Genomic Cloning and Promoter Analysis of Aortic Preferentially
Expressed Gene-1
IDENTIFICATION OF A VASCULAR SMOOTH MUSCLE-SPECIFIC PROMOTER
MEDIATED BY AN E BOX MOTIF*
Chung-Ming
Hsieh
,
Shaw-Fang
Yet
,
Matthew D.
Layne
,
Masafumi
Watanabe
,
Audrey M.
Hong
,
Mark A.
Perrella
§¶, and
Mu-En
Lee
§
**
From the
Cardiovascular Biology Laboratory, Harvard
School of Public Health, the § Department of Medicine,
Harvard Medical School, and the ¶ Pulmonary and Critical Care and
Cardiovascular Divisions, Brigham and Women's Hospital,
Boston, Massachusetts 02115
 |
ABSTRACT |
Aortic preferentially expressed gene-1
(APEG-1) was originally identified as a 1.4-kilobase (kb)
transcript preferentially expressed in differentiated vascular smooth
muscle cells (VSMC). Its expression is markedly down-regulated in
de-differentiated VSMC, suggesting a role for APEG-1 in
VSMC differentiation. We have now determined that APEG-1 is
a single-copy gene in the human, rat, and mouse genomes and have mapped
human APEG-1 to chromosome 2q34. To study the molecular
mechanisms regulating its expression, we characterized the genomic
organization and promoter of mouse APEG-1. APEG-1 spans 4.5 kb in the mouse genome and is composed of five exons. Using reporter
gene transfection analysis, we found that a 2.7-kb APEG-1
5'-flanking sequence directed a high level of promoter activity only in
VSMC. Its activity was minimal in five other cell types. A repressor
region located within an upstream 685-base pair sequence suppressed the
activity of this 2.7-kb promoter. Further deletion and mutation
analyses identified an E box motif as a positive regulatory element,
which was bound by nuclear protein prepared from VSMC. In conjunction
with its flanking sequence, this E box motif confers VSMC-specific
enhancer activity to a heterologous SV40 promoter. To our knowledge,
this is the first demonstration of an E box motif that mediates gene expression restricted to VSMC.
 |
INTRODUCTION |
The de-differentiation of vascular smooth muscle cells
(VSMC)1 from a quiescent and
contractile phenotype to a proliferative and synthetic phenotype is one
of the most prominent features of arteriosclerosis, the leading cause
of death in developed countries (1). These phenotypic changes may
result from vascular injuries caused by smoking, hypercholesterolemia,
hyperhomocysteinemia, hypertension, or trauma (1-6). Owing to, at
least in part, a lack of markers specific for differentiated VSMC, the
molecular mechanisms regulating VSMC differentiation are largely
unknown (2).
Although several gene products have been used as specific markers for
differentiated smooth muscle cells (SMC), such as smooth muscle
-actin, smooth muscle myosin heavy chain, calponin, SM22
, and
caldesmon (7-12), their expression in vivo is not
restricted to VSMC. However, a 0.4-kb segment of the SM22
promoter,
which contains two CArG elements, has been shown recently to confer expression only in the arterial SMC of transgenic mice (13, 14).
Because mutation of the proximal CArG element eliminates all SM22
promoter activity in transgenic animals, this element appears to be
necessary and sufficient for high-level expression restricted to the
SMC lineage (13). The CArG element binds to nuclear proteins such as
serum response factor and YY1. Serum response factor and YY1 are both
expressed ubiquitously; thus it is unclear how the CArG element
regulates arterial SMC-specific expression conferred by the 0.4-kb
segment of the SM22
promoter. This puzzle could be explained by the
presence of arterial SMC-specific transacting factors or co-factors
that have yet to be identified.
Aortic preferentially expressed gene-1 (APEG-1) was cloned
in our laboratory by virtue of its preferential expression in VSMC (15). It is a 1.4-kb message and encodes a 12.7-kDa protein (15).
APEG-1 is expressed in differentiated VSMC in
vivo and is down-regulated rapidly in de-differentiated VSMC
in vitro and in injured arteries in vivo (15).
These data suggest that APEG-1 may serve as a sensitive
marker for VSMC differentiation, and that it may play a role in
regulating growth and differentiation in this cell type.
In this report we show that APEG-1 is a single-copy gene.
The APEG-1 transcription unit spans 4.5 kb in the mouse
genome and contains five exons. Using reporter gene transfection
analysis, we found that 2.7 kb of the APEG-1 5'-flanking
sequence contains potent VSMC-specific promoter activity. Further
deletion and mutation analyses identified an E box motif as a positive
regulatory element, which was bound by nuclear protein prepared from
VSMC. In conjunction with its flanking sequence, this E box motif
confers VSMC-specific enhancer activity to a heterologous promoter.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture and Reagents--
Rat aortic SMC (RASMC) were
harvested from the thoracic aortas of adult male Sprague-Dawley rats
(200-250 g) by enzymatic digestion (16). Bovine aortic endothelial
cells were harvested from bovine aortic endothelium as described (17).
U-2 OS cells were kindly provided by Dr. T.-P. Yao (Dana-Farber Cancer
Institute, Boston, MA). HeLa, HepG2, C2C12, and NIH 3T3 cells were
obtained from the American Type Culture Collection. All cells were
grown in Dulbecco's modified Eagle's medium (JRH Biosciences, Lenexa, KS) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 4 mM L-glutamine, penicillin (100 units/ml), and
streptomycin (100 µg/ml) in a humidified incubator (37 °C, 5%
CO2). RASMC culture medium contained 10 mM
HEPES (pH 7.4). Monc-1 cell culture and differentiation into SMC were
as described (18).
Southern Blot Analysis and Chromosomal Localization of
APEG-1--
Genomic DNA was extracted from cultured mouse embryonic
stem cells, RASMC, and human umbilical vein endothelial cells. Genomic DNA (10 µg) was digested with 50 units of the restriction enzyme XbaI, EcoRI, BamHI,
HindIII, or SacI, separated on a 0.8% agarose gel, denatured, and transferred to NitroPure filters (MSI, Westboro, MA) by using a standard protocol (19). The filters were hybridized with
[
-32P]dCTP-labeled APEG-1 cDNA probes
from the appropriate species (the cDNA probes contain exons 2 to
5). After hybridization, filters were washed at 55 °C in 0.2 × SSC (30 mM sodium chloride, 3 mM sodium
citrate), 0.1% sodium dodecyl sulfate and exposed to Kodak BioMax
films with intensifying screens. To locate APEG-1 on a specific human chromosome, we hybridized a genomic Southern blot membrane containing an EcoRI-digested human monochromosomal
somatic cell hybrid panel (BIOS Laboratories, New Haven, CT) with a
human APEG-1 cDNA probe under the same hybridization and
washing conditions.
Genomic Library Screening and Sequence Analysis--
A 129/SvJ
mouse genomic library in
FIX II vector (Stratagene, La Jolla, CA)
was screened with a mouse APEG-1 cDNA probe. Four
positive clones were purified and subcloned into pUC 18 (Promega, Madison, WI) or pBlueScript II SK (Stratagene) plasmid vectors. The
genomic DNA and exon-intron junctions were sequenced by the dideoxy
chain termination method with primers designed from the mouse
APEG-1 cDNA. Intron sizes were determined by polymerase chain reaction (PCR) with flanking exon primers, and by direct sequencing. The DNA sequences were assembled and analyzed with Sequencher 3.0 software (Gene Codes Co., Ann Arbor, MI) running on a
PowerMacintosh (Apple Computer, Cupertino, CA).
RNase Protection Assay--
A 305-bp mouse APEG-1
fragment (
122 to +183) was amplified by PCR from genomic DNA using
the forward primer 5'-CGTTCgagcTCCACCACTCCAGGG-3' (the lowercase
sequence indicates a SacI site introduced for cloning purposes) and the reverse primer 5'-GAGGCTTTGCACACGGAC-3' and subcloned
into the pCR-Script vector (Stratagene). After linearization this
construct was used to produce an [
-32P]UTP-labeled
antisense riboprobe with T3 RNA polymerase. Gel-purified antisense
riboprobe was hybridized overnight with 20 µg of total RNA from mouse
aorta, heart, or skeletal muscle. Yeast tRNA was also hybridized as a
negative control. RNase A (0.1 unit) and RNase T1 (4 units) were added
subsequently to digest single-strand RNA, and the remaining riboprobe
was separated on an 8% sequencing gel along with a sequencing ladder
using the linearized template and reverse primer. The gel was exposed
to Kodak X-OMAT film overnight with an intensifying screen at
80 °C.
5' RACE (Rapid Amplification of cDNA Ends) Experiment--
A
5' RACE system (Life Technologies, Inc., Manassas, VA) was used
according to the protocol provided by the manufacturer. The nested
primers were R592, 5'-CCATATTCGTTGACCGCC-3' for reverse transcription;
R354, 5'-TTGGGGGTGCCTTGGAAGAAGAGTC-3' for the initial PCR; and R309,
5'-TGGGACTGAGCTTCATGGTAGGGGTTCGG-3' for the nested PCR. One microgram
of total RNA from mouse aorta was used. Chloramphenicol acetyltransferase (CAT) RNA and primers provided by the manufacturer were used as positive controls. The 5' RACE product was cloned into the
pCR-II vector (Invitrogen, Carlsbad, CA) for sequence analysis.
Cloning of the Mouse APEG-1 Promoter and Construction of Reporter
Plasmids--
A 2.7-kb mouse APEG-1 5'-flanking genomic
sequence (bp
2663 to +76) was amplified from genomic DNA by PCR using
the forward primer
2663 (5'-GCGATAGATAACCTGGTGATCC-3') and the
reverse primer +76 (5'-TCAGCCTGGCCAGCCCCACTCACTC-3'). The reporter
plasmid p(
2663/+76) was made by inserting this 2.7-kb fragment into
the SacI and SmaI sites of the pGL3-Basic vector
(Promega). Additional 5'-end deletion constructs were generated by PCR
with the GL2 primer (Promega) and the following primers:
1073
(5'-CTGGAGCtcGGAATCTAAACTC-3'),
479
(5'-CCCTGAgAGcTCTTTGGTTCTC-3'),
355 (5'-GCTGaGCTcTATGGGTCAACAC-3'), and
122 (5'-CGTTCgagcTCCACCACTCCAGGG-3'). (The nucleotides in lowercase in each primer show mutations introduced for cloning purposes.) To generate 3'-deletion and mutation constructs, we paired
the
479 or
122 forward primer with the +38
(5'-TGACCaAGCTtAGGCCCCGCAC-3') or +76Emut
(5'-ggcaagcttTCAGCCTGGCCAGCCCCACTCACTCGCTGACCgtGCTGAGGCCC-3') reverse primer by PCR and subcloned the products into the
SacI and HindIII sites of pGL3-Basic. The 3.3-kb
APEG-1 promoter construct p(
3336/+76) was generated by
subcloning a 3.3-kb fragment of APEG-1 5'-flanking region
(bp
3336 to +76) into the SacI and SmaI sites
of pGL3-Basic. The reporter plasmid p(
3336/+76)Rev was made by
digesting p(
3336/+76) with KpnI to invert a 1.5-kb DNA
fragment (bp
3336 to
1813) within the plasmid. Each construct was
confirmed by sequencing from both orientations.
Transient Transfection and Reporter Activity
Assays--
p(
2663/+76) and pGL3-Control (equal moles) were used to
transfect growing cells on 60-mm dishes with DEAE-dextran for RASMC (20) or LipofectAMINE (Life Technologies, Inc.) for other cell types.
To correct for variability in transfection efficiency, we
co-transfected 1 µg of pCAT3-Control in all experiments. Luciferase and CAT activities were measured 48-60 h after transfection as described (20, 21). To compare APEG-1 promoter activity in different cell types, we expressed the normalized activity of p(
2663/+76) as a percentage of that of pGL3-Control. In the promoter deletion and mutation analyses, the normalized activity of each construct was expressed as a percentage of the activity of
p(
2663/+76). All data are presented as the mean ± S.E.
Nuclear Protein Extraction and Electrophoretic Mobility Shift
Assay--
RASMC and NIH-3T3 nuclear extracts were prepared from cells
grown on 150-mm dishes essentially as described (22). C2C12 cells were
grown in 10% fetal bovine serum then kept in 2% horse serum for 3 days to induce differentiation into myotubes. Protein concentration was
measured by the Bradford dye-binding method (23) with the Bio-Rad
protein assay system (Bio-Rad). For the electrophoretic mobility shift
assay, oligonucleotide probes were synthesized according to the mouse
APEG-1 exon 1 sequences E (5'-GGGCCTCAGCTGGGTCAG-3') and
Emut (5'-GGGCCTCAGCacGGTCAG-3'). An 18-bp E box-containing probe was
also synthesized according to the mouse muscle creatine kinase (MCK)
enhancer sequence (5'-CCCCACACCTGCTGCCT-3'). An unrelated Oct-1
sequence (5'-TTATGCAAAATAATAAAACGTATT-3') was made as a nonspecific
competitor. Double-stranded oligonucleotide (50 pmol) was end-labeled
with [
-32P]ATP by using polynucleotide kinase (New
England Biolabs, Beverly, MA) and purified on a Sephadex G-25 column
(Roche Molecular Biochemicals, Indianapolis, IN). A typical binding
reaction consisted of 8 µg of nuclear extract, DNA probe (20,000 cpm), 250 ng of poly(dI-dC)·poly(dI-dC), 25 mM HEPES (pH
7.9), 40 mM KCl, 0.1 mM EDTA, 1 mM
dithiothreitol, and 10% glycerol. A molar excess (100-fold) of
unlabeled, double-stranded oligonucleotide was added to the reaction to
compete for DNA binding. For mobility supershift experiments, 4 µg of
an anti-E2A protein (E12 and E47) monoclonal antibody (Yae antibody,
Santa Cruz Biotechnology, Santa Cruz, CA) was included in the binding
reaction and incubated on ice for 20 min before the probe was added.
Binding reactions were incubated on ice for 15 min and resolved by 5%
nondenaturing polyacrylamide gel electrophoresis in 0.5 × TBE
buffer (44.5 mM Tris base, 44.5 mM boric acid,
1 mM EDTA) at 4 °C.
 |
RESULTS AND DISCUSSION |
APEG-1 Is a Single-copy Gene and Is Located on Human Chromosome
2q--
We performed Southern analysis with a full-length
APEG-1 cDNA probe to determine whether APEG-1
is a single-copy gene. Hybridization of EcoRI-,
BamHI-, HindIII-, or SacI-digested
genomic DNA from human, rat, and mouse revealed a simple pattern that
suggests that APEG-1 is a single-copy gene in all three
species (Fig. 1A).
To test the possibility that APEG-1 associates with certain
genetic disorders that have been mapped, we determined the chromosomal location of the human APEG-1 gene. Southern hybridization to
an EcoRI-digested human/rodent monochromosomal somatic cell
hybrid panel revealed that APEG-1 is located on human
chromosome 2 (Fig. 1B). This
result was confirmed by using an independent human/rodent somatic cell
hybrid panel (data not shown). To determine the subchromosomal localization of human APEG-1, we carried out a genomic PCR
analysis by using the GeneBridge 4 radiation hybrid panel with specific primers from the human APEG-1 cDNA sequence (24). The
PCR results were then screened against a sequence-tagged site data base
for the human genome (25). APEG-1 mapped to human chromosome
2q34 between markers D2S360 and D2S353 (data not shown). Despite the preferential expression of APEG-1 in VSMC, a search of the
human genome data base did not reveal an association with known
inherited vascular diseases or with a particular gene cluster (26).

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Fig. 1.
Genomic analysis of
APEG-1. A, human, mouse, and rat
genomic DNAs (10 µg) were digested with restriction enzymes and used
in Southern analysis as described under "Experimental Procedures."
Hybridization was carried out with APEG-1 cDNA probes.
The positions of DNA size markers are indicated in kb. B,
monochromosomal somatic cell hybrid Southern analysis. Each lane
contained the EcoRI-digested genomic DNA of a single human
chromosome (1-22, X, and Y) and mouse or hamster
background genomic DNA (asterisk). Human, mouse, hamster,
and an equal mixture of human and mouse (Mix) genomic DNAs
were included as controls. Arrow marks a signal specific to
human APEG-1, present in the positive control lanes
(Human and Mix) and in the lane containing the
genomic DNA of human chromosome 2.
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Genomic Cloning and Organization of APEG-1--
Using a
full-length mouse APEG-1 cDNA probe, we identified
several clones from a 129/SvJ mouse genomic library. These
APEG-1 genomic sequences assembled into a 4.5-kb
organization that contained five exons and four introns (Fig.
2, A and B). The
open reading frame for the APEG-1 protein began in the
second exon and terminated at the 5'-end of the fifth exon (Fig.
2B). The identified exon-intron junctions (Fig.
2B) were all in agreement with the consensus 5' GT and 3' AG
sequences (27).

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Fig. 2.
Mouse APEG-1 genomic
organization. A, diagram of APEG-1 exons (E1
to E5, filled boxes) and introns (lines).
Arrow indicates the transcription start site. Recognition
sites of restriction enzymes SacI (S),
XbaI (X), EcoRI (E), and
BamHI (B) are indicated. B, genomic
sequence of APEG-1 exons (E1 to E5) and partial intron
sequence. Exon sequences are shown in uppercase and intron
sequences (as well as sizes) are shown in lowercase. Index
on the left corresponds to the APEG-1 cDNA
sequence. Single-letter codes (in boldface) show the deduced
APEG-1 peptide sequence. The polyadenylation signal is
underlined.
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Identification of the APEG-1 Transcription Start Site--
To
determine the APEG-1 transcription start site, we performed
RNase protection assays. A 305-bp (
122 to +183) APEG-1
antisense riboprobe encompassing the 5'-untranscribed region and most
of the first exon was hybridized with RNA from mouse aorta, brain, heart, and undifferentiated and differentiated Monc-1 cells (Monc-1 cells are transformed neural crest cells that can be differentiated into VSMC) (18). After RNase digestion (Fig.
3A), three partially protected
riboprobe signals of between 100 and 200 nucleotides were observed
primarily in the mouse aorta and to a lesser extent in the heart and
the differentiated Monc-1 cells (28 and 57% compared with aorta,
respectively). Very little signal was seen in the brain (12%) and the
undifferentiated Monc-1 cells (11%). The most 5' transcription start
site (Fig. 3A, long arrow) for mouse
APEG-1 mapped to a CA nucleotide pair (Fig. 3A,
bent arrow), which is found at many eukaryotic transcription
start sites (28). Two shorter fragments (Fig. 3A,
arrowheads) corresponded to downstream CA pairs and may also
be transcription start sites. In addition, a fully protected riboprobe
(minus sequence from the vector) was found in the aorta, brain, heart,
and differentiated Monc-1 cells (Fig. 3A,
asterisk). This fully protected probe was generated by a
4-kb APEG-1
isoform.2

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Fig. 3.
Analysis of mouse APEG-1
transcription start site by RNase protection assay and 5'
RACE. A, 25 µg of total RNA extracted from mouse
aorta, brain, heart, and undifferentiated and differentiated (5 days)
Monc-1 cells was hybridized to an [ -32P]UTP-labeled
antisense riboprobe as described under "Experimental Procedures."
The transcription start site was identified by running a sequencing
ladder with the reverse primer (+182) on the same gel. Long
arrow marks the largest fragment protected by APEG-1
mRNA, which indicates the transcription start site. It corresponds
to an A nucleotide preceded by a C nucleotide (right).
Two arrowheads indicate shorter fragments that correspond to
downstream CA nucleotide pairs. Asterisk indicates a fully
protected probe that does not contain the vector sequence. It is the
result of a riboprobe hybridizing with a 4-kb APEG-1 isoform
whose transcript shares genomic sequence with APEG-1 (see
text). An RNA size marker is included to indicate the length of the
nucleotide sequence. Riboprobe hybridized with yeast tRNA was used as a
negative control, and the lane without RNase treatment (no RNase) shows
the input of riboprobe in the hybridization (2.5 × 105 cpm/reaction). One additional probe lane (Probe)
(2.5 × 104 cpm) was loaded to indicate the size of
the riboprobe in the absence of hybridization and RNase treatment. The
signal intensity of the primary transcription start site is shown as a
percentage of the intensity of the aorta signal. B, 5' RACE
was used to locate the 5'-end of mouse APEG-1. A 363-bp
fragment was obtained from mouse aortic RNA and cloned for sequencing.
C, 5'-end sequence of the 363-bp product is shown on the
right. The poly(G) sequence was introduced during the 5'
RACE experiment. The 5'-end of mouse APEG-1 is indicated by an
arrow.
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To confirm the transcription start site of APEG-1, we
performed a 5' RACE experiment. We amplified and cloned a 363-bp
cDNA fragment from mouse aortic RNA (Fig. 3B). After we
had removed sequences introduced by the 5' RACE procedure, this 363-bp
product contained a 309-bp APEG-1 cDNA sequence whose 5'
end (Fig. 3C, bent arrow) was the same as the
primary transcription start site (Fig. 3A, long
arrow) identified by the RNase protection assays.
APEG-1 Promoter Region Contains a CArG Motif Specific to Smooth
Muscle Gene Expression--
To identify potential
cis-acting elements that may be important in the regulation
of APEG-1 promoter activity, we cloned and sequenced the
3.3-kb 5'-flanking region (Fig. 4). There
are no upstream TATA-like sequences in the APEG-1
5'-flanking sequence; however, there is a consensus initiator motif
(5'-YYCAYYYYY-3') at the APEG-1 transcription start site
(Figs. 3A and 4, bent arrow) (29, 30). We
identified several cis-acting elements (by their consensus
sequences) that have the potential to regulate APEG-1 promoter activity (Fig. 4). Among them are two CArG (or CArG-like) elements known to regulate expression of SMC-specific genes (13, 31-33). In addition, we identified two Sp1 sites in the proximal promoter region (bp
52 and bp
143 relative to the transcription start site) that may potentiate transcription from the TATA-less APEG-1 promoter (34).

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Fig. 4.
APEG-1 5'-flanking genomic sequence and
potential cis-acting elements. Approximately 3.3 kb of APEG-1 5'-flanking sequence was analyzed for known
cis-acting elements in the TRANSFAC data base (50). DNA
sequence upstream of the APEG-1 transcription start site
(arrow) is shown in lowercase. The first 76 bp of
exon 1 is shown in uppercase. Potential
cis-acting elements are underlined and specified.
The locations of the 5' and 3' promoter deletion constructs are also
indicated.
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The APEG-1 5'-Flanking Region Contains a Potent VSMC-specific
Promoter--
To see whether the APEG-1 5'-flanking
sequence contains a VSMC-specific promoter, we constructed the reporter
plasmid p(
2663/+76) by cloning a 2.7-kb APEG-1 5'-flanking
region and 76 bp of exon 1 into the pGL3-Basic vector. In VSMC (RASMC),
the 2.7-kb APEG-1 5'-flanking region directed a high level
of promoter activity that was similar (86%) to the activity of
pGL3-Control, which contains the potent SV40 promoter/enhancer (Fig.
5). In contrast, this 2.7-kb
APEG-1 sequence directed low levels of promoter activity in
the five other non-SMC that we tested (bovine aortic endothelial cells,
HeLa, U-2 OS, NIH 3T3, and HepG2). Thus, the 2.7-kb APEG-1 5'-flanking region contains potent VSMC-specific promoter activity.

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Fig. 5.
Cell type-specificity of mouse
APEG-1 promoter activity. Mouse APEG-1
promoter construct p( 2663/+76) and pGL3-Control were each transfected
into the indicated cell types, and reporter luciferase activity was
measured as a representation of promoter activity. The difference in
transfection efficiency was corrected by the CAT activity from a
co-transfected pCAT3-Control vector. For each cell type, the corrected
p( 2663/+76) promoter activity is shown as a percentage of that of
pGL3-Control.
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The 2.7-kb APEG-1 Promoter Is Inhibited by a 5' Repressor--
We
reported elsewhere that expression of APEG-1 is
down-regulated in de-differentiated VSMC both in vivo and
in vitro (15). Thus although we had expected VSMC-specific
APEG-1 promoter activity, we were surprised that 2.7 kb of
the APEG-1 5'-flanking region directed high levels of
promoter activity in cultured, and therefore de-differentiated, RASMC.
One explanation for this discrepancy would be the presence of negative
DNA regulatory elements outside the 2.7-kb APEG-1
5'-flanking sequence. To test this possibility, we constructed
p(
3336/+76) and p(
3336/+76)Rev by cloning an additional 685 bp of
APEG-1 5'-flanking sequence into p(
2663/+76) in both
orientations. In comparison with that of p(
2663/+76), the promoter
activity of p(
3336/+76) and p(
3336/+76)Rev was reduced markedly
(Fig. 6). An additional (upstream) 4-kb
DNA sequence did not decrease promoter activity further (data not
shown). Taken together, these results indicate that an
orientation-independent transcription repressor is located between bp
3336 and
2663 5' of the APEG-1 transcription start site.
This APEG-1 transcriptional repressor may explain, at least
in part, the decrease in expression of APEG-1 in
dedifferentiated VSMC (15).

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Fig. 6.
A repressor region is located between bp
3336 and 2663 5' of the APEG-1 transcription start
site. Two 3.3-kb mouse APEG-1 promoter constructs,
p( 3336/+76) and p( 3336/+76)Rev, were made as described under
"Experimental Procedures." The promoter activity of the two
constructs in RASMC was only 20% of the activity of the 2.7-kb
APEG-1 promoter construct p( 2663/+76).
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An E Box Motif Mediates High Levels of APEG-1 Promoter Activity in
RASMC--
To identify the cis-acting element in the
APEG-1 promoter responsible for its potent activity in
RASMC, we made three deletion constructs from the 5' end of
p(
2663/+76). In transient transfection experiments, the three
deletion constructs, p(
1073/+76), p(
479/+76), and p(
355/+76), had
promoter activity similar to that of p(
2663/+76). A fourth deletion
construct, p(
122/+76), showed a 20% reduction in promoter activity
(Fig. 7). These data suggest that most of the APEG-1 promoter activity is contained between bp
122
and +76.

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Fig. 7.
Deletion and mutation analysis of mouse
APEG-1 promoter. Reporter plasmids were
constructed from the APEG-1 promoter as described under
"Experimental Procedures." The four 5' deletion constructs
p( 1073/+76), p( 479/+76), p( 355/+76), and p( 122/+76) were made
from p( 2663/+76). They show that most of the APEG-1
promoter activity is contained within p( 122/+76). The two 3' deletion
constructs p( 479/+38) and p( 122/+38) were made from p( 479/+76)
and p( 122/+76). They show minimal promoter activity. The
p( 479/+76)Emut and p( 122/+76)Emut constructs contain a 2-bp
mutation that changes the E box motif in exon 1 from CAGCTG
to CAGCAC. The diagram on the left shows the
relative lengths of the constructs and the positions of the CArG boxes
(white boxes) and the E box (black ovals). The E
box mutation is indicated by the hatched ovals. Transfection
experiments were repeated at least three times for each construct, and
promoter activity is expressed as a percentage of p( 2663/+76)
activity.
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To further localize the positive cis-acting element, we
generated a series of 3' deletion constructs based on p(
479/+76) and
p(
122/+76). These allowed us to determine whether the presence of the
76-bp exon 1 sequence was important for promoter activity. In
comparison with p(
479/+76), p(
479/+38), and p(
122/+38) both had
much lower promoter activity (16 and 4%, respectively) (Fig. 7). These
results indicated that the sequence between bp +38 and +76 in exon 1 was essential for APEG-1 promoter activity. When we
inspected the sequence between bp +38 and +76 for potential transcription factor-binding sites, we identified an E box motif (CAGCTG) at bp +39 to +44 (Fig. 4). To determine the importance of this
E box motif, we mutated its sequence from CAGCTG to CAGCAC in
p(
479/+76) and p(
122/+76). As demonstrated by transfection experiments with p(
479/+76)Emut and p(
122/+76)Emut, mutation of the
exon 1 E box motif caused a dramatic reduction in APEG-1 promoter activity (Fig. 7). These data indicate that this E box motif
located at the 5'-untranslated region (5'-UTR) is essential for high
level APEG-1 promoter activity in RASMC.
Although not commonly found, transcription regulatory elements have
been documented to locate to the 5'-UTR of a few other genes (35-37).
For instance, the 5'-UTR of the herpes simplex virus type 1 ICP22 gene
and the human integrin
3 gene contain cis-acting elements
that mediate high level expression of these genes (36, 37).
Furthermore, the human A
-globin gene also has regulatory
elements in the 5'-UTR. One of these elements binds to the erythroid
transcription factor GATA-1 and may regulate transcription of the human
A
-globin gene during development (35).
It is noteworthy that one CArG box and one CArG-like box are located at
bp
1531 to
1522 and bp
443 to
434 of the APEG-1 5'-flanking region, respectively (Fig. 4). The CArG box is crucial to
the expression of several other SMC-specific genes (13, 31-33), although there is no known SMC-specific, CArG box-binding protein. In
the case of the APEG-1 promoter, however, deletion of the
CArG and CArG-like boxes did not alter its activity (Fig. 7),
indicating that the two boxes are dispensable. This dispensability
distinguishes APEG-1 from other SMC-specific genes and
suggests the existence of CArG-independent mechanisms of SMC-specific
gene expression. Indeed, we have shown that the CArG-less promoter of
mouse CRP2/SmLIM directs a high level of VSMC-specific
reporter gene expression in transgenic mice (38).
The E Box in APEG-1 Exon 1 Is Not Bound by E12 and E47
Proteins--
Transcriptional regulation via the E box motif CANNTG is
important for the regulation of myogenesis (39), immunoglobulin gene
expression (40), cell development and differentiation (41-44), and
cell proliferation and apoptosis (45). It has been shown that the
ubiquitously expressed E2A gene products E12 and E47 (46)
heterodimerize with tissue-specific transcription factors to regulate
gene expression via the E box motif (47), although an E47 or E12
heterodimerization partner has not been identified in VSMC. We
therefore wanted to determine by electrophoretic mobility shift assay
whether the APEG-1 exon-1 E box was bound by nuclear protein
prepared from VSMC, and whether E12 and E47 were present in the binding
complex. Incubation of a probe containing the APEG-1 E box
(E) but not one containing a mutated E box (Emut) with RASMC nuclear
extract resulted in a DNA-protein complex (Fig.
8A). This DNA-protein complex
was specific because only an excessive amount (100-fold) of unlabeled E
box oligonucleotide could compete with it for binding (Fig.
8A); the mutated E box oligonucleotide and an unrelated
Oct-1 oligonucleotide could not compete with it.

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Fig. 8.
Electrophoretic mobility shift assays.
A, two 18-bp oligonucleotide probes derived from the mouse
APEG-1 exon 1 sequence with an E box (E) or
mutated E box (Emut) motif were end-labeled with
[ -32P]ATP and incubated with or without RASMC (SM)
nuclear extract (N.E.). Arrow indicates the
mobility-shifted probe and its binding nuclear protein complex. Three
competitors (E, Emut, and Oct-1) were added to
the binding reaction at a 100-fold molar excess to demonstrate E
box-binding specificity. B, monoclonal anti-E2A protein
antibody ( -E2A) was added to the binding reaction but was
not able to change the mobility of the E box-binding complex in RASMC
(SM) nuclear extract (N.E.). As a positive
control, a mouse MCK enhancer E box (MCK E box)
was used as probe to incubate with differentiated C2C12 myotube nuclear
extract (C2) in the presence or absence of -E2A. The
MCK E box probe was bound weakly by RASMC nuclear extract.
C, nuclear extracts (N.E.) from RAMSC
(SM), C2C12 myotubes (C2), and NIH-3T3 cells
(3T3) were incubated with the APEG-1 exon 1 E box
to demonstrate that the E box-binding protein is present in all three
cell types.
|
|
To see if E47 or E12 was present in the DNA-protein complex, we
performed mobility shift assays with the Yae monoclonal antibody specific to E47 and E12 (48). Addition of the Yae antibody to the
binding reaction did not, however, cause a mobility supershift or a
disappearance of the DNA-protein complex (Fig. 8B). As a positive control, we used another E box-containing mouse enhancer sequence, MCK, and nuclear proteins extracted from
differentiated C2C12 myotubes (48). The Yae antibody was able to
supershift a distinct binding complex in C2C12 myotubes which is
presumably composed of heterodimeric E2A proteins and MyoD or myogenin
(Fig. 8B). These results indicate that the E12 and E47
proteins are not present in the APEG-1 E box-binding protein complex.
We next tested the specificity of the APEG-1 E box-binding
protein in two additional cell types and found that the same binding complex also existed in NIH-3T3 fibroblasts and C2C12 myotubes (Fig.
8C). This result and the deletion/mutation analyses of the APEG-1 promoter by transfection experiments would suggest
that the E box is essential for APEG-1 promoter activity but
not sufficient to confer SMC specificity. A similar scenario is found
in CArG box-mediated SMC-specific gene expression (13, 31-33). By
electrophoretic mobility shift assay, the CArG box is bound by serum
response factor, whose expression is not cell type restricted. Thus, it appears that additional SMC-specific accessory factors may be required
to interact with either the APEG-1 E box-binding protein or
serum response factor to direct SMC specific transcription.
Although the APEG-1 E box and the MCK E box share
the same MyoD-type E box (49), they formed distinct DNA-protein
complexes when incubated with nuclear extracts of RASMC or C2C12
myotubes (Fig. 8, B and C). For example,
incubation of RASMC nuclear extracts resulted in a more prominent
DNA-protein complex with the APEG-1 E box than the
MCK E box. Furthermore, an E2A-containing complex was
present when C2C12 nuclear extracts were incubated with the MCK but not APEG-1 E box. These observations
indicate that additional E box-flanking sequences are required to
determine the binding preferences of different nuclear proteins in the
same cell type, which may contribute to cell type-specific gene expression.
The APEG-1 E Box Motif and Its Flanking Sequences Confer
VSMC-specific Enhancer Activity to a Heterologous SV40
Promoter--
We cloned (in both orientations) a 74-bp exon 1 sequence
(+2 to +76) containing the APEG-1 E box upstream of the SV40
promoter in pGL3-Promoter. The resulting plasmids pGL3-E1Ebox and
pGL3-E1Ebox.Rev were then transfected into several cell types. The SV40
enhancer in pGL3-Control (positive control) increased SV40 promoter
activity in all cell types tested (Fig.
9). Only in RASMC, in contrast, did the
APEG-1 E box and its flanking sequence (in either
orientation) increase SV40 promoter activity markedly (Fig. 9). These
findings indicate that the E box motif and its flanking sequence are
both necessary and sufficient for a VSMC-specific enhancer.

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|
Fig. 9.
Activation of transcription on a heterologous
SV40 promoter by the APEG-1 E box motif and its
flanking sequences. A 74-bp DNA fragment containing mouse
APEG-1 E1 sequence from bp +4 to +76 was cloned into the
SmaI site of pGL3-Promoter in the correct (pGL3-E1Ebox) and
the reverse (pGL3-E1Ebox.Rev) orientations. The two constructs, along
with pGL3-Promoter and pGL3-Control, were each transfected into the
indicated cell types and their reporter luciferase activities were
analyzed. The diagram on the left shows the constructs with the SV40
promoter (P) and an SV40 enhancer region (En).
Arrowhead indicates orientation of the E box-containing
mouse APEG-1 E1 sequence. Normalized luciferase activity is
presented as a percentage of pGL3-Control activity.
|
|
To our knowledge, this work on the APEG-1 promoter is the
first demonstration that an E box plays an important role in
VSMC-specific gene transcription. Future studies to identify the
trans-acting factors responsible for E
box-dependent APEG-1 promoter activity and
down-regulation of promoter activity by VSMC de-differentiation will
contribute to our understanding of the molecular mechanisms regulating
VSMC-specific gene expression and phenotypic modulation.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Peter D'Eustachio for helpful
suggestions on human genome mapping, Bonna Ith for technical support,
and Thomas McVarish for editorial assistance.
 |
FOOTNOTES |
*
This work was supported by the Bristol-Myers Squibb
Pharmaceutical Research Institute and National Institutes of Health
Grants F32-HL10113 (to M. D. L.), KO8-HL03194 (to M. A. P.), and RO1-GM53249 (to M.-E. L.).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.
We dedicate this work to the late Edgar Haber in gratitude for his
enthusiasm and support of our research.
**
To whom correspondence should be addressed: Harvard School of
Public Health, 677 Huntington Ave., Boston, MA 02115. Tel.: 617-432-4994; Fax: 617-432-0031; E-mail: lee{at}cvlab.harvard.edu.
2
C.-M. Hsieh and M.-E. Lee, unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
VSMC, vascular
smooth muscle cells;
SMC, smooth muscle cells;
RASMC, rat aortic SMC;
APEG-1, aortic preferentially expressed gene-1;
PCR, polymerase chain reaction;
5'-UTR, 5'-untranslated region;
CAT, chloramphenicol acetyltransferase;
MCK, muscle creatine kinase;
bp, base pair(s);
kb, kilobase pairs(s).
 |
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