From the 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
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ABSTRACT |
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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-2 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: CD155 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,
AGM 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-2 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 [ 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).
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-2
expression vector indicates that
overexpression of AP-2
modulated the promoter activity of a
CD155 promoter construct.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
and CD155
,
which are integral membrane proteins; and CD155
and CD155
, 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).
1 and AGM
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).
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-2
. 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-2
in cotransfection experiments was
found to stimulate the activity of the CD155 promoter
approximately 3-fold.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-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-2
(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.
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 II/I plasmids mixed with up to 3 µg of
pSP(RSV)-hAP-2
. 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.
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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 [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-2 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-2 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.
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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-2 full-length clone, AP2A[B,G]5'
and AP2A[B,G]3' for AP-2
[
,
] 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-2
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).
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RESULTS |
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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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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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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 NFB 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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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 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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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 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-2
. Interestingly, rhAP-2
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-2
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-2
is able to bind
consensus binding motifs located within FPI and FPII, but not those
within FPIII.
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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-2
(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-2
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
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-2 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-2
and crude nuclear
extract, a difference is observed (compare Figs. 2 and 5). The 5' and
3' borders of the AP-2
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-2
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-2
oligodeoxynucleotide
(lane 7), and it was not supershifted by the anti AP-2
antibody (lane 9). These results confirm that AP-2
is
indeed the protein from crude HeLa nuclear extracts that binds FPI.
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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 NFB 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-2
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-2
, -
and -
. 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-2
-containing complexes (38) as well as
those complexes that contain AP-2
(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-2
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-2
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 (,
, or
) 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-2
or
, 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-2
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.
|
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-2
in a cotransfection experiment with the BE CD155 promoter
construct. In these experiments the BE construct was transfected with
increasing amounts of an AP-2
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-2
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.
|
The functional contribution of the FPII and FPI AP-2 binding sites
toward the AP-2-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-2
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.
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DISCUSSION |
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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
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
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-2
transcription factor was
originally cloned from HeLa cells (44, 45) and was subsequently shown to contain at least two other family members, AP-2
and AP-2
(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-2 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-2
or crude HeLa
S3 nuclear extracts. However, the area of rhAP-2
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-2
has been
reported by Gao et al. (50) with the rat
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
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 NF
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-2 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-2
-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 -galactosidase under the control of a 3-kilobase pair
CD155 promoter
fragment.3 It is interesting
to note that
-galactosidase expression can be observed in these
CD155 promoter
-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.
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ACKNOWLEDGEMENTS |
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We thank C. Meese for excellent technical
assistance and Helen Hurst for the kind gift of the AP-2 and AP-2
expression vectors.
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FOOTNOTES |
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* 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.
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.
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REFERENCES |
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