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Sp1 and Sp3 Are Important Regulators of AP-2 Gene Transcription¹

Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas 77030

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
AP-2 is a member of the AP-2 transcription factor family, is highly enriched in the trophoblast cell lineage, and is essential for placental development. In an effort to identify factors regulating AP-2 gene expression, we isolated and characterized the promoter and 5'-flanking region of the mouse and human AP-2 genes. The transcription start site of the mouse AP-2 gene was mapped by primer extension and 5' rapid amplification of cDNA ends. Deletion analysis of the 5'-flanking region revealed a 704-base pair (bp) sequence located approximately 6 kilobases (kb) upstream of the transcription start site that is required for enhanced expression in trophoblast cells. Additional gene transfer studies showed that basal promoter activity resides within a highly conserved, approximately 200-bp DNA sequence located immediately upstream of the transcription start site. The conserved region is highly GC-rich and lacks typical TATA or CCAAT boxes. Multiple potential Sp- and AP-2-binding sites are clustered within this region. Electrophoretic mobility shift assays demonstrated that Sp1 and Sp3 bind to three sites in the promoter region of the mouse AP-2 gene. Combined mutation of the three putative Sp sites reduced promoter activity by 80% in trophoblast and nontrophoblast cells, demonstrating the functional importance of these sites in regulating AP-2 gene expression. In summary, we have identified a potential trophoblast cell-specific regulatory element located approximately 6 kb upstream of the murine AP-2 gene transcription start site, and we have shown that Sp1 and Sp3 bind to cis-regulatory elements located in the promoter proximal region and contribute to basal promoter activity.

placenta, trophoblast

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
During mammalian development, the first differentiation event establishes two cell populations, the inner cell mass and the trophectoderm. The inner cell mass contributes primarily to formation of the embryo, whereas the trophectoderm contributes to formation of the trophoblast lineage and development of the placenta. Using the murine adenosine deaminase gene, Ada, as a model to identify signaling pathways controlling gene expression in the trophoblast lineage, Shi and Kellems [1] determined that the transcription factor AP-2 is an essential regulator of Ada gene expression during placental development. AP-2 binds to the placenta-specific footprinting region I of the Ada gene placental enhancer. Mutation of one nucleotide of the AP-2-binding site completely abolished placenta-specific Ada gene expression in a transgenic mouse assay [1]. Other genes implied in placental development and function contain AP-2-binding sites in their regulatory sequences. These genes include those encoding hCG and ß subunits and placental lactogen I [2–4]. The AP-2 gene is activated early in development of the trophoblast lineage and remains highly expressed in all branches of the trophoblast lineage during placental development [1, 5, 6]. AP-2 mRNA is considerably more abundant in the placenta than in any other tissue examined. Thus, available evidence indicates that AP-2 is likely to be a key regulator of trophoblast development and differentiation [1]. This view is supported by recent gene disruption studies in two independent laboratories. AP-2 (-/-) mice died of extraembryonic structure malformation around embryonic Day 7.5 [5, 6]. Development of the embryo proper was rescued to term by the introduction of wild-type cells in the extraembryonic lineages, demonstrating an absolute requirement for AP-2 in placental development. When introducing wild-type embryonic stem cells into AP-2 (-/-) blastocysts in which the developing placenta is AP-2 deficient, embryos derived from AP-2 (-/-) hosts were not rescued to term, further confirming the indispensability of AP-2 in the extraembryonic tissues [5]. Thus, available data indicate that AP-2 is essential in the extraembryonic lineages for early postimplantation development.

At present, no information regarding the tissue-specific or basal transcriptional regulation of AP-2 is available. Information regarding the regulation of AP-2 gene expression may help us to understand how extraembryonic development is established and maintained. To this end, we isolated and characterized the promoter and 5'-flanking regions of the human and mouse AP-2 genes. Gene transfer studies identified a small region approximately 6 kilobases (kb) upstream of the murine AP-2 gene transcription start site that is required for enhanced expression in trophoblast cell lines. A comparison of 5'-flanking sequences between the human and mouse AP-2 genes showed that an approximately 200-base pair (bp) region of promoter proximal sequence is highly conserved and shares 93% sequence identity. In this short region, Sp-1 and Sp-3 transcription factors bind to three cis-acting elements and participate in activation of the AP-2 gene promoter.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Mouse Placenta Library Screening, Genomic Cloning, Southern Blot Analysis, and DNA Sequencing

A mouse placental genomic library FIX II (Stratagene, La Jolla, CA) was screened with a 550-bp probe from the 5' end of mouse AP-2 cDNA [7]. A positive clone was characterized by Southern hybridization using different subfragments of the cDNA. Purified DNA was cut with NotI and SalI, and the resulting fragments were subcloned into pBluescript II vector (Stratagene). Clones were sequenced in both directions. The sequence was deposited to GenBank (accession no. AY196820).

Culture of Human AP-2 Genomic PAC Clone and Purification of PAC Gene

The human AP-2 PAC genomic DNA clone RP4-539E24 was obtained from The Sanger Center (London, England). The clone was streaked on LB plates that contained kanamycin (25 µg/ml). A single clone was grown in 2X YT medium with kanamycin (25 µg/ml) at 37°C overnight. The PAC DNA was isolated using NucleoBond Nucleic Acid Purification Kit (Clontech, Palo Alto, CA). To confirm that it was the correct clone, Southern blot analysis was performed using a fragment of the human AP-2 cDNA [8].

Reporter Gene Constructs

Mouse AP-2 reporter gene constructs were prepared by ligating various AP-2 genomic fragments into the polylinker of pGL3 Basic vector (Promega, Madison, WI), which contains a firefly luciferase gene without a promoter and enhancer. The genomic fragments were prepared by restriction enzyme digestion or by polymerase chain reaction (PCR). The cloning sites of all reporter plasmids were verified by DNA sequencing. An approximately 6500-bp fragment of mouse AP-2 genomic DNA was generated by PCR using sense primer (5'-GACTAGTCGACTCGATCAGGAACCT) with an SpeI linker and an antisense primer (5'-GGGATCCAGCGGTAAATCCA) with a BamHI linker. The approximately 6500-bp PCR fragment was digested with SpeI and BamHI and inserted into pGL3 Basic vector. The construct (-6583/+181)pGL3 Basic was digested with restriction enzymes BglII, SacI, KpnI, and SmaI separately, and isolated fragments containing the luciferase expression gene were self-ligated. Their 5' ends corresponded to nucleotide positions -4290, -689, -233, and -60, and the 3' end corresponded to nucleotide +181 with respect to the transcription start site of the mouse AP-2 gene.

Mouse AP-2 gene fragment (-6583/-4290) was isolated from plasmid (-6583/+181)pGL3 Basic using MluI and BglII restriction enzyme digestion. The fragment (-6583/-4290) was then inserted into the polylinker of Promoter pGL3 vector (Promega), which contains a firefly luciferase gene with SV40 basic promoter but without enhancer. Three deletion constructs were obtained by PCR using high-fidelity pfuTurbo DNA polymerase (Stratagene). Construct (-6583/-4290)/promoter pGL3 was used as template. The sense primers were 5'-GAGAGTAGCAGCAGGTAACA, 5'-GAGTCCTCGGACTCGATTGT, and 5'-GAGTGGCCTCAGTAGTTGTT. Their 5' ends corresponded separately to nucleotide positions -5879, -5583, and -4907. The common antisense primer was 5'-GCTCGGTACCTATCGATAGA.

Human AP-2 reporter gene constructs were prepared using a similar strategy. A fragment of an approximately 4300-bp genomic DNA was generated by PCR using human AP-2 PAC DNA as template. The sense primer was 5'-GATATCGGCGTCCGGCGTCCCCCAA with an NheI linker site, and the antisense primer was 5'-ACGCGTTGTTGTCCAGGCTGGAGTG with an EcoRV linker site. The approximately 4300-bp PCR product was cut with NheI and EcoRV and then inserted into the pGL3 Basic vector. The other constructs were obtained by digesting the (-4139/+166)pGL3 Basic plasmid with SacI, StuI, SmaI, and SacII and then the isolated DNA fragments were self-ligated. The 5' ends of the constructs corresponded to nucleotide positions -988, -295, -130, and -36, and the 3' ends corresponded to nucleotide +166 with respect to the 5' end of the human AP-2 cDNA [8].

RNA Isolation and Northern Blot Analysis

The RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA) in accordance with the manufacturer's suggested protocol. Thirty micrograms of total RNA per lane were loaded on denaturing 1% agarose gels (6% formaldehyde). Blotting and hybridization using ³²P-labeled probes (500 bp of 5' end of the mouse AP-2 cDNA) were performed. Equal loading was verified by the intensity of ethidium bromide staining of the ribosomal RNA.

Primer Extension

An antisense nucleotide (5'-GCTGTCCCAGTCACTGGACGCGCATCGGTGGCTGT) complementary to nucleotides 58–92 of the mouse AP-2 cDNA was radiolabeled at the 5' end with T4 polynucleotide kinase and [-³²P]ATP (3000 Ci/mmol; ICN, Irvine, CA). The radiolabeled primer was added to 20 µg of total RNA from Day 14 placenta or 20 µg of total RNA from Day 14 embryonic kidney in hybridization buffer (0.4 M NaCl and 10 mM PIPES, pH 6.4) and heated for 10 min at 85°C. Hybridization between the radiolabeled oligonucleotide and mRNA was accomplished at 55°C for 16 h. Reverse transcription was performed with 200 U of RNase H reverse transcriptase Superscript II (Invitrogen) according to manufacturer's protocol. The reaction was carried out at 42°C for 1 h. After completion of the reaction, the products were dissolved in a denaturing dye solution and separated on 6% polyacrylamide-urea gel. The size was determined by comparison with a radiolabeled dephosphorylated X174 HinfI marker (Promega).

5' Rapid Amplification of cDNA Ends

A 5' rapid amplification of cDNA ends (RACE) system (Invitrogen) was used according to the manufacturer's instructions. Using total mouse placental RNA (2 µg) as template, first-strand cDNA synthesis was performed using a primer called GSP1 (5'-GGACTGCGAGTATACCACCT) complementary to 418–437 nucleotides of the mouse AP-2 cDNA. Single-stranded cDNA was separated from unincorporated dNTPs and primer using GlassMax spin cartridges (Invitrogen). A 5'-polyC tail was added using dCTP and terminal deoxynucleotidyltransferase. Poly(C)-tailed cDNA was amplified by PCR using an anchor primer supplied with the kit and primer GSP2 (5'-AGTATACCACCTGCTGGTAA) complementary to 410–429 nucleotides of the mouse AP-2 cDNA. The PCR product was used as template for a second round of PCR using nested primers supplied by the manufacturer and primer GSP3 (5'-GTAAGGAGGCGGCTGGTACT) complementary to nucleotides 379–398 of the mouse AP-2 cDNA. The final PCR product was run on a 0.7% agarose gel and then purified using QIAEX II resin (Qiagen, Inc., Santa Clarita, CA). Purified PCR DNA was subcloned into the TA cloning vector (Invitrogen), and individual clones were sequenced.

Cell Culture and Transfection

The human trophoblast cell line JEG3 and human epithelioid carcinoma cell line Hela (ATCC CCL2) were obtained from the American Type Culture Collection (Manassas, VA). Human primary trophoblast cells HTR-8/SVneo were provided by Dr. Charles H. Graham (Department of Anatomy and Cell Biology, Queen's University, Kingston, ON, Canada) [9]. The Hela, JEG3, and NIH3T3 cells were grown in Dulbecco modified eagle medium supplemented with 10% fetal bovine serum and antibiotics. The HTR-8/SVneo cells were cultured in RPMI medium with 10% fetal bovine serum and antibiotics. Approximately 5 x 10⁴ cells were seeded in 24-well culture plates at 24 h before transfection at 37°C in 5% CO₂. The cells were transfected with 0.2 µg of plasmid plus 0.02 µg of pRL-TK vector (Promega) using 0.6 µl of Fugene 6 transfection reagent (Roche, Indianapolis, IN). The pRL-TK containing the renilla luciferase gene under the regulation of a thymidine kinase (TK) promoter served as an internal control for normalizing the expression of the firefly luciferase reporter. After incubation for 48 h, cells were lysed, and luciferase activity was determined using the Dual Luciferase Assay System (Promega). The firefly luciferase reading was normalized against that of the cotransfected renilla luciferase. All the transfections were performed in triplicate and repeated at least three times.

Luciferase Assay

Luciferase assays were performed following the protocol for the Promega luciferase dual system. The transfected cells in 24-well plates were washed twice with PBS and harvested in 100 µl of Passive Reporter Lysis Buffer (Promega). After addition of luciferase assay buffer (100 µl), relative luciferase activity (20 µl of the lysate) was measured for 10 sec in a Monolight luminometer (PharMingen, San Diego, CA).

Site-Directed Mutagenesis

The Sp- and AP-2-binding motifs were mutated using the QuickChange Site-Directed Mutagenesis Kit (Stratagene). The mutagenesis oligonucleotides for Sp-A, Sp-B, and Sp-C motifs were 5'-GGCAGGGTGCGGGGCtaAGCTAGGCAGGGACTGG, 5'-GGCTGCCCCGCaCGGCGCCTGG, and 5'-GCGGCGCCGaCaCtCGGGGAGGAG. All the mutations were confirmed by sequencing. Mutagenesis was performed according to manufacturer's protocols.

Electrophoretic Mobility Shift Assay

The JEG3, HTR-8/SVneo, Hela, and NIH3T3 cells were grown to 80% confluence and used for preparing nuclear extracts by a modification of the method as described by Shi et al. [10]. All the oligonucleotides were synthesized by Sigma Genosis Corp. (The Woodlands, TX). The single-strand complementary oligonucleotides were mixed together in an equimolar ratio, incubated at 97°C for 10 min, and then allowed to anneal overnight as the thermocycler cooled down slowly. After annealing, the probes were radiolabeled by Klenow (Promega) end filling with [-³²P]dCTP (3000 Ci/mmol). Ten micrograms of cellular nuclear extract in 10-µl binding reactions [20 mM Hepes (pH 7.8), 100 mM KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, 2 µg of poly (dI-dC), and 1x complete Protease Inhibitor Cocktail (Roche)] and 50 000 cpm of ³²P-labeled probes were incubated with or without unlabeled competitor oligonucleotides as indicated for 20 min at room temperature. The binding mixtures were resolved on nondenaturing 6% polyacrylamide gels (Invitrogen). For supershift assays, 1 or 2 µg of rabbit polyclonal Sp1, Sp3, and Sp4 and mouse monoclonal AP-2 and AP-2 antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added in the binding reactions and incubated at 4°C for 20 min before electrophoresis. The Sp1 and AP-2 consensus wild type and mutant sequences were obtained from Santa Cruz Biotechnology, Inc. Nonspecific polyclonal rabbit antibodies were used as control.

Statistical Analysis

All transient transfection experiments were performed in triplicate and repeated three times. Statistics were performed when noted using one-way ANOVA followed by the Holm-Sidak multiple-range test. A P value of 0.05 or less was considered to be significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cloning of the Mouse AP-2 Gene and Determination of the Transcription Start Site

The human and mouse AP-2 genes have been mapped to chromosome 20q13.2 and chromosome 2H3-4, respectively [11]. To clone and study transcriptional regulation of the mouse AP-2 gene, genomic fragments containing the 5'-flanking region of the mouse AP-2 gene were obtained. A mouse phage genomic DNA library (Stratagene) was screened using a 500-bp probe from the 5' end of the mouse AP-2 cDNA [7]. Three overlapping mouse genomic clones were obtained. Southern blot analysis using the 5' end of AP-2 cDNA as probe revealed that one 15-kb clone contained approximately 6500 bp of 5'-flanking sequence as well as several exons. The approximately 6500-bp DNA fragment was subcloned into pBluescript vector (Stratagene) and sequenced by standard methods. A gross restriction enzyme map is shown in Figure 1A.

View larger version (54K): [in this window] [in a new window]   FIG. 1. Sequence analysis of the human and mouse AP-2 upstream sequences. A) The phage AP-2 genomic clone contains approximately 6500 bp of the DNA sequence upstream of the transcription start site. Some restriction enzyme sites are indicated: Sm, SmaI; N, NotI; Bg, BglII; K, KpnI; Sc, SacI; Sa, SalI. These restriction enzyme sites were used in constructing AP-2 reporter genes. B) Sequence alignment was performed with the BLAST analysis program (PubMed). The region with high identity is shown. The translation start sites are circled. Potential binding sites for Sp, AP-2, and GATA transcription factors are underlined. The arrow indicates the transcription start site of the mouse AP-2 gene. The sequences were analyzed with programs MatInspector.V2.2 and AliBaba2. Potential Sp-A, Sp-B/AP-2, and Sp-C/AP-2 sites (underlined) were examined further in later experiments

Before evaluating the importance of cis-acting elements in conferring mouse AP-2 gene expression, primer extension and 5' RACE were performed to identify and confirm the transcription start site. In the primer extension experiments, an antisense oligonucleotide with a 5' end located at -92 bp relative to the 5' end of the previously cloned AP-2 cDNA end was used for reverse transcription [12]. A major single band of 110 nucleotides was obtained using total mRNA from mouse placenta but not using the control mRNA from kidney (Fig. 2A). To confirm these results, 5' RACE was performed. The 5' RACE is a procedure for amplification of nucleic acid sequences from an mRNA template between a defined internal site and unknown sequences at the 5' end of mRNA. Comparing the sequences of reverse-PCR products with the genomic AP-2 DNA sequence, five of six clones suggested that 18 more nucleotides were obtained from reverse PCR and that the 18 nucleotides were located upstream of the 5' end of the previously cloned cDNA (Fig. 2B). Consistent results were obtained from both primer extension and 5'-RACE experiments, indicating that the transcription start site of the mouse AP-2 gene is located at an A base 18 nucleotides upstream of the 5' end of the previously cloned cDNA [7].

View larger version (15K): [in this window] [in a new window]   FIG. 2. Identification of the transcription start site of the mouse AP-2 gene by primer extension and 5' RACE. A) An antisense oligonucleotide complementary to nucleotides 58–92 of the mouse AP-2 cDNA sequence was used as a primer in a reverse transcription reaction with embryonic kidney or placental total RNA. A major single band of 110 nucleotides was obtained with total RNA from mouse placenta but not with RNA from kidney. B) 5'-RACE reactions were performed and sequenced as described in Materials and Methods. Three primers were used for reverse transcription and PCR. These were called GSP1, GSP2, GSP3 and were separately complementary to nucleotides of exon 1. One major band was obtained and cloned into the TA cloning vector for subsequent sequence analysis. A comparison with the genomic AP-2 DNA sequence (five of six clones) suggested that 18 more nucleotides are located upstream of the known 5' end of AP-2 cDNA. The sequences of the 18 nucleotides are 5'-ACACACTGTGAGGGTGAG. Thus, primer extension and 5'-RACE experiments obtained consistent results and indicated that the transcription start site of mouse AP-2 gene is located at an A base 18 nucleotides upstream of the known 5' end of AP-2 cDNA.

Cloning of the 5'-Flanking Region of the Human AP-2 Gene

A human AP-2 PAC genomic DNA clone, RP4-539E24, was obtained from The Sanger Center. The clone is approximately 86 kb and contains complete exons and introns of the human AP-2 gene and approximately 14 kb of upstream sequence. Southern blot analysis using a 5' fragment of the human AP-2 cDNA as probe was performed to confirm that it is the correct clone (data not shown).

Sequence Analysis of the Human and Mouse AP-2 Promoter Regions

The AP-2 gene is highly conserved in structure and function during evolution. A comparison of the human and mouse AP-2 5'-flanking regions using the Nucleic Acid Dot Plots Program of Colorado State University (Fort Collins, CO) revealed little similarity in approximately 6500 bp of 5'-flanking sequence, except for a short region of approximately 200 bp in the proximal promoter region (the dot plot is not shown). The promoter proximal 200-bp region has more than 93% identity between the human and mouse AP-2 genes. High sequence similarity was also seen in the nontranslated region of exon 1 (Fig. 1B). The proximal promoters of both species are extremely GC-rich and contain approximately 71% G+C. No typical TATA or CAAT box sequences are found. The presence of putative cis-acting elements within this approximately 200-bp region was evaluated using two transcription factor search programs, MatInspector.V2.2 (http://transfac.gbf.de/cgi-bin/matSearch/matsearch.pl) and AliBaba2 (http://wwwiti.cs.uni-magdeburg.de/grabe/alibaba2/). Sequence analysis revealed that multiple potential Sp- and AP-2-binding sites are concentrated in this short region (Fig. 1B).

Functional Analysis of the Mouse AP-2 Promoter and 5'-Flanking Region

To examine the 5'-flanking region of the mouse AP-2 gene for important transcriptional regulatory elements, we chose two trophoblast cell lines (HTR-8/SVneo and JEG-3) and two nontrophoblast cell lines (Hela and NIH3T3) for use in transient transfection analysis. These cell lines all express the AP-2 gene constitutively, as shown by Northern blot analysis. However, AP-2 mRNA is much more abundant in trophoblast cells than in the nontrophoblast cells (Fig. 3). Thus, these four cell lines should be useful in gene transfer studies to identify trophoblast cell-specific regulatory elements as well as cis-regulatory elements required for basal transcription.

View larger version (122K): [in this window] [in a new window]   FIG. 3. The levels of AP-2 transcripts in trophoblast cells and nontrophoblast cells. The relative abundance of AP-2 mRNAs in trophoblast cells (HTR-8/SVneo, JEG3, and Bewo) and nontrophoblast cells (Hela, NIH3T3, and CosM6) were determined by Northern blot analysis. Thirty micrograms of total RNA were loaded per lane, and 32P-labeled AP-2-specific probe was used for hybridization. The AP-2 transcripts are more abundant in trophoblast cells than in nontrophoblast cells. The lower image is an agarose gel stained with ethidium bromide

For transient transfection experiments, we prepared a series of 5'-deletion constructs with a common 3' end at +181 bp with respect to the transcription start site as +1 (Fig. 4A). In the transfection assays, construct (-6583/+181) showed much higher expression in trophoblast cells compared to nontrophoblast cells. Enhanced expression in trophoblast cells was considerably reduced with construct (-4290/+181), indicating that trophoblast-specific elements may reside in the approximately 2.3-kb region between -4290 and -6583 bp. To further test whether the DNA sequence between -6583 and -4290 bp contains trophoblast-specific elements, we inserted this approximately 2.3-kb fragment into the pGL3 promoter vector that uses the SV40 promoter as the basic promoter. This construct (-6583/-4290)/pGL3 promoter showed significantly enhanced expression in the trophoblast cells (HTR-8/SVneo and JEG3) as compared to the nontrophoblast cells (Hela and NIH3T3) (Fig. 4B). These data suggest that this approximately 2.3-kb DNA fragment contains cis-acting elements that provide for enhanced expression in trophoblast cells. This approximately 2.3-kb fragment was further subdivided, and the resulting fragments were placed upstream of the SV40 promoter in pGL3. Transient transfection experiments were conducted using the trophoblast cell line, HTR-8/SVneo, and the nontrophoblast cell line, Hela, as recipients. The results presented in Figure 4C indicate that trophoblast cell-specific elements reside in the 5' 704 bp of the approximately 2.3-kb fragment, corresponding to positions -6583 to -5879 upstream of the murine AP-2 transcription start site.

View larger version (23K): [in this window] [in a new window]   FIG. 4. Transcriptional analysis of the mouse AP-2 gene promoter and 5'-flanking region in trophoblast cells and nontrophoblast cells. A) Successive deletions of the mouse AP-2 5'-flanking region were fused to the pGL3 Basic reporter gene. The 3-terminus of each deletion construct is nucleotide +181 relative to the transcription start site mapped by primer extension and 5' RACE. Using Fugene 6 transfection reagent, 0.2 µg of each reporter gene plasmid and 0.02 µg of the internal control pRL-TK were cotransfected into HTR-8/SVneo, JEG3, Hela, and NIH3T3 cells. Forty-eight hours after transfection, the cells were harvested, and luciferase activity was measured. Transfection efficiency was normalized to the renilla luciferase activity encoded by pRL-TK. The x-axis shows fold-induction relative to the luciferase activity of empty pGL3 Basic reporter. The values are the mean ± SD of triplicate determinations. Promoter activity of labeled construct is significantly increased over control construct pGL3Basic. *P 0.05. B) A DNA fragment (-6583/-4290) was inserted upstream of the SV40 basic promoter sequence of the pGL3 promoter reporter gene. Transient transfection and luciferase assays were performed as described above. The x-axis shows fold-induction relative to the luciferase activity of the empty pGL3 promoter reporter. The values are the mean ± SD of triplicate determinations. Promoter activity of labeled construct is significantly increased over control construct pGL3promoter. *P 0.05. C) Successive deletions of (-6583/-4290) fragment of the mouse AP-2 gene were obtained by PCR using plasmid (-6583/-4290)/pGL3 Promoter as template. Transient transfection and luciferase assays were performed as described above. The x-axis shows fold-induction relative to the luciferase activity of the empty pGL3 promoter reporter. The values are the mean ± SD of triplicate determinations. Promoter activity of labeled construct is significantly different from control construct pGL3promoter. *P 0.05

Transcriptional analysis of additional 5'-deletion constructs indicated that transcriptional activity increased with DNA sequence shortening from -4290 to -233 bp in trophoblast and nontrophoblast cell lines. Construct (-233/+181) showed the highest transcriptional activity in all cell lines tested. Further reduction in size to -60 bp resulted in a complete loss of transcriptional activity (Fig. 4A). The critical region between -233 and -60 bp contains multiple potential Sp- and AP-2-binding sites that may contribute to promoter activity in both trophoblast and nontrophoblast cells.

Functional Analysis of the Human AP-2gamma; Promoter and 5'-Flanking Region

We prepared five human AP-2 gene 5' constructs. They ranged in size from approximately 200 to approximately 4300 bp and shared a common 3' end located immediately upstream of the translation initiation codon. The 5' end of the human AP-2 cDNA is designated as +1 [8]. Promoter activities of the reporter genes increased as the 5'-flanking region was reduced from -4300 to -295 bp in all cell lines. Construct (-295/+166) showed the strongest promoter activity. Deletion of DNA sequence between -295 and -130 bp abolished the promoter activity (Fig. 5), indicating that this short DNA sequence contains important cis-acting elements.

View larger version (17K): [in this window] [in a new window]   FIG. 5. Transcriptional analysis of human AP-2 gene promoter and 5'-flanking region in trophoblast cells and nontrophoblast cells. Successive 5' deletions of the human AP-2 upstream region were fused to the pGL3 Basic reporter gene. The 3' end of each deletion construct is nucleotide +166 relative to the 5' end of the known human AP-2 cDNA. Using Fugene 6 transfection reagent, 0.2 µg of each reporter gene plasmid and 0.02 µg of the internal control pRL-TK were cotransfected into HTR-8/SVneo, JEG3, Hela, and NIH3T3 cells. Forty-eight hours after transfection, the cells were harvested, and luciferase activity was measured. Transfection efficiency was normalized to the renilla luciferase activity encoded by pRL-TK. The x-axis shows fold-induction relative to the luciferase activity of empty pGL3 Basic reporter. The values are the mean ± SD of triplicate determinations. Promoter activity of labeled construct is significantly increased over control construct pGL3Basic. *P 0.05.

Sp1 and Sp3 Bind to the Promoter Region of the Mouse AP-2 Gene

As described above, the promoter region of the human and mouse AP-2 genes are highly conserved. Multiple potential Sp- and AP-2-binding sites are concentrated in a short stretch between -233 and -60 bp of the mouse AP-2 gene (Fig. 1B). To determine whether members of the Sp or AP-2 family transcription factors bind to these elements, electrophoretic mobility shift assays were performed with three DNA fragments within the region between -233 and -60 bp of the mouse AP-2 gene promoter. The three binding sites are designated as Sp-A, Sp-B, and Sp-C. The results using HTR-8/SVneo nuclear extracts and a probe (-142/-123) containing the Sp-A element showed two major DNA-protein complexes. Complex formation was diminished by the addition of a 50- or 100-fold molar excess of nonradiolabeled identical competitor, but not by the addition of the oligonucleotides in which the Sp-A site was mutated. Furthermore, complex formation was competed with a 50- or 100-fold molar excess of nonradiolabeled Sp1 consensus oligonucleotide, but not by a mutant Sp1 consensus oligonucleotide (Fig. 6A), suggesting that these bands represent specific protein binding to the Sp1 sequence elements. Similar results were obtained using nuclear extracts from JEG3 and Hela cells (data not shown).

View larger version (34K): [in this window] [in a new window]   FIG. 6. Analysis of protein-DNA interactions associated with the (-142/-123) region of the mouse AP-2 gene promoter. A) A 19-bp radiolabeled duplex probe (-142/-123) of the mouse AP-2 gene promoter was incubated with 10 µg of HTR-8/SVneo nuclear extracts in the absence (lane 1) or the presence of a 50- or 100-fold molar excess of unlabeled wild-type (WT) cold probe (lanes 2 and 3, respectively) or mutated cold probe (lanes 4 and 5, respectively). Competition was also performed with a 50- or 100-fold molar excess of unlabeled cold Sp1 consensus sequence (lanes 6 and 7, respectively) or mutant Sp1 consensus sequence (lanes 8 and 9, respectively). Bands A1 and A2 represent specific protein-DNA complexes. B) Supershift assays were performed using 1 µg of rabbit polyclonal Sp1, Sp3, or Sp4 antibodies (Ab). Band is the supershift band of Sp1 antibody at 1 µg (lane 2). Complex A2 is diminished by Sp3 antibody (lane 3). No supershift was seen using nonspecific polyclonal rabbit antibody (lane 5)

We performed a gel-supershift assay using Sp1, Sp3, and Sp4 antibodies to confirm that Sp family members bind to these sites. As shown in Figure 6B, complex A1 was supershifted by Sp1 antibody, and complex A2 was diminished by Sp3 antibody. These results indicate that complex A1 is formed from Sp1 binding, whereas complex A2 results from Sp3 binding. The Sp4 antibody did not supershift or diminish any complex. Similar results were obtained using nuclear extracts from JEG3 and Hela cells (data not shown).

Four DNA-protein complexes (B1-B4) were formed using fragment (-107/-86) containing the Sp-B site and HTR-8/SVneo nuclear extracts. The formation of the four complexes was reduced by the competition of the nonradiolabeled wild-type identical probe and by a probe containing the Sp1 consensus sequence, but not by the nonradiolabeled mutant probe or the probe with mutant Sp1 consensus sequence. Cold probes of both wild-type AP-2 and mutant AP-2 consensus sequences did not decrease the abundance of the complexes (Fig. 7A). These results suggest that these complexes represent specific protein binding to the Sp sequence elements, but not to the putative AP-2 sequence elements in this probe. In supershift assays, Sp1 and Sp3 antibodies, but not antibodies to Sp4, AP-2, or AP-2, reduced the formation of the complexes (Fig. 7B). The Sp1 antibody decreased the abundance of the complexes greater than Sp3 antibody. These data suggest that transcription factors Sp1 and Sp3, but not AP-2 and AP-2, are involved in the formation of specific DNA-protein complexes.

View larger version (40K): [in this window] [in a new window]   FIG. 7. Analysis of protein-DNA interactions associated with the (-107/-86) region of the mouse AP-2 gene promoter. A) A 22-bp radiolabeled duplex probe (-107/-86) of the mouse AP-2 gene promoter was incubated with 10 µg of HTR-8/SVneo nuclear extracts alone (lane 1) or in the presence of a 50- or 100-fold molar excess of unlabeled wild-type (WT) cold probe (lanes 2 and 3, respectively) or mutated cold probe (lanes 4 and 5, respectively). Competition was also performed with a 100-fold molar excess of unlabeled cold Sp1 and AP-2 consensus sequences (lanes 6 and 8, respectively) or mutant Sp1 and AP-2 consensus sequences (lanes 7 and 9, respectively). Bands B1, B2, B3, and B4 represent specific protein-DNA complexes. B) Supershift assays were performed using 2 µg of rabbit polyclonal Sp1, Sp3, and Sp4 antibodies (Ab) and mouse monoclonal AP-2 and AP-2 antibodies. The Sp1 antibody diminished the formation of the four specific complexes (lane 2). The Sp3 antibody decreased the abundance of complexes B1 and B2 (lane 3). No supershift was observed using nonspecific polyclonal rabbit antibody (lane 7).

Three DNA-protein complexes were formed using a DNA fragment (-75/-50) containing the Sp-C site and HTR-8/SVneo nuclear extracts. The nonradiolabeled wild-type probe and a probe containing the Sp1 consensus sequence diminished the abundance of the complexes, but the nonradiolabeled mutant probe and the probe with a mutant Sp1 consensus sequence showed no effect. Cold probes with both wild-type AP-2 and mutant AP-2 consensus sequences did not reduce the formation of the complexes (Fig. 8A). These data suggest that these complexes represent specific protein binding to the Sp sequence elements, but not to the AP-2 consensus sequences. Complex C3 was decreased by both wild-type and mutant Sp-C probe, but not by Sp1 and AP-2 consensus sequence probes, indicating that complex C3 resulted from interaction with nucleotide sequences other than Sp1-binding elements in the 26-bp Sp-C probe. Supershift assays showed that Sp1 antibody decreased the abundance of complex C1 and C2 and that Sp3 antibody diminished complex C2 exclusively (Fig. 8B), suggesting that Sp1 and Sp3 are involved in the formation of complexes C1 and C2.

View larger version (33K): [in this window] [in a new window]   FIG. 8. Analysis of protein-DNA interactions associated with the (-75/-50) region of the mouse AP-2 gene promoter. A) A 26-bp radiolabeled duplex probe (-75/-50) of the mouse AP-2 gene was incubated with 10 µg of HTR-8/SVneo nuclear extracts alone (lane 1) or in the presence of a 100-fold molar excess of unlabeled wild-type (WT) cold (-70/-50) probe, Sp1, and AP-2 consensus sequences (lanes 2, 4, and 6, respectively) or mutated cold sequences (lanes 3, 5, and 7, respectively). Bands C1, C2, and C3 represent specific protein-DNA complexes. B) Supershift assays were performed using 2 µg of rabbit polyclonal Sp1, Sp3, or Sp4 antibodies (Ab). The Sp1 antibody decreased formation of complexes C1 and C2 (lanes 2 and 3, respectively). The Sp3 antibody diminished complex C2. No supershift was observed using nonspecific polyclonal rabbit antibody (lane 5)

Sp1-Binding Sites Are Critical for the Promoter Activity of the Mouse AP-2 Gene

Deletion of the region between -233 and -60 bp of the mouse AP-2 gene resulted in a drastic decrease in promoter activity in all the trophoblast and nontrophoblast cells tested (Fig. 4A). The electrophoretic mobility shift assays showed that transcription factors Sp1 and Sp3 bind to the three conserved cis-elements in this region (Figs. 6–8). To test the functional importance of the three Sp-binding sites, we made a series of point mutations that disrupted the three cis-elements. In Sp-A (-142/-123) site, GG was mutated to AT. In the Sp-B site (-107/-86), C was substituted with A. Three Cs were mutated to three As in the Sp-C site (-75/-50) (Fig. 9A). The transfection studies revealed that mutational alteration of any of the three sites resulted in an approximately 20%–50% reduction in promoter activity when compared with that of the wild-type construct (-233/+181). Mutation of the Sp-A site decreased the promoter activity more than mutations at the other two sites, indicating that the Sp-A site is more important than the Sp-B and Sp-C sites. Promoter activity was decreased by 60% with combined mutations of Sp-A and Sp-B sites. Double mutation of Sp-B and Sp-C sites resulted in a 25% decrease. Triplet mutation of Sp-A, Sp-B, and Sp-C sites reduced promoter activity by more than 80%, suggesting that Sp-A, Sp-B, and Sp-C sites may function additively in the control of the AP-2 gene promoter activity (Fig. 9B). Promoter activities of all the deletion constructs are significantly decreased compared with the wild-type construct (P 0.05). These data suggest that all three Sp-binding sites contribute significantly to the promoter activity of the mouse AP-2 gene and that Sp-A is the most important site.

View larger version (25K): [in this window] [in a new window]   FIG. 9. Functional analysis of Sp sites in the promoter region of the mouse AP-2 gene promoter. A) Mutagenesis probes for disruption of the three potential Sp-binding sites. Potential Sp cis-elements in Sp-A, Sp-B, and Sp-C sites were mutated by point mutagenesis as described in Materials and Methods. B) Each construct (0.2 µg) was cotransfected with pRL-TK (0.02 µg) into HTR-8/SVneo cells (similar results were obtained using JEG3, Hela, and NIH3T3 cells). The x-axis shows the percentage luciferase activity of each mutated plasmid with respect to the luciferase activity of the wild-type (WT) construct (-233/+181). Diamonds indicate the Sp-A site, circles represent the Sp-B site, and stars represent the Sp-C site. X's represent introduced mutations. The values shown are the mean ± SD of triplicate determinations. Promoter activity of labeled construct is significantly decreased compared with wild-type construct (-233/+181). *P 0.05

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In the present study, we cloned and characterized the promoter and 5'-flanking regions of the mouse and human AP-2 genes as an initial step in understanding the regulation of AP-2 gene expression in the trophoblast cell lineage during placental development. Primer extension and 5' RACE experiments showed that the major transcription start site of the mouse AP-2 gene is located 241 nucleotides upstream of the ATG translation initiation codon. An atypical TATA box is located approximately 30 bp upstream of the transcription start site, suggesting that this sequence is involved in positioning the transcription initiation complex. Sequence comparison of approximately 6500 bp of 5'-flanking sequence revealed that the mouse and human AP-2 genes share little similarity except for a short, approximately 200-bp promoter proximal region that has 93% sequence identity between the human and mouse AP-2 genes. The conserved region is located immediately upstream of the transcriptional initiation site and has features of housekeeping genes, such as high GC content and the absence of canonical TATA or CCAAT box sequences. Gene transfer assays showed that the approximately 200-bp conserved region contains AP-2 promoter activity in both trophoblast and nontrophoblast cells. Within this region are three major Sp transcription factor-binding sites that contribute to AP-2 promoter activity.

Three Sp-binding sites (Sp-A, Sp-B, and Sp-C) were analyzed in detail by mutagenesis and electrophoretic mobility shift assays. Two protein-DNA complexes were formed with DNA corresponding to site Sp-A (-142/-123). Supershift assays indicated that one complex is composed of Sp1 and that the other complex contains Sp3. Mutational analysis showed that site Sp-A is important for the transcriptional activity of the mouse AP-2 promoter. Four protein-DNA complexes are formed with DNA corresponding to site Sp-B (-107/-86). It is noteworthy that consensus Sp1 oligonucleotides competed for site Sp-B complex formation better than authentic AP-2 promoter sequence. These results suggest that the binding proteins have a higher affinity for the consensus Sp1 sequence than for the murine AP-2 promoter sequence at this site. Analysis with Sp antibodies showed that Sp1 and Sp3 are involved in these four protein-DNA complexes. The Sp1 has a higher affinity than Sp3 for site Sp-B. Mutational analysis showed that site Sp-B is important for AP-2 promoter activity. Three specific bands were formed with DNA corresponding to site Sp-C (-75/-50). Consensus Sp1 oligonucleotides competed complexes C1 and C2, indicating that C1 and C2 complexes are Sp1 specific. Nucleotides outside of Sp1-binding elements in probe (-75/-50) may be involved in the formation of the C3 complex. Supershift assays with Sp antibodies indicated that Sp1 and Sp3 are involved in the protein-DNA complexes that form at site Sp-C. Mutational analysis indicated that site Sp-C contributes to the transcriptional activity of the mouse AP-2 gene. Two well-conserved AP-2-binding sites are located in sites Sp-B and Sp-C of the mouse AP-2 promoter, and they overlap with the Sp sites. However, our competition assays and supershift assays with AP-2 consensus sequences and AP-2 and AP-2 antibodies failed to show that AP-2 proteins are involved in the formation of the DNA-protein complexes discussed above.

Sp1 is a ubiquitous DNA-binding protein, with three zinc fingers at its C-terminus that activates the transcription of many cellular and viral genes [13]. The Sp1 recognizes and binds to GC-boxes. Other transcription factors (Sp2, Sp3, and Sp4) have been cloned and characterized that have similar structural and transcriptional properties as Sp1 [14, 15]. Both Sp1 and Sp3 are ubiquitously expressed, whereas Sp4 has a restricted distribution in neuronal cells and certain epithelia [16]. Both Sp1 and Sp3 are expressed in trophoblast cells of different species and regulate a variety of genes [17, 18]. Thus, our findings that Sp1 and Sp3, but not Sp4, function in the AP-2 promoter activity are consistent with the expression patterns of the Sp transcription factors in the placenta. Mutation of one or two critical nucleotides in Sp-binding sites of the mouse AP-2 gene promoter sequence reduced promoter activity by 20%–50%, respectively. Mutation of Sp-A from GGGCGG to GGGCta resulted in a 50% decrease of promoter activity, indicating that this site is the most important of the three Sp sites. Combined mutation of the three sites, Sp-A, Sp-B, and Sp-C, reduced the promoter activity by more than 80%, suggesting that all three sites contribute to transcriptional activity. Indeed, these three protein-binding sites are clustered in an approximately 80-nucleotide stretch of DNA just upstream of the transcription start site. Because all these Sp sites are well conserved in human and mouse, they most likely have similar functions in the regulation of human AP-2 gene expression.

Four AP-2 genes, AP-2, AP-2ß, AP-2, and AP-2, have been characterized in mammalian species [7, 19–21]. They exhibit partially overlapping but distinct expression patterns during development. Expression of AP-2 in the embryo was higher than that in the placenta, and AP-2ß is barely detected in either the embryo or the placenta. The mRNA of AP-2 is the most abundant AP-2 mRNA in the placenta, and it is highly expressed in all trophoblast lineages throughout placental development [1]. The results of gene disruption studies indicate that AP-2 family members have distinct, divergent roles. The disruption of either AP-2 or AP-2ß genes showed embryonic phenotypes, with placentas largely unaffected. The AP-2 (-/-) mice displayed multiple organ malformations, including the neural tube, eye, face, forelimbs, body wall, and cardiovascular system [22–25]. The AP-2ß (-/-) mice showed kidney defects [26]. In contrast, the AP-2 gene is required for early placental function. In placental development, AP-2 has a major and unique function, and it is not required for embryonic and adult viability [5]. The abundant expression of AP-2 in all trophoblast lineages throughout placental development [1, 5, 6], the requirement for AP-2 in the regulation of other placental genes [2–4], and the impaired placental development resulting from AP-2 gene disruption [5, 6] together provide strong evidence that AP-2 plays an important role in placental development.

The AP-2 gene is highly expressed in the trophoblast lineage during placental development [1, 5, 6]. We attempted to identify regulatory sequences responsible for trophoblast cell-specific expression using a transient transfection strategy in trophoblast cells (JEG3 and HTR-8/SVneo) and nontrophoblast cells (Hela and NIH3T3). Northern blot assays showed that AP-2 transcripts are considerably more abundant in trophoblast cells than in nontrophoblast cells, a feature consistent with the important biological functions carried out by AP-2 in these cells. A luciferase reporter plasmid (-6583/+181) showed approximately 3-fold more activity in trophoblast cells compared with nontrophoblast cells. Additional studies showed that an approximately 2.3-kb fragment (-6583/-4290) contains a trophoblast-specific enhancer when inserted upstream of the SV40 basal promoter in the pGL3 promoter vector. This construct displayed enhanced luciferase activity in trophoblast cells relative to nontrophoblast cells, indicating that the (-6583/-4290) fragment contains trophoblast cell-specific regulatory elements. Further analysis of this approximately 2.3-kb fragment revealed that the trophoblast cell-specific elements reside in the 5' proximal 704 bp located from 5.8 to 6.5 kb upstream of the transcription start site of the AP-2 gene. Multiple GATA transcription factor-binding sites are located in the 704-bp DNA sequence. The GATA factors are expressed in trophoblasts and are required for trophoblast-specific transcriptional regulation of the mouse placental lactogen I gene [27] and the mouse Ada gene [1]. Highly conserved GATA-binding sites in this region are good candidates with which to begin identifying the critical trophoblast-specific factors. Thus, our results indicate that the AP-2 gene may contain a distal enhancer element that functions in concert with the conserved promoter elements to achieve enhanced expression in trophoblast cells. In future studies, transgenic mouse assays will be used to assess the importance of this region in the control of AP-2 gene expression during placental development.

	FOOTNOTES

 
¹ Supported by a grant from the National Institutes of Health (HD34130) to R.E.K.

² Correspondence: Rodney E. Kellems, Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston, 6431 Fannin, Houston, Texas 77030. FAX: 713 500 0652; rodney.e.kellems{at}uth.tmc.edu

Received: 21 January 2003.

First decision: 9 February 2003.

Accepted: 23 May 2003.

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