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INTRODUCTION |
The human polio virus receptor protein, which has recently been
given the designation CD155, is a highly glycosylated 80-kDa type Ia
single pass transmembrane cell surface protein that belongs to the
immunoglobulin superfamily (1-5). CD155 was originally cloned based on
its ability to serve as the cellular receptor for polio virus (1)
(reviewed in Refs. 6 and 7). CD155 belongs to a subgroup of genes
within the immunoglobulin superfamily; these genes share a V-C2-C2
domain structure as well as primary sequence identity. Thus far,
cDNAs have been cloned that encode two new human molecules related
to CD155, named PRR1 and PRR2 (polio
virus receptor related) (8, 9). Other CD155-related proteins possessing
the V-C2-C2 immunoglobulin domain structure appear to be relatively
conserved during evolution (10). Koike et al. (10) have
shown that African green monkeys possess polio virus receptors encoded
by two related but distinct genes AGM
1 and
AGM2 (African Green Monkey receptor). Two genes,
mPRR2, also known as MPH (mouse polio virus
receptor homologue), and Tage4 that are related to CD155,
also exist in the mouse (11-13).
Members of the new CD155 gene family have emerged as
cellular receptors for animal viruses. As pointed out earlier, CD155 is
the receptor for all three serotypes of the polio virus. There is no
available evidence for an alternative receptor except, perhaps, for
mouse-adapted polio viruses (14). hPRR1 has recently been identified as
a receptor for the -herpes viruses (15, 16), and hPRR2 and mPRR2
have been identified as receptors for the pseudo-rabies virus (17).
The biological importance of CD155 and its relatives has slowly begun
to be elucidated. CD155, as well as mPRR2, are expressed during
embryogenesis.1 Recently,
immunohistochemistry was used to determine that CD155 is expressed in
regions of the developing human central nervous system, such as the
notochord, floor plate, neural tube and the optic system.1
Surprisingly, in situ hybridization analysis of mPRR2
mRNA indicated that this gene was also expressed in these
structures of embryonic mice.2 Many cell adhesion
molecules belonging to the immunoglobulin superfamily important for the
development of the CNS3 are
expressed during embryogenesis in the floor plate and optic system
(19-21). Recently, it has been reported that both mPRR2 (22) and hPRR2
(23) possess activity as homotypic adhesion molecules. In addition,
cell adhesion activity has also has been demonstrated for hPRR1 (24).
This may suggest that some of the members of the CD155 gene
family may be adhesion molecules involved in the development of the
CNS.
We have cloned the promoter region of the CD155 gene (25).
Our initial analyses have mapped a 280-bp core promoter fragment that
is high in GC nucleotide content, lacks TATA and CAAT boxes, harbors a
region of multiple transcriptional start sites, and appears to contain
determinants required for cell type-specific promoter activity (25,
26). Indeed, we have used a transgenic mouse system to determine that
the 3.0-kilobase CD155 promoter is capable of directing
reporter gene expression to the appropriate anatomical structures where
CD155 is expressed during embryogenesis.1 We have been
interested in identifying the cis-acting elements and
trans-acting factors that could potentially play a role in the tissue-specific activity of the CD155 promoter.
Moreover, we have hoped that our studies would identify target elements or factors that could be involved in orchestrating the activity of the
CD155 promoter during embryonic development. We have
recently reported the presence of three cis-acting elements
within the CD155 core promoter region referred to as FPI,
FPII, and FPIII (25). Members of the activator protein-2 (AP-2)
transcription factor have been found to bind to FPI and FPII and are
able to activate reporter gene expression driven by the
CD155 core promoter. Here, we report the characterization of
a fourth cis-acting element (FPIV) within the
CD155 core promoter that is essential for basal promoter
activity. This element is bound by a nuclear protein that is present in
the nuclear extracts prepared from murine embryos of gestational stages
where the CD155 promoter is active in vivo. We
have determined that the transcription factor is nuclear respiratory factor-1 (NRF-1). NRF-1, a regulatory protein present in the nuclear extracts of murine embryos and many established cell lines, binds to
FPIV and functions as a potent transcriptional activator of the
CD155 core promoter. Interestingly, NRF-1 belongs to a
family of developmentally expressed transcription factors (27). In view
of these observations, we will discuss the potential significance of
the NRF-1/FPIV interaction relative to the in vivo activity of the CD155 promoter.
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MATERIALS AND METHODS |
Cell Culture
The HeLa (cervical carcinoma), HepG2 (hepatocellular carcinoma),
HEK293 (embryonic kidney), SK-N-MC (neuroblastoma), HTB15 (glioblastoma), and Ntera-2/clone D1 (teratocarcinoma) cell lines were
grown in Dulbecco's modified Eagle's medium, 10% fetal bovine serum.
Nuclear Extract Preparation and DNase I Footprint Assays
Mouse embryonic nuclear extracts were prepared using a
modification of the method of Tamura et al. (28). Briefly,
embryos of defined gestational age were harvested and homogenized in a solution containing 10 mM Hepes, 15 mM KCl, 1 mM EDTA, 2.2 M sucrose, and 5% glycerol. The
homogenate was layered over a solution containing 10 mM
Hepes, 15 mM KCl, 1 mM EDTA, 2.0 M
sucrose, and 10% glycerol and centrifuged at 24,000 rpm for 1 h.
The resulting pellet of nuclei was harvested and resuspended in a
solution containing 10 mM Hepes, 550 mM KCl,
0.1 mM EDTA, 3 mM MgCl2, and 10%
glycerol to lyse the nuclei and strip the chromatin of DNA-binding
proteins. After the chromatin was pelleted by centrifugation at 40,000 rpm for 1 h, the supernatant containing the nuclear proteins was
harvested and concentrated by ammonium sulfate precipitation. Nuclear
extracts from human cell lines for use in EMSA experiments were
prepared according to the procedure by Schreiber et al.
(29). DNase I footprinting was performed as described previously (25).
Briefly, end-labeled DNA probes were generated via the polymerase chain reaction (PCR), using an oligonucleotide primer carrying a 5'-terminal [
-32P]ATP label. PCR was performed under standard
conditions using 10 ng of pGL2-H template, 25 pmol of labeled and
unlabeled primers, and 1.2 units 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
100,000 cpm of labeled probe which was incubated in a 50 µl binding
reaction containing 2 µg of poly[d(I-C)] and nuclear protein. After
a 30-min incubation on ice, 50 µl 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. One µl of DNase I (100 ng/ml) was then added and
incubated another minute at room temperature. The reactions were then
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 DNA was
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, the CD155 core promoter spanning nucleotides 
343
to 58 upstream of the CD155 translation initiating ATG codon (Fig.
1A; (26)), was carried out using the megaprimer mutagenesis
technique of Picard et al. (30). To generate a megaprimer
for each mutant construct, 100 ng of pGL2-BE plasmid was amplified in a
reaction containing 50 pmol of the 4532 flanking primer, 50 pmol of
mutagenic primer, 5 µl of 10x buffer, 2 µl of 10 mM
dNTP mix, and 0.5 µl of Taq-polymerase (2.5 unit,
Stratagene) in a total reaction volume of 50 µl. 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 BE fragment,
100 ng of pGL2-BE plasmid was amplified in a reaction containing 50 pmol of either 4529 flanking primer, 1-2 µg of megaprimer, 5 µl of
10x buffer, 2 µl of 10 mM dNTP mix, and 0.5 µl of
Pfu-polymerase (2.5 unit, Stratagene). The primers used for
megaprimer PCR reactions were: 4532, 5'-GGCGCTAGCGCCGCCTCTTCTAGTG-3'; 4529, 5'-GCCAGATCTGCTCGCTCTGCCGCGG-3'; IV (1),
5'-CGCCTGCGCAGGACTAGTCCGGCGCTCAGT-3'; IV (2),
5'-GCGCTGCGCCTGACTAGTTCTCTCCCGGCG-3'; IV (3),
5'-CCCCGCGCGCTGACTAGTCGCAGGTCTCTC-3'; and 
TR,
5'-GGCCCTCCCCGCGCGAGGTCTCTCCCGGCG-3'.
Transfection and Harvest of Cells for Dual Luciferase Assays
All cell lines were transfected by the calcium phosphate
procedure. Each transfection mixture for the linker scan series of constructs was composed of 18 µg of wild type or mutant BE reporter constructs and 1 µg of pRL-TK (standard to the measure of efficiency of transfection). The compositions of the co-transfection experiments was 9.0 µg of the BE or BE TR, mixed with up to 1.5 µg of
pcDNA3(NRF-1). Co-transfections were supplemented with empty
pcDNA3 to keep the amount of backbone plasmid constant for each
experiment. 50 µl of 2.5 M CaCl2 was added to
the DNA mixtures that were subsequently diluted to a total volume
of 500 µl with Tris/EDTA buffer. These solutions were then separately
combined dropwise with 500 µl of ice-cold 2x HBSS and incubated ten
minutes at room temperature. Half of the precipitates were then added
to a separate 6-cm plate of tissue culture cells (~105
cells), and the plates were incubated at 37 °C. 4 h later the medium was removed, and a solution of 20% glycerol in HBSS added. Following 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.
Electrophoretic Mobility Shift Assays
The oligodeoxynucleotides used for EMSA were: FPIVs,
5'-GAGAGACCTGCGCAGGCGCAGCG-3'; FPIVas,
5'-GCGCGCTGCGCCTGCGCAGGTCT-3'; IV (3)s, 5'-GAGAGACCTGCGACTAGTAGCG-3';
and IV (3)as, 5'-GCGCGCTGACTAGTCGCAGGTCT-3'.
Oligo Association--
One nmol of each the coding and noncoding
oligodeoxynucleotide were reassociated in a volume of 50 µl using a
thermocycler. Settings were 5 min at 95 °C and 1 h each at
65 °C, 60 °C, 55 °C, 50 °C, 45 °C, and 40 °C. The
oligodeoxynucleotides were designed to possess a G as the 5'-protruding nucleotide.
Labeling--
Ten pmol of reassociated oligodeoxynucleotide was
end-labeled by a fill in reaction using CombiPol Polymerase (InViTek).
In a volume of 20 µl, the buffer, 0.5 µl of enzyme, 50 µCi of
[-32P]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 G50
chromatography (Nick columns, Amersham Pharmacia Biotech). Usually more
than 50% of label were found to be incorporated into the oligodeoxynucleotide.
Shift Assay--
A binding reaction containing 1 µl of 10×
incubation buffer (50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 5 mM EDTA, 5 mM DTT, 25% glycerol), 1.5 µg of poly(dI-dC)(dI-dC), various concentrations of
competitor or antibody (goat anti-NRF-1 antiserum was the kind gift of
Richard Scarpulla), and 4 µl of cell extract was prepared and
preincubated for 10 min at room temperature. After preincubation 1 µl
of labeled oligodeoxynucleotide corresponding to 100 fmol were added,
and the incubation continued for another 20 min. Samples were loaded
onto a 6% 1/2× TBE polyacrylamide gel. After the electrophoresis (200 V) the gel was fixed in 10% acetic acid/30% methanol for 30 min and dried.
One-Hybrid Expression Cloning of a FPIV-binding Protein
The Matchmaker one-hybrid system kit
(CLONTECH) was used to screen an E9/E10.5 mouse
embryo library for the cDNAs of FPIV-binding proteins.
Generation of the 4xFPIV-Reporter Strain--
Oligonucleotides
consisting of four tandem repeats of the FPIV sequence were associated
and then were cloned into pcDNA3. The identity of the cloned insert
was verified by sequence analysis. The four tandem repeats were then
subcloned into the yeast integration vectors pHISi-1 and pLacZi. These
resulting vectors, p4xFPIV-HISi-1 and p4xFPIV-LacZi, were then
linearized, transformed sequentially into the YM4271 yeast strain, and
grown on the proper medium to select for colonies harboring the
plasmids that had recombined in their proper genetic loci. The
resulting doubly integrated yeast strain, YM4271(His, LacZ 4xFPIV), was
found to possess very low background expression of the integrated HIS3
and LacZ reporter genes and was suitable for use in a library screen
(data not shown). Library Screen
Competent YM4271(His, LacZ
4xFPIV) cells were transformed with 20 µg of an E9/E10.5 mouse embryo
library using the procedure outlined by the Matchmaker one-hybrid
system protocol (CLONTECH). The cells that were
transformed with the library were then plated onto SD/-His/-Leu/+15
mM 3-aminotriazole selection plates and incubated at 30°C
for 3 days. Colonies that grew under these selection conditions were
then streaked onto fresh SD/-His/-Leu/+15 mM
3-aminotriazole selection plates and were then subjected to colony to
assay the expression of the LacZ reporter gene. Colonies that were His
and LacZ positive were candidates for expressing library clones that encoded proteins that could bind to the 4xFPIV-target element. Therefore, the library plasmids from the His+ and LacZ+ colonies obtained from the library screen were isolated from the yeast, transformed into Escherichia coli DH5 cells,
and subjected to sequence analysis.
RNA Isolation, RT-PCR, Cloning of NRF-1 Expression Vectors, and
in Vitro Transcription/Translation
Total cellular RNA for use in RT-PCR was isolated from 3 × 107 SK-N-MC or HeLa cells according to the TRIzol protocol (Life Technologies, Inc.). Reverse transcription 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 (NRF RT) 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 DTT, 1 µl of 10 mM
dNTP mixture, 1 µl of RNAsin (25 unit), 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. Following 10 min at 42 °C, 20 µl of 1 M TrisHCl pH 7.5 were added and the RT stocks frozen at
20 °C. The oligodeoxynucleotides used were: NRF RT, 5'-CATTTGATTGCACCTCTGCAAACG-3'; NRF 5'-NTR,
5'-GGATATTTGTTTAATGAATGTGGTATGC-3'; NRF ATG,
5'-TAGAAGCTTATGGAGGAACACGGAGTGACCC-3'; NRF TGA,
5'-GGCTCTAGATCACTGTTCCAATGTCACCACC-3'; NRF,
5'-CTGTCTAGATCACTGTGATGGTACAAGATGAGCTATACTATG-3'.
The full-length NRF-1 cDNA was amplified by nested PCR. For the
first PCR reaction, 1 µl of RT stock was taken as template and mixed
with 2 µl of 10 mM dNTP, 0.5 µl of primers each (50 pmol of NRF 5'-NTR and NRF TGA), 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
Following a denaturation step at 94 °C, 2 min, 35 cycles were
done at 94 °C, 45 s; 55 °C, 45 s; and 72 °C, 90 s. For the second PCR reaction, 1 µl of the first PCR reaction was
amplified with 50 pmol each of the NRF ATG and NRF TGA primers with the
above amplification conditions. Products were separated on a 1.0%
agarose gel. The full-length product was observed when both HeLa and
SK-N-MC total RNA were used as template for RT. The SK-N-MC full-length product was cut out and gel-purified. Following digestion with HindIII and XbaI restriction enzymes, the
fragment was cloned into the pcDNA3 expression vector. The
full-length insert was sequenced and found to be identical to the
published open reading frame. A putative dominant-negative form of
NRF-1 was cloned by amplifying the full-length cDNA with the NRF
ATG and NRF primers. The resulting product encoding the first 304 amino acids of NRF-1 was cloned into the HindIII and
XbaI sites of pcDNA3. In vitro translation of
the NRF-1 cDNA was carried out using the TNT-coupled rabbit
reticulocyte system as per the manufacturer's instructions (Promega).
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RESULTS |
Mapping of the FPIV cis-Acting Element--
Our previous studies
identified three functional cis-acting elements (FPI-III)
within the CD155 core promoter (for schematic locations see
Fig. 1A) (25). All three of
these cis-acting elements are located within a 280-bp
genomic DNA fragment, named BE (see the borders of the BE fragment,
Fig. 1A), that harbors full ability to direct the expression
of a reporter gene when transfected into tissue culture cell lines that
naturally express CD155 (25, 26). Serial deletion analysis of the
CD155 promoter was used in our original experiments to map
the functional boundaries of the BE fragment (26). The exact 5'-borders
of two of the serial deletion constructs, named B and C, are shown in
Fig. 1, A and B. The promoter activity of the C
construct has been found to be greatly reduced when compared with that
of B (26), an observation suggesting the existence of a fourth
cis-acting element within this area of the CD155
promoter. To investigate this possibility further, we utilized two
BssHII restriction sites that flank the B-C region to
generate a fine scale deletion within this area (for location of the
BssHII restriction sites see Fig. 1B). The BssHII construct was transiently transfected into the
HeLa and HEp-2 cell lines, and its ability to direct the expression of the luciferase reporter gene was determined. Strikingly, an 80% reduction of promoter activity was observed when the 50 bp in the
vicinity the C construct border were removed (Fig. 1C) or replaced in an inverted orientation (data not shown). These results suggest that a strong cis-element resides within this
area.
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Fig. 1.
Overview of CD155 promoter
region and mapping of the FPIV cis-acting
element. A, schematic of the 5'-flanking region of the
CD155 gene. Numbering, A of ATG+1.
Parentheses indicate the region of transcriptional
initiation. Open box, transcribed region; gray
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
oval with roman numerals, locations of the footprints
seen in this and previous studies. The horizontal lines
illustrate the 5'- and 3'-borders of deletion constructs used in this
study and our previous studies. B, sequences of the
5'-region of the CD155 core promoter. The bold
sequence represents the borders of FPIV (see Fig. 2).
Brackets under the sequence represent where the wild type
promoter sequence was replaced by a SpeI restriction site in
linker scanning mutation studies. The bent arrows represent
the 5'-borders of the B and C deletion constructs. The
underlined sequences represent the positions of the
BssHII restriction sites. C, luciferase activity
of the BssHII deletion construct. The BssHII
restriction fragment was removed for the pGL2-BE promoter construct to
generate the BssHII deletion construct. This construct
was transfected into the HeLa and HEp-2 cell lines. Reporter gene
expression of the wild type BE fragment was normalized to 100%, and
the activity of the BssHII construct was expressed
relative to that level. The average Reniella luciferase corrected RLU
values of these transfections were 103,870 for HEp-2 and 73,153 for
HeLa.
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To map the location of any potential nuclear protein binding sites we
subjected the 5'-portion of the CD155 core promoter to DNase
I footprint analysis. The choice of the nuclear extract was guided by
the following observation. Analysis of the CD155 promoter/LacZ transgene indicated that murine embryos of the
E10.5-E14.5 post conception stages express the LacZ reporter gene in a
cell type-specific manner within the developing CNS.1
Therefore, nuclear extracts of embryos from the E10.5-E14.5
gestational stages could conceivably contain the full complement of
trans-acting factors required to produce this expression
pattern. Because our main aim was to identify transcription factors
that could potentially contribute toward the in vivo
activity of the CD155 promoter, we used nuclear extracts
prepared from E10.5 murine embryos for DNase I footprint experiments.
When footprint analysis was performed with 75 µg of extract, a single
protected region, called FPIV, was observed that was located from 282
to 264 base pairs upstream of the initiator ATG of the
CD155 gene (see Fig. 2, for
relative locations see Fig. 1, A and B).
Interestingly, FPIV is located within the boundaries of the
BssHII restriction fragment that we had discovered to be
essential for core promoter activity. When footprinting experiments
were carried out using nuclear extracts prepared from human cell lines,
a partial footprint was also observed overlapping with that produced
from E10.5 extract (data not shown). These results suggest that a
nuclear protein binding site exists within the 5' of the
CD155 core promoter.
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Fig. 2.
DNase I footprint analysis of the 5'-portion
of the BE core promoter fragment. The 5'-portion of BE core
promoter fragment (see Fig. 1A) labeled on the noncoding
strand was incubated in the absence (0) or presence of 75 µg of E10.5
embryonic nuclear extract. After allowing the nuclear protein to bind
to the probe, the labeled promoter fragments in the reactions were
digested with DNase I. The resulting digested DNAs were isolated and
electrophoresed on 10% sequencing gels. Brackets indicate
the FPIV region of protection from nucleotide 282 to 264 of the
core promoter.
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Several data base searches for transcription factor binding motifs
using the FPIV region yielded no significant homology to known
transcription factor binding sites. However, a 12-base pair tandem
repeat sequence, GCGCAGGCGCAG, showed significant homology to identical
motifs found in a variety of other human promoters (data not shown).
Linker Scanning Mutagenesis of the FPIV Region--
The DNase I
footprint experiment (Fig. 2) identified a segment of the
CD155 upstream sequence that is likely to harbor a
cis-element required for promoter function. To more
precisely map this element, a series of linker scan mutations was
generated throughout this protected sequence. Each mutant promoter
construct contained 6 base pairs 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,
HEp-2, SK-N-MC, and HTB15 cell lines (Fig.
3). Of the three linker replacement
mutations, the IV (3) mutant construct displayed a greater than 80%
reduction in promoter activity in all the cell lines tested. This
result indicated that this mutation disrupts a sequence required for the basal activity of the CD155 core promoter.
Interestingly, the IV (3) mutation is located in the center of the
tandem repeat motif mentioned above (GCGCAGGCGCAG to GCGactagtCAG, see Fig. 1B) that we found by homology searching to be contained
in other human promoters. We also generated a construct that lacked this 12-base pair motif. When this construct, called TR, was transfected into the cell lines, the reporter gene activity was as
severely reduced as in the IV (3) mutant promoter (see Fig. 3). We
conclude that a functional cis-acting element resides within boundaries of the FPIV region. Linker scanning and fine scale deletion
mutations suggest that a 12-base pair tandem repeat motif is likely to
represent this important regulatory element.
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Fig. 3.
Linker scanning mutagenesis of the FPIV
region. Results of transient transfection of the FPIV linker scan
series into the HeLa, SK-N-MC, HTB15, and HEp-2 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 6-well plates
were transfected by the calcium phosphate method with 9 µg of
promoter construct. Co-transfection of 500 ng of Reniella luciferase
vector, pRL-SV40 (Promega), was used to monitor transfectional
efficiency. Eighteen hours 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 and HEp-2 transfections, and two transfections for HTB 15. The
average Reniella luciferase corrected RLU values of the BE core
promoter fragment in these sets of transfections was 50,858 for HeLa,
78,797 for SK-N-MC, and 162,831 for HTB15. TR, construct
lacking the 12-base pair motif.
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EMSA Analysis of FPIV Binding Activity--
To further study
protein binding to FPIV, we performed EMSA experiments. For these
studies, nuclear extracts of human cell lines were used to analyze the
characteristics of the FPIV binding activities. All the nuclear
extracts tested possessed several binding activities for the FPIV probe
(see Fig. 4A, lanes
2-10). Competition analysis using a 250-fold molar excess of
unlabeled FPIV probes (containing either a wild type or mutated tandem
repeat sequence) was used to dissect which of the observed complexes were specifically binding to the tandem repeat motif. One set of the
complexes showed a competition profile expected for a nuclear protein
binding to the FPIV probe (see Fig. 4A, complex marked with
arrows). The addition of a 250-fold molar excess of the wild type cold competitor reduced the intensity of this complex, whereas the
addition of an equal amount of a FPIV probe that harbored the IV (3)
linker mutation did not. Interestingly, this specific complex was
present in all of the cell line nuclear extracts tested. When
electrophoresed for a longer time, this complex resolved into a doublet
of two closely migrating bands (see Fig.
5A).
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Fig. 4.
Electrophoretic mobility shift analysis of
FPIV binding activities from the nuclear extracts of human cell lines
and murine embryos. 10 µg of the indicated nuclear extracts were
incubated with radiolabeled FPIV probe in a buffer containing 10 mM Tris-HCl, pH 7.5, 75 mM KCl, 1 mM EDTA, 1 mM DTT, and 5% glycerol. Binding
reactions were incubated 30 min on ice and then were electrophoresed on
a 8% TBE polyacrylamide gel. The addition of 250× FPIV (wt) or IV(3)
competitor oligonucleotides are indicated at the top of the
panel. The FPIV(3) competitor is a probe that contains the third FPIV
linker scan mutation incorporated within its sequence (see Fig.
1B). Two arrows indicate the specific complex. An
asterisk indicates nonspecific complexes.
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Fig. 5.
Biochemical confirmation of the yeast
one-hybrid system screen. A, comparison of in
vitro translated NRF-1 with native FPIV binding activity. An
aliquot of in vitro translated NRF-1 and HeLa cell nuclear
extract was incubated with radiolabeled FPIV probe in a buffer
containing 10 mM Tris-HCl, pH 7.5, 75 mM KCl, 1 mM EDTA, 1 mM DTT, and 5% glycerol. Binding
reactions were incubated 30 min on ice and then were electrophoresed on
a 8% TBE polyacrylamide gel. The addition of 250× FPIV (wt) or IV(3)
competitor oligonucleotides are indicated at the top of the
panel. Two arrows indicate the specific complexes produced
by both the in vitro translated NRF-1 and the HeLa nuclear
extract. B, supershift analysis of FPIV binding activities
from the nuclear extracts of human cell lines and murine embryos. 10 µg of the indicated nuclear extracts were incubated with radiolabeled
FPIV probe in a buffer containing 10 mM Tris-HCl, pH 7.5, 75 mM KCl, 1 mM EDTA, 1 mM DTT, and
5% glycerol. Binding reactions were incubated 30 min on ice and then
were electrophoresed on a 8% TBE polyacrylamide gel. One µl of
anti-NRF-1 goat polyclonal antiserum was added the binding reaction of
lanes 3, 5, 7, 9,
11, and 13. Double arrows indicate the
location of either NRF-1 or that of NRF-1 that has been supershifted by
the addition of antiserum.
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The combination of EMSA experiments and our genetic analyses suggested
that we had identified an as yet unknown transcription factor or
factors required for the activity of the CD155 core promoter
in tissue culture cell lines. Nuclear extracts prepared from embryos of
the E10.5, E12.5, and E14.5 gestational stages possessed FPIV binding
activities that appeared to have a similar mobility to those observed
with those from human cell lines (see Fig. 4A, lanes
11-19). Importantly, these complexes also shared identical
competition characteristics when exposed to a 250-fold molar excess of
the wild type or mutant FPIV probes. These data suggest that murine
embryo nuclear extracts possess FPIV binding activity and that this
binding activity is identical in migration and competition
characteristics to those observed in human cell lines. This result
represents an important link between our genetic and biochemical
analyses of the core promoter in tissue culture, on one hand, and
CD155 promoter activity in vivo, on the other. The mutations within the FPIV cis-acting element greatly
reduce reporter gene expression indicating that the transcription
factor or factors interacting with this region are required for the
basal activity of the CD155 promoter. Because this binding
activity is present in embryonic nuclear extracts, we conclude that
this factor may play an important role in the regulation of
CD155 promoter activity during embryogenesis.
A One-hybrid Screen Identifies Nuclear Respiratory Factor-1 Binding
to FPIV--
We next attempted to determine the identity of the
protein(s) that could bind to FPIV. To accomplish this task, we used
the yeast one-hybrid system, a method suitable to isolate the cDNAs of a DNA-binding protein whose binding site has been characterized by
biochemical methods. Briefly, a yeast strain was generated where the
expression of the HIS3 and LacZ reporter genes
was placed under of control promoters in which the essential upstream
activator site was replaced by four tandem repeats of the FPIV sequence (for details see "Materials and Methods"). In the absence of
protein binding to the synthetic upstream activator site, the promoter controlling the expression of the reporter was inactive (data not
shown). The reporter strain was then transformed with a library consisting of cDNAs from an E9/E10.5 mouse embryo fused to the HSV
VP16 activation domain. Any cDNA that possessed an open reading frame encoding a protein with the capability to bind FPIV would lead to
the activation of the HIS3 and LacZ genes. The
one-hybrid screen yielded seven His3+ and LacZ+ colonies (data not
shown) that were candidates to harbor cDNAs encoding DNA-binding
protein(s) with FPIV binding activity. The library plasmids of these
seven colonies were isolated, transformed into E. coli
DH5 cells, and produced in bulk for sequencing of the cDNA
inserts. The inserts of those four colonies that were most robustly
positive for LacZ expression during the library screen all contained
overlapping regions of an identical sequence (data not shown). This
sequence was then entered into BLAST (31) searches to determine if it harbored homology with any known open reading frames. The results of
BLAST homology searches indicated that the sequences of the cDNA
inserts were identical to the DNA binding domain of murine NRF-1. This
strongly suggested that NRF-1 might indeed represent a FPIV-binding
protein. The optimal binding site of NRF-1 has been determined to be
GCGCATGCGCAG (32), which differs by only one base pair from the tandem
repeat sequence of FPIV. As we had mentioned earlier, the tandem repeat
sequence of FPIV was identical to motifs in other human promoters.
Indeed, NRF-1 has been shown to bind to motifs in the promoters of the
human ubiquinone-binding protein and GPAT-AIRC, two sequences that are
identical to our FPIV tandem repeat (33, 34).
Confirmation of NRF-1 Binding to FPIV--
To biochemically
corroborate the result of our yeast one-hybrid experiment, the
full-length human NRF-1 cDNA was cloned by RT-PCR (see "Material
and Methods"). The NRF-1 cDNA was subcloned into a mammalian
expression vector that also contained a phage T7 promoter for in
vitro transcription by T7 RNA polymerase. This cDNA was then
used to program an in vitro transcription/translation system
to produce NRF-1 protein for EMSA experiments. Indeed, the NRF-1
cDNA produced binding complexes with a FPIV probe that migrated in
a manner identical to the native FPIV binding activity in HeLa nuclear
extracts (see Fig. 5A, compare lanes 2 and
5). Interestingly, the in vitro translated NRF-1
protein produced both bands of the doublet that we had previously
observed, a result suggesting both complexes contain NRF-1. The reason
for the formation of the doublet bond is as yet unknown but appears to
relate to post translational protein modification. In vitro
translated NRF-1 protein also displayed competition characteristics
identical to the native FPIV binding activity of HeLa nuclear extract
when exposed to a 250-fold molar excess of cold wild type and mutant FPIV probes (see Fig. 5A, compare lanes 3 and
4 to lanes 6 and 7). Experiments with
radiolabeled mutant FPIV competitors as probes for EMSA corroborated
these results. Neither the in vitro translated NRF-1 nor the
native binding activity present in nuclear extracts could bind to
probes that possessed alterations in the GCGCAGGCGCAG motif (data not shown).
Further evidence supporting the hypothesis that NRF-1 binds to FPIV was
obtained by supershift analysis. Anti-NRF-1 polyclonal serum was added
to EMSA binding reactions, and the resulting complexes were then
resolved by native polyacrylamide gel electrophoresis (see Fig.
5B). Both bands of the doublet were supershifted by the
addition of the antiserum, an observation indicating that both
complexes were antigenically related to NRF-1 (Fig. 5B, see lanes 3, 5, 7, 9,
11, and 13). Taken together the results of our competition and supershift experiments provide direct biochemical evidence confirming the findings from our one-hybrid system screen.
The Importance of the NRF-1 Interaction for CD155 Promoter
Function--
Our results suggest that NRF-1 binds to the
CD155 promoter and that binding is detectable in cell lines
and murine embryos where the CD155 promoter is active. We
next wanted to determine the functional significance of this
interaction on core promoter activity. Our first attempt to address
this question was to overexpress the NRF-1 transcription factor in the
presence of the core promoter in co-transfection experiments. In these
experiments the BE construct was transfected with 750 ng of an NRF-1
expression vector into the HepG2, HTB15, SK-N-MC and Ntera-2 cell
lines. Remarkably, overexpression of NRF-1 stimulated CD155
core promoter activity 3-5-fold in all four of the cell lines tested
(see Fig. 6B). The TR
mutant construct was also co-transfected with 750 ng of NRF-1 expression vector and was activated to a much lower extent by the
overexpression than that we had observed for the wild type BE construct
(see Fig. 6B). These results confirm the interaction of
NRF-1 with the CD155 core promoter. Moreover, they suggest that NRF-1 is a potent activator of CD155 promoter activity
and that full activation of the core promoter requires an intact NRF-1 binding site.
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Fig. 6.
The overexpression of full-length and
dominant-negative NRF-1 modulates reporter gene expression driven by
the CD155 core promoter. A, domain
structure of NRF-1 proteins used in overexpression studies. The
full-length NRF-1 protein contains 503 amino acids (aa). The
N terminus of NRF-1 harbors DNA binding, dimerization, and nuclear
localization signal domains, whereas the C terminus harbors the
bipartite hydrophobic activation domain. The putative dominant-negative
dominant-negative NRF-1 used in these studies is comprised of only the
first 304 amino acids of the NRF-1 protein. B, affect of
co-transfection of a full-length NRF-1 expression vector on the
promoter activity of the wild type and a FPIV mutant BE promoter
construct in the HepG2, SK-N-MC, HTB 15, and Ntera-2 cell lines. Each
cell line was seeded in 6-well tissue culture plates and were
transfected by the calcium phosphate method with 4.5 µg of the BE or
TR promoter construct with 750 ng of pcDNA3(NRF-1).
Transfections were filled in with pcDNA3 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 the promoter
construct co-transfected with only pcDNA3 was set to 100% (control
promoter activity) and the level of activation caused by co-transfected
of pcDNA3(NRF-1) is expressed relative to that 100%. The absolute
level of reporter gene activities directed by the BE and TR
promoters was as described in Fig. 3. Results are the mean + S.D. of
triplicate transfections for HepG2, SK-N-MC, HTB 15, and one
transfection for Ntera-2. The average Reniella luciferase corrected RLU
values of these transfections were 38,433 (BE) and 8033 (TR) for
HepG2, 10,179 (BE) and 1931 (TR) for SK-N-MC, 120,000 (BE) and
23,500 (TR) for HTB15, and 35,744 (BE) and 5905 (TR) for Ntera-2.
C, co-transfection of the dominant-negative NRF-1 into
HepG2, SK-N-MC, and HTB 15 cell lines. Each cell line was seeded in a
6-well tissue culture plate and was transfected by the calcium
phosphate method with 4.5 µg of the BE and up to 2.0 µg of
pcDNA3(NRF-1). 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 the BE construct
co-transfected with only pcDNA3 was set to 100% (control promoter
activity), whereas the activity of each construct co-transfected with
the indicated amount of pcDNA3(NRF-1) is expressed relative to
that 100%. Results are the mean + S.D. of triplicate transfections.
The average Reniella luciferase corrected RLU values of the BE core
promoter fragment in these sets of transfections was 137,128 for HEp-2,
95,501 for SK-N-MC, and 581,303 for HTB15.
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We also investigated the requirement for NRF-1 on core promoter
activity using a dominant-negative approach. The first 304 amino acids
of NRF-1 have been proposed to comprise the DNA binding, dimerization,
and nuclear localization signal domains, but they lack the bipartite
hydrophobic activation domains of this protein (see Fig.
6A). Virbasius et al. (27) and
Gómez-Cuadrado et al. (35) have shown that this
N-terminal portion of NRF-1 possesses functional DNA binding
properties. Therefore the NRF-1 protein that lacks the C terminus could
display a dominant-negative activity if it were to competitively
inhibit the wild type protein from binding to a NRF-1 binding site.
Indeed, Gugneja et al. (36) have shown that overexpression
of this portion of NRF-1 could inhibit luciferase expression driven by
four tandem repeats of an NRF-1 binding site. We transfected the BE
construct with an increasing amount of a vector expressing this
putative dominant-negative NRF-1 into the HEp2, HTB15, and SK-N-MC cell
lines. Overexpression of the truncated NRF-1 decreased CD155
core promoter activity in a dose-dependent manner in the
three cell lines tested to a level 50-60% of wild type activity (see
Fig. 6C). These results suggest that the truncated NRF-1
protein used in this set of experiments exerts a dominant-negative
activity, a finding that may be of use in the elucidation of the
biological functions of the NRF-1 transcription factor. In addition,
they support our hypothesis that the binding of NRF-1 is needed for
optimal activity of the CD155 core promoter. These data are
in agreement with the activities of our FPIV linker scan mutation
constructs, which lack NRF-1 binding to FPIV.
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CONCLUSIONS |
We have uncovered a new genetic element of the CD155
promoter that, together with the previously identified AP-2-responsive elements (25), contributes to the control of the expression of CD155
polypeptides. These CD155 proteins of which the splice variants
CD155 and CD155
serve as receptors for polio virus in humans (1,
2, 7) belong to a new group of Ig-like polypeptides with the general
extracellular structure V-C2-C2. These proteins are found in humans,
monkeys and rodents.1 A protein named hPRR1 (8), which is
related to CD155, has recently been shown to function as a receptor for
-herpes viruses (15, 16). The viral receptor functions of CD155 and
of the other CD155-related polypeptides is the best understood aspect of the biology of these molecules, whereas little is known about their
nonpathogenic functions. It has recently come to light that both the
PRR1 and PRR2 relatives of CD155 possess cell adhesion activity.
However, the function(s) of CD155 is unknown. Our studies that have
focused on the mechanism of regulation of expression of the
CD155 gene are also aimed at gaining insight into the
nonpathologic function of the CD155 polypeptide.
CD155 is expressed in the developing human CNS; the anatomical location
of expression includes the notochord, floor plate, neural tube, and
optic system.1 Furthermore, making use of transgenic mice
expressing a reporter gene, we have recently shown that the
CD155 promoter is active during embryogenesis.1
In this system, the CD155 promoter directed the expression
of 
-galactosidase to regions of the developing CNS at midgestation that matched the authentic expression of the CD155 protein.
Interestingly, the expression was observed only between E10 and E12.5
postconception, and it subsequently disappeared to an undetectable
level.1 This expression profile of CD155 is strikingly
similar to the expression of other molecules of the immunoglobulin
superfamily that are known to be cell adhesion molecules with functions
in the developing CNS (19-21). Therefore, given the fact that other members of the CD155-related gene family are cell adhesion molecules, it is conceivable that CD155 may possess similar functions. This possibility is now being investigated in our laboratory.
Of special interest to us were cis-acting elements and
trans-acting factors that could participate in the
tissue-specific activity of the CD155 promoter. The core
promoter region of the CD155 gene harbors basal and cell
type-specific activities (26). In the past, we have characterized three
cis-acting elements (FPI, -II, and -III) that are located
within the core promoter that are likely to be involved in the
regulation of expression of the CD155 gene (25). Two of
these elements (FPI and FPII) are bound by the developmentally
expressed protein AP-2, a transcription factor that has been described
to participate in the regulation of gene expression in the developing
lens, retinal ganglion cell layer, and neural tube (37-39). The
developmentally regulated expression of AP-2 overlaps temporally and
spatially with that of CD155, an observation we interpret to suggest
that the AP-2 transcription factors may influence the CD155 expression
profile during embryogenesis.
Further studies have now revealed that reporter gene expression was
lost upon deletion of sequences in the 5'-terminal part of the core
promoter (constructs B, C, and BssHII, see Fig. 1, A and B). This suggested the presence of a fourth
cis-acting element within the first 50 bp of the core
promoter that was essential for basal activity. A DNase I footprint,
called FPIV, was detected within this region 282 to 264 nucleotides
upstream of the ATG translation initiation codon of the
CD155 gene. It was of particular interest that FPIV could
also be readily detected when footprinting experiments were carried
with nuclear extracts prepared from E10.5 murine embryos. This
observation suggested that a trans-acting factor existed in
the extract prepared during this phase of embryogenesis and that this
factor could interact with FPIV of the core promoter. Mutagenesis of
FPIV identified a 12-base pair tandem repeat, GCGCAGGCGCAG, required
for basal promoter activity.
The essential role of the tandem repeat was then confirmed by EMSA
analyses, using authentic probes or mutated derivatives thereof and a
variety of extracts. All nuclear extracts that we tested, including
those that were prepared from murine embryos, possessed identically
shifted complexes that bound to the tandem repeat motif of FPIV (see
Fig. 4A). These results suggested that the FPIV binding
activity was present at times when the CD155 promoter is
active during embryogenesis.
A yeast one-hybrid system screen of a mouse embryo cDNA library
then revealed a protein that was identical in nucleotide sequence to the murine NRF-1 transcription factor. Indeed, EMSA experiments using in vitro translated human NRF-1 or anti-NRF-1
polyclonal antiserum confirmed that the authentic NRF-1 protein was
indistinguishable from the native FPIV binding activities that we had
observed in gel shift experiments.
Evans and Scarpulla (33) originally cloned NRF-1 as a factor that could
bind to the rat somatic cytochrome c promoter. These authors
have suggested that NRF-1 plays an integral role in the transcription
of mitochondrial genes that are encoded in the nucleus. Subsequently,
NRF-1 has been shown to regulate many other genes, such as the genes
for eIF2, tyrosine aminotransferase, chicken histone h5, and CXCR4.
This suggests a wider range of genes can be regulated by this
transcription factor than originally envisioned (32, 35, 40-42).
Indeed, Virbasius et al. (27) and Gómez-Cuadrado et al. (35) have both proposed that NRF-1 may be more
accurately described as a regulator of cell growth, when considering
this wider range of promoters it appears to regulate. NRF-1 is a member of a family of transcription factors (32, 35, 43). NRF-1 homologues
have been cloned from human, mouse, rat, chicken, and zebrafish (35,
43, 44). Two proteins related to NRF-1, called Erect Wing and P3A2,
have been cloned from drosophila and sea urchin, respectively (18, 45),
although, so far, only P3A2 and NRF-1 have been directly shown to be
DNA-binding proteins. The highest region of homology between NRF-1,
Erect Wing, and P3A2 is located in their putative DNA binding domains,
which share ~54% amino acid identity. Interestingly, both Erect Wing
and P3A2 are developmentally expressed, an observation suggesting that they function during embryogenesis (18, 45). Recently, the expression
pattern and possible function during embryogenesis of a protein termed
"not really finished" (Nrf), the zebrafish homologue to NRF-1, has
been studied in detail (44). The Nrf protein of zebrafish shares 91%
amino acid identity with human NRF-1, an observation illustrating that
this protein family is highly conserved during vertebrate evolution.
Becker et al. (44) have suggested that Nrf plays a critical
role in the development of the retina of zebrafish. This was based on a
genetic analysis of zebrafish whose nrf gene was
inactivated; zebrafish that were homozygous for retroviral insertion at
the nrf locus displayed a larval lethal phenotype with
perturbations in the formation of the neural retina during development.
At early stages of wild type zebrafish development, Nrf RNA is
expressed strongly in the developing CNS, most intensely in the optic system.
Later in zebrafish development, Nrf RNA is expressed in the retinal
ganglion cell layer, optic nerve, and optic tract regions of the
developing eye (44). Genetic inactivation of the nrf locus
inhibits Nrf RNA expression in mutant zebrafish leading to increased
apoptosis in the retina and optic tectum. This, in turn results in the
disruption of retinal formation. If mammalian NRF-1 shares a similar
expression profile with its zebrafish homologue, the report of Becker
et al. (44) is highly informative for our finding that the
CD155 promoter is regulated by this transcription factor.
The expression profile of zebrafish Nrf suggests its function in
development is to regulate the expression of genes required for proper
retinal development. The CD155 promoter is active in exactly
these anatomical structures,1 suggesting that potential
regulation by NRF-1 could play a role in directing promoter activity to
these locations. Interestingly, Erect Wing is expressed in all
developing neurons of drosophila embryos, an observation suggesting
that expression in the CNS may also be an evolutionary conserved
feature among NRF-1-related proteins (18).
The cell type- and tissue-specific activity of the CD155
promoter in transgenic embryos indicates that this promoter fragment harbors cis-acting elements specific for directing gene
expression to a select subset of structures within the developing CNS.
The CD155 promoter can be used as a model for understanding
the mechanism of gene expression in the above mentioned embryonic
structures. Our studies have elucidated some of the
cis-elements of the CD155 promoter and have
revealed the nature of some of the trans-acting factors,
which could exert affects on the CD155 expression pattern. Concomitantly, a picture is emerging of how these factors may cooperate
to orchestrate CD155 promoter activity, especially during embryogenesis. Therefore, it is of particular interest to learn about
the significance of FPIII and the identification of its putative
trans-acting binding partner(s). This problem is currently under investigation. We hope to build on our genetic analyses of this
promoter by generating additional transgenic mouse lines where reporter
gene expression is directed by mutant CD155 promoter fragments. These in vivo experiments should lead to an
improved understanding of the roles that the NRF-1 and AP-2
transcription factors play in the transcriptional regulation of
CD155 expression.
§,
,
TOP
ABSTRACT
INTRODUCTION
Supported by Grant BE1886/1-2 from the Deutsche Forschungsgemeinschaft.
