Identification and Characterization of the cis-Acting Elements of the Human CD155 Gene Core Promoter*

David SoleckiDagger §, Eckard WimmerDagger , Martin Lipp, and Günter Bernhardtparallel

From the Dagger  Department of Molecular Genetics and Microbiology, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794 and the  Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rossle-Strasse 10, 13122 Berlin, Germany

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The CD155 protein is the founding member of a new group of related molecules within the immunoglobulin superfamily sharing a V-C2-C2 domain structure and significant amino acid identity. We have recently isolated the promoter of the CD155 gene so that we may determine the transcription factors that regulate its expression and possibly gain insight into the cell biology of this gene. Here we report the mapping of three cis-elements within the CD155 core promoter, designated FPI, II, and III. The results of linker scanning mutagenesis suggest that all three of these cis-elements are required in varying degrees for the promoter activity of the core promoter fragment. The relative contribution of each region ranked in the following order: III > II > I. Interestingly, footprint and electrophoretic mobility shift assays show that FPIII binding activity is much reduced in a human cell line that does not express CD155. Additionally, protein binding to FPI and FPII was also investigated. DNase I footprinting using recombinant hAP-2alpha indicated that this transcription factor bound to both the FPI and FPII regions of the CD155 core promoter fragment. Electrophoretic mobility shift assays and supershift analysis confirmed the binding of AP-2 from crude nuclear extracts to FPI and to FPII. Lastly, cotransfection of the CD155 promoter with an AP-2alpha expression vector indicates that overexpression of AP-2alpha modulated the promoter activity of a CD155 promoter construct.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The study of the mechanism by which poliovirus (PV)1 binds to and enters a host cell has led to the discovery of a group of related genes belonging to the immunoglobulin superfamily. The founding member of this group of related genes, the human poliovirus receptor (hPVR)/CD155, was cloned based on its ability to mediate the attachment of PV to host cells (1). The CD155 gene encodes glycoproteins with three extracellular immunoglobulin domains designated V-C2-C2 (1-6). Alternate splicing of the CD155 primary transcript gives rise to four isoforms: CD155alpha and CD155delta , which are integral membrane proteins; and CD155beta and CD155gamma , which are presumably secreted because they lack the exon encoding a transmembrane domain (2). The involvement of the viral receptor activity of the CD155 protein in both poliovirus attachment and the pathogenesis of poliovirus infections has been the subject of intense investigation (for review, see Refs. 7-9). The amino-terminal V type immunoglobulin domain of the integral membrane splice variants of CD155 serves as the PV binding moeity. Receptor binding then leads to virion destabilization and virus entry into host cells (7, 10-18). In addition, transgenic mice expressing the CD155 protein develop a syndrome very similar to human poliomyelitis when infected by PV, an observation suggesting that the CD155 protein is a major determinant of the tissue tropism displayed by PV (19-21).

Since the discovery of the CD155 gene, many genes possessing the V-C2-C2 domain architecture have been cloned from mouse, monkey, and man. In keeping with the fact that poliomyelitis is a disease that strictly affects primates, two homologous genes of CD155 which function as viral receptors were cloned from the African green monkey, AGMalpha 1 and AGMalpha 2 (22). A mouse relative, called MPH, has been isolated and characterized (23). In addition, two human genes, PRR1 (poliovirus receptor-related) and PRR2 have been described which share approximately 52 and 51% homology to the CD155 extracellular domains (24, 25). Interestingly, the MPH protein is more closely related to PRR2 than to CD155. Thus, MPH may not be the functional homolog of CD155, for which reason we will refer to it as mouse PRR2. In addition a CD155-related gene, the putative tumor antigen Tage4 was identified in rat and mouse (26, 27).

Until recently, the functions attributed to the molecules belonging to this subfamily have been obscure. Akoi et al. (28) have reported that mouse PRR2 can serve as a homotypic cellular adhesion molecule that cannot bind any other members of the CD155-related gene family. Furthermore, CD155 has been reported to be physically associated with CD44 on monocytes (29). Interestingly, two publications by Chadeneau et al. (26, 30) report that the rat and mouse Tage4 antigens are highly expressed in neoplastic tissue, but little expression is observed in normal tissues, suggesting that Tage4 is a tumor antigen. Taken together, these studies suggest that the CD155 subfamily of genes may possess important biological activities such as representing a new group of homotypic cellular adhesion molecules or as diagnostic markers in the study of neoplasia.

We have recently reported the cloning of the promoter region of the CD155 gene. A characterization of the CD155 promoter region will allow us to determine the cis-acting elements and trans-acting factors that regulate the expression of the CD155 gene and possibly provide insight into the biology of the CD155 protein. Our previous work determined that the CD155 promoter activity resides within an approximately 280-bp genomic DNA fragment that lacks TATA and CAAT boxes and is rich in GC nucleotide content. Three major and several minor transcriptional start sites have been identified within an approximately 60-bp region of this segment of genomic DNA (31). Interestingly, promoter constructs containing the 280-bp CD155 core promoter were inactive in Raji cells, a cell line that did not express endogenous CD155 mRNA.

In this report we have extended our analyses of the CD155 promoter. By DNase I footprinting we have identified three functional cis-acting elements (FPI-FPIII) within the CD155 core promoter and addressed their importance for basal promoter activity by linker scanning mutagenesis. We have also identified a cis-element in FPIII which is required for CD155 promoter activity and may be involved in the tissue-specific expression of CD155. We demonstrate in footprinting experiments that recombinant hAP-2alpha can bind two adjacent AP-2 binding motifs within the core promoter. Mutation of these potential binding motifs, either singly or in tandem, resulted in a reduction of core promoter activity. These mutations also abrogated the binding of hAP-2alpha . Electrophoretic mobility shift assays (EMSAs) confirmed AP-2 binding to FPI and FPII when the experiments were carried out using crude nuclear extracts. Lastly, overexpression of AP-2alpha in cotransfection experiments was found to stimulate the activity of the CD155 promoter approximately 3-fold.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell Culture

Cells from HeLa (cervical carcinoma), HEp-2 (epidermoid carcinoma), HepG2 (hepatocellular carcinoma), Saos-2 (osteocarcinoma), MDA-MB 435 (breast carcinoma), HEK293 (embryonic kidney), SK-N-SH (neuroblastoma), SK-N-MC (neuroblastoma), and HTB15 (glioblastoma) were grown in Dulbecco's modified Eagle's medium, 10% fetal bovine serum. Raji (Burkitt's lymphoma), BL99 (Burkitt's lymphoma), IARC 549 (B-lymphoblast), U937 (histiocytic lymphoma), K562 (chronic myelogenous leukemia), HL60 (promyelocytic leukemia), CEM (T-lymphoblastic leukemia), and Jurkat (T-lymphoblastic leukemia) cells were grown in RPMI 1640, 10% fetal bovine serum.

DNase I Footprint Assays

Nuclear extract preparation and DNase I footprinting were performed as described by Dynan et al. (32) with the following modifications. End-labeled DNA probes were generated via the polymerase chain reaction (PCR), using oligonucleotides that were end-labeled with [gamma -32P]ATP by polynucleotide kinase. PCR was performed under standard conditions using 10 ng of pGL2-H template, 25 pmol labeled and unlabeled primers, and 1.2 units of Taq polymerase. Radiolabeled PCR products were subjected to electrophoresis on a 10% native polyacrylamide gel, the bands were visualized by autoradiography, and a selected band was excised from the gel and passively eluted. DNase I protection assays were performed using 105 cpm of labeled probe that was incubated in a 50-µl binding reaction containing 2 µg of poly(dI-dC) and nuclear protein or recombinant human AP-2alpha (Promega Corp.). After a 30-min incubation on ice, 50 ml of a solution (room temperature) of 5 mM CaCl2 and 10 mM MgCl2 was added to each reaction and incubated for 1 min at room temperature. 1 µl of DNase I (6-100 ng/µl) was added and incubated another min at room temperature. The reactions were terminated by the addition of 90 µl of stop solution (0.2 M NaCl, 0.03 M EDTA, 1% SDS, and linear polyacrylamide as a carrier for ethanol precipitation). The mixture was then phenol/chloroform extracted twice, and the DNAs were ethanol precipitated. The samples were then electrophoresed on 10% polyacrylamide sequencing gels with a sequencing reaction as a marker.

Site-directed Mutagenesis

Site-directed mutagenesis of the BE CD155 promoter fragment was carried out using the megaprimer mutagenesis technique (33). To generate a megaprimer for each mutant construct, 100 ng of pGL2-BE plasmid was amplified in a reaction containing 50 pmol of either 4529 or 4532 flanking primer, 50 pmol of mutagenic primer, 5 µl of 10 × buffer, 2 µl of 10 mM dNTP mix, and 0.5 µl of Taq polymerase (2.5 units, Stratagene) in a total reaction volume of 50 µl. Reactions to generate FPIII megaprimers utilized 4529 as the first flanking primer, whereas FPII and FPI utilized 4532. PCR amplification conditions were 94 °C, 30 s; 55 °C, 45 s; 72 °C, 45 s for 35 cycles. All megaprimers were then gel purified. To extend a megaprimer to generate to a full-length 280 bp, 100 ng of pGL2-BE plasmid was amplified in a reaction containing 50 pmol of either 4529 or 4532 flanking primer (4529 for FPIII constructs and 4532 for FPII/FPI constructs), 1-2 µg of megaprimer, 5 µl of 10 × buffer, 2 µl of 10 mM dNTP mix, and 0.5 µl of Pfu polymerase (2.5 units, Stratagene).
<AR><R><C><UP>4532</UP></C><C><UP>5′-GGC<UNL>GCTAGC</UNL>GCCGCCTCTTCTAGTG-3′</UP></C></R><R><C><UP>4529</UP></C><C><UP>5′-GCC<UNL>AGATCT</UNL>GCTCGCTCTGCCGCGG-3′</UP></C></R><R><C><UP>III</UP>(<UP>1</UP>)</C><C><UP>5′-GCCAGCCTGG<UNL>ACTAGT</UNL>CACCCCGCGC-3′</UP></C></R><R><C><UP>III</UP>(<UP>2</UP>)</C><C><UP>5′-CTGGGTGGCC<UNL>ACTAGT</UNL>GCGCCTGGCG-3′</UP></C></R><R><C><UP>III</UP>(<UP>3</UP>)</C><C><UP>5′-GCCCACCCCG<UNL>ACTAGT</UNL>GCGGGACTGG-3′</UP></C></R><R><C><UP>III</UP>(<UP>4</UP>)</C><C><UP>5′-CCGCGCCTGG<UNL>ACTAGT</UNL>TGGCCGCCAAC-3′</UP></C></R><R><C><UP>III</UP>(<UP>5</UP>)</C><C><UP>5′-CTGGCGGGAC<UNL>ACTAGT</UNL>CCAACTCCCC-3′</UP></C></R><R><C><UP>III</UP>(<UP>6</UP>)</C><C><UP>5′-GACTGGCCGC<UNL>ACTAGT</UNL>CCCTCCGCTC-3′</UP></C></R><R><C><UP>III</UP>(<UP>7</UP>)</C><C><UP>5′-CCGCCAACTC<UNL>ACTAGT</UNL>GCTCCAGTCAC-3′</UP></C></R><R><C><UP>II</UP>(<UP>1</UP>)</C><C><UP>5′-GGGAAGGGGAA<UNL>ACTAGT</UNL>CTTCTTCAAGC-3′</UP></C></R><R><C><UP>II</UP>(<UP>2</UP>)</C><C><UP>5′-GGGGTGGGAA<UNL>ACTAGT</UNL>TACCCACTTCTTC-3′</UP></C></R><R><C><UP>II</UP>(<UP>3</UP>)</C><C><UP>5′-CCAGTGCCTGGGG<UNL>ACTAGT</UNL>GGGGAATACCC-3′</UP></C></R><R><C><UP>II</UP>(<UP>4</UP>)</C><C><UP>5′-GCTCCTCCAGTGC<UNL>ACTAGT</UNL>TGGGAAGGGG-3′</UP></C></R><R><C><UP>I</UP>(<UP>1</UP>)</C><C><UP>5′-GGTCCTGGAATC<UNL>ACTAGT</UNL>GGGCCGCTCCTC-3′</UP></C></R><R><C><UP>I</UP>(<UP>2</UP>)</C><C><UP>5′-GGAATCCCCGGG<UNL>ACTAGT</UNL>CTCCTCCAGTGC-3′</UP></C></R><R><C><UP>I</UP>(<UP>3</UP>)</C><C><UP>5′-GCTCAGGTCCT<UNL>ACTAGT</UNL>CCCGGGGGGCCG-3′</UP></C></R></AR>
<UP><SC>Sequence I</SC></UP>

Transfection and Harvest of Cells for Dual Luciferase Assays

The HTB15 (human glioblastoma), HeLa (human cervical carcinoma), SK-N-MC (human neuroblastoma), and HepG2 (human hepatocellular carcinoma) cell lines were transfected by the calcium phosphate procedure. Each transfection for the linker scan series of constructs was composed of 18 µg of wild type or mutant BE plasmid and 0.5 µg of pRL-TK (standard to the measure of transfectional efficiency). The composition of the cotransfection experiments was 9 µg of the BE, BE II(4), BE I(I), or BE Delta II/I plasmids mixed with up to 3 µg of pSP(RSV)-hAP-2alpha . Cotransfections with less than 3 µg of expression vector were supplemented with pSP(RSV) to keep the amount of backbone plasmid constant for each experiment. 50 µl of 2.5 M CaCl2 was added to the DNA mixtures, and then they were diluted to a total volume of 500 µl with TE buffer. These solutions were separately combined dropwise with 500 µl of ice-cold 2 × Hanks' balanced saline solution and incubated for 10 min at room temperature. Half of the precipitates were added to a separate plate of tissue culture cells, and the plates were incubated at 37 °C. 4 h later the medium was removed, and a solution of 20% glycerol in Hanks' balanced saline solution was added. After a 3-min incubation at 37 °C, 3 ml of medium was added, and the supernatant was removed again and replaced by fresh medium with serum. All transfected cells were harvested 18 h post-transfection, and cell extracts (usually 200-400 µl) were made using the reporter lysis buffer from Promega.

EMSAs

The oligodeoxynucleotides used for EMSAs were as follows.
<AR><R><C><UP>FPIs</UP></C><C><UP>5′-GGAGCGGCCCCCCGGGGATTCCAGGA-3′</UP></C></R><R><C><UP>FPIas</UP></C><C><UP>5′-GGTCCTGGAATCCCCGGGGGGCCGCT-3′</UP></C></R><R><C><UP>FPIIs</UP></C><C><UP>5′-GAAGAAGTGGGTATTCCCCTTCCCACCCCAGGCACT-3′</UP></C></R><R><C><UP>FPIIas</UP></C><C><UP>5′-GAGTGCCTGGGGTGGGAAGGGGAATACCCACTTCTT-3′</UP></C></R><R><C><UP>FPII</UP>(<UP>short</UP>)<UP>s</UP></C><C><UP>5′-CCTTCCCACCCCAGGCACT-3′</UP></C></R><R><C><UP>FPII</UP>(<UP>short</UP>)<UP>as</UP></C><C><UP>5′-CCAGTGCCTGGGGTGGGAA-3′</UP></C></R><R><C><UP>FPIIIs</UP></C><C><UP>5′-GGTGGCCCACCCCGCGCCTGGCGGGACTGGCCGCCAACTCCCCTCCGCTCCAGTCA-3′</UP></C></R><R><C><UP>FPIIIas</UP></C><C><UP>5′-GTGACTGGAGCGGAGGGGAGTTGGCGGCCAGTCCCGCCAGGCGCGGGGTGGGCCA-3′</UP></C></R></AR>
<UP>Sequence II</UP>
The AP-2 consensus oligodeoxynucleotide was purchased from Santa Cruz Biotechnology.

Oligoassociation-- 1 nmol of each the coding and noncoding oligodeoxynucleotides was reassociated in a volume of 50 µl using a thermocycler. Settings were: 5 min at 95 °C and 1 h each at 65, 60, 55, 50, 45, and 40 °C. The oligodeoxynucleotides were designed to possess a G as 5'-protruding nucleotide.

Labeling-- 10 pmol of reassociated oligodeoxynucleotide was end labeled by a fill-in reaction using a thermostable polymerase (Thermoprime, Dianova). In a volume of 20 µl, the buffer, 0.5 µl of enzyme, 50 µCi of [alpha 32-P]dCTP, 1 µl of 25 mM MgCl2, and the oligodeoxynucleotide were incubated at 40 °C for 10 min, 45 °C for 10 min, and 50 °C for 20 min. The labeled oligodeoxynucleotide was purified by Sephadex G-50 chromatography (Nick columns, Amersham Pharmacia Biotech). Usually more than 50% of label was found to be incorporated into the oligodeoxynucleotide.

Shift Assay-- Nuclear extracts were prepared according to the procedure by Schreiber et al. (34). In a total of 17 µl, 5 × incubation buffer (50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 5 mM EDTA, 5 mM dithiothreitol, 25% glycerol), 1.5 µg of poly(dI-dC), various concentrations of competitor or antibody (anti-AP-2alpha antibody, Santa Cruz Biotechnology) when indicated, and 4 µl of cell extract were preincubated for 10 min at room temperature. After preincubation 3-4 µl of labeled oligodeoxynucleotide corresponding to 100 fmol was added and the incubation continued for another 20 min. Samples were loaded onto a 6% Tris-glycine polyacrylamide gel (5 × Tris-glycine: 250 mM Tris, 1.9 M glycine, 10 mM EDTA). After the electrophoresis (160 V) the gel was fixed in 10% acetic acid and 30% methanol for 30 min and dried.

RNA Isolation, RT-PCR, and Cloning of an AP-2alpha Expression Vector

For Northern blotting and RT-PCR, total cellular RNA was isolated from 3 × 107 cells according to the TRIzol protocol (Life Technologies, Inc.). mRNA was extracted from 107 cells using the Quick Prep mRNA purification kit from Amersham Pharmacia Biotech. RT was done with Superscript Reverse Transcriptase from Life Technologies, Inc. using 10 µg of RNA as template and 1 pmol of gene-specific 3'-primer (AP-233) or 1 µg of oligo(dT12-18) in a reaction volume of 12 µl. The mixture was heated to 80 °C for 5 min and then allowed to cool to 42 °C. 4 µl of 5 × reaction buffer, 2 µl of 0.1 M dithiothreitol, 1 µl of 10 mM dNTP mix, 1 µl of RNasin (25 units), and 1 µl of reverse transcriptase were added and the reaction incubated for 90 min at 42 °C. The reaction was stopped by adding 20 µl of 0.4 M NaOH. After 10 min at 42 °C 20 µl of 1 M Tris-HCl, pH 7.5, was added and the reverse transcriptase stocks frozen at -20 °C. Oligodeoxynucleotides used were as follows.
<AR><R><C><UP>AP233 </UP>(<UP>for RT</UP>)</C><C><UP>5′-TCGAGGCGGGTGCAGAGTCG-3′</UP></C></R><R><C><UP>AP23 </UP>(<UP>for PCR</UP>)</C><C><UP>5′-CGGAATTCGGAGAGCCTCACTTTCTGTGC-3′</UP></C></R><R><C><UP>AP25 </UP>(<UP>for PCR</UP>)</C><C><UP>5′-CGGAATTCATGAAAATGCTTTGGAAATTGACG-3′</UP></C></R><R><C><UP>AP2A5′</UP></C><C><UP>5′-GGTCTAGACCAGAGGCAGAGCCAGGAGT-3′</UP></C></R><R><C><UP>AP2A3′</UP></C><C><UP>5′-CGCTCGAGTTCTTAATTACAGTTTGATCTGG-3′</UP></C></R><R><C><UP>AP2B5′</UP></C><C><UP>5′-GGTCTAGAGTGGGTTCGGAAGCCGGCTC-3′</UP></C></R><R><C><UP>AP2B3′</UP></C><C><UP>5′-CGCTCGAGTCATCATTAGAGAAGTCACC-3′</UP></C></R><R><C><UP>AP2G5′</UP></C><C><UP>5′-GGTCTAGAGCTGCCCTCGCACCACGGG-3′</UP></C></R><R><C><UP>AP2G3′</UP></C><C><UP>5′-CGCTCGAGCATGGAAATGGGACCTTTGCGA-3′</UP></C></R></AR>
<UP><SC>Sequence III</SC></UP>

For PCR, 1 µl of reverse transcriptase stock was used and mixed with 2 µl of 10 mM dNTP, 5 µl of primers each (50 pmol of AP23 and AP25 for the AP-2alpha full-length clone, AP2A[B,G]5' and AP2A[B,G]3' for AP-2alpha [beta ,gamma ] subtype-specific PCR), 5 µl of 10 × buffer, 10 µl of 5 × optimizer buffer, 1 µl of CombiPol Polymerase (InViTek), and water to a total volume of 50 µl. Cycling conditions were a denaturation step at 94 °C, 2 min, 35 cycles with 94 °C, 45 s; 55 °C, 45 s; 72 °C, 90 s (45 s for subtype-specific PCR), and a final extension step for 10 min at 72 °C. Products were separated on a 1.2% agarose gel. The HeLa full-length product was cut out and purified. After digestion with EcoRI the fragment was cloned into the expression vector pSP(RSV) (a kind gift from Helen Hurst). The AP-2alpha full-length insert was sequenced and found to be identical to the published open reading frame. Likewise, one clone each of the AP-2 subtype-specific PCR products was cloned and verified by sequencing.

Flow Cytometry

Cells were incubated on ice with monoclonal anti-CD155 antibody D171 (35) in a 96-well plate for 20 min in a total volume of 100 µl, washed twice with 100 µl of phosphate-buffered saline (1% fetal bovine serum), and incubated with R-phycoerythrin-labeled donkey anti-mouse IgG antibody (Dianova) for another 20 min. Cells were washed twice and then analyzed using a flow cytometer (Becton Dickinson).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

cis-Acting Elements of the CD155 Core Promoter-- By transfecting several CD155-positive cell lines with reporter plasmids containing 5' and 3' serial deletions of CD155 upstream sequence, we have determined regions required for the expression of a reporter gene (31). These experiments identified a 280-bp genomic DNA fragment that possessed full promoter activity when transfected into all cell lines that we have previously determined to express CD155 (BE-fragment, see Fig. 1A). In Raji cells as well as in all other Burkitt's lymphoma (BL) cell lines tested, the endogenous CD155 locus is transcriptionally inactive (31). When BE reporter constructs were transfected into Raji cells, only background levels of luciferase reporter gene activity were detected, an observation indicating that this cell line was unable to support the expression of the reporter gene (31). Therefore, it seemed likely that the BE core promoter fragment may harbor cis-element(s) required for basic promoter activity which may also confer cell type-specific expression to the CD155 gene.


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Fig. 1.   Overview of CD155 promoter region. Panel A, schematic drawing of the 5'- flanking region of the CD155 gene. In numbering, A of ATG = +1. Brackets from -203 to -152 indicate region of transcriptional initiation. Open box, transcribed region. Stippled box, coding region of exon 1. The border of the BE minimal promoter segment used in footprinting and linker scanning studies extends from -343 to -58 upstream of the ATG codon. Black ovals with Roman numerals show the locations of the footprints seen in HeLa and Raji cell nuclear extracts (see Fig. 2). Panel B, sequences of footprinted regions. Boxed sequences indicate potential transcription factor binding motifs for AP-2 and NFkappa B, respectively. Brackets under sequences represent where a wild type CD155 promoter sequence was replaced by an SpeI restriction site in linker scanning mutation studies. The arrow in FPIII indicates the location of a major transcriptional start site mapped by rapid amplification of cDNA ends.

To identify portions of the BE core promoter fragment which would interact with nuclear proteins, we performed DNase I footprint analyses using the nuclear extracts of CD155-negative Raji cells and CD155-positive HeLa S3 cells (Fig. 2). The nuclear extracts of HeLa S3 cells produced three protected regions, designated FPI, FPII, and FPIII (for relative location, see Fig. 1A; for sequences of protected regions, see Fig. 1B). Interestingly, the protection in the FPIII region is absent when the footprint reactions were carried using Raji nuclear extracts. When nuclear extracts of other cell lines that express CD155 were examined, footprint patterns identical to that of HeLa S3 were observed (data no shown). More specifically, the FPIII binding activity was always detected when extracts from CD155-expressing cell lines were used in the footprint assays. The results of these experiments identify three regions of the CD155 core promoter which are bound by nuclear proteins and are therefore candidates to harbor potential cis-acting elements. In addition, they suggest that one of the binding activities for the CD155 promoter is either not present or may be present in much reduced abundance in the extracts of cells such as Raji cells which do not express CD155.


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Fig. 2.   DNase I footprint assays of the BE minimal hPVR promoter fragment. The BE minimal promoter fragment (see Fig. 1A) labeled on the noncoding strand was incubated in the absence (0) or presence of the indicated amount of Raji or HeLa S3 nuclear extracts. After allowing the nuclear protein to bind the BE promoter fragment, the reactions were digested with DNase I. The resulting digested DNAs were isolated and electrophoresed on 10% sequencing gels. Regions of protection, FPI (at -128 to -104), FPII (at -167 to -139), and FPIII (at -243 to -193), are indicated by brackets.

Computer searches were performed to determine which potential transcription factor binding motifs are located in the footprinted regions (see Fig. 1B). Most notably, putative AP-2 binding motifs are scattered over all three protected regions. In addition, potential binding sites for PuF are found in FPII and FPIII, whereas FPII contains a potential NFkappa B site. FPII also contains an overlapping GC box adjacent to the AP-2 site (not shown).

Linker Scanning Mutagenesis of the Footprinted Regions-- To address the functional significance and map precisely the cis-acting elements located in the FPI-FPIII regions of the BE promoter fragment, a series of linker scan mutations was generated throughout the protected sequences. Each mutant promoter construct contains 6 bp of wild type CD155 promoter sequence replaced by a SpeI restriction enzyme site within the context of the BE fragment (for locations, see Fig. 1B). The panel of mutant promoter constructs was transfected into the HeLa, SK-N-MC (human neuroblastoma), and HTB15 (human glioblastoma) cell lines (Fig. 3). Particular mutations within all three protected regions showed reduced promoter activity. Moreover, the activity profile of the constructs was similar for all three cell lines tested (see Fig. 3). These results indicate that each of the footprinted regions harbors functional cis-acting elements, and the activity patterns of the constructs suggest that the three cis-acting elements exert similar functions in the three CD155-expressing cell lines tested.


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Fig. 3.   Linker scanning mutagenesis of the three protected regions of the BE CD155 promoter fragment. Results of transient transfection of the FPI, FPII, and FPIII linker scan series into the HeLa, SK-N-MC, and HTB15 cell lines. The nomenclature of mutant constructs was the footprint mutated followed by the specific linker insertion of a series (see Fig. 1B for locations of mutation going 5' to 3'). Cells seeded in six-well plates were transfected by the calcium phosphate method with 9 µg of promoter construct. Cotransfection of 250 ng of Reniella luciferase vector, pRL-SV40 (Promega), was used to monitor transfectional efficiency. 18 h post-transfection, cells were harvested, and firefly and Reniella luciferase activity contained in the cytoplasmic extracts of transfected cells was determined using the Dual Luciferase Reporter System (Promega). The activity of the wild type BE promoter construct was set to 100%, and the promoter activities of the linker scan mutant constructs are expressed relative to the wild type BE activity. Results are the mean ± S.D. of four HeLa transfections, three SK-N-MC transfections, and two transfections for HTB15.

Profound reductions in promoter activity were observed when mutations were located within a 13-bp segment of FPIII (see Figs. 1B and 3). The III(5) and III(6) promoter constructs possessed promoter activities 4-fold lower than that of the wild type BE fragment. It should be noted that the III(6) linker replacement disrupts one of the major transcriptional start sites mapped by rapid amplification of cDNA ends in our previous work (31). All three of these major transcriptional start sites bear homology to an initiator-like element (36). To test if the loss of promoter activity seen in the III(6) construct could be attributed to the initiator-like sequence, the CA core of all three potential initiator motif was changed to GC, a mutation that was previously shown to disrupt initiator activity (37). The activity of this promoter construct was equal to that of the BE promoter construct (data not shown). This result indicates that the III(5) and III(6) mutations do not elicit their loss in expression of the luciferase reporter gene by disruption of an initiator-like motif in the vicinity of the 5' most transcriptional start site.

Reductions of promoter activity were also observed with single linker replacements in FPII and FPI. The II(4) construct possessed an activity 55% that of wild type, whereas an activity of 60-80% of wild type was seen with the I(1) construct. The close proximity of FPI and FPII, and the II(4) and I(1) linker replacements, led us to speculate that these two cis-acting sequences may act together to contribute functionally toward CD155 promoter activity. To test whether there was an additive effect upon mutating both sites simultaneously a construct was cloned in which the region including both the II(4) and I(1) linker replacements were deleted. The Delta FPII/I construct possessed an activity approximately 40-45% that of the wild type BE promoter construct when transfected into the HeLa S3 and HTB15 cell lines (see Fig. 3). Taken together, these results indicate that FPI and FPII possess cis-acting sequences that contribute significantly toward CD155 promoter activity. These sequences may possibly contribute in an additive manner because deletion of both sites leads to a linear reduction in promoter activity.

EMSA of FPIII Binding Activity-- Footprinting and linker scanning analysis suggested that FPIII plays an important role in the transcription of the CD155 gene. To link the biochemical evidence of the footprinting results with the genetic evidence of the FPIII linker scan mutations, EMSA was used to characterize the FPIII binding activities present in the nuclear extracts of HeLa and Raji cells.

The nuclear extracts of both cell lines contain binding activities for the FPIII probe which migrate identically (see Fig. 4). In both cases, the intensity of the specific complexes increased as more of each nuclear extract was added to the binding reactions (see Fig. 4, lanes 2-4 for Raji and 7-9 for HeLa). The addition of a 50-fold molar excess of unlabeled probe significantly reduced the appearance of one of the gel shifted bands originating from each nuclear extract (Fig. 4, lanes 5 and 10; the specific band is marked with an arrow). We would like to note that we do not consider the slower migrating complex (see Fig. 4, marked by an asterisk) a specific binding activity. This complex is incompletely competed by the addition of the 50-fold molar excess of unlabeled probe (see Fig. 4, lanes 5 and 10). In addition, in many of our competition experiments we have observed a significantly lower reduction in the intensity of this particular band (data not shown), indicating that this complex is nonspecific in nature.


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Fig. 4.   EMSA of FPIII binding activities from the nuclear extracts of the HeLa and Raji cell lines. 1, 5, and 10 µg of HeLa and Raji nuclear extracts were incubated with radiolabeled FPIII probe in a buffer containing 10 mM Tris-HCl, pH 7.5, 75 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, and 5% glycerol. Binding reactions were incubated for 30 min on ice and then were electrophoresed on an 8% Tris-glycine polyacrylamide gel. The addition of 50 × FPIII (wild type) or III(m) competitor oligonucleotides is indicated at the top of the panel. The FPIII(m) competitor is a probe that contains both the III(5) and III(6) mutations incorporated within its sequence. An arrow indicates the specific complex. An asterisk indicates nonspecific complexes.

At each amount of nuclear extract tested the intensity of the retarded complexes formed by HeLa extract was significantly higher than those formed from Raji extracts. This result indicates that there is a much greater abundance of FPIII binding activity contained within the nuclear extracts of HeLa cells than prepared from Raji. Indeed, the intensity of the specific band produced by 1 µg of HeLa extract is very similar to the intensity of the shifted complex produced by 10 µg of Raji extract (see Fig. 4, compare lanes 4 and 7). This result may explain why FPIII was absent from Raji nuclear extracts when we initially carried out footprinting experiments. FPIII does not become fully visible until 45-75 µg of HeLa nuclear extract is used. The results of our EMSA experiments suggest that these amounts of Raji extract may not possess sufficient FPIII binding activity to elicit a footprint under the experimental conditions used.

The III(5) and III(6) linker scan mutations resulted in a large reduction in promoter activity as measured by our reporter gene assays. It was of interest to determine if the sequences altered by these linker scanning mutations played any role in the binding of the activities that we detected with our FPIII EMSA. The addition of the 50-fold molar excess of a cold probe harboring both the III(5) and III(6) mutations was not able to compete away the FPIII binding activities of Raji and HeLa extracts detected in this assay (competitor III(m); see Fig. 4, lanes 6 and 11). Taken together these results provide evidence supporting the hypothesis that the FPIII(5) and III(6) linker scan mutations disrupt the sequences required for nuclear protein binding to the FPIII region. Consensus oligonucleotides for NF-1 and C/EBP did not compete for the binding of the FPIII probe from both HeLa and Raji extracts (data not shown). In addition, we could not observe a supershift of the FPIII complexes when an anti-TFIID antibody was added to the EMSA reactions (data not shown), suggesting that FPIII does not represent the preinitiation complex of transcription. Therefore, the identity of the FPIII binding factor(s) remains unknown.

AP-2 Binding to FPI and FPII-- We have also carried out experiments designed to determine the identity of the trans-acting factors that bind the FPI and FPII regions of the CD155 promoter region. The II(4) and I(1) linker replacements as well as the Delta FPII/I deletion depicted in Fig. 1B disrupt potential AP-2 binding sites that led to an observable phenotype (Fig. 3). This suggests that members of the AP-2 transcription factor family are candidates to interact with either FPI or FPII. To assess whether an AP-2 family member could bind FPI or FPII, the BE fragment was labeled on the noncoding strand and subjected to footprint analysis using recombinant human AP-2alpha . Interestingly, rhAP-2alpha protected both the FPII and FPI regions of the BE CD155 core promoter fragment (see Fig. 5, lanes 2-4). In contrast, the FPIII sequence was not protected even when high amounts of AP-2alpha were used (data not shown). This was surprising because there are multiple potential AP-2 binding sites within this region (see Fig. 1B). These results indicate that AP-2alpha is able to bind consensus binding motifs located within FPI and FPII, but not those within FPIII.


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Fig. 5.   Recombinant human AP-2alpha binds the FPI and FPII regions of the CD155 promoter. The BE, BE 2(4), BE 1(1), and BE 1(2) promoter fragments were labeled on the noncoding strand and incubated in the presence or absence of rhAP-2alpha protein (Promega). The amount of AP-2alpha added to each binding reaction is indicated in footprint units (FPU), an arbitrary measurement defined by the manufacturer. The binding reactions were subjected to DNase I digestion and the isolated DNA fragments separated by electrophoresis on 10% sequencing gels. The regions of protection in the vicinity of FPI and FPII are -128 to -104 and -156 to -132, respectively. The schematic on the right of the figure indicates the relative position of FPI and FPII generated by footprinting experiments using the crude nuclear extract from the HeLa and Raji cell lines. wt, wild type.

The II(4), I(1), and I(2) linker replacement mutations that we had generated in FPII and FPI disrupted the potential AP-2 binding motifs in these two protected regions. To assess whether the loss in promoter activity which we observed with our mutant promoter constructs could be caused by the loss of the ability to bind AP-2, we subjected the II(4), I(1), and I(2) promoter fragments to footprint analysis with rhAP-2alpha (Fig. 5, lanes 5-7). Mutation of the AP-2 binding motif of either footprint caused the loss of AP-2 binding to that particular footprint but not for the neighboring region. This indicates that AP-2alpha binding to each region is independent of binding to the AP-2 consensus motif of the neighboring region. From these data and those of the activities of the mutant promoter constructs, we suggest that losses of promoter activity of the II(4), I(1), and Delta FPII/I could indeed be the result of loss of AP-2 binding to these promoters.

The borders of the FPI region of protection resulting from binding of recombinant AP-2alpha and the factor(s) from HeLa nuclear extracts are indistinguishable. However, on close comparison of the borders of the FPII region of protection produced by AP-2alpha and crude nuclear extract, a difference is observed (compare Figs. 2 and 5). The 5' and 3' borders of the AP-2alpha FPII appear to be shifted 11 nucleotides toward FPI. We entertain two explanations for this phenomenon. First, a combination of AP-2 and another unknown protein may produce the distinctive footprint observed when footprint experiments are carried out with crude nuclear extract or, second, the protein binding FPII from crude nuclear extracts may not be an AP-2 family member. EMSAs were performed using radiolabeled oligodeoxynucleotides corresponding to either FPI or FPII sequences (protected by HeLa extracts; see Fig. 1B). A FPI oligonucleotide was able to produce a specific shift when using HeLa extracts (Fig. 6A, lane 2), which could be competed by both FPI as well as a consensus AP-2alpha oligodeoxynucleotide (containing the sequence 5'-GCCCGCGG-3') (lanes 3 and 4). Moreover, the AP-2-induced shift could be supershifted using a polyclonal anti AP-2 antibody (lane 5). In contrast, the shifted complex showed a different mobility when using Raji nuclear extracts (Fig. 6A, lane 6). This shift could be competed by an excess of a cold FPI probe (lane 8) but not the AP-2alpha oligodeoxynucleotide (lane 7), and it was not supershifted by the anti AP-2 antibody (lane 9). These results confirm that AP-2alpha is indeed the protein from crude HeLa nuclear extracts that binds FPI.


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Fig. 6.   EMSA of FPI and FPII binding activities from the crude nuclear extracts of the HeLa and Raji cell lines. Panel A, nuclear extracts of HeLa and Raji cells were incubated with radiolabeled FPI probe in a buffer containing 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, and 5% glycerol. Binding reactions were incubated for 30 min at room temperature and then were electrophoresed on a 6% Tris-glycine polyacrylamide gel. The addition of an anti-AP-2 polyclonal anti-body or 50 × various competitors is indicated at the top of the panel. Panel B, nuclear extracts of HeLa and Raji cells were incubated with the radiolabeled FPII(GAAGAAGTGGGTATTCCCCTTCCCACCCCAGGCAC) probe with the above binding conditions. Binding reactions were incubated for 30 min at room temperature and then electrophoresed on a 6% Tris-glycine polyacrylamide gel. The addition of an anti-AP-2 polyclonal antibody or 50 × various competitors is indicated at the top of the panel. An arrow indicates specific complexes. Panel C, nuclear extracts of HeLa and Raji cells were incubated with the radiolabeled FPII(short, CCTTCCCACCCCAGGCACT) probe with the above binding conditions. Binding reactions were incubated for 20 min at room temperature and then electrophoresed on a 4% 1/2 × TBE polyacrylamide gel. The addition of an anti-AP-2 polyclonal antibody or 100 × various competitors is indicated at the top of the panel. An arrow indicates specific complexes. An asterisk indicates nonspecific complexes.

We observed a more complex pattern of protein interactions with FPII. When EMSAs were performed with a probe encompassing the entire FPII sequence a specific shift of differing mobilities with HeLa and Raji nuclear extracts was detected (Fig. 6B, lanes 2 and 7). Surprisingly, the same anti-AP-2 antibody used above failed to supershift this complex formed from HeLa extract (lane 3), an observation indicating that the protein binding the full-length probe was not AP-2. The specific band could be competed to some extent by an excess of cold FPI oligodeoxynucleotide (lane 5), but this competition was not as strong as that seen with the cold full-length FPII probe (lane 4). Raji nuclear extracts contained ample binding activity (lane 7) which could be competed efficiently by excess FPII oligodeoxynucleotide only (lane 8).

The apparent discrepancy regarding AP-2 binding to FPII between our footprint and EMSA experiments prompted us to examine more closely protein binding at this region. A shorter FPII probe was generated which differed from the original full-length one by the deletion of the upstream NFkappa B consensus binding motif (see Fig. 1B). A different pattern of shifted complexes was observed with the shorter probe (Fig. 6C). Again, HeLa and Raji extracts produced complexes of differing mobility (compare lanes 3 and 7). In addition, a complex formed by rhAP-2alpha protein migrated differently compared with those formed from both nuclear extracts (lane 2). However, the addition of the anti-AP-2 antibody resulted in a supershift of the HeLa- but not the Raji-derived complex (lane 6). There are three known members of the AP-2 transcription factor family, AP-2alpha , -beta and -gamma . The peptide that was used in the generation of the anti-AP-2 antibody is well conserved among all three AP-2 proteins. This anti-AP-2 antibody has been reported to supershift AP-2gamma -containing complexes (38) as well as those complexes that contain AP-2beta (39).2 Therefore, it is reasonable to assume that this antibody should be able to supershift all AP-2-containing complexes. Indeed, the fact that the HeLa complex migrates differently than that of rhAP-2alpha yet is recognized by the antibody suggests that the binding protein is a member of the AP-2 family. It is unclear if there is a difference in the binding site specificity of the various AP-2 family members. The results of our competition analysis suggest that this might be the case. The appearance of the HeLa complex could be reduced by the addition of a 100-fold molar excess of cold short FPII probe, but to a much less extent by the consensus AP-2alpha oligodeoxynucleotide (CCCCAGGC AP-2 motif versus GCCCGCGG; compare lanes 4 and 5). Taken together these experiments provide evidence that there are multiple proteins interacting with the FPII region of the CD155 promoter. Moreover, an AP-2-like factor from crude HeLa nuclear extract is able to bind an FPII probe only when an upstream transcription factor binding site is deleted. Possibly, the unknown protein that binds the full-length FPII probe is able to inhibit AP-2 binding.

AP-2 and CD155 Expression in Various Cell Lines-- The results of our footprint and EMSAs suggested that members of the AP-2 transcription factor family bind the FPI and FPII region of the CD155 promoter. The fact that Raji cells express neither AP-2 nor CD155 prompted us to examine the correlation between the expression of AP-2 family members and CD155. Several cell lines of different origin were tested for CD155 expression by flow cytometry. These same cell lines were also analyzed for the expression of all three AP-2 family members by RT-PCR and gel supershift with an FPI probe, using a polyclonal anti-AP-2 antibody (see Table I). RT-PCR was used to dissect which of the three AP-2 family members (alpha , beta , or gamma ) are expressed in a particular cell line, by using primers specific for each isoform. The supershift analysis was used to confirm the presence of AP-2 protein binding activity. Preliminary Northern blotting experiments indicate that AP-2 mRNAs are expressed at a low level (data not shown), a result in agreement with other studies in the literature (40). Interestingly, except for HepG2, K562, and U937, cell lines expressing high amounts of surface-bound CD155 also scored positive for AP-2 (see Table I: HeLa, HEp-2, MDA-MB 435, HEK293, HTB15, and HL60). Similarly, cell lines expressing low or undetectable levels of CD155 failed to express AP-2alpha or gamma , as detected by RT-PCR and by supershift when using the anti-AP-2 antibody in gel shift assays (see Table I: K562, CEM, IARC 549, BL64, BL99, and Raji). However, a faint RT-PCR band representing an AP-2alpha transcript was detected in the Jurkat cell line, indicating that AP-2 may be expressed at a low rate in these cells. This was supported by supershift of the FPI binding complex produced using Jurkat nuclear extracts. Taken together, the expression of CD155 and AP-2 family members correlates, with the exception of the HepG2, U937, and K562 cell lines. Notably, three of the four cell lines (U937, HL60, and K562) which express CD155 but score negative for AP-2 binding activities, belong to the class of hematopoietic precursor cell lines. It is possible that in this particular case CD155 promoter regulation follows a different pathway.

                              
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Table I
Analysis of the expression of CD155 and the members of the AP-2 family of transcription factors in various cell lines
Analysis of the expression of CD155 and the members of the AP-2 family of transcription factors in various cell lines. Cell surface expression of CD155 antigen was determined by flow cytometry in two to three independent experiments for each cell line tested. A rough quantitation indicates the amount of surface-bound CD155 as a 25% step gradient where ++++ equals 75-100% of the signal obtained with HeLa cells (for 435 cells, 127%). Expression of AP-2 alpha , beta , or gamma  mRNA was determined by RT-PCR; the presence of functional AP-2 protein was monitored by supershift of the FPI EMSA complex. The identity of each of the cell lines is given under "Materials and Methods."

AP-2 Transactivates the CD155 Promoter-- The results of linker scan mutations, protein binding assays, and analysis of CD155 and AP-2 expression provide circumstantial evidence that AP-2 family members may be involved in the regulation the CD155 gene expression. To assess more directly whether AP-2 transcription factor family members can modulate the activity of the CD155 promoter, we overexpressed AP-2alpha in a cotransfection experiment with the BE CD155 promoter construct. In these experiments the BE construct was transfected with increasing amounts of an AP-2alpha expression vector into the HTB15 or HepG2 cell lines. As can be seen in Fig. 7A, an increase of approximately 3-fold was observed when cotransfection experiments were carried out using HepG2 cells, a cell line known to be deficient for endogenous AP-2 activity (Ref. 41 and Table I). On the other hand, overexpression of AP-2alpha in HTB15 cells led to no change in promoter activity. These results reflect findings reported previously in the literature (42, 43). Briefly, it has been found that only in cells lacking AP-2 can an AP-2-responsive promoter be transactivated by overexpression of this transcription factor. In contrast, in cells that already express AP-2 (e.g. HTB15 cells), the overexpression of AP-2 may have no effect or may even lead to a decline in the promoter activity of an AP-2-responsive promoter, a phenomenon that has been coined "self-interference" (42). Taken together, the results of these cotransfection experiments suggest that activity of the CD155 core promoter is modulated in a manner that is consistent with regulation by AP-2.


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Fig. 7.   Panel A, effect of cotransfection of an AP-2alpha expression vector on the promoter activity of the BE CD155 minimal promoter fragment in the HepG2 and HTB15 cell lines. HepG2 and HTB15 cells that were seeded in six-well tissue culture plates were transfected by the calcium phosphate method with 9.0 µg of the BE promoter construct (driving the expression of luciferase; Ref. 31) with up to 3.0 µg of pSP(RSV)-AP-2alpha . Transfections with less than 3.0 µg of pSP(RSV)-AP-2alpha were filled in with pSP(RSV) to keep the amount of expression vector backbone in each reaction at a constant level for all experiments. Transfected cells were harvested 18 h post-transfection, and the luciferase activity contained within the cytoplasmic extract of transfected cells was determined using the luciferase reporter system (Promega). The activity of BE promoter construct cotransfected with only pSP(RSV) was set to 100% (control promoter activity), and the activity of the BE fragment cotransfected with pSP(RSV)-AP-2alpha is expressed relative to that 100%. Results are the mean ± S.D. of triplicate transfections. Panel B, cotransfection of pSP(RSV)-AP-2alpha with the wild type and mutant BE promoter constructs. HepG2 cells that were seeded in six-well tissue culture plates were transfected by the calcium phosphate method with 9.0 µg of the BE, II(4), I(1), or Delta II/I promoter constructs and 3.0 µg of pSP(RSV)-AP-2alpha . Transfected cells were harvested 18 h post-transfection, and the luciferase activity contained within the cytoplasmic extract of transfected cells was determined using the luciferase reporter system (Promega). The activity of each promoter construct (BE, II(4), I(1), or Delta II/I) cotransfected with only pSP(RSV) was set to 100% (control promoter activity), whereas the activity of each construct cotransfected with 3.0 µg of pSP(RSV)-AP-2alpha is expressed relative to that 100%. Results are the mean ± S.D. of quadruplicate transfections.

The functional contribution of the FPII and FPI AP-2 binding sites toward the AP-2alpha -induced activation was also assessed (Fig. 7B). Mutation of each site singly resulted in an approximately one-half reduction of the activation. Furthermore, deletion of both AP-2 binding sites resulted in a complete abolishment of the AP-2alpha activation. These results indicate that both AP-2 binding sites contribute in an additive manner toward activation. This result is in agreement with the effect of our linker replacement mutation on basal promoter activity and is consistent with the independence of AP-2 binding to each footprinted region we observed with our footprinting experiments.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have extended our study of the genetic organization and function of the promoter controlling the CD155 gene. This human gene expresses four glycoproteins through splice variation of which two function as a cellular receptor for PV (7). The non-pathological function of CD155 is unknown. Using DNase footprint analyses of a minimal promoter fragment, we have identified three potential cis-acting elements (FPI, -II, and -III) which we propose are involved in the regulation of expression of the CD155 gene. Importantly, we have been able to observe a variety of differences in the nuclear protein interactions to all three elements when we analyzed the nuclear extracts of CD155-negative Raji cells and CD155-positive HeLa S3 cells. These differences may be of importance for the understanding of the factors participating toward the cell type and tissue-specific expression of the CD155 gene.

The footprint designated FPIII is located at -243 to -193 nucleotides upstream of the ATG translation initiation codon of exon 1 of the CD155 gene (Fig. 1). Although FPIII contains putative AP-2 and PuF binding sites, these factors are unlikely to play a decisive role in transcriptional regulation mediated by FPIII because linker scanning mutagenesis effecting these putative binding sites did not significantly lower the expression of a reporter gene. Rather, a region identified by the III(5)/III(6) mutations (see Fig. 1B), located downstream of the putative AP-2 and PuF binding sites, appears to be a strong cis-acting element of the promoter. Indeed, the genetic and biochemical data we have obtained implicate the 13-bp region (III(5)/III(6)) as being essential for nuclear protein binding at FPIII and CD155 promoter activity (Figs. 3 and 4). The III(5)/III(6) region bears no apparent homology to binding motifs of known transcription factors or to portions of known promoter regions.

Footprint and EMSAs have led to the interesting observation that nuclear extracts prepared from Raji cells, a BL cell line that does not express the CD155 protein, possess a much reduced quantity of FPIII binding activity. Because FPIII binding is required for the expression of a reporter gene, at least when analyzed in the context of the minimal CD155 promoter, the apparent weak FPIII binding activity in Raji cells may account for the lack of CD155 expression in these cells. We consider it possible, therefore, that the FPIII region plays a decisive role in basic as well as the cell type-specific activity of the CD155 promoter. The identity of the FPIII-binding factor(s), as well as the reason why its activity is much less abundant in Raji cell nuclear extracts, is at present unknown. Future experiments will focus on determining the identity of the FPIII binding activity.

The two footprints FPI and FPII are located -128 to -104 and -167 to -139 nucleotides, respectively, upstream of the ATG translation initiation codon of the CD155 gene. The II(4), I(1), and Delta II/I mutations mapping to FPI and/or FPII have resulted in a significant reduction of reporter gene expression controlled by the BE promoter fragment when the corresponding plasmids were transfected into the CD155- and AP-2-expressing cell lines (HeLa, SK-N-MC, and HTB15 (see Fig. 3 and Table I). In the three II(4), I(1), and Delta II/I mutations, potential AP-2 binding sites were disrupted, either singly or in tandem (see Fig. 1B). AP-2 transcription factor(s) are therefore candidate trans-acting factors to interact with the CD155 promoter. The AP-2alpha transcription factor was originally cloned from HeLa cells (44, 45) and was subsequently shown to contain at least two other family members, AP-2beta and AP-2gamma (38, 40, 46, 47). AP-2 factors bind as dimers to cis-elements of responsive genes in a sequence-specific fashion, although considerable variations to the proposed consensus binding sequence have been observed (5'-GCCNNNGGC-3'; (48, 49)).

Footprint analysis of the BE promoter fragment using rhAP-2alpha showed that the potential AP-2 motifs within both the FPI and FPII regions represent functional binding sites in an in vitro binding assay. Generally, the protection of the FPI region is identical when the protein sources for footprinting assays are rhAP-2alpha or crude HeLa S3 nuclear extracts. However, the area of rhAP-2alpha protection in the FPII region appears to be smaller and shifted toward FPI (compare Figs. 2 and 5). A similar observation between a footprint produced by nuclear extract versus a footprint produced by rhAP-2alpha has been reported by Gao et al. (50) with the rat alpha 1B-adrenergic receptor promoter. Gao et al. (50) interpreted their result by hypothesizing that another protein(s) in addition to AP-2 was binding to the rat alpha 1B-adrenergic receptor promoter. Indeed, our EMSA using a full-length FPII probe clearly identifies an unknown protein in HeLa S3 extracts which binds to FPII. Interestingly, AP-2 binding to FPII probe could only be observed when a probe missing a potential NFkappa B binding site was used in EMSA experiments (Fig. 6C). We therefore conclude that FPII may bind AP-2 in the context of other factors, whereas FPI clearly interacted with AP-2 alone, as shown by EMSA and supershift analyses (see Fig. 6, A-C). It is possible that a factor binding to the 5'-half of FPII limits the proper access of AP-2 toward its binding site, thus controlling the AP-2 influence on the CD155 promoter.

An examination of the expression of both CD155 surface antigen and the members of the AP-2 family members shows some correlation in that in 8 out of 12 cell lines CD155 expression and AP-2 binding to FPI co-vary (Table I). Of all adherent cell types tested only HepG2 lacks any AP-2 activity despite expressing CD155 to a certain extent. It should be noted that we have observed three cell lines (U937, HL60, and K562) in which the CD155 protein is expressed without apparent binding activity of any of the known AP-2 factors. Currently we have no explanation for this phenomenon apart from the assumption that in these hematopoietic precursor cell lines CD155 regulation may not require AP-2 proteins.

Cotransfection experiments of an AP-2alpha expression vector plus BE promoter fragment provided direct evidence that the AP-2 family members can modulate the activity of the CD155 promoter (see Fig. 7, A and B). The cotransfection experiments revealed that the activity of the BE could be increased or decreased depending upon the endogenous expression of AP-2 in the two cell lines tested. Thus, transcription factors belonging to the AP-2 family are likely to be involved in the regulation of the expression of the CD155 gene. Indeed, both FPII and FPI binding sites appear to function in an additive manner toward AP-2alpha -induced activation in the context of the CD155 promoter.

It should be noted that expression of the AP-2 proteins in mice occurs at distinct stages during mouse embryogenesis (40, 51). Expression was mainly observed in neural crest cells and their derivatives, namely epithelial cells and cells of the CNS (spinal cord). In humans, CD155 is expressed at very low levels in many tissues, but the major target cells in PV pathogenesis are motor neurons of the spinal cord. A pattern of CD155 expression and PV pathogenesis similar to that in humans has been observed in CD155 transgenic mice (19-21). Recently, our laboratory has generated transgenic mouse lines that express beta -galactosidase under the control of a 3-kilobase pair CD155 promoter fragment.3 It is interesting to note that beta -galactosidase expression can be observed in these CD155 promoter beta -galactosidase transgenic mice primarily during embryogenesis in the developing spinal cord, in a spatial location overlapping AP-2 expression. The potential modulation of the CD155 promoter by the AP-2 family of transcription factors may be of importance for the in vivo expression of the CD155 gene during human embryogenesis.

    ACKNOWLEDGEMENTS

We thank C. Meese for excellent technical assistance and Helen Hurst for the kind gift of the AP-2beta and AP-2gamma expression vectors.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant AI39485.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Member of the graduate program in Molecular and Cellular Biology, SUNY at Stony Brook, and recipient of a grant from the Deutscher Akademischer Austauschdienst.

parallel Supported by Grant BE1886/1-1 from the Deutsche Forschungsgemeinschaft. To whom correspondence should be addressed. Tel.: 49-30-9406-3330; Fax: 49-30-9406-2887.

The abbreviations used are: PV, poliovirus; bp, base pair(s); EMSA, electrophoretic mobility shift assay; PCR, polymerase chain reaction; RSV, Rous sarcoma virus; RT, reverse transcription; BL, Burkitt's lymphoma; NF, nuclear factor.

2 D. Solecki, E. Wimmer, M. Lipp, and G. Bernhardt, unpublished observations.

3 M. Gromeier, D. Solecki, and E. Wimmer, submitted for publication.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

  1. Mendelsohn, C. L., Wimmer, E., and Racaniello, V. R. (1989) Cell 56, 855-865[Medline] [Order article via Infotrieve]
  2. Koike, S., Horie, H., Ise, I., Okitsu, A., Yoshida, M., Iizuka, N., Takeuchi, K., Takegami, T., and Nomoto, A. (1990) EMBO J. 9, 3217-3224[Abstract]
  3. Zibert, A., Selinka, H. C., Elroy-Stein, O., Moss, B., and Wimmer, E. (1991) Virology 182, 250-259[Medline] [Order article via Infotrieve]
  4. Bibb, J. A., Bernhardt, G., and Wimmer, E. (1994) J. Gen. Virol. 75, 1875-1881[Abstract]
  5. Bibb, J. A., Bernhardt, G., and Wimmer, E. (1994) J. Virol. 68, 6111-6115[Abstract]
  6. Bernhardt, G., Bibb, J. A., Bradley, J., and Wimmer, E. (1994) Virology 199, 105-113[CrossRef][Medline] [Order article via Infotrieve]
  7. Wimmer, E., Harber, J. J., Bibb, J. A., Gromeier, M., Lu, H.-H., and Bernhardt, G. (1994) in Cellular Receptors for Animal Viruses (Wimmer, E., ed), pp. 101-127, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  8. Freistadt, M. (1994) in Cellular Receptors for Animal Viruses (Wimmer, E., ed), pp. 445-461, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  9. Koike, S., Aoki, J., and Nomoto, A. (1994) in Cellular Receptors for Animal Viruses (Wimmer, E., ed), pp. 463-480, Cold Spring Harbor Laboratory, Cold Sring Harbor, NY
  10. Freistadt, M. S., and Racaniello, V. R. (1991) J. Virol. 65, 3873-3876[Medline] [Order article via Infotrieve]
  11. Koike, S., Ise, I., and Nomoto, A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4104-4108[Abstract]
  12. Selinka, H. C., Zibert, A., and Wimmer, E. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3598-3602[Abstract]
  13. Zibert, A., Selinka, H. C., Elroy-Stein, O., and Wimmer, E. (1992) Virus Res. 25, 51-61[CrossRef][Medline] [Order article via Infotrieve]
  14. Morrison, M. E., He, Y.-J., Wien, M. W., Hogle, J. M., and Racaniello, V. R. (1994) J. Virol. 68, 2578-2588[Abstract]
  15. Aoki, J., Koike, S., Ise, I., Sato-Yoshida, Y., and Nomoto, A. (1994) J. Biol. Chem. 269, 8431-8438[Abstract/Free Full Text]
  16. Bibb, J. A., Witherell, G., Bernhardt, G., and Wimmer, E. (1994) Virology 201, 107-115[CrossRef][Medline] [Order article via Infotrieve]
  17. Bernhardt, G., Harber, J. J., Zibert, A., deCrombrugghe, M., and Wimmer, E. (1994) Virology 203, 344-356[CrossRef][Medline] [Order article via Infotrieve]
  18. Harber, J., Bernhardt, G., Lu, H. H., Sgro, J. Y., and Wimmer, E. (1995) Virology 214, 559-570[CrossRef][Medline] [Order article via Infotrieve]
  19. Ren, R. B., Costantini, F., Gorgacz, E. J., Lee, J. J., and Racaniello, V. R. (1990) Cell 63, 353-362[Medline] [Order article via Infotrieve]
  20. Koike, S., Taya, C., Kurata, T., Abe, S., Ise, I., Yonekawa, H., and Nomoto, A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 951-955[Abstract]
  21. Gromeier, M., Lu, H.-H., and Wimmer, E. (1995) Microbiol. Pathog. 18, 253-267
  22. Koike, S., Ise, I., Sato, Y., Yonekawa, H., Gotoh, O., and Nomoto, A. (1992) J. Virol. 66, 7059-7066[Abstract]
  23. Morrison, M. E., and Racaniello, V. R. (1992) J. Virol. 66, 2807-2813[Abstract]
  24. Lopez, M., Eberle, F., Mattei, M.-G., Gabert, J., Birg, F., Bardin, F., Maroc, C., and Dubreuil, P. (1995) Gene (Amst.) 155, 261-265[CrossRef][Medline] [Order article via Infotrieve]
  25. Eberle, F., Dubreuil, P., Mattei, M. G., Devilard, E., and Lopez, M. (1995) Gene (Amst.) 159, 267-272[CrossRef][Medline] [Order article via Infotrieve]
  26. Chadeneau, C., LeMoullac, B., and Denis, M. G. (1994) J. Biol. Chem. 269, 15601-15605[Abstract/Free Full Text]
  27. Chadeneau, C., LeMoullac, B., LeCabellec, M., Mattei, M., Meflah, K., and Denis, M. G. (1996) Mamm. Genome 7, 636-637[CrossRef][Medline] [Order article via Infotrieve]
  28. Aoki, J., Koike, S., Asou, H., Ise, I., Suwa, H., Tanaka, T., Miyasaka, M., and Nomoto, A. (1997) Exp. Cell Res. 235, 374-384[CrossRef][Medline] [Order article via Infotrieve]
  29. Freistadt, M. S., and Eberle, K. E. (1997) Mol. Immunol. 34, 1247-1257[CrossRef][Medline] [Order article via Infotrieve]
  30. Chadeneau, C., LeCabellec, M., LeMoullac, B., Meflah, K., and Denis, M. G. (1996) Int. J. Cancer 68, 817-821[CrossRef][Medline] [Order article via Infotrieve]
  31. Solecki, D., Schwarz, S., Wimmer, E., Lipp, M., and Bernhardt, G. (1997) J. Biol. Chem. 272, 5579-5586[Abstract/Free Full Text]
  32. Dynan, W. S. (1987) in Genetic Engineering (Setlow, J. K., ed), pp. 75-87, Plenum Press, New York
  33. Picard, V., Ersdal-Badju, E., Aiqin, L., and Bock, S. C. (1994) Nucleic Acids Res. 22, 2587-2591[Abstract]
  34. Schreiber, E., Matthias, P., Müller, M. M., and Schaffer, W. (1989) Nucleic Acids Res. 17, 6419[Medline] [Order article via Infotrieve]
  35. Nobis, P., Zibirre, R., Meyer, G., Kühne, J., Warnecke, G., and Koch, G. (1985) J. Gen. Virol. 66, 2563-2569[Abstract]
  36. Kraus, R. J., Murray, E. E., Wiley, S. R., Zink, N. M., Loritz, K., Gelembiuk, G. W., and Mertz, J. E. (1996) Nucleic Acids Res. 24, 1531-1539[Abstract/Free Full Text]
  37. Javahery, R., Khachi, A., Lo, K., Zenzie-Gregory, B., and Smale, S. T. (1994) Mol. Cell. Biol. 14, 116-127[Abstract]
  38. McPherson, L. A., Baichwal, V. R., and Weigel, R. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4342-4347[Abstract/Free Full Text]
  39. Lee, B. S., Krits, I., Crane-Zelkovic, M. K., and Gluck, S. (1997) J. Biol. Chem. 272, 174-181[Abstract/Free Full Text]
  40. Moser, M., Imhof, A., Pscherer, A., Bauer, R., Amselgruber, W., Sinowatz, F., Hofstadter, F., Schule, R., and Buettner, R. (1995) Development 121, 2779-2788[Abstract/Free Full Text]
  41. Chiu, R., Imagawa, M., Imbra, R. J., Bockoven, J. R., and Karin, M. (1987) Nature 329, 648-651[CrossRef][Medline] [Order article via Infotrieve]
  42. Kannan, P., Buettner, R., Chiao, P. J., Yim, S. O., Sarkiss, M., and Tainsky, M. A. (1994) Genes Dev. 8, 1258-1269[Abstract]
  43. Duan, C., and Clemmons, D. R. (1995) J. Biol. Chem. 270, 24844-24851[Abstract/Free Full Text]
  44. Mitchell, P. J., Wang, C., and Tjian, R. (1987) Cell 50, 847-861[Medline] [Order article via Infotrieve]
  45. Williams, T., Admon, A., Lüscher, B., and Tjian, R. (1988) Genes Dev. 2, 1557-1569[Abstract]
  46. Bosher, J. M., Totty, N. F., Hsuan, J. J., Williams, T., and Hurst, H. C. (1996) Oncogene 13, 1701-1707[Medline] [Order article via Infotrieve]
  47. Oulad-Abdelghani, M., Bouillet, P., Chazaud, C., Dolle, P., and Chambon, P. (1996) Exp. Cell Res. 225, 338-347[CrossRef][Medline] [Order article via Infotrieve]
  48. Williams, T., and Tjian, R. (1991) Genes Dev. 5, 670-682[Abstract]
  49. Williams, T., and Tjian, R. (1991) Science 251, 1067-1071[Medline] [Order article via Infotrieve]
  50. Gao, B., Spector, M. S., and Kunos, G. (1995) J. Biol. Chem. 270, 5614-5619[Abstract/Free Full Text]
  51. Mitchell, P. J., Timmons, P. M., Hebert, J. M., Rigby, P. W., and Tjian, R. (1991) Genes Dev. 5, 105-119[Abstract]


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