PU.1 and USF Are Required for Macrophage-specific Mannose
Receptor Promoter Activity*
Brian S.
Egan
,
Kirk B.
Lane§, and
Virginia L.
Shepherd
¶
**
From the Departments of
Biochemistry,
§ Pathology, and ¶ Medicine, Vanderbilt University and
the
Department of Veterans Affairs Medical Center,
Nashville, Tennessee 37212
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ABSTRACT |
In the current study we report the isolation of
854 base pairs of the rat mannose receptor promoter. Analysis of the
sequence revealed one Sp1 site, three PU.1 sites, and a potential TATA box (TTTAAA) 33 base pairs 5' of the transcriptional start site. The
tissue specificity of the promoter was determined using transient transfections. The promoter was most active in the mature macrophage cell line NR8383 although the promoter also showed activity in the
monocytic cell line RAW. No activity was observed in pre-monocytic cell
lines or epithelial cell lines. Mutation of the TTTAAA sequence to
TTGGAA resulted in a 50% decrease in activity in transient transfection assays suggesting that the promoter contains a functional TATA box. Using electrophoretic mobility shift assays and mutagenesis we established that the transcription factors Sp1, PU.1, and USF bound
to the mannose receptor promoter, but only PU.1 and USF contributed to
activation. Transient transfections using a dominant negative construct
of USF resulted in a 50% decrease in mannose receptor promoter
activity, further establishing the role of USF in activating the rat
mannose receptor promoter. Comparison of the rat, mouse, and human
sequence demonstrated that some binding sites are not conserved. Gel
shifts were performed to investigate differences in protein binding
between species. USF bound to the rat and human promoter but not to the
mouse promoter, suggesting that different mechanisms are involved in
regulation of mannose receptor expression in these species. From these
results we conclude that, similar to other myeloid promoters,
transcription of the rat mannose receptor is regulated by binding of
PU.1 and a ubiquitous factor at an adjacent site. However, unlike other
myeloid promoters, we have identified USF as the ubiquitous factor, and
demonstrated that the promoter contains a functional TATA box.
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INTRODUCTION |
The maturation of phagocytes during the latter stages of myeloid
cell differentiation is accompanied by the appearance of a number of
myeloid-restricted proteins. One of these proteins, the mannose
receptor, is absent from monocytes but is expressed on mature
macrophages, appearing very late in the differentiation process (1, 2).
The mannose receptor is a 175-kDa type I transmembrane protein and is a
member of the C-type lectin family (3). The protein has been purified
from a variety of species (4, 5), and the murine and human cDNA
sequences have been reported (6, 7). Several in vivo
functions have been proposed for the macrophage mannose receptor:
endocytosis of extracellular peroxidases and hydrolases during the
resolution phase of inflammation (8), phagocytosis of unopsonized
pathogens (9), and antigen capture for eventual presentation to T cells
(10).
Studies in our laboratory for the past several years have focused on
the mechanisms involved in regulation of expression of the mannose
receptor in macrophages. Mannose receptor expression is a marker of the
mature macrophage, although expression has also been reported on
dendritic cells (11) and retinal pigmented epithelial cells (12).
Mannose receptor protein is absent in freshly isolated monocytes and
appears during in vitro differentiation in the presence of
colony-stimulating factors (13). Recent studies from a number of
laboratories have begun to shed some light on the mechanisms that
regulate myeloid-restricted gene expression. Studies of the promoters
of these genes have revealed a number of potential factors that
modulate myeloid-specific gene expression, and a pattern of features
required for activation of these genes during myeloid differentiation
has emerged (14, 15). Control of tissue specificity typically resides
in approximately 150 bp1 5'
of the major transcriptional start site. All of the myeloid promoters
contain consensus binding sites for PU.1, a member of the ets family of
transcription factors (16). A role for this factor in myeloid cell
differentiation has been supported by the findings that macrophage
development is restricted in PU.1 null mice, and that PU.1 expression
is up-regulated during commitment of multipotential stem cells to the
myeloid lineage (17). The activity of a number of myeloid promoters
requires an intact PU.1 site (18-26), and the PU.1 sites are in close
proximity to a site that binds a second transcription factor. Binding
elements for four of these factors have been identified to date: AML1,
a member of the Runt/PEBP2/CBF family; C/EBP
or C/EBP
; Sp1; and
AP-1 (14). Binding of both PU.1 and this second factor are involved in
controlling maximal activity. In addition, the promoters for many of
these myeloid-restricted proteins lack a TATA box and have multiple
transcriptional start sites.
Some information on factors involved in mannose receptor regulation has
come from the recent reports of the isolation and partial
characterization of the promoters for the human and murine mannose
receptor genes (27, 28). Potential regulatory mechanisms were
identified in the murine sequence, and myeloid restricted activity was
demonstrated for the human promoter. Both promoters contain PU.1 sites,
and one of these sites appears to be involved in murine promoter
activity. Using transient transfection of mannose receptor-negative
myeloid cells, Eichbaum et al. (28) reported that activity
of the murine promoter required binding of PU.1 and Sp1 at sites within
the first 200 bp. What has hampered further studies on a complete
description of factors involved in mannose receptor transcriptional
regulation is the inability to efficiently transfect primary
macrophages. We have recently described mannose receptor
expression in a rat alveolar macrophage cell line (29), and in the
current study we have used this cell line to examine potential
factors involved in mannose receptor transcriptional regulation.
Studies of the control of mannose receptor expression are important for
two reasons: first, the mannose receptor is a key phagocytic receptor
involved in first-line host defense against a variety of invading
pathogens, including Mycobacterium tuberculosis (30),
Candida albicans (31), Pseudomonas aeruginosa
(32), and Pneumocystis carinii (33). Second, the mannose
receptor is expressed only on the mature macrophage (1, 2) making this
a unique model system to study the mechanisms involved in monocyte to
macrophage maturation. In the current study we report the isolation of
854 bp of the rat mannose receptor promoter and investigate the
elements within this fragment that contribute to mannose receptor
expression in mature macrophages. We have focused on sites that are
potentially involved in myeloid-restricted expression of the rat
mannose receptor, and have extended the findings from both the human
and murine systems to include regulation and function of the mannose
receptor promoter in a mannose receptor-positive macrophage cell line.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
NR8383 cells were obtained from Dr. R. Helmke
(University of Texas Health Science Center) and maintained in Ham's
F-12 medium with 15% fetal bovine serum (Life Technologies, Inc.). The
murine macrophage cell line RAW264, the human monocytic line HL60, and the human epithelial line A549 were obtained from the American Type
Tissue Culture Collection (ATCC, Rockville, MD). RAW and HL60 cells
were maintained in RPMI 1640 with 10% fetal bovine serum and
antibiotics. A549 epithelial cells were maintained in F-12K
supplemented with 10% fetal bovine serum and antibiotics. Rat bone
marrow-derived macrophages were prepared from Sprague-Dawley rats as
described previously (34) by culture for 5-7 days in the presence of L
cell-conditioned medium. Rat bone marrow macrophages were maintained
following differentiation in Dulbecco's modified Eagle's medium
containing 10% fetal bovine serum.
Promoter Isolation--
The strategy for isolation of the rat
mannose receptor promoter was a modification of the procedure used for
cloning the human mannose receptor promoter (27) as follows: a
synthetic oligonucleotide of known sequence (Integrated DNA
Technologies) was ligated to the ends of rat genomic DNA pieces
generated by digestion with HincII. This oligonucleotide
formed an "anchor" sequence to which a complementary primer could
be hybridized as primer 1 for generation of mannose receptor promoter
fragments by PCR using a mannose receptor-specific oligonucleotide as
primer 2. The digests contained genomic DNA (1 µg), in a buffer
containing 10 mM ATP and 10 mM dithiothreitol.
Double-stranded, dephosphorylated synthetic oligonucleotide and T4 DNA
ligase were added to the DNA plus restriction enzyme mixture, and
incubated overnight at 37 °C. These conditions favored the direction
of complete digestion of the DNA at the respective restriction
endonuclease sites, with a synthetic oligonucleotide ligated onto each
blunt end. Any genomic DNA that re-ligated would again be cut by the
restriction enzyme. At the completion of the overnight incubation, a
PCR reaction was set up by addition of a mannose receptor-specific 3'
primer derived from the partial rat cDNA sequence obtained in our
laboratory (5'-CCAAAGAATACTGCCCAAGTCCCCGG-3') and a single-stranded
oligonucleotide complementary to the synthetic oligonucleotide
(5'-TCCAGGCCAGGGAAATTGGAGCACAGG-3') using the Expand Long Template PCR
system (Boehringer Mannheim). The reaction mixture was incubated at
95 °C for 2 min, followed by 30 cycles of 92 °C for 20 s and
68 °C for 5 min. This procedure yielded a fragment of approximately
2.2 kilobases. This fragment was further digested with
BglII/SpeI resulting in a fragment of
approximately 900 bp. This fragment was subcloned into pGEM-T (Promega)
and sequenced using vector-based primers.
Primer Extension--
An oligonucleotide derived from the rat
mannose receptor cDNA corresponding to positions +23 to +46
(5'-CCGGGTTGCCCCTTGGCTTAGTCC-3') was 5'-end labeled with
[
-32P]ATP and T4 polynucleotide kinase (Promega). A
primer with the 5'-end at the predicted +1 position of the mannose
receptor mRNA (5'-AGTCAGACGGCTCCCAGACC-3') was used as a control to
verify that no extension would occur from this site. Total RNA was
isolated from rat bone marrow macrophages using TriReagent (Molecular
Research Center, Inc) according to the manufacturer's instructions.
Labeled primer and 5 µg of RNA were incubated in Tris-HCl, pH 8.3, containing 0.15 M KCl and 1 mM EDTA for 90 min
at 65 °C. Extension of the primer was initiated by addition of avian
myeloblastosis virus reverse transcriptase in Tris-HCl buffer, pH 8.3, with 5.5 mM dithiothreitol, 10 mM
MgCl2, actinomycin D (7 µg), and 150 µM dNTP mixture. This mixture was incubated at 42 °C for 1 h, then terminated by addition of salmon sperm DNA and RNase A. Nucleic acid
was then extracted with phenol/chloroform/isoamyl alcohol and
precipitated with ethanol. The resulting pellet was collected by
centrifugation and analyzed on a 9% polyacrylamide, 7 M
urea sequencing gel.
Plasmid Constructs--
The 854-bp mannose receptor promoter was
cloned into the pGL2 basic vector (Promega) containing the luciferase
cDNA after BglII/SspI digestion (MR854). The
proximal 656-bp promoter fragment (MR656) was generated by
HindIII digestion of the 854-bp promoter with subsequent
ligation into a HindIII-digested pGL2 vector. A construct
containing the proximal 228 bp of the mannose receptor promoter (MR228)
was generated by digestion of the 854-bp construct with SmaI
and PvuII, followed by re-ligation. The
108 (MR108) and
48 (MR48) constructs were generated by PCR using the MR656 as a
template, the appropriate sense primers, and a vector-based antisense
primer. The products were digested with HindIII, and ligated
into the pGL2 basic vector at the HindIII/SmaI
site. Mutations in the mannose receptor promoter were introduced using
a PCR-based approach as described by Ho et al. (35).
Complimentary sense and antisense primers containing the desired
mutations were obtained from Integrated DNA Technologies (Coralville,
IA). The products of the PCR reactions containing the mutations were
isolated, digested with the appropriate restriction enzymes, and
ligated into MR854 following removal of the specific mutated region.
The dominant negative USF1 expression vector,
bTDU1, was
a kind gift from Dr. Raymondjean (Institut Cochin de Genetique Moleculaire).
Transfection and Reporter Gene Assays--
Electroporation was
used for transfection of the cells lines under the following
conditions: mannose receptor promoter-luciferase DNA (5 µg) and
CMV-Renilla-luciferase (pRL-CMV, Promega) (1 µg) were
added to 250 µl of serum-free media. RAW cells were transfected at a
concentration of 107 cells/reaction at 280 V and 960 µfarads. NR8383 cells were transfected at a concentration of 5 × 106 cells/reaction at 250 V and 960 µfarads. A549
cells (5 × 106) were transfected at 250 V and 960 µfarads. NR8383 and HL60 cells were also transfected using
DEAE-dextran as follows: cells were washed with phosphate-buffered
saline, then resuspended in trypsin/EDTA for 3 min at room temperature.
The cells were collected by centrifugation, and suspended in 1 ml of 25 mM Tris, pH 7.4, containing 0.14 M NaCl, 5 mM KCl, and 0.7 mM
K2HPO4. DNA (10 µg) and 50 µl of 4 mg/ml
DEAE-dextran were added, and the mixture incubated at room temperature
for 20 min. The transfected cells were then plated into 100-mm tissue
culture dishes for 24 h in the media used for routine culture.
Firefly and Renilla luciferase activities were measured
using the dual luciferase reporter assay system (Promega) according to
the manufacturer's instructions. The pGL2-basic vector (Promega) was
used as a negative control in transfection assays. The pRL-CMV vector
was used to correct for transfection efficiency. Data are expressed as
the luciferase activity for each sample normalized to the
Renilla luciferase activity.
Electrophoretic Mobility Shift Assays (EMSA)--
Nuclear
extract was isolated from cell preparations as follows: 107
cells were suspended in 300 µl of lysis buffer A (10 mM
HEPES, pH 7.9, containing 10 mM KCl, 0.1 mM
EDTA, 0.1 mM EGTA, 0.4% Nonidet P-40, 1 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 5 µg/ml leupeptin, and 500 ng/ml pepstatin), and
placed on ice for 10 min. After centrifugation for 1 min at 14,000 rpm,
the pellet containing the nuclei was washed once with buffer A, then
suspended in 200 µl of buffer B (20 mM HEPES, pH 7.9, with 0.4 M NaCl, 1 mM EDTA, 1 mM
EGTA, 1 mM dithiothreitol, plus levels of protease
inhibitors as in A) and vortexed for 15 min. Cell debris was removed by
centrifugation and nuclear protein was quantified using the Bio-Rad
Protein Assay (Bio-Rad).
Single-stranded oligonucleotides (Integrated DNA Technologies) were
annealed and 5' nucleotides were added using Klenow (Promega). The
resulting double-stranded oligonucleotides were end-labeled using T4
polynucleotide kinase (Promega) and [
-32P]ATP (NEN
Life Science Products Inc.). Labeled oligonucleotide (2 × 105 cpm), single-stranded nonspecific DNA (200 ng), nuclear
extract (10 µg), and binding buffer (3 µl) (20 mM
Tris-HCl, pH 7.5, with 20% glycerol, 5 mM
MgCl2, 2.5 mM EDTA, 2.5 mM
dithiothreitol, 250 mM NaCl, 0.25 mg/ml poly(dI-dC)) were
incubated at room temperature for 20 min in a total reaction volume of
15 µl. A 200-fold excess of unlabeled double-stranded oligonucleotide
was used in competition assays. For supershift analysis, 2 µg of
anti-USF1, anti-USF2, anti-Sp1, or anti-PU.1 antibodies (Santa Cruz)
were added immediately after the addition of labeled probe. Samples
were electrophoresed in a 5% polyacrylamide nondenaturing gel in
0.5 × TBE at 150 volts. The gels were dried and bands visualized
by autoradiography. Sequences of double-stranded oligonucleotide probes
used for the gel shifts analyses are listed in Table
I.
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RESULTS |
Identification of the Rat Mannose Receptor Transcriptional Start
Site--
Primer extension analysis using rat bone marrow macrophage
RNA was performed using a probe derived from the rat cDNA to
determine the transcriptional start site of the rat mannose receptor
gene. As shown in Fig. 1, a major
extended product of 46 bp was found, with minor products of 52 and 58 bp upstream of the 5'-end of the probe. The start site of
1 indicated
in Fig. 2 for the rat mannose receptor is
based on the 46-bp extension product, and numbering of all
oligonucleotides and positions of consensus sequences used in the
current paper refer to this start site. The determined rat start site
is 23 bp 5' of the start site reported by Taylor et al. (7)
for the human mannose receptor and by Harris et al. (6) for
the murine mannose receptor, and 21 bp 3' of the start site reported by
Rouleux et al. (27) for the human mannose receptor
gene. The reasons for these discrepancies are not known, although
multiple start sites have been reported for other myeloid-restricted transcripts (14).

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Fig. 1.
Identification of the transcriptional start
site in the rat mannose receptor gene by primer extension. Total
RNA was prepared from rat bone marrow macrophages, then incubated with
an end-labeled oligonucleotide derived from the rat mannose receptor
cDNA spanning the region from +23 to +46 bp
(5'-CCGGGTTGCCCCTTGGCTTAGTCC-3'). The DNA was extended using avian
myeloblastosis virus reverse transcriptase, then analyzed on a 9%
polyacrylamide sequencing gel. Lanes 3 and 4 show
the products of the extension reaction. Lanes 1 and
2 show that no extension occurred using a primer with the
sequence 5'-AGTCAGACGGCTCCCAGACC-3' which was designed with the 3'-end
at the predicted start site. A sequencing ladder is shown on the
left for size identification of the products. The site
labeled 46 is the major product, with minor products
indicated at 52 and 58 bp.
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Fig. 2.
Sequence of the 854-bp promoter region of the
rat mannose receptor gene. The mannose receptor promoter region
was isolated using PCR amplification of rat genomic DNA. The DNA was
subcloned in pGEM-T and sequenced using vector-based primers. Consensus
sites for PU.1 and Sp1, and a consensus E box motif are highlighted by
the boxed areas. A potential TATA box is
underlined.
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Isolation and Characterization of the Rat Mannose Receptor Promoter
Sequence--
The rat mannose receptor 5'-flanking region was isolated
using a PCR-based method similar to that reported for the isolation of
the human mannose receptor promoter (27). The resulting 854-bp fragment
was cloned into pGEM-T and sequenced (Fig. 2). Southern analysis of rat
genomic DNA using probes derived from the promoter sequence, and PCR
using primers from both promoter and rat cDNA sequence confirmed
that this fragment was the 5'-flanking region of the rat mannose
receptor (data not shown). A comparison of the first 250 bp of the rat
promoter sequence to both the human and murine mannose receptor
promoter sequences is shown in Fig. 3.
There is almost complete identity of murine and rat sequences over the
first 150 bp, with a homology over the entire 250 bp of 87%. There is
lower homology between the rodent sequences and the human, with very
little agreement upstream of position
150. The homology of rat to
human over the 250-bp sequence is 56%. The highest degree of homology
among the three sequences occurs between
30 and
150, suggesting
that important regulatory sites may be located within this region.
Careful analysis of the first 250 bp of the rat sequence revealed a
potential TATA box at
33, a core Sp1-binding site (CCGCCC) at
position
91, and three potential PU.1-binding sites in the non-coding
strand at
18 (GAGGAA),
106 (CAGGAA), and
163 (GAGGAA) which are
conserved in the mouse and human sequences.

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Fig. 3.
Comparison of the proximal 250 bp of the rat,
murine, and human mannose receptor promoters. Sequences of the
mannose receptors promoters from rat, mouse, and human are shown with
identical residues marked by the dots. PU.1, Sp1, and E box
motifs in each sequence are in bold. The human sequence is
from Rouleux et al. (27), and the murine sequence is from
Eichbaum et al. (28).
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Analysis of Rat Mannose Receptor Promoter Activity in Mannose
Receptor-positive and Mannose Receptor-negative Macrophages and
Non-myeloid Cells--
To determine if the 854-bp 5'-region of the rat
mannose receptor gene directs cell-specific expression, promoter
activity was determined in mannose receptor-positive macrophages,
mannose receptor-negative macrophages, and mannose
receptor-negative non-macrophage cell lines using a reporter gene
assay. In Fig. 4A, NR8383
macrophages (open bars) and A549 epithelial cells
(solid bars) were transfected with the pGL2-MR854, -MR656,
-MR228, -MR108, -MR48, and promoterless pGL2 basic vectors. PU.1 sites
are represented by the closed ovals, and the Sp1 sites by
the open ovals. Cells were co-transfected with pRL-CMV as an
internal control for transfection efficiency. The 228-bp fragment was
almost as active as the 854-bp promoter in NR8383 cells, suggesting
that this region contains the necessary myeloid-specific sites as
suggested by Clarke and Gordon (14) for myeloid-restricted gene
expression. Interestingly, there was a slight decrease in activity with
the MR656 construct. This supports the observation by Rouleux et
al. (27) that the region from
250 to
750 may contain a
repressor site that is overcome by the additional 5' bases in MR854. In
addition, Eichbaum et al. (28) reported that sequences 5' of
200 bp in the murine promoter reduced activity. The MR108 construct
showed approximately 50% of the activity of MR228. MR108 still
contains two PU.1 sites and the Sp1 site, but maximal activity may
require the upstream PU.1 site at
163. MR48 still exhibited some
activity in NR8383 cells when compared with basic vector alone,
suggesting that the one remaining PU.1 site (
18 bp) also contributes
to macrophage-specific expression from this promoter. Mannose receptor
promoter activity in A549 cells (Fig. 4A) and Hep2 cells
(data not shown) was comparable to levels observed when these cells
were transfected with empty vector, supporting the conclusion that the
mannose receptor promoter is active only in myeloid cell types.

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Fig. 4.
Transient transfection assays of mannose
receptor promoter-luciferase truncated constructs. The 854-bp
mannose receptor promoter construct (MR854) was cloned into
the pGL2 basic vector. Truncated fragments were produced from this
construct by restriction digest and each was subcloned into the pGL2
vector. Cells were transfected with 5 µg of mannose receptor
promoter-pGL2, and 1 µg of CMV-Renilla-luciferase to
control for transfection efficiency. DNA was introduced by
electroporation (NR8383, RAW, and A549 cells) or DEAE-dextran (HL60
cells) using the conditions described under "Experimental
Procedures." Cells were then plated in complete medium in 100-mm
tissue culture dishes for 24 h prior to assaying luciferase
activity. Panel A shows a comparison of the relative light
units for firefly luciferase normalized to Renilla
luciferase for each mannose receptor construct in both NR8383
macrophages and A549 epithelial cells. Data are the average of
duplicate determinations and are representative of four separate
experiments (MR854, MR656, and MR228) or two separate experiments
(MR108 and MR48). Panel B shows the relative activity for
the MR854 construct in NR8383 macrophages, RAW264 monocytes, and HL60
myeloid cells. Data are the average of triplicate determinations ± S.D. and are representative of three separate experiments.
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In Fig. 4B activity of the MR854 construct is compared in
cell lines representative of various stages of myelopoiesis. NR8383 cells express mannose receptor and are representative of mature macrophages, RAW cells are monocyte-like and do not express mannose receptor, and HL60 cells are pre-monocytic cells that also do not
express mannose receptor. Activity in NR8383 cells was 5-fold greater
than activity in RAW cells, while the construct was completely inactive
in HL60 cells. These results suggest that mannose receptor promoter
activity is maximally active in mature mannose receptor-positive macrophages, shows less activity in monocytes, and is inactive in
pre-monocytic cells.
The Mannose Receptor Promoter Contains a Functional TATA
Box--
Based on the position of the start site in the rat gene, a
potential TATA box (TTTAAAA) was identified at
33 bp. Rouleux et al. (27) suggested that the sequence at
27 in the human sequence (
48 in the rat sequence) of TAAATT might be a potential TATA
box, while Eichbaum et al. (28) reported that the murine promoter was TATA-less. To determine if the sequence at
33 bp in the
rat promoter is a functional TATA box, we prepared mannose receptor
promoter-luciferase constructs with the TTTAAA sequence mutated
to TTGGAA or changed to a consensus TATA sequence (TATAAA). NR8383 cells were transiently transfected with the wild type
MR854 or mutated constructs. As shown in Fig.
5, insertion of the TATAAA sequence
increased activity by 25% compared with the activity of the wild type
sequence (TTTAAA). However, activity of the TTGGAA mutant was reduced
by approximately 50%, suggesting that the proposed TATA box (TTTAAA)
in the rat mannose receptor promoter is functional but not essential.
It has been suggested that a characteristic of myeloid-restricted
promoters is the absence of a TATA box (14). However, neutrophil
elastase (36) and proteinase-3 (26) promoters contain the sequence
TATAA, and the CD14 promoter has a potential TATA box (CATAAA)
30 bp
upstream of the start site (37). A recent study by Hoopes et
al. (38) examined the binding of TATA-binding protein to a variety
of potential TATA-box sequences. They classified the sequences CATAAA
(CD14) and TTTAAA (mannose receptor) as intermediate TATA boxes
compared with the strong TATAAA sequence. The observation that mutation
of the TTTAAA sequence in the rat mannose receptor promoter reduced
promoter activity by 50% (Fig. 5) suggests that this sequence may bind
TATA-binding protein with an intermediate affinity to enhance
transcription. In addition, the demonstration that this site plays a
role in promoter activity supports the major transcriptional start site
identified by primer extension analyses. Since this TTTAAA sequence is
conserved across rat, mouse, and human, the major start site in the
mouse and human promoters may be the same as in the rat promoter, and
the TTTAAA sequence may function as a TATA box in these promoters.

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Fig. 5.
Transient transfection assays of mannose
receptor-promoter-luciferase constructs containing the wild type,
consensus, and mutated TATA box sequences. The 854-bp mannose
receptor promoter construct (MR854) was cloned into the pGL2 basic
vector. Mutations were introduced into this construct using a PCR-based
method. Cells were transfected with 5 µg of mannose receptor
promoter-pGL2 and 1 µg of CMV-Renilla-luciferase to
control for transfection efficiency. DNA was introduced by
electroporation using the conditions described under "Experimental
Procedures." Cells were then plated in complete medium in 100-mm
tissue culture dishes for 24 h prior to assay of luciferase
activities. Results are expressed as the relative light units for
firefly luciferase normalized to Renilla luciferase. Data
are the average ± S.D. of triplicate determinations, and are
representative of two separate experiments. *, p < 0.05 for TTGGAA compared with TTTAAA.
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Characterization of Binding of Transcription Factors to the Rat
Mannose Receptor Promoter by EMSA--
Based on the above
observations, we chose to examine more closely the region within the
228-bp fragment that contains conserved sequences known to bind
transcription factors involved in myeloid-restricted expression of
other genes (14, 15), and that contains sites conserved in the rat,
human, and murine mannose receptor promoters. A double-stranded
oligonucleotide was prepared spanning the region from
108 to
71
(108/71). This region contains a consensus PU.1 site (CAGGAA) on the
non-coding strand (PU106), and a core Sp1 site (CCGCCC) on the coding
strand (Sp91). To assess which sites in the rat mannose receptor
promoter are potentially functional, we performed EMSAs using nuclear
extracts from mannose receptor-positive NR8383 cells. In the
experiments shown in Fig. 6, nuclear
extract from NR8383 cells was incubated with labeled oligonucleotides and competitors as indicated. Labeled 108/71 oligonucleotide was bound
by three major proteins from nuclear extracts as shown in lane
1. The top band was identified as Sp1 by competition with a
consensus Sp1 oligonucleotide (lane 2), and by partial
supershift using an Sp1 antibody (lane 3). The bottom band
was identified as PU.1 by competition with a consensus PU.1
oligonucleotide (lane 4) and a supershift of the complex
with anti-PU.1 antibody (lane 5). Competition with an
oligonucleotide mutated at the Sp1 site (CCGCCC
CCGAGC) (S91M)
reduced PU.1 but not Sp1 binding (lane 6), and competition
with an oligonucleotide mutated at the PU.1 site (CAGGAA
CACCAA)
(P106M) inhibited the binding of Sp1 but not PU.1 (lane 7).
A labeled oligonucleotide mutated at the PU.1 site (P106M) did not bind
PU.1 (lane 8) and a labeled Sp1 mutant (S91M) did not bind
Sp1 (lane 9). These results demonstrate that both PU.1 and
Sp1 bind to the rat mannose receptor promoter, and suggest that these
two factors may play a role in the regulation of mannose receptor
promoter activity.

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Fig. 6.
Electrophoretic mobility shift assays.
Nuclear extracts were prepared from NR8383 cells as described under
"Experimental Procedures," and incubated with end-labeled
double-stranded probes as indicated in Panel B. Probe 108/71
spans the 108 to 71 region of the rat mannose receptor promoter;
probe 106M has a mutated PU.1 site (CC residues at 103 and 102
mutated to GG); probe 91M has a mutated Sp1 site (CC residues at 87
and 86 changed to AG). Single-stranded oligonucleotide probes were
annealed and end-labeled. The labeled probes (2 × 105
cpm) were then incubated with single-stranded nonspecific DNA (200 ng),
and nuclear extract (10 µg) in a total volume of 15 µl in Tris
buffer with EDTA, dithiothreitol, and poly(dI-dC) for 20 min at room
temperature. Samples were electrophoresed in a 5% polyacrylamide
nondenaturing gel. The gels were dried and bands were visualized by
autoradiography. Sequences of competitor oligonucleotides are listed in
Table I. Panel A, positions of Sp1 and PU.1 are indicated.
The arrow marks the position of the supershifted
antibody-protein-DNA complex. The asterisk marks the
position of a prominent band seen in all gel shift using the labeled
108/71 probe. Lanes 1-7 are reactions containing labeled
108/71 probe plus nuclear extract with the following additions:
lane 1, buffer; lane 2, excess unlabeled Sp1
consensus oligonucleotide (Table I); lane 3, anti-Sp1
antibody; lane 4, excess unlabeled PU.1 consensus
oligonucleotide (Table I); lane 5, anti-PU.1 antibody;
lane 6, excess unlabeled S91M oligonucleotide; lane
7, excess unlabeled P106M oligonucleotide. Lanes 8 and
9 are nuclear extract plus labeled P106M
oligonucleotide or S91M oligonucleotide, respectively.
|
|
A prominent band that migrated slightly faster than the Sp1 complex was
seen in all gels (Fig. 6, lane 1). Several oligonucleotides were used as competitors in order to determine the sequence to which
the unidentified factor was binding. First, two truncated oligonucleotides were made spanning the regions from
108 to
82 (108/82) and
96 to
71 (96/71). As shown in Fig.
7, competition with the 96/71
oligonucleotide completely inhibited binding of this factor to labeled
108/71 (lane 2), while competition using the 108/82 fragment
did not compete with the unknown factor (lane 3), narrowing
the region for binding to
82 to
71. A potential ets site exists in
this sequence between
74 and
71 (GGAT) (39). The addition of a cold
108/71 oligonucleotide with 75GG74 mutated to
75CC74 was still capable of competing for the
unknown factor (lane 4), thus ruling out the possibility
that this protein was binding the potential ets site. Based on the
above competitions we determined that the third factor was binding
somewhere in the sequence CATGTGACA (
83 to
74). This stretch
contains a sequence (CATGTG) that fits the core hexamer sequence CANNTG
(E box) that is bound by the basic helix loop helix transcription
factor family (40). Competition using the CATGTG sequence (U83,
lane 5) showed conclusively that the third factor was indeed
binding the E box. A number of different transcription factors are
capable of binding the E box including TFE3, TFEB, TFEC, USF, Max, Myc,
Mad, and Mxi (41-48). Wang and Sul (49) have shown that USF is capable
of binding the specific sequence CATGTG which is present in the mannose
receptor promoter (49). We therefore chose to examine the binding of
USF to the 108/71 sequence using antibodies against both isoforms, USF1
and USF2, in EMSAs. In Fig. 7, lane 6, the USF1 antibody
completely supershifted the unknown protein. Lane 7 shows
that antibody to USF2 partially shifted the unknown protein. These
results suggest that USF1 homodimers and USF1/USF2 heterdimers may be
the active forms that bind to the mannose receptor promoter at the
CATGTG sequence.

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Fig. 7.
Electrophoretic mobility shift analysis for
identification of USF binding. Gel shift assays were run as
described in the legend to Fig. 6. The labeled probe used for all lanes
was 108/71. The positions of the Sp1, PU.1, and USF bands are noted.
The arrows mark the position of the anti-USF·USF·DNA
complexes. Oligonucleotides used as competitors in lanes
2-5 are shown in Table I and Panel B. Each reaction
contained the labeled 108/71 oligonucleotide plus nuclear extract with
the following additions: lane 1, buffer; lane 2, excess unlabeled probe spanning 96 to 71; lane 3, excess
unlabeled probe spanning 108 to 82; lane 4, excess
unlabeled E74M oligonucleotide; lane 5, excess unlabeled E
box consensus oligonucleotide (U83; Table I). Lanes 6 and
7 contain antibodies against USF1 (lane 6) and
USF2 (lane 7).
|
|
Functional Effects of PU.1, Sp1, and USF Binding on Mannose
Receptor Promoter Activity--
To assess the functional importance of
binding of PU.1, Sp1, and USF to the mannose receptor promoter, the
following mutations were inserted in MR854: PU163 (161CC to
161GG), PU106 (104CC to 104GG),
Sp91 (87CC to 87AG), and U83 (80TG
to 80CC). The effects of these mutations on MR854 promoter
activity in transfection assays using NR8383 cells are shown in Fig.
8. Mutation of the PU.1 site at
106 or
the E box at
83 resulted in an approximately 80% reduction in
mannose receptor promoter activity. Mutation of the Sp1 site had only a
slight effect on promoter activity, while mutation in the PU163 site
reduced mannose receptor activity by approximately 25%. Since Sp1 had
been found to function in activation of the mouse promoter, we examined
the possibility that the Sp1 and PU.1 sites in the rat promoter acted cooperatively. However, mutation at both the Sp1 site and the PU106
site showed no additional decrease in promoter activity compared with
the single PU106 mutation. These data suggest that the PU.1 site at
106 and the E box at
83 are critical for rat mannose receptor
promoter activity.

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Fig. 8.
Transient transfection assays to determine
the contribution of the PU.1, Sp1, and USF sites to mannose receptor
promoter activity. NR8383 cells were transfected with full-length
854 mannose receptor-luciferase constructs in pGL2, or with constructs
containing site mutations in the PU.1-106 site (second
bar), the Sp1-91 site (third bar), the PU.1-106 and
Sp1-91 sites (fourth bar), the PU.1 site at 163
(fifth bar), or the USF site at 83 (sixth bar).
Relative light units were normalized to the activity of the
co-transfected CMV-Renilla-luciferase construct. DNA was
introduced as described in the legend to Fig. 4 and under
"Experimental Procedures." Results are expressed as the percent of
activity of the MR854 construct as 100% and are representative of four
separate experiments. Error bars are the S.D. of triplicate
or quadruplicate determinations. *, p < .05 for the
second, fourth, fifth, and sixth bars compared
with control (first bar).
|
|
To further establish the role of USF in regulating mannose receptor
promoter activity we used a dominant negative USF1 expression construct
(
bTDU1) in transient transfections of NR8383 cells. The
bTDU1 is an expression vector encoding a USF1 protein
lacking the basic region which is essential for DNA binding (50). This protein retains the helix loop helix and leucine-zipper domains and can
still dimerize with both USF1 and USF2 thus serving as a dominant
negative of both factors. As shown in Fig.
9, MR854 promoter activity is reduced by
50% when co-transfected with the
bTDU1 construct. This
does not seem to be a general effect since the activity of the internal
control (pRL-CMV) was nearly equal in the presence or absence of the
dominant negative (data not shown).

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Fig. 9.
Effect of a dominant negative USF on MR854
promoter-reporter expression. NR8383 cells were transfected by
electroporation as described in the legend to Fig. 4 and under
"Experimental Procedures" with 5 µg of mannose receptor
promoter-pGL2, 1 µg of CMV-Renilla-luciferase, and 4 µg
of pGL2 basic vector (open bar), or 5 µg of mannose
receptor promoter-pGL2, 1 µg of CMV-Renilla-luciferase,
and 4 µg of the bTDU1 vector (hatched bar).
Activity is expressed in relative light units (RLU) for
firefly luciferase normalized to Renilla luciferase. Data
are the average of triplicate determinations ± S.D., and are
representative of two separate experiments. p = 0.0002 for comparison of MR854 (open bar) to MR854+ bTDU1
(hatched bar).
|
|
Species-specific Differences between the Rat, Mouse, and Human
Mannose Receptor Promoters--
Comparison of the results of the gel
shift assays and transfection experiments presented in this study to
the results from the murine mannose receptor promoter study reported by
Eichbaum et al. (28) reveals some significant differences.
First, activity of the murine mannose receptor promoter was found to
involve two PU.1 sites at
164 and
177 (
150 and
163 in the rat
sequence). The murine PU164 site bound PU.1 in gel shifts, and mutation
of the core sequence reduced activity in transfection assays by 50%. This binding site is not conserved in either the rat or human sequences, suggesting that this site may not be critical for mannose receptor promoter activity. No binding of PU.1 to the P177 site (
163
in the rat) was reported in the murine study, but mutation of the core
sequence reduced promoter activity to basal levels. Mutation of this
site in the rat promoter reduced activity by only 25%. This site is
also conserved in the human mannose receptor sequence, but the role of
this site in the human promoter has not yet been examined. More
interestingly, murine mannose receptor promoter activity appeared to
require Sp1 binding. However, in our study, mutation of the Sp1 site
had no effect on mannose receptor promoter activity in mannose
receptor-positive cells, either by itself or in conjunction with a
mutation of the PU.1-106 site. The Sp1 core-binding site is not
completely conserved in the human sequence and Sp1 does not appear to
contribute to activation of the rat promoter, suggesting that other
sites in the rat and human promoters may be more important for maximal
activity. This theory is supported by the presence of an E box at
83
in the rat and human sequence. In order to determine if sequence
differences in the
108/
71 region result in differences in protein
binding between species, we performed EMSAs using species-specific
oligonucleotides. Fig. 10 shows, as
demonstrated above, that PU.1, Sp1, and USF bind the
108/
71 rat
sequence (lane 1). In contrast only PU.1 and Sp1 bind to the
mouse sequence. As we anticipated, the mouse promoter does not bind
USF. The human sequence (lane 3) also binds all three
factors although the G to A change in the Sp1 binding element results
in a decrease in the intensity of the Sp1 band. Our conclusions from
these experiments are that the PU.1 site at
106 is required for rat
promoter activity, and that PU.1 acts together with the USF at
83 to
regulate rat mannose receptor gene expression. In contrast, since USF
does not bind to the mouse promoter, Sp1 may substitute as an activator
in this species.

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Fig. 10.
Comparison of protein binding to the
108/ 71 region of the rat, mouse, and human mannose receptor
promoters. EMSAs were run using the labeled probes spanning the
region from 108 to 71 in the rat (R), mouse
(M), or human (H) promoter sequence as shown in
Fig. 3 plus nuclear extracts from NR8383 cells as described in the
legend to Fig. 6 and under "Experimental Procedures." Positions of
the SP1-, USF-, and PU.1·DNA complexes are noted in the figure.
|
|
 |
DISCUSSION |
The process of maturation of macrophages is controlled by a
complex array of factors that are turned on sequentially as cells move
from the bone marrow into tissues. Considerable work in the past decade
has contributed significantly to our understanding of the factors that
are involved as cells move through the maturation process to fully
differentiated macrophages. The mannose receptor is expressed only on
the end product of this differentiation cascade, the mature tissue
macrophage, and thus is an excellent model gene to study the factors
that control the transition from monocyte to mature macrophage. In the
present study we report the isolation and characterization of the
promoter region of the rat mannose receptor gene, and describe factors
that are involved in regulating expression of this gene in mannose
receptor-positive macrophages.
The rat mannose receptor 5'-flanking region contains multiple consensus
sequences for the myeloid-specific PU.1 transcription factor. In
addition, a potential binding site for the ubiquitous factor Sp1 is
located just 3' of PU.1-106. The presence of a second site bound by a
ubiquitous factor is characteristic of myeloid-restricted expression.
We examined the role of these sites in mannose receptor promoter
activity by transient transfection assays using mannose receptor
promoter-luciferase constructs and by gel shift assays. We focused our
studies on the region from
108 to
71 that contains binding sites
for PU.1 and Sp1. The PU.1 site is conserved in the rat, murine, and
human mannose receptor promoters, while the Sp1 site is completely
conserved in both rat and mouse sequences (27, 28). In addition, these
sites are located within the first 108 bp of the mannose receptor
promoter and are in close proximity to each other. All of these
features have been suggested to be important for control of
myeloid-restricted expression (14).
In gel shift assays using the labeled 108/71 oligonucleotide, we
observed both Sp1 and PU.1 binding (Fig. 6, lane 1). These factors were competed by excess unlabeled consensus oligonucleotides and the protein-DNA complexes were supershifted with the appropriate antibodies. PU.1 binding was not seen in extracts from non-myeloid cells such as A549, Rat II, or HeLa cells (data not shown), while Sp1
binding was observed in all cells examined, supporting the reported
expression patterns of these factors (51, 52).
PU.1 is a member of the ets family of transcription factors (16). PU.1
binding has been reported to be required for activity of a number of
other myeloid promoters including the promoters for growth factor
receptors (18, 19), integrins (20, 21), Fc receptors (22, 23), the
scavenger receptor (24), myeloperoxidase (25), and human proteinase-3
(26). The rat mannose receptor promoter contains three PU.1 sites
within the first 163 bases 5' of the transcriptional start site. We
examined the function of two of these sites by transfection assays
using site-directed and truncated mutants of the MR854 construct.
Mutation of the PU.1-106 site reduced promoter activity by
approximately 80% of MR854, while mutating the PU.1 site at
163
resulted in a 25% decrease. Deletion of the 5' 622 bp resulting in
MR228 had little effect on promoter activity compared with MR854. This
region contains the three PU.1 sites, and appears to contain all of the
required sequences for maximal activity of the mannose receptor
promoter. Removal of an additional 124 5' bp (MR108) which includes one PU.1 site reduced activity by approximately 50%. Removal of the region
containing the PU.1-106 site (MR48) further reduced activity to 25%
of MR854. The MR48 construct retained myeloid activity and showed no
activity in non-myeloid cells. These results suggest that all three
PU.1 sites contribute to the control of myeloid-specific transcription
driven by the mannose receptor promoter.
One characteristic feature of most myeloid promoters studied to date is
the cooperation between the PU.1 site and another factor bound to a
nearby site (14). Sp1 has been found to function as this second factor
in a number of myeloid-specific promoters including the tyrosine kinase
tec (53), CD11b (54), and c-fes (55). Sp1 was shown to be
important in the mouse mannose receptor promoter, although the distance
between the identified functional PU.1 site (at
163 in the rat
sequence numbering) and the Sp1 site is significantly greater than is
typical for myeloid promoters. It is possible that in the murine
promoter the
106 PU.1 site is active in regulating promoter activity
and cooperates with Sp1 binding at
91 to produce maximal activity,
although this has not been tested. We examined the role of Sp1 binding
in the regulation of rat mannose receptor promoter activity by
transfection of MR854 with a mutation in the Sp1 binding sequence.
Surprisingly, mutations within this site showed only a slight decrease
(10%) in mannose receptor promoter activity and a mutation in the Sp1 site did not further decrease the reduced activity of the PU.1-106 mutant, suggesting that Sp1 does not play a role in the regulation of
rat mannose receptor promoter activity. It is possible that this
difference between the rat and murine studies is due to the longer
promoter used for our studies or the use of mannose receptor-positive macrophages. However, another possible explanation is that an additional factor is functional in the rat mannose receptor promoter as
discussed below.
Binding of a third protein to the 108/71 oligonucleotide was detected
in our gel shift analyses. Through competition using portions of the
108/71 oligonucleotide, protein binding was localized to the E box
(CATGTG) between positions
84 to
79. Mutation of the
80TG
79 to CC resulted in an oligonucleotide
that did not bind this factor (data not shown), suggesting that these
two bases are required for binding. The identity of this factor as the
upstream stimulatory factor (USF) was confirmed by use of specific
anti-USF1 and anti-USF2 antibodies to supershift the USF·DNA complex.
In transient transfection assays, mutation of the TG residues resulted
in loss of approximately 80% of promoter activity, and expression of a
dominant negative USF decreased mannose receptor promoter activity by
50%, suggesting that USF binding is required for a functional rat
mannose receptor promoter.
USF belongs to the family of basic helix loop helix transcription
factors (40). Although these factors are ubiquitously expressed, an
increasing number of studies have appeared in the past several years
demonstrating a role for USF in the regulation of tissue-specific
genes. USF binding has been shown to be required for human
immunodeficiency virus type-1 enhancer activity in T cells (56) and for
regulation of expression of the surfactant protein-A gene (57).
Involvement of USF in myeloid cell differentiation was suggested by
Kreider et al. (58) and Suzow and Friedman (25) reported
that deletion of the E box in the myeloperoxidase gene reduced promoter
activity by 5-fold. It is not yet known how USF might contribute to
myeloid-specific control. In a recent study Feinman et al.
(23) reported that USF bound to an E box region in the Fc
RIII
promoter and that binding of both USF and PU.1 were required for
promoter activity. The PU.1 and USF proteins interacted to form a high
affinity DNA-binding complex that appeared to be responsible for the
myeloid specific activity. These authors further speculated that
myeloid specificity resulted from post-translational modifications of
either or both factors. This is supported by the finding that
phosphorylation of Sp1 and PU.1 are increased as myeloid cells
differentiate (52, 59). A second mechanism suggested by Sieweke
et al. (56) suggests that ets-1 and USF-1 interact to form a
complex, resulting in exposure of the respective DNA-binding domains of
each factor. According to both of these models a ternary complex would
exist that should be detectable by EMSA. We did not detect such a
complex in our assays, although the possibility remains that under the
specific conditions of our assay this complex was not detectable. A
third model recently proposed by Rigaud et al. (60) might
explain the mutually exclusive binding that we observed in gel shift
assays with the rat mannose receptor promoter probe. In this model the
tissue-specific factor PU.1 binds transiently to the promoter and
induces a conformational change in the chromatin which in turn exposes
the binding element of a ubiquitous factor, in this case USF.
Studies of myeloid promoters, including the mannose receptor promoter,
have shown that a variety of ubiquitous factors such as Sp1 and USF
interact with the myeloid-specific factor PU.1 to control
transcription, suggesting that these ubiquitous factors share a common
function. In support of this hypothesis, Pugh and Tjian (61) have
proposed that Sp1 is involved in the assembly of the basal initiation
factors, particularly through interactions with TFIID. The role of Sp1
in pre-initiation complex formation has been demonstrated on both
TATA-containing and TATA-less promoters (62). Similar experiments have
shown that USF can function in the same manner, through interactions
with TFIID (63). This interaction appears to mediate the binding of
TFIID to the promoter, even in the absence of a functional TATA box. A
comparison of binding elements in the rat, human, and murine mannose
receptor promoter sequences shows complete conservation of the core Sp1 site in mouse and rat while the USF site is present only in the rat and
human sequence. Furthermore, the Sp1 and USF sites are nearly
overlapping suggesting that the function of these factors is
position-dependent. The fact that the ubiquitous factor Sp1 is critical for mouse mannose receptor promoter activity while USF is
required for rat promoter activity suggests that these two factors may
be performing the same function in the two different species: namely,
recruitment and stabilization of the pre-initiation complex.
In the present study we have demonstrated that maximal tissue specific
activity is contained within the proximal 228 base pairs of the rat
mannose receptor promoter. This region contains three PU.1 sites and a
functional USF site. We propose that all three PU.1 sites are required
for maximal expression. Mutation of PU.1-106 has the most marked
effect, decreasing activity by approximately 80%. Mutation of the E
box, which binds the ubiquitous factor USF, also decreases activity by
approximately 80% while the expression of a dominant negative USF
decreases activity by 50%. This finding is surprising in light of a
recent study on the murine mannose receptor promoter which demonstrated
a requirement for the ubiquitous factor Sp1. We demonstrate by EMSA
that protein binding to the promoters of the rat, mouse, and human
mannose receptor promoters are indeed different, with the most obvious difference being the absence of USF binding to the mouse promoter. We
speculate that USF and Sp1 provide an essential and overlapping function that is required for activation from these promoters. Further
studies will be required to determine the role that these transcription
factors play in the different species.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grants HL55977 (to V. L. S.) and EY02853 (to Dr. B. McLaughlin, University of Louisville School of Medicine).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF121966.
**
To whom correspondence should be addressed: Dept. of Veterans
Affairs Medical Center/Research Service; 1310 24th Ave. South, Nashville, TN 37212. Tel.: 615-327-4751 (ext: 5499); Fax: 615-321-6305; E-mail: virginia.l.shepherd{at}vanderbilt.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
bp, base pair(s);
CMV, cytomegalovirus;
MR, mannose receptor;
USF, upstream stimulatory
factor;
EMSA, electrophoretic mobility shift assays;
PCR, polymerase
chain reaction.
 |
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