From the Departments of Pathology and Molecular
Biology and Pharmacology, Washington University School of Medicine,
St. Louis, Missouri 63110
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ABSTRACT |
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We previously noted that the initial receptor by
which murine osteoclast precursors bind matrix is the integrin
The osteoclast is a physiological polykaryon, related to, but
distinct from, foreign body giant cells (1). Both multinucleated cells
are derived by fusion of macrophages in a process apparently requiring
attachment of the mononuclear precursors to matrix (2, 3). Cell-matrix
interactions are mediated by several classes of receptors, including
the family of heterodimers known as integrins (4-6). The integrin
We have demonstrated that murine osteoclast precursors initially
express and utilize the integrin In order to study the molecular mechanism by which integrin
Cloning of a Full-length Murine Integrin Isolation of Murine Integrin Primer Extension Analysis--
Primer extension was performed
using total RNA prepared from mouse osteoclast precursors (bone marrow
macrophages) and a 30-mer oligonucleotide complementary to a sequence
23 bp downstream of the 5'-end of the longest cDNA clone (Fig. 1).
Excess oligonucleotide, end-labeled with [32P]ATP, was
hybridized for 90 min at 65 °C with total RNA (previously denatured
for 5 min at 95 °C). Annealed primer was extended with SuperScript
II reverse transcriptase (Life Technologies, Inc.) at 42 °C for 60 min. The reaction product was treated with DNase-free RNase at 37 °C
for 15 min to remove the RNA templates, and the resulting mixture was
separated on a denaturing gel, with an unrelated and previously defined
marker to establish the size of specific bands.
S1 Nuclease Analysis--
S1 nuclease experiments were performed
using total RNA prepared from mouse bone marrow macrophages and a
single-stranded 70-mer oligonucleotide comprising a sequence
complementary to the first 44 bases of the 5'-end of the longest
cDNA clone, the contiguous 16-base genomic sequence immediately
upstream of the 5'-end of the longest clone, and a 10-base nonspecific
sequence (as a control for nuclease activity; see Fig. 3B).
The oligonucleotide, end-labeled with [32P]ATP, was mixed
with excess total RNA, and the mixture was denatured at 75 °C for 10 min and then incubated overnight at 55 °C. The annealed
oligonucleotide was treated with S1 nuclease (Boehringer Mannheim) at
37 °C for 60 min, and the resulting products were separated on a
denaturing sequencing gel, using an unrelated and previously defined
marker to establish the size of specific bands.
Construction of a
Deletion mutants of the 1-kb fragment were made by using the
exonuclease III/mung bean nuclease kit from Stratagene. The presence of
two unique restriction sites in the multiple cloning sites region, one
generating a 3'-overhang (KpnI) and another generating a
5'-overhang (Mlu I), made it possible to use this method.
Following religation, the sizes of all mutants were determined by
sequencing, using a common primer derived from vector sequence. The
series of mutants generated in this manner is as follows: pGL3( Cell Culture and Transient Transfection--
FDC-P1/MAC11 cells
were cultured in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) containing 10% fetal bovine serum (FBS) with 5%
WEHI cell conditioned medium as a source of interleukin-3. Prior to
transfection, cells were counted and spun down at 1400 × g for 7 min. The medium was then removed, and cells were
resuspended in Opti-MEM (Life Technologies, Inc.) at a concentration of
8.75 × 106 cells/ml. Four hundred microliters of this
cell suspension were transiently transfected, by electroporation at 325 V and 950 microfarads in Gene Pulser II (Bio-Rad) in a 0.4-cm Gene
Pulser cuvette, with 20 µg of reporter plasmid and 0.5 µg of
CMV- Electrophoretic Mobility Shift Assays (EMSAs)--
Nuclear
extracts for EMSAs were prepared as follows. Bone marrow macrophages
were isolated and cultured in minimum Eagle's medium containing 10%
heat-inactivated FBS with daily supply of recombinant mouse macrophage
colony-stimulating factor (R & D Systems; 10 ng/ml) for 2 days in
150-mm Petri dishes (1 × 107 cells were plated in one
dish). Then, the cells were treated with mouse recombinant GM-CSF (R & D Systems) for 48 h. For controls, PBS containing 0.1% BSA was
used. The cells were washed three times with cold PBS and incubated
with 20 ml of PBS containing 5 mM EDTA and 5 mM
EGTA for 30 min on ice. Cells from two plates were scraped with rubber
policemen, pooled, spun down, resuspended in 1.5 ml of cold PBS, and
transferred to 2-ml microcentrifuge tubes. The cells were pelleted in a
microcentrifuge for 30 s, media were removed and the cells were
resuspended in 500 µl of hypotonic lysis buffer (10 mM
Hepes-KOH, pH 7.9, 10 mM KCl, 1.5 mM
MgCl2, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride; dithiothreitol and
phenylmethylsulfonyl fluoride were added freshly). Cells were lysed for
15 min on ice, at which time 32 µl of 10% Nonidet P-40 were added to
the suspension, followed by vortexing of the tube for 15 s and
incubating on ice for 10 min. Nuclei were spun down and resuspended in
100 µl of nuclear extraction buffer (20 mM Hepes-KOH, pH
7.9, 420 mM NaCl, 1.2 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 0.5 mM dithiothreitol,
0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM
4-(2-aminoethyl)-benzenesulfonyl fluoride, 5 µg/ml pepstatin, and 5 µg/ml leupeptin). Dithiothreitol, phenylmethylsulfonyl fluoride,
4-(2-aminoethyl)-benzenesulfonyl fluoride, pepstatin, and leupeptin
were added freshly to the buffer. The extract was kept for 20 min on
ice and spun down in a microcentrifuge. The supernatant (nuclear
extract) was aliquoted, quickly frozen in a dry ice/ethanol bath, and
stored at
All oligos used for gel shift assays were synthesized by Life
Technologies, Inc. Oligos were end-labeled with 32P by T4
polynucleotide kinase (Life Technologies, Inc.). 1 × 105 cpm of probe were incubated with 2 µg of nuclear
extracts in a 20-µl volume of binding reaction (10 mM
Tris-Cl, pH 7.5, 100 mM NaCl, 10% glycerol, 50 ng/ml
poly(dI-dC)) on ice for 30 min. In competition experiments, different
amounts of cold competitors were premixed with equal counts of hot
probe before being added to the binding mixture. The binding mixture
was separated at 300 V for 3.5 h by 4% high ionic strength native
polyacryamide gel with an acrylamide:bis ratio of 80:1 as described
(17), using a Hoefer SE 600 series gel unit. The gels were transferred
to 3 M blotting paper, dried, and exposed to film with an
intensifying screen at Site-directed Mutagenesis--
The point mutations were
introduced in the context of the 1-kb promoter (pGL3-1kb+)
using a site-directed mutagenesis kit (Stratagene). Oligos used
are 5'-CCCACAAGTGGtTtAGCTGTtAtAGCCACCTGGG-3' and
5'-CCCAGGTGGCTaTaACAGCTaAaCCACTTGTGGG-3'. Lowercase letters indicate the mutation sites. The oligos were purified by polyacrylamide gel electrophoresis. PCR was performed in a 50-µl volume with Pfu polymerase (Stratagene), 10 ng of pGL3-1kb+ (template),
and 125 ng of each oligo under the following conditions: 1 cycle at 95 °C for 30 s; 16 cycles at 95 °C for 30 s, 55 °C
for 1 min, and 68 °C for 12 min; and parking at 4 °C. The PCR was
treated with Dpn I (10 units) for 60 min at 37 °C. XL1-Blue
supercompetent cells were transformed with the Dpn I-treated PCR
mixture as described in the instruction manual and plated on Ampicillin
plates. Plasmids were prepared from individual colonies and sequenced
to confirm the correctness of the introduced mutations.
Northern Analysis--
Mouse bone marrow macrophages were
isolated and cultured in minimum Eagle's medium containing 10%
heat-inactivated FBS with daily supply of recombinant mouse macrophage
colony-stimulating factor (10 ng/ml; R & D Systems) for 2 days. Cells
were treated with recombinant mouse GM-CSF (10 ng/ml; R & D Systems)
for 6 h (PBS containing 0.1% BSA was used for the control). Total
RNA was prepared from these cells using Trizol Reagent (Life
Technologies, Inc.). For cycloheximide treatment experiments,
macrophages were treated with GM-CSF (10 ng/ml) 30 min after the
addition of cycloheximide (5 µg/ml). Cells then were cultured for
6 h before total RNA was isolated. 10 µg of each sample was
separated on 1% agarose gel, and Northern analysis was performed using
NorthernMax kit (Ambion, Austin, TX). A 1.4-kb mouse DNA Sequence Analysis--
Sequence analysis was performed using
the Genetic Computer Group sequence analysis software.
Molecular Cloning of a Full-length Murine Isolation of Murine Identification of the Transcriptional Start Site--
To identify
the 5'-end of the Sequence Analysis and Activity of Promoter Region--
A 1-kb
genomic fragment, derived from the original 9-kb genomic cDNA by
restriction digestion and shown, by Southern analysis, to include the
5'-UTR of the GM-CSF Represses Transcriptional Activity of the
To further localize the region responsible for GM-CSF-mediated
inhibition of Identification of a 19-bp Sequence within the 109-bp Region
Mediating the GM-CSF-dependent Inhibition of
These EMSA-derived data provide information only on the capacity of the
sequence to bind nuclear proteins in vitro. In order to
determine whether the 19-bp fragment defined by our EMSA studies is
indeed functional in mediating GM-CSF inhibition of
We compared our 19-bp fragment with consensus sequences for
transcription factors in a data base (Sitesdata.gcg) obtained from the
National Center for Biotechnology Information (anonymous FTP site:
ftp://ncbi.nlm.nih.gov/repository/TFD/datasets/), using the Findpattern
program of the Genetic Computer Group sequence analysis package. We
found that this 19-bp sequence contains putative binding sites for
the transcription factors E2A (25), estrogen receptor (26), and
GT-IIBa-SV40 (27).
Inhibition of the During authentic osteoclastogenesis, expression of the integrin
The coding region of the human integrin We were concerned because our EMSA experiments had been performed using
BMMs as a source of protein, whereas our transfections used exclusively
the FDC-P1 cell line. To address this issue, we repeated the EMSA
studies using oligos I-IV and nuclear proteins from FDC-P1 cells
treated with either GM-CSF or PBS-containing medium as control. In all
instances, the pattern of bands was identical to that obtained using
BMMs (data not shown), results that buttress the approach of using
primary cells and transformed lines to dissect the mechanism of GM-CSF
regulation of the We initially attempted to determine whether the 19-bp GM-CSF-responsive
element in the GM-CSF is a member of a cytokine superfamily that utilizes a number of
major pathways, including those involving c-Myc (36), STAT5 (36), IRS-2
(37), and Ras (36), to transduce receptor-mediated signaling from the
cell surface to the nucleus. STAT proteins are transcription factors
that, in their inactive form, reside in the cytoplasm (38-40). Binding
of a wide range of cytokines to their cognate plasma membrane receptor
activate members of the Janus kinase family, the major substrates of
which are STAT proteins. Phosphorylated STATs dimerize and translocate
to the nucleus, where they almost always activate transcription (40). STAT5, which exists in both mice and humans as two isoforms, STAT5A and
STAT5B (41, 42), in other circumstances mediates
GM-CSF-dependent transcription (40). Although the
v
5 and that granulocyte-macrophage
colony-stimulating factor (GM-CSF) decreases expression of this
heterodimer by suppressing transcription of the
5 gene.
We herein report cloning of the
5 integrin gene promoter
and identification of a GM-CSF-responsive sequence. A 13-kilobase (kb)
genomic fragment containing part of the
5 gene was
isolated by screening a mouse genomic library with a probe derived from
the most 5'-end of a murine
5 cDNA. A combination of
primer extension and S1 nuclease studies identifies two transcriptional start sites, with the major one designated +1. A 1-kb subclone containing sequence
875 to + 110 is transcriptionally active in a
murine myeloid cell line. This 1-kb fragment contains consensus binding
sequences for basal (Sp1), lineage-specific (PU.1), and regulatable
(signal transducer and activator of transcription) transcription
factors. Reflecting our earlier findings, promoter activity is
repressed in transfected myeloid cells treated with GM-CSF. Using
deletion mutants, we localized a 109-base pair (bp) promoter region
responsible for GM-CSF-inhibited
5 transcription. We
further identified a 19-bp sequence within the 109-bp region that binds
GM-CSF-induced nuclear proteins by gel shift/competition assays.
Mutation of the 19-bp sequence not only ablates its capacity to bind
nuclear proteins from GM-CSF-treated cells, in vitro, but
the same mutation, when introduced in the 1-kb promoter, abolishes its
ability to respond to GM-CSF treatment. Northern analysis demonstrates
that cycloheximide treatment abrogates the capacity of GM-CSF to
decrease
5 mRNA levels. In summary, we have
identified a 19-bp cis-element mediating GM-CSF-induced down-regulation
of
5 by a mechanism requiring protein synthesis.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
v
3 plays a critical role in osteoclastic bone resorption (7-10). In contrast, the integrin
v
5, although closely related to
v
3 in structure and sharing many of the
same target ligands (4), is not expressed on mature bone resorbing cells (11).
v
5 and
not
v
3 for attachment to matrix (12). In
this circumstance,
v
3 and
v
5 levels rise and fall, respectively,
during formation of bone-resorbing polykaryons. The reciprocal changes
in the two functional integrins are replicated by treating osteoclast
precursors with GM-CSF,1 a
cytokine that is important for osteoclastogenesis (13-15). Reflecting their surface heterodimers,
5 and
3
mRNAs fall and rise, respectively, in response to GM-CSF, whereas
those of
v are unaltered. GM-CSF-mediated inhibition of
5 expression reflects decreased transcriptional activity
of the
5 gene (12).
5 gene is regulated by the cytokine, we have isolated a
5 integrin genomic fragment containing the
transcriptional start sites and a portion of the regulatory domain of
the gene. We demonstrate that the cloned sequence exhibits promoter
activity and responds to GM-CSF in the same manner as the intact
promoter in osteoclast precursors. Finally, we identify a 19-bp
sequence within the
5 promoter that mediates the GM-CSF
responsiveness of this gene. This sequence does not contain consensus
sequences for any transcription factor known to be regulated by GM-CSF
and, as such, represents a novel GM-CSF response element.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
5
cDNA--
Human
5 integrin cDNA (provided by
Dr. Eric Brown, Washington University School of Medicine) was labeled
with digoxigenin to screen a 5'-stretch mouse kidney cDNA library
(CLONTECH), and three clones were isolated and
sequenced by dideoxynucleotide chain termination (16). Because none
contained a translational start site, the most 5' sequence available
was used to screen a mouse brain cDNA library by the GeneTrapper
Screening Method (Life Technologies, Inc.). Several new clones were
isolated and completely sequenced.
5 Subunit Genomic
Clones--
A mouse genomic library in FIX II vector (Stratagene) was
screened with a 230-bp digoxigenin-labeled fragment derived from the
most 5'-end of the longest cDNA clone. One positive clone, identified after examining 500,000 plaques, was purified by secondary and tertiary screenings. Phage DNA was prepared from the clone and was
digested with NotI, resulting in two DNA fragments of 4 and
9 kb. Both fragments were subcloned into a pBluescript plasmid. Southern blot analysis showed that the 9-kb fragment positively reacted
with the 230-bp probe derived from the most 5'-end of the longest
cDNA clone.
5 Promoter-luciferase Reporter
Plasmid and Deletion Mutants--
Sequence analysis indicates that
there is a AccI site in the 5'-UTR of the full-length clone.
A 30-mer oligo complementary to a sequence upstream of the
AccI site was synthesized and end-labeled with digoxigenin.
The 9-kb genomic fragment was digested with AccI, and the
products were separated in an agarose gel, transferred to
nitrocellulose, and hybridized with the 30-mer probe. A 1-kb fragment
from the digestion reacted with the probe. Based on our screening
strategy, this 1-kb fragment, which should contain the transcriptional
start site, was subcloned into pGL3-basic plasmid containing the
luciferase reporter gene, to construct a reporter plasmid named
pGL3-1kb(+). A control reporter plasmid, pGL3-1kb(
), was also
obtained by placing the 1-kb
5 promoter fragment in antisense orientation in front of the luciferase gene.
796), with a sequence from
796 to +110; pGL3(
726), from
726 to +110; pGL3(
633), from
633 to +110; pGL3(
483), from
483 to +110; pGL3(
340), from
340 to +110; pGL3(
172), from
172 to +110; pGL3(
63), from
63 to +110; pGL3(
28), from
28 to +110; and pGL3(+10), from +10 to +110.
-gal as a control plasmid for transfection efficiency.
Transfected cells were grown in Dulbecco's modified Eagle's medium
containing 10% FBS with 5% WEHI cell conditioned medium in 6-well
tissue culture dishes for 16 h. Cell lysates were prepared, and
luciferase activities were measured using the luciferase assay kits
from Promega, with normalization to
-gal activities measured
separately. In experiments utilizing GM-CSF treatment, FDC-P1/MAC11
cells were cultured in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) containing 10% FBS with 5 ng/ml recombinant mouse
(R & D Systems). When cell density reached 5 × 105/ml, recombinant mouse GM-CSF (R & D Systems) was added
to the cell culture at 10 ng/ml. For controls, PBS containing 0.1% BSA was used. After 24 h, cells were counted and spun down at
1400 × g for 7 min. The culturing medium was then
removed and saved, and the cells were resuspended in Opti-MEM (Life
Technologies, Inc.) at a concentration of 12.5 × 106
cells/ml. 0.4 ml of this cell suspension was transiently cotransfected by electroporation at 325 V and 950 microfarads in Gene Pulser II
(Bio-Rad) in a 0.4-cm Gene Pulser cuvette, with 20 µg of reporter plasmid and 0.5 µg of CMV-
-gal as a control plasmid for
transfection efficiency. The transfected cells were grown in 6-well
tissue culture dishes with 3 ml of the saved culturing medium per well for 16 h. Luciferase and
-gal activities were measured as
described previously.
70 °C. Protein concentration of nuclear extracts was
determined using the Micro BCA kit (Pierce).
70 °C.
5
cDNA fragment was labeled by BrightStar Psoralen-Biotin labeling
kit (Ambion) and used as a probe in the Northern analysis (30 ng/ml).
The signal was detected using BrightStar nonisotopic detection system
(Ambion). The blot was stripped and reprobed with 1.3-kb rat GAPDH
probe (30 ng/ml, labeled in the same way as the
5 probe)
to normalize loading. To analyze the intensity of each band on the
autoradiograms, the x-ray films were subjected to image analysis using
ISS SepraScan 2001 (Integrated Separation Systems, Natick, MA).
5 mRNA levels were normalized to the GAPDH signal.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
5
cDNA--
Because integrin cDNA sequences are highly conserved
across species (18, 19), we labeled a human
5 cDNA
(20-22) and used it as a probe to screen more than 1,000,000 plaques
derived from a 5'-stretch mouse kidney cDNA library
(CLONTECH), which yielded three positive clones.
Phage DNA was prepared, and the inserts were subcloned into pBluescript
plasmid. Sequence analysis revealed that all three clones are
incomplete, each terminating at +400, based on the human
5 sequence. Because we lacked sequence coding for the
5'-end of the cDNA, we used the GeneTrapper method (Life Technologies, Inc.) to rapidly screen a large number of clones. This
approach, using as a probe the 5' sequence derived from our first round
of cloning, resulted in isolation of several new clones, the longest
containing 294 bp of the 5'-untranslated region, the entire coding
sequence, and 443 bp of the 3'-untranslated region, including a
polyadenylation sequence (Fig. 1)
(sequence submitted to GenBankTM; accession number
AF022110). The deduced amino acid sequence is more than 90% identical
to the human
5 protein (not shown).
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Fig. 1.
Nucleotide and amino acid sequence of a
cDNA for mouse integrin 5 subunit
(GenBankTM accession number AF022110). A 3104-bp
murine integrin
5 cDNA clone was completely
sequenced. This sequence comprises a 294-bp 5'-untranslated region
(1-294), a 2367-bp coding region (295-2661), and a 443-bp
3'-untranslated region (2662-3104). The translational start (ATG) and
stop (TGA) codons are underlined. The deduced amino acid
sequence is shown below the nucleotide data. The
polyadenylation sequence is double underlined. Location of
the primer (PE primer) used for the primer extension
experiment (Fig. 2) is also shown below its complementary
sequence (bp 24-54).
5 Integrin Subunit Genomic
Clones and Sequence Analysis of the 5'-Upstream Region--
Primer
extension was performed to determine whether the largest
5 cDNA clone contains the complete 5'-untranslated
region. The size of oligonucleotide used as primer (30 bp), plus the
amount of untranslated sequence 5' of the ligation site of this primer (23 bp) would predict formation of a product of at least 53 bp (Fig.
1). A single band corresponding to a 59-mer oligonucleotide was
detected (Fig. 2), indicating that the
available
5 cDNA clone lacks 6 bases at its 5'-end.
A 230-bp fragment derived from the most 5'-end of this cDNA was
labeled and used to screen a mouse genomic library. After screening
500,000 plaques, one positive clone was identified, from which phage
DNA was prepared. Restriction digestion analysis reveals the phage
contains a 13-kb insert. A restriction map for the 13-kb was created by
restriction digestion and Southern analysis (Fig.
3A). Digestion of the phage
DNA with NotI yielded two fragments, 4 and 9 kb. Both
fragments were subcloned into pBluescript plasmid. By Southern
analysis, a probe containing the translational start site hybridized to
the 9-kb clone. Sequencing of a portion of this 9-kb fragment, using
primers (initially based on one vector arm and subsequently based on
generated sequences), provided approximately 1000 bases representing,
in the 3' to 5' orientation, a 30-bp intron, 70 bp coding for the known
23-amino acid leader sequence, and a portion of the 5'-untranslated
region. The combined results yielded a region subsequently identified as the proximal regulatory domain of the
5 integrin gene
(see Fig. 3B). Given the contiguous nature of the regulatory
domain, 5' untranslated region and leader sequences, which end in the canonical AG sequence typical of intron/exon splice boundaries, this
genomic fragment contains the first exon, which codes for the entire
5'-UTR and leader peptide, and the first base of the following codon.
The size of the first intron, of which we have sequenced only 30 bp, is
unknown.
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Fig. 2.
Identification of the 5
cDNA 5'-UTR by primer extension analysis. Total RNA prepared
from murine bone marrow macrophages was hybridized with an end-labeled
30-mer oligonucleotide complementary to a sequence 23 bp downstream of
the 5'-end of the longest
5 cDNA clone (see Fig. 1).
Annealed primer was extended with Superscipt II reverse transcriptase
(Life Technologies, Inc.). The reaction products were treated with
DNase-free RNase, separated on a sequencing gel, transferred onto
filter paper, dried, and exposed to x-ray film. A single band, the size
of which corresponded to a 59-mer oligonucleotide, was detected
(lane 2). Lane 1 is an unrelated sequencing
reaction used as a DNA marker.
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Fig. 3.
Molecular cloning of a promoter for murine
integrin 5 subunit gene. A, restriction
map of a 13-kb genomic clone containing part of the
5
integrin gene. Exon 1 is indicated by a hatched box.
B, DNA sequence of the 1-kb integrin
5
promoter region (GenBankTM accession number AF022111).
Transcription factor binding sites are underlined. The
109-bp GM-CSF-responsive region, identified by transfections with the
deletion mutants (see Fig. 7), is double underlined. The
19-bp GM-CSF-responsive sequence (identified in Figs. 9-11) is
indicated by asterisks. Location of the oligos used for the
primer extension experiment (PE primer) and the S1 nuclease
assay (S1 nuclear assay oligo) (see Fig. 4) are also shown.
Arrow a indicates the major transcriptional site (designated
+1), corresponding to band A in Fig. 4. Arrow b
indicates an alternative transcriptional start site, corresponding to
band B in Fig. 4. Arrow c shows the
transcriptional start site identified by primer extension experiment
(Fig. 2). Arrow d marks the end of the sequenced cDNA
clone (Fig. 1).
5 cDNA we turned to S1 nuclease
protection (Fig. 4). Using this
technique, two bands were detected, suggesting that this gene may have
two transcriptional start sites, a documented feature of TATA-less
promoters (23). The more prominent band, corresponding to a
transcriptional start site that is 5 bp more upstream than that
predicted by primer extension (Figs. 2 and 3B), identifies a
major transcriptional start site, designated +1 (Fig. 3B).
The discrepancy between this result and that obtained by primer
extension (Fig. 2) probably reflects secondary structure near the
GC-rich transcriptional start site inhibiting primer extension.
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Fig. 4.
Confirmation of the position of
5 cDNA 5'-UTR by S1 nuclease assay. Total RNA
prepared from BMMs was subjected to S1 nuclease protection assay using
an end-labeled 70-mer oligonucleotide containing a sequence
complementary to the first 44 bases of the 5'-end of the longest
5 cDNA clone, a sequence complementary to the
16-base genomic sequence immediately upstream of the 5'-end of the
longest clone, and a 10-base nonspecific sequence (see Fig.
3B). The hybridized and digested mixture was separated on a
denaturing sequencing gel, transferred to a membrane, dried, and
autoradiographed. Lane 1, unrelated sequencing reaction used
as DNA marker; lane 2, labeled oligonucleotide hybridized
with the total RNA, without subsequent S1 nuclease treatment. The
arrow indicates the location of the intact oligonucleotide.
Lane 3, bands A and B represent a 55- and a 54-bp oligo, respectively. This result suggests that two
transcriptional start sites may exist.
5 cDNA, was subcloned into pGL3-basic, which contains the reporter gene luciferase. The resultant 1-kb plasmid, pGL3-1kb (+), contains 875 bp upstream and 110 bp downstream of the transcriptional start site. The putative 875-bp promoter includes no TATA box, but two Sp1 sites are located at
27 to
18 and
192 to
283. Putative STAT binding sites are found at
528 to
520
and
402 to
393, whereas PU.1 recognition sequences are present at
778 to
773,
772 to
767,
504 to
499, and +68 to +73 (Fig.
3B). To determine whether the cloned region is an active
promoter, we transfected FDC-P1/MAC11 cells with either the 1-kb
fragment pGL3-1kb(+) or pGL3-1kb(
), in which the same genomic
fragment was placed, in the same reporter, in the antisense orientation. As controls, we transfected the promoterless pGL3-basic and pGL3-promoter (SV40 promoter). Luciferase activity was normalized to
-gal content of cell extracts reflecting transfection with CMV-
-gal. As seen in Fig. 5, the 1-kb
5 upstream fragment is transcriptionally active in
murine myeloid progenitor cells. To locate the region essential for
transcriptional activity, FDC-P1/MAC11 cells were transfected with
pGL3-basic, pGL3-1kb(
), pGL3-1kb(+), or a series of nested deletion
constructs of the
5 promoter, pGL3(
726),
pGL3(
340), pGL3(
172), pGL3(
63), pGL3(
28), and pGL3(+10). The
results indicate that basal promoter activity is regulated by a region
located between
28 and +10 (Fig. 5), a sequence containing a
consensus domain for the basal transcription factor Sp1. Additional
sequences may act as modest enhancers (e.g. pGL3(
172)) or
silencers (e.g. pGL3(
340)) of basal
5
integrin transcription.
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Fig. 5.
The cloned 1-kb 5 integrin
promoter is transcriptionally active when transfected into murine
myeloid progenitor cell line FDC-P1/MAC11 cells. Cells were
transfected with the plasmids pGL3-SV40, pGL3-basic (promoterless),
pGL3-1kb(
), pGL3-1kb(+), pGL3(
726), pGL3(
340), pGL3(
172),
pGL3(
63), pGL3(
28), pGL3(+10), and CMV-
-gal, with luciferase
activity normalized to that of
-gal. The experiment was repeated
three times, and a representative result is shown. Each bar
is the mean of three replicates ± S.D.
5
Integrin Proximal Promoter Region via a 109-bp Region Not Encompassing
STAT Binding Domains--
GM-CSF transcriptionally down-regulates the
5 integrin gene in mouse marrow osteoclast precursors
(12). Because FDC-P1/MAC11 cells are GM-CSF-responsive (24), we
transiently transfected this line with pGL3-1kb(+) and CMV-
-gal
reporter constructs. Cells were exposed to vehicle or GM-CSF (10 ng/ml
for 24 h) prior to transfection. Luciferase assay demonstrates
that the 1-kb promoter fragment contains sequences that mediate GM-CSF
inhibition of
5 transcription (Fig.
6).
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Fig. 6.
The 1000-bp proximal 5
promoter confers GM-CSF-mediated repression of integrin
5 gene expression. FDC-P1/MAC11 cells were
transfected with pGL3-promoter (SV40), pGL3-1kb(+), or CMV
-gal,
with luciferase activity normalized to that for
-gal. The experiment
was repeated three times, and a representative result is shown. Each
bar is the mean of three replicates ± S.D.
5 integrin gene expression, we repeated
our transfection assays, using a series of
5 promoter
deletion mutants. Once again cells were treated with vehicle or GM-CSF
prior to transfection. Whereas the activity of constructs pGL3(
796),
pGL3(
726), pGL3(
633), pGL3(
483), pGL3(
340), and pGL3(
172) is
inhibited by GM-CSF, that of the genomic fragment lacking
172 to
63
is not (Fig. 7). Thus, we have identified
a 109-bp sequence mediating GM-CSF-dependent repression of
the
5 integrin gene. This promoter region (
172 to
63) does not contain the two STAT-like sequences located at
528 to
520 and
402 to
393 (Fig. 3B).
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Fig. 7.
Localization of a 109-bp region mediating
GM-CSF-dependent repression of the integrin
5 gene transcription. FDC-P1/MAC11 cells were
cultured, transfected with a series of deletion constructs of the
5 promoter, and assayed as described in Fig. 6. The
experiment was independently repeated three times, and a representative
result is shown. Each bar is the mean of three
replicates ± SD.
5 Gene Transcription--
To identify a smaller
sequence mediating GM-CSF responsiveness, we synthesized four
overlapping oligos (oligo I, II, III, and IV) spanning the previously
defined 109-bp region (Fig.
8A). These short DNA fragments
were end-labeled with 32P and used for EMSA studies.
Nuclear extracts used were prepared from bone marrow macrophages
treated for 48 h with GM-CSF (10 ng/ml) or PBS containing 0.1%
BSA (as control). GM-CSF treatment did not alter the pattern of
proteins binding to oligos I, II and IV (Fig. 8B). In
contrast, oligo III bound different nuclear proteins, derived from
cytokine-treated, as compared with control, cells (Fig. 8B).
Thus, untreated extracts, incubated with oligo III, generate a major
band (Fig. 8B, labeled C) and a minor band (labeled B). When proteins from GM-CSF-treated cells were
used, band C disappeared, band B increased in intensity, and a
prominent new band (labeled A) appeared. This result
suggests, but does not prove, that alterations in protein binding by
bases within oligo III may mediate the inhibitory effect of GM-CSF on
transcription of the
5 gene. To obtain additional
information as to the identity of the bases responsible for the
specific EMSA patterns seen in Fig. 8, we synthesized 7 deletion
mutants of oligo III (Fig. 9A) and assessed their capacity to compete with labeled oligo III in EMSA
(Fig. 9, B and C). When untreated nuclear
extracts were used, mutants a, b, and g, but not mutants c-f, were
efficient competitors (Fig. 9B), a finding that suggests
that a 16-bp sequence (bp 8-23, Fig. 9A) can bind the
protein complexes B and C. When GM-CSF-treated nuclear extracts were
used as source of protein, mutants a and g again competed efficiently,
whereas b-f did not (Fig. 9C). This result indicates that a
19-bp sequence (bp 8-26, Fig. 9A) binds the proteins
present in bands A and B. Taken together, the studies using the various
deletion mutations of oligo III, itself derived from the regulatory
region of the murine
5 gene, allow us to conclude that a
19-bp sequence (bp 8-26, Fig. 9A) is responsible for
binding of both basal and GM-CSF-induced nuclear proteins.
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Fig. 8.
Identification of 34-bp sequence within the
109-bp region binding GM-CSF-induced nuclear proteins.
A, schematic locations of the four overlapping oligos (I,
II, III, and IV) spanning the 109-bp region. B, EMSA with
oligos I, II, III, and IV and nuclear extracts from GM-CSF-treated (48 h) or untreated BMMs. Nuclear extracts were incubated with 1 × 105cpm of each oligo labeled with 32P,
separated by polyacrylamide gel electrophoresis, dried, and
autoradiographed.
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Fig. 9.
Identification of a 19-bp sequence within the
34-bp oligo III as sufficient for binding the GM-CSF-induced
nuclear proteins. A, deletion mutants based on oligo
III (a-g). B, EMSA/competition assays with the
deletion mutants shown in A. A 20× or 100× excess of each
unlabeled mutant oligo was premixed with 1 × 105 cpm
of labeled oligo III, prior to EMSA, performed as described in Fig.
8B. C, EMSA/competition assays, as performed in
B, but using nuclear extracts from GM-CST-treated
cells.
5
gene transcription, we first generated a set of point mutations (Fig. 10A) in oligo III that
abolished its capacity to compete in EMSA for binding of nuclear
proteins derived from GM-CSF-treated or untreated BMMs (Fig.
10B). The mutant oligo III (Fig. 10B,
M) failed to compete for binding of the nuclear proteins in
bands A, B, and C (lanes 4, 5, 9, and 10),
whereas wild-type oligo III (III) did so efficiently
(lanes 2 3, 7, and 8). These findings show that
the 19-bp sequence containing the indicated point mutations did not
bind the nuclear proteins in bands A, B, and C. We then introduced the
same point mutations in context of the 1kb promoter (pGL3-1kb(+))
and named the resulting plasmid mpGL3-1kb(+). When mpGL3-1kb(+)
was transfected into FDC-P1/MAC11 cells, its activity was no longer
inhibited by GM-CSF treatment, whereas the wild-type reporter continued
to respond (Fig. 11). Thus, we have
identified a cis-element in the
5 promoter that mediates
down-regulation of transcription of this gene by GM-CSF.
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Fig. 10.
Point mutations in the 19-bp
5 integrin promoter sequence abolish its
ability to bind nuclear proteins from both GM-CSF-treated and untreated
BMMs. A, identity of the four point mutations
introduced into oligo III. The 19-bp sequence identified in Fig. 9 is
boxed; the mutated nucleotides are in lowercase
letters, with their wild-type counterparts above or
below. B, EMSA/competition assays were repeated
as described in Fig. 9, B and C, with wild-type
oligo III (III) or the point mutated oligo III
(M).
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Fig. 11.
Mutation of the 19-bp sequence in the 1-kb
5 promoter abrogates its capacity to mediate GM-CSF
responsiveness. The same four point mutations described in Fig.
10A were introduced in the context of the 1-kb
5 promoter, generating the construct mpGL3-1kb(+).
GM-CSF-treated or untreated FDC-P1/MAC11 cells were transfected with
wild-type promoter (pGL3-1kb(+)) or the mutant form, mpGL3-1kb(+),
with CMV-
-gal plasmid as control and assayed as described in Fig. 6.
These studies were performed four times, with a representative result
shown. Each bar is the mean of three replicates ± SD.
5 Gene by GM-CSF Requires Protein
Synthesis--
GM-CSF treatment of BMMs for at least 6 h dampens
5 gene transcription, thus decreasing
5 mRNA levels (12). To investigate whether
inhibition of
5 transcription by GM-CSF is dependent on
protein synthesis, we exposed bone marrow macrophages to cycloheximide (5 µg/ml) for 30 min prior to treating the cells with GM-CSF for 6 h. Total RNA was isolated, and Northern analysis demonstrated that cycloheximide treatment abrogates the inhibitory effect of GM-CSF
on the
5 gene (Fig. 12).
Thus, GM-CSF down-regulation of
5 gene transcription
requires protein synthesis.
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Fig. 12.
Inhibition of 5 transcription
by GM-CSF is dependent on protein synthesis. A,
Northern analysis. BMMs were treated with the protein synthesis
inhibitor cycloheximide at 5 µg/ml (lanes 3 and
4) for 30 min prior to addition of PBS containing either
0.1% BSA (lanes 1 and 3) or GM-CSF at 10 ng/ml
(lanes 2 and 4). Six hours later, total RNA was
prepared, and Northern analysis was performed using
5
and GAPDH cDNAs probes. B, the intensity of
5 and GAPDH mRNA signals was quantitated using ISS
SepraScan 2001 (Integrated Separation Systems, Natick, MA), and
5 mRNA levels were normalized to GAPDH. The results
are displayed graphically.
DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
v
5 falls in parallel with appearance of
v
3, events mimicked by treatment of
osteoclast precursors with GM-CSF (12). Blunting of
v
5 expression in these cells involves
transcription suppression of the
5, but not the
v, gene. Because GM-CSF, in other circumstances, invariably stimulates gene expression, our observations represent a
novel role for the cytokine, namely transcriptional inhibition.
3 gene contains
14 exons within 46 kb of genomic DNA (28). The 5'-UTR resides in a
separate exon, separated by an uncloned intron (29). Because the
3 and
5 integrin cDNAs are highly
homologous (20), the
5 integrin gene, like that of
3 (28), may bear a large first intron. If this is so, it
would be difficult to isolate a genomic clone containing the promoter
region of the murine
5 integrin gene without identifying
the 5'-untranslated end of the cDNA. With this in mind, our first
exercise was to clone a full-length
5 murine cDNA.
Using the most 5'-untranslated sequence as a probe, we screened a mouse
genomic library and isolated a 13-kb fragment containing part of the
coding region. A combination of primer extension and S1 nuclease
analysis identified two potential transcriptional start sites. Similar
to other integrin promoters (30-33), the
5 gene does
not contain a classical TATA element (25) in the vicinity of the
transcriptional start site. The first kb of the
5
promoter, in contrast, accommodates four consensus sites for the B and
myeloid cell-specific transcription factor, PU.1, which plays an
essential role in osteoclastogenesis (34). Most importantly, the 1-kb fragment bearing the putative transcriptional start site contains a
functional promoter as it enhances transcription at least 15-fold in
transfected cells. Deletion analysis of the initial 1-kb promoter shows
that basal promoter activity resides between
28 and +10, a region
that contains a consensus recognition site for Sp1. Because SP1 can act
as an initiator of transcription in genes lacking a TATA box (35), the
finding of a consensus SP1 binding site suggests that it may function
in this role in the
5 promoter. Most importantly, by a
combination of transient transfection studies and EMSAs, using both
deletion and mutation of the regulatory region of the integrin
5 gene, we have identified a potentially novel 19-bp
sequence responsible for GM-CSF-dependent transcriptional inhibition.
5 gene.
5 promoter can mediate
GM-CSF-dependent inhibitory effects on heterologous
promoters, subcloning three tandem repeats of the 19-bp sequence
upstream of the thymidne kinase minimal promoter. However, when this
reporter plasmid was transfected into FDC-P1/MAC11 cells not treated
with GM-CSF, only background luciferase activity was detected (data not
shown). When the same three copies of the 19 bp sequence were subcloned in front of the SV40 promoter (pGL3-promoter from Promega), high promoter activity was detected in transfected FDC-P1/MAC11 cells. However, GM-CSF treatment does not decrease the activity of this construct (data not shown). SV40 promoter is a strong TATA-box based
viral promoter, whereas integrin
5 promoter is
TATA-less. Such differences may contribute to the failure of the 19-bp
sequence to mediate the inhibitory effect of the cytokine on the SV40
promoter. Thus, it is likely that the characterized GM-CSF-responsive
sequence functions in a promoter-specific manner.
5 promoter contains two STAT-like sequences, they do not
reside in the GM-CSF-responsive sequence and thus play no role in the
mechanism by which the cytokine modulates expression of the gene. To
show that the nuclear proteins binding to the 19-bp sequence are indeed
not STAT5 proteins, we performed EMSA experiments with anti-STAT5A and
anti-STAT5B antibodies and found, as expected, that these reagents fail
to supershift the bands (data not shown). Thus, we conclude we have
identified a GM-CSF response element binding transcription factor(s)
other than STATs. The fact that this response element does not bind proteins known to be involved in transcriptional control by GM-CSF suggests that we may have identified a novel response element.
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FOOTNOTES |
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* 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) AF022110 for murine 5 cDNA and Afo22111 for
genomic sequences.
§ To whom correspondence should be addressed: Dept. of Pathology, Washington University School of Medicine, Barnes-Jewish Hospital North, 216 S. Kingshighway, St. Louis, MO 63110. Tel.: 314-454-8463; Fax: 314-454-5505; E-mail: rossf{at}medicine.wustl.edu.
The abbreviations used are:
GM-CSF, granulocyte-macrophage colony-stimulating factor; UTR, untranslated
region; STAT, signal transducer and activator of transcription; BMM, bone marrow macrophage; EMSA, electrophoretic mobility shift assay; UTR, untranslated region; kb, kilobase(s); bp, base pair(s); FBS, fetal
bovine serum; -gal,
-galactosidase; PBS, phosphate-buffered
saline; BSA, bovine serum albumin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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REFERENCES |
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