alpha} gene. In this study we determined that the 5'-flanking region of the human AP-2{alpha} gene has an enhancer element between nt-1279 and -1139 that contains a critical Ets1 DNA-binding site.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid constructs

All constructs used luciferase (Luc) as the reporter for assaying transcriptional activation of the promoters under study. The orientation and sequence of all constructs were confirmed by DNA sequence analysis. A 2.0-kb promoter fragment spanning nt -1728 to +286 with respect to the major mRNA initiation site was amplified using the 5' primer GTC GAC CAT TTT CTG GAC GGA GGG CGG CGC and the 3' primer CCG TCA ATT TCC AAA GCT TTT TCA TGG ATC GGC GTG. Genomic DNA provided by Dr. David Repaske (Children’s Hospital Medical Center, Cincinnati, OH) was used as the PCR template. The PCR product was gel purified after being digested by HindIII and then ligated into the SmaI and HindIII sites of pGL3-basic (Promega Corp., Madison, WI) to generate pAP-2(-1728/+286)-Luc. By using unique internal EcoRI, SacII, and XbaI sites, smaller 5' promoter fragments were constructed from pAP-2(1728/+286)-Luc reporter to generate pAP-2(-1515/+286)-Luc, pAP-2(-1027/+286)-Luc, and pAP-2(-152/+286)-Luc. Both nt -1279/+286 and nt -1139/+286 fragments were amplified by PCR using the 5' primers GCA GAG AGA AAA GAC AGA GGT AAC TTT and TAA AAC CGA AAG GGA TAA AAG CCC CAG, respectively, and the 3' primer CCG TCA ATT TCC AAA GCT TTT TCA TGG ATC GGC GTG. The PCR product was ligated into the SmaI and HindIII sites of pGL-basic after digestion with HindIII to generate pAP-2(-1279/+286)-Luc and pAP-2(-1139/+286)-Luc. To create the pAP-2(-1279/-1139)-AP-2(-152/+283) construct, the 140-bp fragment was amplified by PCR using the 5' primer GCA GAG AGA AAA GAC AGA GGT AAC TTT and the 3' primer GGA TTT TTT TTT AAG TAA GAA AAA AAA AAA and ligated into the KpnI site of pAP-2(-152/+286)-Luc. The same PCR fragment was inserted into the KpnI site of pGL3-promoter (Promega Corp.) vector to create pAP-2(-1279/-1139)-simian virus 40 (SV40)-Luc. The expression vector for mouse Ets1 was provided by Dr. Koichi Yoshida (Sapporo University School of Medicine, Sapporo, Japan).

Mutants of two PEA3 binding sites, one insulin upstream factor 1 (IUF1), and one Ets1 binding site were created in the pAP-2(-1279/1139)-AP2(-152/+286) construct using the Quick Change Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Mutated base pairs were confirmed by DNA sequencing. The oligonucleotides used are as follows, with the mutated nucleotides underlined (the complementary strand sequence is not shown): mutant polyomavirus enhancer activator-3 (mtPEA3), 5'-CTT TCT TTT GGA AGT CAA CGA GGA AGC TCA GCC AGG-3'; mtEts1, 5'-CTT TTG GAG GAA AAC GAT GTA TCT CAG CCA GGA AAA-3'; mtPEA3, 5'-GAG GAA GCT CAG CCA TGT TAA TTA ATT ACT CTG TAT TTC-3'; and mtIUF1, 5'-CTG TAT TTC CTA GTG TCA CGC TCG GTC CTT TTT CTT TTT-3'.

Cell culture

Third trimester placentas were obtained from women with normal pregnancies and deliveries. The protocol for obtaining placentas was approved by the human investigation committees of the University of Cincinnati and the Children’s Hospital Medical Center. Cytotrophoblast cells were isolated by enzymatic disaggregation, purified by negative CD-9 selection, and grown in DMEM with 10% fetal bovine serum (FBS) (9). Human choriocarcinoma JEG3 cells (HTB-36, American Type Culture Collection, Manassas, VA) were grown in MEM with 2.0 mM L-glutamine, 1.5 g/liter sodium bicarbonate, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, and 10% FBS. HepG2 cells (American Type Culture Collection, HB-8065) were grown in MEM with 2.0 mM L-glutamine, 1.5 g/liter sodium bicarbonate, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, and 10% FBS.

Transfection studies

Transient transfection studies of trophoblast cells were performed in triplicate by the liposome method described by Cheng et al. (9). Each sample contained 5 µg reporter plasmid and 0.5 µg pRL-thymidine kinase (TK)-Luc (Promega Corp.). In cotransfection experiments in JEG3 cells 4.0 µg of the reporter plasmid and 1.5 µg of the expression plasmid pSG5 (empty vector) or pEts1-SG5 were used. The cells were harvested 48 h after transfection. The results represent the average of three independent transfection assays normalized to pRL-TK-Luc using a dual Luc reporter assay system (Promega Corp.).

Deoxyribonuclease I (DNase I) footprinting

DNase I footprinting was performed as previously described (15). A 5'-end-labeled probe of a 259-bp AP-2{alpha} promoter region was generated by PCR using pAP-2(-1728/+286)-Luc as template with the 5' primer GCA ACT CTG AGG CGG GAC CTA GTG T and the 3' primer GGT CAG TCC GGC CTT TTG GAC TCC T.

Gel mobility shift assays

Nuclear extracts of primary human placental cells and HepG2 were prepared as previously described (9). Nuclear extract protein concentrations were determined using the Bradford protein assay (Bio-Rad Laboratories, Inc., Hercules, CA). Gel shift assays were performed as previously described (9). The 32P-labeled synthetic double-stranded oligonucleotide (5'-AAA ACG AGG AAG CTC AGC CA-3') encompassing the Ets1 of the AP-2{alpha} promoter region (nt -1242/-1223) was used. Binding reactions (24 µl) contained 20 mM Tris (pH 7.6), 50 mM KCl, 2 mM MgCl2, 1 mM dithiothreitol, 10% glycerol, 40 ng poly[d(I-C)], 50,000 cpm 32P-labeled probe, and 5 µg nuclear extract. The binding reactions were incubated for 20 min at room temperature and then loaded onto a 5% polyacrylamide gel in 0.5x TBE (Tris-borate-EDTA). In competition assays unlabeled probes were incubated with the nuclear extracts for 10 min before the addition of labeled probe. For supershift analysis, nuclear extracts were incubated with appropriate antibody before the addition of the radiolabeled probe. An antiserum to human Ets1 was a gift from Dr. James Hagman (National Jewish Center, Denver, CO).

Semiquantitative RT-PCR

Total RNA was respectively isolated from normal placental cells and HepG2 hepatoma cells. First strand cDNA synthesis was performed using SuperScript II reverse transcriptase (Life Technologies, Inc., Grand Island, NY) and oligo(deoxythymidine), according to the manufacturer’s protocol. Primer sequences used for detection of Ets1 transcripts were 5'-GAT GTC CCA CTA TTA ACT CCA AGC AGC-3', which corresponds to Ets1 nucleotide sequence in exon II, and 5'-CGT CTG ATA GGA CTC TGT GAT GAA GC-3', which corresponds to nucleotide sequence in exon V of the Ets1 gene. The PCR resulted in a predicted product of 480 bp. Primers for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were 5'-cca tgg aga agg ctg ggg-3' and 5'-caa agt tgt cat gga tga cc-3', which resulted in a predicted PCR product of 192 bp. The PCR reactions were spiked with 0.1 µl [{alpha}-32P]deoxy-CTP (3000 Ci/mM). The optimal number of PCR cycles required for linear amplification for each set of primers was determined. Total amplification in each reaction primer set (Ets1 plus GAPDH) was kept below saturation levels to permit the two products to remain within the exponential range. GAPDH required 18–20 cycles, whereas Ets1 required 25 cycles. The radiolabeled PCR products were separated by a 6% PAGE at 225 V for 3 h. The gel was then transferred to 3M paper, dried, and quantified using a PhosphorImager and ImageQuant 1.2 software (Molecular Dynamics, Inc., Sunnyvale, CA).

Western blot analysis

Equal amounts (35 µg) of nuclear extract from normal trophoblast cells that were cultured for 3 d or from HepG2 cells were subjected to 12.5% SDS-PAGE. The blot membrane was blocked for 30 min in 5% instant nonfat dry milk, which was dissolved in Tris (hydroxymethyl) aminomethane-buffer saline containing 0.05% Tween 20 (TBST). Ets1 antiserum was diluted 1:5000 in milk-TBST and incubated overnight at room temperature. The blot was washed four times (5 min each wash) with TBST and incubated for another 1 h with secondary antiserum (horseradish peroxidase-coupled donkey antirabbit IgG antiserum), which was diluted 1:5000 in milk-TBST. The blot was washed four times with TBST and developed with a chemiluminescence kit (Pierce Chemical Co., Rockford, IL).

Statistical analysis

Statistical differences between the two groups were determined by t test, and multiple comparisons were performed by one-way ANOVA or repeated measures ANOVA together with post hoc pairwise comparisons. The values were expressed as the mean ± SD, and P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Deletion analysis of the 5'-flanking region of the human AP-2{alpha} gene

To delineate the putative DNA sequences that are important in the regulation of AP-2{alpha} gene expression, transient transfection studies were performed in trophoblast cells using fragments of the 5'-flanking region of the AP-2{alpha} gene extending from nt -1728/+286 (Fig. 1Go). The fragment of the 5'-flanking region of the reporter construct pAP-2(-1728/+286)-Luc supported a high level of reporter expression, which was 13-fold higher than the promoterless vector (pGL3-basic). Progressive deletions of 5' sequences to nt -1279 did not significantly reduce promoter strength. However, deletion of 140 bp of the 5'-flanking region from nt -1279 to -1139 resulted in a 60% decrease in Luc activity. Further deletion to nt -152 had no effect on promoter strength. These data indicate that the 140-bp fragment contains a regulatory region that enhances AP-2{alpha} gene expression in trophoblast cells.



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  		FIG. 1. Deletion analysis of the 5'-flanking region of the human AP-2{alpha} promoter. Fragments of the 5'-flanking region were ligated upstream of a Luc reporter gene (pGL3-basic) and transiently transfected into primary cultures of human trophoblast cells along with a pRL-TK-Luc plasmid. Luc activities of the plasmids containing the AP-2{alpha} fragments were normalized to pRL-TK-Luc values. The results represent the mean of five experiments (±SD). *, P < 0.01.

 
Heterologous promoter studies

o examine further whether the 140-bp fragment can act as an enhancer, the fragment was subcloned directly upstream of a minimal AP-2{alpha} promoter (nt -152/+286) linked to a Luc gene. In normal trophoblast cells, the promoter activity of the chimeric gene was 2.5-fold greater than the activity of the minimal AP-2{alpha} promoter (Fig. 2Go). The 140-bp fragment had a similar effect on a heterologous promoter. When the fragment was subcloned directly upstream of the SV40 promoter linked to a Luc gene, the promoter activity of the chimeric gene was 2-fold greater than the activity of the SV40 promoter. Taken together, these data strongly suggest that there is an enhancer located between nt -1279 and -1139 bp of the AP-2{alpha} gene.



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  		FIG. 2. Effect of the AP-2{alpha} enhancer on the activities of a minimal AP-2{alpha} promoter and a heterologous SV40 promoter. The 140-bp enhancer element of the AP-2{alpha} gene was cloned upstream of the minimal AP-2{alpha}, and the SV40 promoters were coupled to a Luc reporter gene. The chimeric constructs were then transfected into primary cultures of trophoblast cells along with a pRL-TK-Luc. In each instance, Luc activity was normalized to the control pRL-TK-Luc values. The results represent the mean of three experiments (±SD). *, P < 0.01.

 
DNase I footprinting of the AP-2{alpha} enhancer element

To identify whether putative elements of the 140-bp fragment interact with transcription factors in trophoblast and HepG2 cells, DNase I footprinting analyses were performed with these nuclear extracts using a 259-bp fragment of the AP-2{alpha} gene (nt -1326/nt -1068 bp). HepG2 cells were studied because these cells, unlike trophoblast cells, do not express AP-2{alpha}. As shown in Fig. 3AGo, four protected regions (E1, E2, E3, and E4) were observed in the experiment using trophoblast cell nuclear extract. However, there was no protected region using a HepG2 cell nuclear extract. Both E1 and E4 are beyond the 140 bp of AP-2{alpha} enhancer region. E2 spans approximately 27 bp (nt -1245 to -1218) and contains two hypersensitive sites, and E3 spans 28 bp (nt -1211 to -1184) and also contains two hypersensitive sites. Computer analysis of the AP-2{alpha} enhancer region demonstrated that E2 contains two PEA3 consensus elements and one Ets1 binding site, and E3 contains a consensus element for IUF1. The sequence of the AP-2{alpha} enhancer element (nt -1279 to -1130) is presented in Fig. 3BGo with the footprinting regions underlined.



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  		FIG. 3. DNase I footprinting analysis of the human AP-2{alpha} enhancer element. A, A 5' end-labeled 32P-labeled oligonucleotide corresponding in sequence to the 140-bp enhancer was incubated with BSA (B; lanes 2–4), nuclear extracts of primary placental cells after 3 d of culture (P; lanes 5–7), or nuclear extracts of HepG2 cells (H; lanes 8–10). The triangles above the lanes indicate an increasing amount of DNase I enzyme. G+A sequencing reactions were used as a marker (lane 1). The four regions protected are designated elements E1, E2, E3, and E4. Asterisks indicated DNase I-hypersensitive sites. B, Nucleotide sequence of the 140-bp enhancer of the human AP-2{alpha} gene. The four distinct footprint elements are underlined and named (elements E1, E2, E3, and E4).

 
Mutational analysis of the enhancer

To determine which consensus binding sites of E2 and E3 regions are important for the regulation of AP-2{alpha} gene expression, site-directed mutagenesis of the putative PEA3, IUF1, and Ets1 binding sites were performed, and the promoter activities of the mutated constructs were compared with that of the wild-type construct. Mutations of the PEA3-binding site in E2 and the IUF1-binding site in E3 had no effect on AP-2{alpha} promoter activity. However, mutation of the Ets1-binding site on E2 inhibited promoter activity in the normal trophoblast cells by more than 30% (Fig. 4Go).



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  		FIG. 4. Effect of mutations in the Ets1 and PEA3 motifs on the human AP-2{alpha} enhancer on transcriptional activity. The pAP-2(-1279/-1139)-AP(-152)-Luc and the derived constructs with mutations in PEA3- or the Ets1-binding sites were transiently transfected in cultured trophoblast cells along with pRL-TK-Luc. In each instance, Luc activity was normalized to the control pRL-TK-Luc values. Thick lines indicate minimal AP-2{alpha} promoter (nt -152/+286) to which the AP-2 enhancer is linked. The results represent the mean of three experiments (±SD). *, P < 0.05.

 
Gel shift and supershift studies of the enhancer

To determine which putative binding sites in E2 bind to Ets1, a gel shift assay was performed using double-stranded oligonucleotides spanning the E2 sequence and nuclear extracts from normal trophoblast (Fig. 5AGo) or HepG2 cells (Fig. 5BGo). Figure 5AGo shows that a specific DNA-protein complex was formed when a 32P-labeled E2 oligonucleotide probe was reacted with a trophoblast cell nuclear extract (lane 1). The formation of the complex was competed by an unlabeled E2 oligonucleotide (lane 2), but not by an oligonucleotide containing a mutated Ets1 binding site (lane 3). The complex formed by the E2 oligonucleotide and the nuclear extract was supershifted by a specific Ets1 antiserum (lane 4). Figure 5BGo shows that a specific DNA-protein complex was also formed when the same E2 oligonucleotide probe was reacted with a nuclear extract from HepG2 cells (lane 5). The formation of the complex was competed by an unlabeled E2 oligonucleotide (lane 6), but not by an oligonucleotide containing a mutated Ets1-binding site (lane 7). However, the complex was not supershifted by a specific Ets1 antiserum (lane 8), suggesting that a transcription factor(s) other than Ets1 also binds to the Ets1-binding site.



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  		FIG. 5. Gel shift and supershift analyses of the AP-2{alpha} enhancer. A gel shift assay was performed using a 32P-labeled oligonucleotide corresponding in sequence to E2 and nuclear extract from normal trophoblast (A) or HepG2 cell (B). Competition studies were performed using a 100-fold excess of unlabeled E2 oligonucleotide (lanes 2 and 6) or an E2 oligonucleotide with a mutated Ets1 binding site (lanes 3 and 7). Supershift analysis was performed by incubating the placental nuclear extract incubated with 32P-labeled E2 oligonucleotide in the presence of an antiserum to Ets1 (lanes 4 and 8). The supershifted complex detected with the Ets1 antiserum is indicated by the arrow.

 
Transfection studies with Ets-1

To investigate the effect of the transcription factor Ets1 on gene expression driven by the enhancer element, we performed transient transfection experiments in JEG3 choriocarcinoma cells. JEG3 cells were studied because these cells have less endogenous Ets1 than normal trophoblast cells (data not shown). As shown in Fig. 6Go, Ets1 stimulated AP-2{alpha} enhancer activity by about 4-fold. However, the mutant reporter construct that eliminated the Ets1 binding site (mt) caused only a 1.5-fold stimulation by Ets1. These data suggest that the transcription factor Ets1 is a regulator of the AP-2{alpha} enhancer element.



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  		FIG. 6. Effect of Ets1 overexpression on the AP-2{alpha} enhancer. JEG3 choriocarcinoma cells were cotransfected with a plasmid expressing Ets1 (pEts1-SG5) and a Luc reporter plasmid containing the wild-type (wt) enhancer (pAP-2{alpha}-enahncer/AP-2(-152/+286)Luc) or a plasmid containing the enhancer with a mutation (mt) in the Ets1-binding site (pAP-2{alpha}-mt/AP-2(-152/+286)Luc). TK-Luc was cotransfected into the cells to account for transfection efficiency, and the Luc activity in each well was normalized to the Luc activity of pRL-TK-Luc. The results represent the mean ± SD of three experiments.

 
RT-PCR and Western blot analysis of Ets-1 in trophoblast cells

Previous studies indicated that Ets1 is expressed in endothelial cells of villous trophoblast and in extravillous trophoblast cells of the first trimester of human pregnancy (16). To determine whether Ets1 is expressed in trophoblast cells from term placenta, we examined Ets1 mRNA and protein levels in primary cultures of human trophoblast cells from term placenta by RT-PCR and Western blot techniques. As shown in Fig. 7Go, Ets1 mRNA and Ets1 protein are strongly expressed in trophoblast cells.



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  		FIG. 7. Ets1 is strongly expressed in primary trophoblast cells after 3 d of culture (P), but not in HepG2 cells (H). Total RNA isolated from d 3 normal trophoblast or HepG2 cells was subjected to RT-PCR analysis (A), and nuclear extracts from these cells were used for Western blot assays (B). Shown is a representative autoradiograph of three replicate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several lines of evidence indicate that the AP-2{alpha} gene contains a distal enhancer element that functions in concert with conserved elements that map to the proximal promoter region to achieve maximal expression in trophoblast cells. Deletion analysis revealed that deletion of a 140-bp fragment of the 5'-flanking region from nt -1279 to -1139 results in a 60% decrease in Luc activity. Furthermore, ligation of the 140-bp fragment immediately upstream of the minimal AP-2{alpha} promoter or the SV40 promoter significantly enhances the promoter activities of the two genes.

Additional investigations indicate that the activity of the enhancer is regulated at least in part by the transcription factor Ets1. Ets has been shown to regulate a wide spectrum of biological functions, including cell proliferation, differentiation, development, hemopoiesis, apoptosis, metastasis formation, tissue remodeling, angiogenesis, and cell transformation (17, 18). A recent literature survey identified over 200 Ets-targeted genes, and the number of genes regulated by Ets1 is increasing. As Ets1 proteins bind to the same DNA-binding site, it has been suggested that the expression of specific Ets target genes in vivo is regulated by tissue-specific expression of a particular Ets protein (17). DNase I footprint analysis indicated that nuclear extracts of trophoblast cells protected two regions on the enhancer, E2 and E3. The E2 region contains two consensus PEA3-binding sites. However, site-directed mutagenesis of these two sites did not diminish enhancer activity, indicating that these sites are not important for enhancer activity. In contrast, site-directed mutagenesis of the Ets1 site on the E2 region inhibited enhancer activity by about 30%, indicating that Ets1 is a mediator of enhancer activity. In addition, overexpression of Ets1 stimulated enhancer activity of the wild-type enhancer, but did not significantly stimulate a mutant enhancer element containing substitutions in the Ets-binding site.

Supershift analysis indicated that Ets1 binds to the Ets-binding site on the AP-2{alpha} enhancer. As the entire complex formed by the interaction of Ets1 and a nuclear extract of trophoblast cells was not supershifted, the possibility that the Ets-binding site may bind factors in addition to Ets1 cannot be excluded. It is now known that the Ets family of transcription factors consists of more than 30 members conserved from Caenorhabditis elegans to the human (19). The family members contain a conserved DNA-binding domain of about 85 amino acids, known as the Ets domain, which confers the ability to recognize a sequence containing a purine-rich core motif GGAA/T Ets binding site. Although Ets proteins may bind to DNA as monomers to activate transcription, the family members are also known to interact with other transcription factors (20). For example, the Ets1 transcription factor is able to cooperate with AP-1 (21), acute myeloblastic leukemia (AML)-1 (22), human transcription factor (TFE)3 (23), mothers against decapentaplgic (Smad)3 (24), and specific protein (SP)1 (25) transcription factors for transcriptional activation. Additional studies are necessary to determine whether other transcription factors bind to Ets1 in the placenta and if an additional transcription factor(s) other than Ets1 binds to the Ets1 site.

In summary, we have identified a 140-bp enhancer element on the 5'-flanking region of the AP-2{alpha} gene that contains an Ets binding site that is critical for trans-activation. Ets1 is a component of the complex of transcription factors that activate the enhancer element.


   Acknowledgments
 
We thank Dr. James Hagman (National Jewish Center, Denver, CO) for the gift of Ets1 antiserum, and Dr. Koichi Yoshida (Sapporo University School of Medicine, Sapporo, Japan) for the Ets1 expression vector.


   Footnotes
 
This work was supported by NIH Grant HD-07447.

Abbreviations: AP-2, Activating protein-2; DNase I, deoxyribonuclease I; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hCRH, human chorionic gonadotropin; IUF1, insulin upstream factor 1; Luc, luciferase; mtPEA3, mutant polyomavirus enhancer activator-3; nt, nucleotide; SV40, simian virus 40; TBST, Tris (hydroxymethyl) aminomethane-buffer saline containing 0.05% Tween 20; TK, thymidine kinase.

Received November 21, 2002.

Accepted April 4, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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