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INTRODUCTION |
Type I collagen is the most abundant protein of the bone
extracellular matrix, accounting for 90% of the matrix protein content (1). It is a heterotrimer made of two
1(I) chains and one
2(I)
chain (2). The
1(I) and
2(I) chains are encoded by two distinct
genes that are expressed most highly in two cell types: the fibroblast
and the osteoblast. Moreover, the expression of these two genes is
often regulated by identical transcription factors (3-7). The
type I collagen genes are expressed in osteoblastic cells at
all stages during development and throughout life (8), suggesting that
the factor(s) controlling their expression in these cells could also
control osteoblast differentiation and function. Another possibility,
which is not exclusive of the previous one, is that different
transcription factors may control their expression in osteoblasts at
various stages of development and of postnatal life. Thus, the
elucidation of the molecular mechanisms controlling
1(I)
and
2(I) collagen gene expression in osteoblasts is of
critical importance in understanding how osteoblast differentiation and, thereby, bone matrix deposition by differentiated osteoblasts is
regulated. Ultimately, these studies may shed light on the pathogenesis
of genetically acquired bone diseases and help design appropriate
therapies for some of these diseases.
The critical role that Cbfa1, a Runt-related osteoblast-specific
transcription factor, plays in osteoblast differentiation and function
has been demonstrated in mouse and in human using both molecular and
genetic approaches (9-14). Cbfa1 was identified as a key regulator of
osteoblast-specific gene expression through its binding to the OSE2
element of the mouse Osteocalcin genes 1 and 2 (OG1 and OG2) (9) and other genes expressed in
osteoblasts. The early and cell-specific expression of this gene
together with its biological role in vivo as a factor
required for osteoblast differentiation (9-11), indicate that Cbfa1
must control the expression of multiple target genes that are expressed
earlier than Osteocalcin. Conceivably, these target genes
could include the
1(I) and
2(I) collagen
genes that are expressed early during development. This hypothesis was
confirmed indirectly by the observation that expression of a dominant
negative form of Cbfa1 in differentiated osteoblasts leads to a
decrease in expression of the type I collagen genes in
vivo (14). To date, no osteoblast-specific cis-acting
elements to which Cbfa1 may bind have been identified in these genes.
Two groups have extensively studied the regulation of expression of the
1(I) collagen gene in osteoblasts and have identified a
region in the promoter of the rat and mouse
1(I) collagen
gene that plays an important role in this regulation of expression (15,
16). The sequence of this region bears no homology to a Cbfa1-binding
site, and several homeobox-containing proteins can bind to this
sequence and affect
1(I) collagen expression. However, a
cell-specific transcription factor binding to this region has not yet
been identified. Moreover, no osteoblast-specific cis-acting
element has yet been identified in the
2(I) collagen promoter. Given the large size of these genes and their expression at
multiple stages of osteoblast differentiation, it is likely that
several distinct osteoblast-specific cis-acting elements, besides those already described (15, 16), contribute to the expression
of the type I collagen genes in osteoblast progenitors and/or in fully differentiated osteoblasts. Consistent with this hypothesis, we noticed the existence of two Cbfa1-binding sites (OSE2s)
in the mouse
1(I) collagen promoter and one OSE2 in the mouse
2(I) collagen gene that are conserved among
multiple species. The functional importance of these sites has never
been studied before. Here we present evidence suggesting that Cbfa1 is
one of the factors controlling osteoblast-specific expression of both type I collagen genes.
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EXPERIMENTAL PROCEDURES |
DNA Constructs--
For DNA transfection and generation of
transgenic mice, multimers of double-stranded oligonucleotides (see
Table I) were cloned into the SmaI site upstream of a
chimeric pK1-luc reporter plasmid containing the
1(I)
collagen
86 minimal promoter (4) fused to a luciferase gene. The
expression plasmid was pCMV-Osf2 (9).
Site-specific mutations were created in the intact promoter by using
polymerase chain reaction-directed mutagenesis (17) on a construct
containing 2.4 kb1 of the
1(I) collagen promoter upstream of a luciferase reporter gene. Sole presence of the desired mutations was verified by sequencing.
Cell Culture and DNA Transfection--
COS7, NIH 3T3, HeLa,
C2C12, and 10T1/2 cells were cultured in Dulbecco's minimal essential
medium (Life Technologies, Inc.), 10% fetal bovine serum (Life
Technologies, Inc.). ROS 17/2.8 cells were cultured in Dulbecco's
minimal essential medium-F12 medium (Life Technologies, Inc.), 10%
fetal bovine serum (Life Technologies, Inc.). Twenty hours before
transfection, cells were plated at a density of 5 × 105 cells/dish and allowed to grow under normal culture
conditions. For cotransfection experiments, we used 5 µg of Cbfa1
expression vector or empty vector, 5 µg of reporter plasmid or empty
vector, and 2 µg of pSV
-gal vector using the calcium phosphate
coprecipitation procedure (17). Transfection conditions were identical
to those used in the cotransfection experiments, except that 5 µg of
reporter plasmid were used. Twenty hours following transfection, cells were washed in phosphate-buffered saline and incubated in medium an
additional 24 h. C2C12 cells were changed to media containing 10%
horse serum (Life Technologies, Inc.) and allowed to incubate for
48 h. Cells were collected by scraping into 0.25 M
Tris-HCl, pH 7.8, and lysed by three freeze-thaw cycles.
-Galactosidase and luciferase assays were carried out as described
previously (18).
-Galactosidase assay results were used to normalize
the luciferase assay results for transfection efficiency. All DNA transfection experiments were repeated at least three times in triplicate.
Electrophoretic Mobility Shift Assays--
Nuclear extract from
ROS 17/2.8 cells, primary osteoblasts, and other tissues were prepared
as described previously (19) from 4-day-old wild-type mice and stored
at
80 °C until use. Glutathione S-transferase-Cbfa1 was
purified from transformed Escherichia coli bacteria using
glutathione beads as described previously (17). Double-stranded
oligonucleotides (see Table I) were end-labeled and purified as
previously described (19). 5 fmol of labeled oligonucleotide was
incubated with 7 µg of ROS 17/2.8 nuclear extract or 0.1 µg of
recombinant Cbfa1 protein.
2(I) collagen EMSA
experiments used twice the amount of extract or protein. The incubation
mix for nuclear extract binding assays consisted of binding buffer (100 mM Tris-HCl, pH 7.5, 200 mM NaCl, 4 mM EDTA, 2 mM DTT, 0.2% Nonidet P-40, 10%
glycerol, 5 µg/ml leupeptin, 5 µg/ml pepstatin) (20), 2 µg of
poly(dI-dC), and 0.5 fmol of single-stranded bottom strand
oligonucleotide. Incubation took place at room temperature for 5 min.
Supershift experiments were carried out as described above, except that
the ROS17/2.8 nuclear extract was preincubated for 10 min at room
temperature with an antibody against Cbfa1 in binding buffer prior to
their incubation with the labeled oligonucleotide for 10 min at room temperature.
For recombinant protein binding assays, the incubation mix consisted of
binding buffer (20 mM Tris-HCl, pH 8.0, 10 mM
NaCl, 3 mM EGTA, 5 mM DTT, 0.05% Nonidet P-40)
and 1 µg of bovine serum albumin. Incubation took place at room
temperature for 5 min, followed by the addition of 1 µl of loading
buffer (2 mM Tris-HCl, pH 8.0, 5% glycerol, 0.025% xylene
cyanol, 0.025% bromphenol blue).
The reactions were run on 5% polyacrylamide gel, 0.25× TBE (89 mM Tris base, 89 mM boric acid, 2 mM EDTA, pH 8.0) for 90 min at 160 V. The gels were then
dried and exposed to film at
80 °C.
Generation and Analysis of Transgenic Mice--
The plasmids
described above containing multimers of the
1AB or
1mutAmutB
oligonucleotides were digested, and the insert was purified by two
rounds of agarose gel electrophoresis. Linear DNA inserts were injected
into the pronuclei of fertilized B6D2F1 (Charles River Laboratory)
mouse eggs, which were reimplanted in the oviduct of pseudo-pregnant
CD1 foster mothers (Jackson Laboratories). Transgenic animals were
identified by Southern blots of tail genomic DNA. The transgenic mice
expressing the p4
1AB-luc construct were analyzed as follows: Organs
from 4-week-old F1 animals were dissected and homogenized on ice in a
buffer containing 100 mM potassium phosphate (pH 7.8) and 1 mM dithiothreitol (DTT). Protein homogenates were
centrifuged, and supernatants were assayed for luciferase activity
according to standard procedures (19). Protein levels were measured
using the Bio-Rad protein assay. Relative luciferase activities were
expressed as luciferase light units per 100 µg of protein expressed
as a percentage of the activity in bone.
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RESULTS |
The
1(I) collagen Promoter Contains Two Conserved OSE2
Sites--
DNA sequence inspection identified three potential OSE2
sites in the promoter of the murine
1(I)
collagen gene. Two are located side-by-side at positions
1347
and
1338 in the mouse gene (Fig. 1A), and a third site is found
at
372 in the mouse gene (Fig. 1A). These potential OSE2
sites were termed
1A,
1B, and
1C, respectively (Fig.
1B). The presence of
1A and
1C, but not of
1B, at
approximately the same location in the
1(I)
collagen promoter sequences of rat and human (Fig. 1A)
suggested a biological role for these sites and led us to study these
two regions. To determine whether Cbfa1 could bind to the OSE2-like
sequences in the mouse
1(I) collagen promoter,
we generated double-stranded oligonucleotides to be used in DNA binding
assays (Table I). One of them, called
1AB, contains the
1A and
1B sites and their surrounding
sequences. A second one, termed
1wtAmutB, contains the wild-type
1A site and a mutated
1B site. A third oligonucleotide, called
1mutAwtB, carries a mutated
1A sequence and a wild-type
1B
sequence. A fourth oligonucleotide,
1mutAmutB, contains mutations in
both the
1A and the
1B sites. Two other oligonucleotides, termed
1C and
1mutC, were generated to test the binding activity of the
1C site. The mutations introduced into all the oligonucleotides mentioned above have previously been shown to abolish binding of
nuclear extract or recombinant Cbfa1 to the OSE2 sequence present in
the Osteocalcin (OG2) promoter
(OSE2OG2) (19). These double-stranded oligonucleotides were
then used as probes in electrophoretic mobility shift assays (EMSA)
using either ROS 17/2.8 nuclear extract or recombinant Cbfa1 as a
source of protein.

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Fig. 1.
Type I collagen OSE2 sites are well conserved
across species. A, three OSE2 sites are present in the
mouse 1(I) collagen promoter, but only the
1A site and the 1C site are conserved across species.
B, diagram of the 1(I) and
2(I) collagen promoters and the OSE2 sites of
each. The arrow indicates the start site of transcription
(+1). The position indicated for each element is relative to the start
site of transcription. C, 2(I)
collagen OSE2 sequence is conserved across vertebrate species for
which the sequence is available: h, human; m,
mouse; r, rat; c, chicken; C.f.,
Canis familiaris; B.t., Bos taurus;
R.c., Rana catesbeiana.
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Cbfa1 Binds to an OSE2 Site in the Mouse
1(I) collagen
Promoter--
The complex formed upon incubation of ROS 17/2.8 nuclear
extract with
1AB migrated at the same location as the complex formed upon incubation of ROS 17/2.8 nuclear extract with the
OSE2OG2 oligonucleotide (Fig.
2A, lanes 1 and
2), although it was of weaker intensity. In contrast, no
protein-DNA complex was observed when using
1mutAmutB as a probe
(Fig. 2A, lane 5).

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Fig. 2.
Cbfa1 binds to the
1A site in the 1(I)
collagen promoter. DNA binding was analyzed by EMSA.
A, labeled oligonucleotides OSE2OG2 (lane
1), 1AB (lane 2), 1wtAmutB (lane 3),
1mutAwtB (lane 4), and 1mutAmutB (lane 5)
were incubated with ROS 17/2.8 nuclear extract. The arrow
indicates the complex of interest. B, labeled 1AB was
used as a probe and incubated with nuclear extract from primary
osteoblasts (lane 1), brain (lane 2), kidney
(lane 3), lung (lane 4), muscle (lane
5), and spleen (lane 6). The arrow indicates
the complex containing Cbfa1. C, supershift EMSA was
performed using an antiserum against Cbfa1 (lane 2), or
nonspecific antiserum (lane 1) using 1AB as a probe. The
arrow indicates the complex of lower mobility observed after
incubation with the anti-Cbfa1 antibody. D, EMSA using
recombinant Cbfa1 as a source of protein and 1AB (lane
1), 1wtAmutB (lane 2), 1mutAwtB (lane
3), and 1mutAmutB (lane 4) oligonucleotides as
probes.
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To determine which of the two upstream OSE2 sites was binding to this
factor, we used
1wtAmutB oligonucleotide or
1mutAwtB oligonucleotide as probes in EMSA. Incubation of labeled
1wtAmutB with ROS 17/2.8 nuclear extract generated a protein-DNA complex that
had the same mobility as the one observed when using
1AB oligonucleotide as a probe and was of stronger intensity (Fig. 2A, lane 3). In contrast, when using
1mutAwtB
oligonucleotide as a probe, we observed only a weak binding of ROS
17/2.8 nuclear extract to the DNA (Fig. 2A, lane
4).
To determine whether this osteoblast-specific factor binding to the
1A OSE2-like element was indeed Cbfa1, we performed three types of
experiments. First, we asked whether the factor present in ROS 17/2.8
nuclear extract and binding to the
1A site was expressed only in
osteoblasts. For that purpose, we prepared nuclear extract from primary
osteoblasts and several other tissues and used them in EMSA. As shown
in Fig. 2B, the factor binding to the
1A oligonucleotide
was present only in primary osteoblast nuclear extract and not in
nuclear extract of other tissues. Next we performed supershift
experiments using anti-Cbfa1 antibody or a nonspecific antiserum.
Incubation of the nuclear extract with an antibody against Cbfa1 prior
to addition of labeled
1AB oligonucleotide led to the formation of a
second protein-DNA complex of slower mobility (Fig. 2C,
lane 2), whereas a nonspecific serum had no effect (Fig.
2C, lane 1), demonstrating that the protein-DNA complex formed upon incubation of the labeled
1AB with ROS 17/2.8 nuclear extract contains Cbfa1, because this antibody is specific for
Cbfa1 (14). Third, we asked whether recombinant Cbfa1 could bind to
1AB but not to
1mutAmutB (Fig. 2D, lanes 1 and 4). Incubation of labeled
1wtAmutB oligonucleotide
with recombinant Cbfa1 resulted in the formation of a protein-DNA
complex (Fig. 2D, lane 2), whereas incubations
using labeled
1mutAwtB oligonucleotide did not (Fig. 2D,
lane 3). Taken together, these results indicate that the
1A site, a site conserved in multiple species, is the major binding site for Cbfa1 in this region of the
1(I)
collagen promoter.
The
1A Site Acts as an Osteoblast-specific Activator of
Transcription in Tissue Culture Experiments and in Vivo--
We
further addressed the functional relevance of the
1A and
1B sites
using two additional approaches. First, to examine the effect of the
1A site on activity of a 2.4-kb promoter fragment, a site-specific
mutation was generated in the
1A site via polymerase chain reaction
and introduced into a 2.4-kb
1(I) collagen
promoter-luc chimeric gene. These 2-bp mutations resulted in a 54%
decrease in promoter activity when tested in DNA transfection
experiments in ROS17/2.8 cells (Fig.
3A). The same mutations did
not reduce the activity of this 2.4-kb
1(I) collagen promoter-luc
chimeric gene in other cell lines of nonosteoblastic nature of
mesenchymal and nonmesenchymal origin (Fig. 3A). This result
further suggests that this cis-acting element is active only
in osteoblasts. Mutations in the
1B site did not affect the activity
of the 2.4-kb fragment of the
1(I) collagen
promoter used in this study (Fig. 3A).

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Fig. 3.
The 1A site can
activate transcription and is required for the full activity of the
1(I) collagen promoter.
A, constructs containing 2.4 kb of the
1(I) collagen promoter with or without
mutations were transfected into several cell types. 1AB (black
bars), 1mutAwtB (gray bars), and 1wtAmutB
(white bars) promoters were cloned upstream of a luciferase
reporter gene and used in DNA transfection experiments in ROS 17/2.8
cells, C2C12 cells, 10T1/2 cells, NIH 3T3 cells, and HeLa cells. Values
represent percentage activity compared with the wild-type promoter.
1mutAwtB displays lower transcriptional activity only in ROS 17/2.8
cells. B, multimers of the oligonucleotides used for EMSA
were placed upstream of a minimal 1(I)
collagen promoter fused to a luciferase reporter gene. These
constructs were transfected into COS7 cells in the presence of a
recombinant Cbfa1 expression construct (dark bars), or an
empty vector (open bars). Values represent -fold activation
in relation to an empty reporter vector and are the average of at least
three experiments done in triplicate. C, wild-type
(dark bars) and double-mutant (open bars)
constructs from DNA transfection experiments were used to generate
transgenic mice. Luciferase activity per 100 µg of protein was
determined for several tissues, and the data are expressed in terms of
percentage activity compared with that of bone.
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Second, in DNA cotransfection assays performed in COS7 cells, a cell
line that does not express Cbfa1 (21), exogenous Cbfa1 transactivated a construct containing four copies of the
1AB oligonucleotide fused to a minimal
1(I)
collagen promoter-luc chimeric gene, p4
1AB-luc (Fig.
3B). Indeed, cotransfection of p4
1AB-luc with a
recombinant Cbfa1-expressing vector resulted in a 180-fold activation,
whereas an empty expression vector had no effect. This level of
transactivation is similar to what we observed when using a vector
containing six copies of the OSE2OG2 as a reporter (Fig.
3B). The minimal
1(I) collagen
promoter fragment has virtually no transactivation ability on its own
(4). When using a vector containing multimers of
1wtAmutB
oligonucleotides cloned upstream of the minimal
1(I) collagen promoter fragment, we observed a
120-fold increase in luciferase activity, indicating that the
1A
site is the main contributor to the transactivating function of this
region of the
1(I) collagen promoter. This is consistent with the observation that only the
1A site is able to
bind Cbfa1 in vitro. The slight decrease in activity seen
with the loss of the
1B site may indicate a synergistic effect of these two sites in this type of experiment. The activity of constructs containing four copies of
1mutAmutB, or of
1mutAwtB upstream of
the minimal
1(I) collagen promoter-luc
chimeric gene (Fig. 3B) could not be increased upon
cotransfection with the Cbfa1-expressing vector, thus demonstrating the
specificity of the effect observed. In this set of experiments, we used
multimers of the
1(I) collagen OSE2 sites, because Cbfa1 does not
transactivate the
1(I) collagen promoter
fragment in this type of assay. This is a consistent feature of Cbfa1
biology, indeed, we observed weak transactivation of the OG2 promoter
when cotransfected with Cbfa1 (9), compared with the strong
transactivating effect of Cbfa1 observed when using 6OSE2-luc (9).
Third, we asked whether these OSE2 sites could confer
bone-specific expression to a reporter gene in vivo. For
that purpose, we generated transgenic mice containing two of the
constructs used in the above transfections, p4
1AB-luc and
p4
1mutAmutB-luc. In transgenic mice harboring p4
1AB-luc,
luciferase activity could be detected in bone but neither in other
tissues expressing type I collagen, nor in tissues which do not express
type I collagen (Fig. 3C). As expected, given their
respective sizes, the expression of p4
1AB-luc was considerably lower
than that of
1(I) collagen (data not shown). The p4
1mutAmutB-luc
construct was not expressed in bone or any other tissue (Fig.
3C). Taken together with the results of the mutagenesis of
the
1A site and of the
1B site, these results indicate that Cbfa1
contributes to the expression of
1(I) collagen
in osteoblasts through the
1A site.
Cbfa1 Binds to the OSE2 Site Located at
372 bp in the Mouse
1(I) collagen Promoter--
As mentioned at the beginning of
"Results," there is a third OSE2 sequence in the
1(I) collagen promoter, located at
372bp in
mouse (site
1C, Fig. 1A). This site
is also conserved across species, and we first asked whether this
OSE2-like sequence could be bound by Cbfa1 in EMSA. Labeled
1C
oligonucleotides were incubated with ROS 17/2.8 nuclear extract as
described above, leading to the formation of a protein-DNA complex that
migrated at the same location as that formed upon incubation of ROS
17/2.8 nuclear extract with labeled OSE2OG2 (Fig.
4A, lanes 1 and
3). However, the protein-DNA complex was of weak intensity
compared with that we observed when using OG2OG2 or even
1wtAmutB oligonucleotides as probes (Fig. 4A, lanes
1 and 2). No complex of this size was observed after
incubation of labeled
1mutC with ROS 17/2.8 extract (Fig.
4A, lane 3).

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Fig. 4.
The 1C site is bound
by Cbfa1 but is unable to activate a minimal 1(I)
collagen promoter. A, EMSA was performed
using labeled OSE2OG2 (lane 1), 1C
(lane 3), or 1mutC (lane 4) oligonucleotides
and ROS 17/2.8 nuclear extract as a source of protein. The
arrow indicates complex of interest. Supershift EMSA was
performed using an antiserum against Cbfa1 (lane 6) or
nonspecific antiserum (lane 5). The arrow
indicates the complex of lower mobility formed after incubation with
the anti-Cbfa1 antibody. B, EMSA using recombinant Cbfa1 as
a source of protein and 1C (lane 1) or 1mutC
(lane 2) oligonucleotides as a probe. C,
multimers of 1C and 1mutC oligonucleotides were placed upstream
of a minimal 1(I) collagen promoter. These
constructs were used in cotransfection assays in COS7 cells with a
Cbfa1-expressing vector (dark bars) or an empty vector
(open bars). Values represent -fold activation in relation
to an empty reporter vector and are an average of at least three
experiments done in triplicate.
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To show that Cbfa1 was part of this protein-DNA complex, supershift
experiments were performed using an anti-Cbfa1 antibody, a labeled
1C oligonucleotide, and ROS17/2.8 nuclear extract as a source of
protein. The incubation of ROS 17/2.8 nuclear extract with an antibody
against Cbfa1 prior to the addition of labeled oligonucleotide led to
the formation of a slower mobility complex. This complex was specific,
because it was not observed when using a nonspecific antiserum (Fig.
4A, lanes 5 and 6). EMSA experiments using recombinant Cbfa1 provided further evidence that this site could
bind, albeit weakly, Cbfa1, because Cbfa1 was able to bind to
1C
oligonucleotide but not to
1mutC oligonucleotide (Fig. 4B, lanes 1 and 2). These results
indicate that Cbfa1 is able to bind only weakly to the
1C site in
the mouse
1(I) collagen promoter, suggesting
that this OSE2 site may not play a critical role. To test this
hypothesis, we cloned four copies of wild-type or mutated
1C
oligonucleotides upstream of the minimal
1(I) collagen promoter fragment-luciferase chimeric gene used in Fig. 3A. As seen in Fig. 4C, in DNA transfection
experiments in COS7 cells, neither wild-type nor mutant
1C
constructs could increase the activity of this reporter gene upon
cotransfection with a Cbfa1 expression vector. This lack of an overt
role for the
1C site is consistent with the rather poor binding of
Cbfa1 to this site. Taken together, these results indicate that the
1C site is not a critical cis-acting element in
controlling the osteoblast-specific expression of the mouse
1(I) collagen gene.
Cbfa1 Binds to an OSE2 Site in the Mouse
2(I) collagen
Gene--
Because
1(I) and
2(I) collagen genes are often coregulated, we
next asked whether Cbfa1 was also regulating the expression of
2(I) collagen. Sequence analysis of the
2(I) collagen promoter uncovered the existence
of several potential OSE2 sites. Only one of these, located in the
first exon of the gene, is present at the same location in the
2(I) collagen gene of multiple vertebrate species (Fig. 1C). For this reason, this site was studied
further. First, DNA binding was studied. EMSA was performed using a
labeled double-stranded oligonucleotide containing the OSE2 element
(
2A) as a probe and ROS 17/2.8 nuclear extract as a source of
protein. Incubation of labeled
2A oligonucleotide with ROS 17/2.8
nuclear extract resulted in the generation of a protein-DNA complex
migrating at the same location as the protein-DNA complex formed upon
incubation of ROS 17/2.8 nuclear extract with labeled
OSE2OG2 and
1wtAmutB (Fig.
5A, lanes 1 and
2). This protein-DNA complex was specific, because it did
not form upon incubation of ROS 17/2.8 nuclear extract with an
oligonucleotide containing a mutation in this OSE2 sequence (
2mutA)
(Fig. 5A, lane 3). Because the binding of nuclear
extract to
2A oligonucleotide was weak, despite using a 2-fold
higher amount of ROS 17/2.8 nuclear extract, we also used recombinant
Cbfa1 protein in EMSA. The incubation of recombinant Cbfa1 with
2A
oligonucleotide, again using a 2-fold higher amount of Cbfa1 compared
with that used to see binding of Cbfa1 to
1wtAmutB, resulted in the
formation of a protein-DNA complex (Fig. 5B, lane 1). This complex did not form upon incubation of Cbfa1 with
2mutA oligonucleotide (Fig. 5B, lane 2). These
data show that the conserved OSE2 sequence present in the
2(I) collagen gene can bind Cbfa1, albeit more
weakly than the
1A site.

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Fig. 5.
Cbfa1 binds an OSE2 site in
2(I) collagen, and this
site can activate transcription. A, EMSA was performed
using labeled OSE2OG2 (lane 1), 2A
(lane 2), or 2mutA (lane 3) oligonucleotides
and ROS 17/2.8 nuclear extract. B, EMSA using recombinant
Cbfa1 as a source of protein and 2A (lane 1) or 2mutA
(lane 2) oligonucleotides as a probe. C,
multimers of 2A and 2mutA oligonucleotides were placed upstream
of the minimal promoter constructs used for 1(I)
collagen transfections. These constructs were used in
cotransfection assays in COS7 cells with a Cbfa1-expressing vector
(dark bars) or with an empty vector (open bars).
Values represent -fold activation in relation to an empty reporter
vector and are an average of at least three experiments done in
triplicate.
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Cbfa1 Can Activate Transcription through the
2A Site--
To
study the function of the
2A site, cotransfection assays were
performed in COS7 cells. In this assay, exogenous Cbfa1 transactivated
a construct containing a multimer of four
2A oligonucleotides fused
to a minimal
1(I) collagen promoter-luc
chimeric gene, p4
2A-luc (Fig. 5C), producing an
~15-fold increase in luciferase activity. This effect was specific,
because the construct containing a multimer of six
2mutA
oligonucleotides (Fig. 5C) produced no activity. The
relatively weak increase in luciferase activity compared with the
effect observed with multimers of the
1wtAmutB oligonucleotide is
consistent with the weaker binding of Cbfa1 to the
2A sequence.
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DISCUSSION |
Taken together, our data provide evidence indicating that Cbfa1 is
one of the transcription factors contributing to the expression of the
two Type I collagen genes in osteoblasts in vivo.
Moreover, along with CBF (3, 4, 18) and Sp1 (5, 7), Cbfa1 belongs to a
growing group of transcription factors accounting for the coordinated
regulation of both genes. The finding that Cbfa1 favors type I
collagen expression in osteoblasts is consistent with the absence
of bone extracellular matrix in Cbfa1-deficient mice and with
the marked decrease of Type I collagen expression in
transgenic mice overexpressing a dominant negative form of Cbfa1 in
osteoblasts (10, 11, 14). These results broaden the spectrum of
transcription factors able to regulate type I collagen gene
expression at various stages of osteoblast differentiation and overall
increase our understanding of type I collagen genes' regulation.
The data presented here indicate that there is a clear functional
hierarchy between the different Cbfa1-binding sites, or OSE2 sites,
present in the
1(I) collagen promoter.
Clearly, the
1A element is the most potent activator of expression
of all the OSE2 elements we studied in this promoter. These findings do
not exclude the possibility that OSE2 sites present further upstream in
the promoter and/or elsewhere in the gene may also contribute to the
osteoblast-specific expression of the
1(I) collagen gene. Conceivably, one of these as of yet uncharacterized OSE2 sites may bind Cbfa1 with a higher affinity and act as a more
powerful osteoblast-specific cis-acting element. We did not identify any conserved consensus OSE2 sites by examining the DNA sequence of the region located between
1540 and
1656 that has been
previously shown to be required for osteoblast expression (25). This
reinforces the hypothesis that other cell-specific transcription
factors must contribute to osteoblastic expression of the type I
collagen genes.
Although Cbfa1 can bind to a site present in the
2(I) collagen gene and the expression of this
gene is decreased in transgenic mice expressing a dominant negative
form of Cbfa1, the level of activation observed in cotransfection
experiments with the
2A construct was lower than that seen when
using the
1AB constructs based on the
1(I)
collagen promoter. At least two explanations could account for
this observation. First, and most importantly, the binding of ROS
17/2.8 nuclear extract to the
2A site was weaker than its binding to
the
1A site of
1(I) collagen, indicating that this site has a lower affinity to Cbfa1. Second, considering the
numerous OSE2 sites present in the
2(I)
collagen promoter, it is likely that, for this gene and for the
1(I) collagen gene as well, some of the other
OSE2 sites act alone or in concert with the conserved OSE2 site to
control its expression in osteoblasts in vivo.
If Cbfa1 is one positive regulator of type I collagen
expression in osteoblasts, it is clear from the above data that it is not the only one. Indeed, Cbfa1 expression is initiated in osteoblast progenitors after type I collagen expression can be noticed
in mesenchymal cells. Moreover, at least one other
cis-acting element has been shown to be implicated in
osteoblast-specific expression of the
1(I)
collagen gene in mouse and rat (15, 16). Members of the DLX family
of homeobox proteins are able to bind to this sequence and to activate
transcription (22). Another homeobox-related protein, MSX2, can bind to
this sequence and repress expression of the
1(I)
collagen gene (23), and recent genetic evidence has demonstrated
that MSX2 is upstream of Cbfa1 (24). These homeobox proteins are likely
to be expressed earlier than Cbfa1 and may even control its expression,
directly or indirectly. It is tempting to speculate that these homeobox
proteins, possibly with other regulatory proteins, act early during the
specification of mesenchymal progenitor cells to the osteoblast lineage
and that Cbfa1 is required for osteoblast differentiation and for the
maintenance of the osteoblast phenotype. This hypothesis will be more
easily testable when mice deficient for several DLX proteins are
available. Regardless, the observation that Cbfa1 binds to and
regulates the activity of both type I collagen genes in
osteoblasts further illustrates how important Cbfa1 is in osteoblast physiology.