From the Department of Medicine, Division of Rheumatology, Thomas Jefferson University, Jefferson Medical College, Philadelphia, Pennsylvania 19107
Received for publication, August 7, 2002, and in revised form, October 9, 2002
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ABSTRACT |
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The COL9A1 gene contains two promoter
regions, one driving expression of a long The chondrocyte is responsible for the precise production of
several different types of cartilage tissues in the developing vertebrate; including growth plate cartilage, articular cartilage, and
the cartilage of the ear and trachea. In each of these situations, the
elaboration of a complex and extensive extracellular matrix, which is
the main functional component of cartilaginous tissues, is crucial. The
expression of extracellular matrix molecules by chondrocytes must,
therefore, be tightly and coordinately controlled at the level of both
synthesis and degradation to ensure that the matrix is properly
constructed and maintained. Part of this control occurs at the level of
the regulation of chondrocyte-specific gene expression. The best
studied gene in this regard is the COL2A1 gene, which gives
rise to the main fibrillar collagen in the cartilage matrix. The
expression of the COL2A1 gene is controlled through transcription factors that interact with both the promoter and the
chondrocyte-specific enhancer located within the first intron (1-4).
Recent work has shown that both positive and negative factors interact
with the COL2A1 gene to regulate its expression (2-8).
Positive regulation of the COL2A1 gene during chondrocyte differentiation is afforded by the interaction of members of the Sry-type HMG box
(SOX)1 family of
transcription factors with a specific region of the intronic enhancer
(5-8). Three SOX factors, L-SOX5, SOX6, and SOX9, have
been shown to cooperatively activate the expression of the
COL2A1 gene, and SOX9 has been shown to be essential for normal skeletogenesis (6, 9, 10).
Despite the wealth of information concerning the control of expression
of the COL2A1 gene, relatively little is known about the
transcriptional regulation of other chondrocyte-specific collagen genes, including those encoding collagens that interact with type II
collagen, such as types IX and XI (11, 12). Collagen IX is a member of
a subfamily of collagens termed fibril-associated collagens with
interrupted-triple helices (FACITs) that also include collagens XII,
XIV, XVI, and XIX (13, 14). Collagen IX is a heterotrimeric molecule
composed of three polypeptide chains ( The purpose of this study was to examine the regulatory elements
located within the proximal promoter region of the human COL9A1 gene in chondrocytic cells. We found that a 976-bp
promoter fragment from the human COL9A1 gene was able to
drive expression of reporter genes in RCS cells. We also found that
this promoter region can be transactivated to high levels by SOX9 in
nonchondrocytic NIH/3T3 and human chondrosarcoma cells (HTB). We show
that the SOX9 transactivation depends on two of five putative
SOX/Sry-binding sites by mutational analysis and that a third region
that does not contain any obvious SOX/Sry-binding sites is also
important for full promoter activity. Finally, we provide evidence that SOX9 can interact with one of the functionally active aforementioned SOX/Sry-binding sites.
Cell Culture--
Rat chondrosarcoma cells (RCS) were a kind
gift from Dr. Benoit de Crombrugghe (23). HTB human chondrosarcoma
cells (SW1353) and NIH/3T3 fibroblasts were obtained from American Type
Culture Collection. The cells were cultured in Dulbecco's modified
Eagle's medium (DMEM, high glucose), 10% fetal bovine serum
supplemented with L-glutamine (2 mM),
penicillin (100 units/ml), streptomycin (100 µg/ml), fungizone (2.5 µg/ml), and ascorbic acid (50 µg/ml). Cells were split at 70-80%
confluence, and the medium was replaced every 3-4 days.
COL9A1-Luciferase Constructs--
The human COL9A1
proximal promoter region and first intron were obtained by PCR using a
P1 phage clone (P1-A) that contains the 5' region of the human
COL9A1 gene (a kind gift from Dr. Lena Ala-Kokko; ref. 20)
and the following primers. For the promoter region: primer DS37,
5'-GATCGAATTCAGTAGGGGGCTTGATGTTA-3' (forward) and primer DS38,
5'-GATCCTCGAGTTCCCAGTTGATTTTCTTTG-3' (reverse), and for the first
intron: primer DS39, 5'-GATCGTCGACAAGACAATAACCCTGGAAAGA-3' (forward)
and primer DS40, 5'-GATCAAGCTTTGAAACAGGAGTCCCCGCAGA-3' (reverse). The
primers for the promoter region contained an EcoRI and a
XhoI site, respectively, and the primers for the first
intron contained a SalI and a HindIII site,
respectively, to facilitate cloning. The PCR-generated promoter
fragment (976 bp) contained the region from Transient Transfection and Luciferase Assays--
Seven to ten
µg of the various COL9A1 reporter constructs, 2 µg of
the SOX expression vectors (a kind gift from Dr. Veronique Lefebvre,
6), and 2 µg of the pCMV Nuclear Extracts and Electrophoretic Mobility-shift
Assays--
Nuclear extracts were prepared according to the method of
Dignam et al. (24) using the CellLytic NuCLEAR extraction
kit (Sigma-Aldrich). Briefly, cells were placed in hypotonic buffer (10 mM HEPES [pH 7.9], 1.5 mM MgCl2,
10 mM KCl, and 0.5 mM dithiothreitol) and
incubated on ice for 15 min. Igeapal CA-630 was added to a final
concentration of 0.6%, and the mixture was vortexed vigorously for
10 s. Nuclei were recovered by centrifugation at 3,300 × g for 30 s at 4 °C and extracted in buffer
containing 20 mM HEPES pH 7.9, 0.42 M NaCl,
25% glycerol, 1.5 mM MgC12, 0.2 mM
EDTA, and 0.5 mM dithiothreitol for 30 min at 4 °C by
gentle shaking. The extract was then centrifuged for 15 min at
25,000 × g, and the supernatant was frozen at
Electrophoretic mobility-shift assays (EMSAs) were carried out as
previously described with minor modifications (25). Briefly, binding
reactions consisted of 12.5 mM Hepes, pH 7.9, 50-100
mM NaCl, 5% glycerol, 2 mg/ml bovine serum albumin,
2 µg poly(dG-dC), 0.1 mM EDTA, 0.1 mM
dithiothreitol, 1 ng of 32P-end labeled
double-stranded oligonucleotide probe, and 10-15 µg of nuclear
protein. Binding reactions were incubated for 30 min at 21 °C and
then loaded onto 4% acrylamide-0.25× Tris borate-EDTA gels and
electrophoresed at 200 V for 2 h. For competition analyses, 50-fold excess of cold-competitor probe was included in the binding reaction. For super-shift analyses, 3 µl of a rabbit polyclonal anti-SOX9 antibody (a kind gift from Dr. Veronique Lefebvre; Ref. 6)
was included in the binding reaction.
Human COL9A1 Promoter Activity in Different Cells and the
Effect of the First Intron on COL9A1 Promoter Activity--
A
luciferase reporter gene construct (846Luc) containing a 976-bp
COL9A1 gene fragment that includes 846 bp of the promoter and 130 bp of the first exon (up to but not including the ATG initiation codon) (Fig. 1A)
was transfected into RCS, HTB chondrosarcoma, and NIH/3T3 cells to
determine the relative promoter activity as compared with the pGL3basic
vector (no promoter). The 846Luc construct exhibited a high level of
promoter activity in the chondrocytic RCS cells (~18-fold over the
vector alone, Fig. 1B), however, in HTB and NIH/3T3 cells,
the COL9A1 promoter activity was substantially lower (Fig.
1B). To examine whether the first intron of the human COL9A1 gene contains transcriptional regulatory elements, an
807-bp fragment containing the first intron (excluding the splice-site sequences at the 5' and 3' ends) was inserted into the 846Luc construct
to obtain 846LucInt. The two constructs, 846Luc and 846LucInt, were
then separately transfected into RCS cells. As can be seen in Fig.
1C, inclusion of the COL9A1 first intron fragment did not result in any significant difference in promoter activity.
Transactivation of the COL9A1 Promoter by SOX9--
SOX
transcription factors have been shown to be important for the
regulation of chondrocyte-specific gene expression. Sequence analysis
of the proximal promoter region of the human COL9A1 gene with the MatInspector program (26) revealed five putative
SOX/Sry-binding sites (Fig.
2A), whereas analysis of the
first intron did not reveal any recognizable SOX/Sry-binding sites. To
determine whether SOX9 regulates the activity of the COL9A1
gene, either the 846Luc or the 846LucInt construct were co-transfected
with a SOX9 expression construct into HTB and NIH/3T3 cells. SOX9
overexpression in these cells activated the 846Luc and 846LucInt
constructs by ~25- to 30-fold (Fig. 2, B and
C). However, there was no difference in activation
between the two constructs in response to SOX9, indicating that the observed transcriptional activation depends on the
proximal-promoter region and that the first intron did not have any
influence on the transcriptional activity of the gene under these
conditions.
Deletion Analysis of the COL9A1 Proximal Promoter Region--
To
delineate which regions of the COL9A1 promoter region are
responsible for promoter activity and SOX9 transactivation, a series of
COL9A1-promoter-luciferase 5'-deletion constructs were employed (Fig. 3A). The
deletion constructs were generated such that the five putative
SOX/Sry-binding sites, designated A-E, were sequentially removed
(Figs. 2A and 3A). To determine the effect of the
deletions on promoter activity, the full-length construct (846Luc) and
the five deletion constructs (588Luc, 560Luc, 357Luc, 167Luc, and
107Luc) were transfected into RCS cells (Fig. 3B). Deletion
of up to 286 bp from the 5' end, including the two distal putative
SOX/Sry-binding sites (588Luc and 560Luc, sites A and
B in Fig. 3A) did not result in any substantial
changes in activity in RCS cells (Fig. 3B). However, when
the region containing the three distal putative SOX/Sry sites was
removed (357Luc, sites A-C in Fig.
3A), the promoter activity was reduced to 20% of control (Fig. 3B). Deletion of the region encompassing the four
distal sites (167Luc, sites A-D in Fig.
3A) resulted in slightly increased expression as compared
with the 357Luc construct (sites A-C deleted) and an overall
expression level of 31% of control (Fig. 3B). Finally, deletion of the region encompassing all five sites (107Luc in Fig.
3A) resulted in a 5.5-fold decrease in promoter activity (9% of control, Fig. 3B). We conclude that the region
encompassing the SOX/Sry-binding sites C-E is important for full
promoter activity in RCS cells.
To determine which of the putative SOX/Sry sites within the proximal
promoter region were responsible for the observed transactivation by
SOX9, the various deletion constructs were co-transfected into NIH/3T3
cells along with a SOX9 expression vector. Fig.
4A shows the results of this
experiment. All of the deletion constructs except for the 107Luc
construct, which does not contain any SOX/Sry-binding sites, were
transactivated by SOX9 overexpression. The full-length 846Luc construct
displayed the strongest response to SOX9 co-transfection, exhibiting a
~30-fold increase in activity, whereas the deletion constructs that
contained at least one putative SOX/Sry-binding site (588Luc, 560Luc,
357Luc, and 167Luc) displayed a 15- to 20-fold increase in activity
over control.
Two other SOX proteins, L-SOX5 and SOX6 have been shown to
cooperate with SOX9 in the transcriptional activation of the
COL2A1 gene through the intronic enhancer region (6). To
determine whether L-SOX5 and SOX6 play a role in the
activation of the COL9A1 proximal promoter region, the
846Luc construct was co-transfected into NIH/3T3 cells with
L-SOX5, SOX6, and SOX9 expression constructs. Fig.
4B demonstrates that neither L-SOX5 or SOX6
alone or in combination activate the COL9A1 proximal
promoter. Interestingly, when L-SOX5, SOX6, and SOX9 were
co-transfected together with the 846Luc construct, transactivation of
the promoter was attenuated as compared with co-transfection with SOX9
alone (Fig. 4B).
Mutations in the SOX/Sry-binding Sites Suppress
Transcriptional Activation--
To define more precisely the role of
the putative SOX/Sry-binding sites within the COL9A1
proximal promoter region, point mutations were introduced into sites C,
D, and E (Fig. 5A). In each
case, a 4-bp change was introduced into the core SOX/Sry-binding sites
employing PCR-based mutagenesis. The intact (wild-type) promoter
construct (846Luc) and each of the mutant constructs (CmutLuc, DmutLuc,
and EmutLuc) were transfected separately into RCS cells to assess the
effect of the mutations on promoter activity. Fig. 5B
demonstrates that mutations in site C had no effect on COL9A1 promoter activity. In contrast, mutations in sites D
and E reduced the promoter activity to 17 and 36% of control,
respectively. Similarly, in co-transfection experiments with a SOX9
expression vector, the wild-type 846Luc and CmutLuc constructs were
transactivated ~30- to 35-fold over control (Fig. 5C),
whereas the DmutLuc and EmutLuc constructs were activated to only 1/3
of those levels (10- to 12-fold, Fig. 5C) by SOX9
co-transfection. These results demonstrate that the SOX/Sry-binding
sites D and E are necessary for full promoter activity in RCS cells and
for full transactivation by SOX9 in NIH/3T3 cells.
Although the results from the deletion analysis indicated that the
region encompassing site C was important for full activity (construct
357Luc, Fig. 3B), the mutational studies were contradictory because they showed that site C itself was dispensable for promoter activity (CmutLuc construct, Fig. 5, B and C). To
clarify these results, either an 85-bp fragment encompassing site C or
a 96-bp fragment encompassing site D (see
brackets in Fig. 2A) were inserted into the
pGL3promoter vector, which contains the SV40 promoter (pGL3proC and
pGL3proD, Fig. 6A). When these
constructs were transfected into RCS cells, the pGL3proC construct
displayed essentially the same activity as the pGL3promoter vector
alone, whereas the pGL3proD construct was ~16-fold more active (Fig.
6B). We conclude that along with SOX/Sry binding sites D and
E, there must be another positive-acting element within the promoter
region from SOX9 Specifically Binds to SOX/Sry-binding Site
D--
To determine whether SOX9 interacts with the putative
SOX/Sry-binding site-D, nuclear extracts were prepared from NIH/3T3 cells that had been transfected with SOX9 expression constructs, and
electrophoretic mobility shift assays were performed. Fig. 7A shows the sequence of the
SOX/Sry-binding site D probe used in these experiments. Fig.
7B shows that specific binding to probes containing
SOX/Sry-binding site D or to a probe containing the SOX binding sites
from the COL2A1 gene enhancer region were detected only in
NIH/3T3 cells that had been transfected with a SOX9 expression construct and not in control cells. Competition analysis revealed that
a specific DNA-protein complex forms with SOX/Sry-binding site-D in
nuclear extracts from RCS cells, and this complex can be competed for
with excess wild-type SOX/Sry-binding site-D probe and excess probe
containing the SOX-binding sites from the human COL2A1 gene
but not with a mutant SOX/Sry-binding site-D probe (Fig.
7C). Finally, the DNA-protein complex that forms with the wild-type SOX/Sry-binding site-D probe and nuclear extract from NIH/3T3
cells transfected with a SOX9 expression construct can be supershifted
with anti-SOX9 antibodies (Fig. 7D).
We demonstrate here that the proximal-promoter region of the human
COL9A1 gene can drive expression of a reporter gene in chondrocytic RCS cells but not in the nonchondrocytic HTB and NIH/3T3
cell lines. We also showed that the COL9A1 proximal-promoter region can be transactivated by SOX9 and that the transactivation depends, in part, on two SOX/Sry-binding sites (sites D and E). Also,
full promoter activity depends on a region located between An interesting difference between the regulation of the
COL9A1 versus the COL2A1 promoter was
observed when the L-SOX5 and SOX6 expression constructs
were included in the co-transfection experiments. L-SOX5
and SOX6 did not stimulate the transcriptional activity of our
COL9A1 promoter construct either alone or in combination. Even more intriguing was the finding that when the L-SOX5
and SOX6 vectors were co-transfected along with the SOX9 expression vector, the activity of the COL9A1 promoter was attenuated.
In contrast to this, L-SOX5 and SOX6 have been shown to
stimulate the transcriptional activity of the COL2A1
enhancer either alone or in combination (6). Furthermore, when
L-SOX5 and SOX6 are expressed along with SOX9 in
co-transfection assays, the activity of the COL2A1 gene was
stimulated in an enhancer-dependent fashion to higher
levels than that observed with the individual SOX factors (6). It is
interesting to speculate that this might be a mechanism by which the
chondrocyte could achieve different levels of COL9A1 and
COL2A1 gene expression utilizing the same transcription
factors. Indeed, a similar type of regulation has been observed with
respect to the COL2A1 gene. The transcription factor cKrox
was shown to interact with multiple sites within the enhancer region,
resulting in up-regulation of the gene in differentiated chondrocytes,
whereas cKrox-binding sites located in the promoter were responsible
for down-regulation of the gene in de-differentiated chondrocytes (2).
However, the COL2A1 gene is also regulated by other
negatively acting transcription factors. The transcription factor
The structure of the promoter for COL9A1 is reminiscent of
that of the COL11A2 gene. A cluster of five SOX/Sry-binding
sites were also characterized in the proximal promoter region of the COL11A2 gene, located between As mentioned above, the arrangement of the SOX/Sry-binding sites within
the COL9A1 proximal-promoter region differ from those found
in the COL11A2 promoter and also from the SOX/Sry-binding sites located in the COL2A1 intronic enhancer (5-8,
27-30). In the case of the COL11A2 gene, the five
SOX/Sry-binding sites are found within a region of ~130 bp (27).
However, the distal three sites are within 45 bp, and the proximal two
sites are separated by only 3 bp. Similarly, the four SOX/Sry-binding
sites within the COL2A1 intronic enhancer are spaced over a
48-bp region (5-8). In contrast to this, the five SOX/Sry-binding
sites within the COL9A1 promoter span a region of 565 bp.
The transient-transfection studies presented here rule out sites A and
B as being important for full promoter activity; however, the remaining
three SOX/Sry-binding sites are scattered over a 415-bp stretch, still
substantially less compact than the COL11A2 and
COL2A1 genes. Whether this arrangement has any bearing on
the relative levels of expression of these genes or the developmental
timing of their expression remains to be determined, as does the
placement of the SOX/Sry-binding sites within the proximal-promoter
region versus an enhancer region or both.
1(IX) chain in cartilage
(upstream) and one driving expression of a shorter chain in the cornea
and vitreous (downstream). To determine how the chondrocyte-specific
expression of the COL9A1 gene is regulated, we have begun
to characterize the upstream chondrocyte-specific promoter region of
the human COL9A1 gene. Transient-transfection analyses
performed in rat chondrosarcoma (RCS) cells, human chondrosarcoma (HTB)
cells, and NIH/3T3 cells showed that the COL9A1 promoter
was active in RCS cells but not HTB or NIH/3T3 cells. Inclusion of the
first intron had no effect on promoter activity. In
transient-transfection analyses with promoter deletion constructs, it
was found that full promoter activity in RCS cells depended on the
region from
560 bp to +130 bp relative to the transcriptional start
site (+1). Sequence analysis of the region from
890 bp to the
transcriptional start predicted five putative SOX/Sry-binding sites.
Mutation analysis revealed that two of three putative SOX/Sry binding
sites within the
560 to +130 bp region are responsible for most of the COL9A1 promoter activity in RCS cells. Co-transfection
experiments with a SOX9 expression plasmid revealed that a construct
containing the five putative SOX/Sry-binding sites was transactivated
20- to 30-fold in both HTB and NIH/3T3 cells. Further co-transfection experiments showed that two of the SOX/Sry-binding sites located within
the
560 to +130 bp region were required for full transactivation. However, mutation and deletion analyses indicated that a region from
560 to
357 bp, which does not contain any other conspicuous SOX9
sites, is also important for full promoter activity. DNA-protein binding assays and super-shift analysis revealed that SOX9 can form a
specific complex with one of the SOX/Sry-binding sites with in the
560 to +130 region.
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1(IX),
2(IX), and
3(IX))
each containing three collagenous domains (COL1-3) interrupted by four
non-triple-helical domains (NC1-NC4) (15, 16). The NC1, NC2, and NC3
domains are of similar size in all three chains, whereas the NC4 domain
of the
1(IX) chain is much larger than in the
2(IX) and
3(IX)
chains (17, 18). Indeed, the NC4 domain of the
1(IX) chain is
encoded by exons 1-8 in the COL9A1 gene, which are
expressed through the use of the cartilage-specific promoter region
studied in the present work (19, 20). A second promoter, located
between exons 6 and 7, is utilized in other tissues to express a short
form of the
1(IX) chain that lacks the large NC4 domain (19, 20). Interestingly, mice that lack type IX collagen develop normally but
exhibit a late-onset form of joint degeneration similar to osteoarthritis, suggesting a stabilizing role for type IX collagen in
cartilage (21, 22).
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
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846 to +130 relative to
the transcriptional start site, and the first intron fragment (807 bp)
contained from position +1037 to +1843 in the human COL9A1
gene sequence (excluding the splice-site sequences) (20). The promoter
fragment was cloned into the NheI-XhoI sites of
the pGL3basic luciferase-reporter vector (Promega, Madison, WI) to
generate the full-length 846Luc promoter construct. To generate the
846LucInt construct, the intron fragment was cloned into the
HindIII-SalI sites of 846Luc. The various
COL9A1 promoter-deletion constructs were generated by PCR
using different forward primers (see below) in combination with the
same reverse primer (DS38, see above). For construct 588Luc,
5'-AATGATTGTTGGGTGTTAGAC-3'; for construct 560Luc,
5'-GATCGCTAGCCTTTGATACCTCATTT-3'; for construct 357Luc,
5'-GTGGGCACATTTTTACTGGA-3'; for construct 167Luc,
5'-TTCCCCTGTAAATCCCTCCTTC-3', and for construct 107Luc, 5'-GATCGCTAGCCTGGGCTCAGAGCGCT-3'. In the case of the primers for constructs 560Luc and 107Luc, NheI sites were placed into
the 5' end to facilitate cloning. The deletion-PCR products for 588Luc, 357Luc, and 167Luc were cloned into the SmaI-XhoI
sites of pGL3basic, and the PCR products for 560Luc and 107Luc were
cloned into the NheI-XhoI sites of pGL3basic. For
the pGL3ProC construct, an 85-bp PCR product encompassing the region
from
559 to
475 was cloned into the pGL3promoter vector (Promega),
which contains the SV40 promoter linked to the luciferase reporter
gene. The pGL3ProD construct was made in a similar manner by inserting
a 96-bp PCR fragment encompassing the region from
356 to
261 into
the pGL3promoter vector. All PCR products were verified by sequencing
on an ABI automatic DNA sequencer (PerkinElmer Life Sciences).
reporter vector
(Clontech), as a control for transfection
efficiency, were co-transfected into either RCS, HTB, or NIH/3T3 cells
by the calcium phosphate precipitation method using the Profection kit
(Promega). The cells were plated 24 h before transfection at a
density of 5 × 105 cells per 10 cm2 dish.
The DNA-CaPO4 precipitate was left on the cells for 16-18 h, after which the cells were washed 3 times with phosphate-buffered saline followed by the addition of fresh media. Forty-eight h after
transfection, the cells were harvested and luciferase assays were
performed with a luciferase assay kit (Promega) and a Turner Designs TD
20/20 luminometer (Turner Designs, Sunnyvale, CA).
-Galactosidase
assays were performed spectrophotometrically with a
-galactosidase
enzyme assay system (Promega). Protein concentrations in the cell
lysates were determined using the Coomassie Blue protein assay
(Pierce). Luciferase activity was normalized to both
-galactosidase activity and protein concentration. Transfection data represent at
least two independent experiments each performed in triplicate, except
where indicated.
70 °C. All buffers contained a protease inhibitor mixture (2 mM 4-(2-aminoethyl) benzenesulfonylfluoride, 1.4 pM trans-epoxysuccinyl-L-leucylamido
[4-guanidinobutane], 130 pM bestatin, 1 µM
leupeptin, and 0.3 pM aprotinin; Sigma-Aldrich).
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Relative activity of the human COL9A1
promoter in RCS, HTB, and NIH/3T3 cells. A,
schematic illustration of the human COL9A1 promoter and
promoter-intron luciferase-reporter constructs. The 846Luc construct
contains an 846-bp COL9A1 gene fragment encompassing the
promoter region and part of the first exon (not including the ATG
translational start codon) and the 846LucInt construct contains an
807-bp gene fragment encompassing the first intron (not including the
5'- and 3'-splice site sequences). B, activity of the 846Luc
construct in RCS, HTB, and NIH/3T3 cells. The cells were co-transfected
with 10 µg of the 846Luc construct or the pGL3basic empty vector and
2 µg of the pCMV plasmid. C, effect of the first intron
on promoter activity in RCS cells. The 846Luc or 846LucInt constructs,
or the pGL3basic empty vector were co-transfected with the pCMV
plasmid into RCS cells. Transfected cells were incubated for 48 h
and then luciferase and
-galactosidase activities were determined as
described under "Materials and Methods." Data are
presented as average -fold difference of luciferase activity
versus control (pGL3basic) vector ± S.D., or as
average percent of control (pGL3basic) ± S.D.
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Fig. 2.
SOX9 transactivates the human COL9A1
proximal promoter region in NIH/3T3 and HTB cells.
A, sequence of the human COL9A1proximal promoter
region. Shown in bold and underlined are the 5 putative SOX/Sry-binding sites designated A-E. Brackets
denote the regions that were used to construct the pGL3proC and
pGL3proD constructs in Fig. 6. Shown below the sequence for comparison
is the HMG-SOX/Sry-binding site consensus sequence (5). B,
transactivation of the 846Luc and 846LucInt reporter constructs in HTB
cells by co-transfection with a SOX9 expression vector. Ten micrograms
of either 846Luc or 846LucInt were transfected into HTB cells with 2 µg of pBluescript or a SOX9 expression vector and 2 µg of pCMV .
C, transactivation of 846Luc and 846LucInt by
co-transfection with a SOX9 expression vector in NIH/3T3 cells. NIH/3T3
cells were transfected as in B. Transfected cells were
incubated for 48 h, and luciferase and
-galactosidase
activities were determined as described in "Materials and Methods."
Data are presented as average -fold difference of luciferase activity
versus control (846Luc, no SOX9) ± S.D.
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Fig. 3.
Deletion analysis of the human
COL9A1 proximal promoter region in RCS cells.
A, schematic illustration of the various 5'-deletion
constructs employed in the analyses. The deletion constructs were
generated as described under "Materials and Methods." B,
relative luciferase activity of the COL9A1 promoter-deletion
constructs in RCS cells. RCS cells were transfected with 10 µg of
each deletion construct and 2 µg of the pCMV plasmid. Transfected
cells were incubated for 48 h, and luciferase and
-galactosidase activities were determined as described under
"Materials and Methods." Data are presented as average percent of
control (846Luc) ± S.D. Data represent 3-4 experiments performed
in triplicate.
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Fig. 4.
Transactivation of
COL9A1-promoter-deletion constructs by SOX9 in NIH3T3
cells. A, the various COL9A1
promoter-deletion constructs (Fig. 3A) were co-transfected
into NIH/3T3 cells with 2 µg of pBluescript vector or with a SOX9
expression vector and 2 µg of the pCMV plasmid. B,
transactivation of the 846Luc construct in NIH/3T3 cells by
L-SOX5 and SOX6. NIH/3T3 cells were co-transfected with 7 µg of the 846Luc construct and either 2 µg of pBluescript or
expression vectors for L-SOX5, SOX6, or SOX9 and 2 µg of
the pCMV
plasmid. Transfected cells were incubated for 48 h,
and luciferase and
-galactosidase activities were determined as
described under "Materials and Methods." Data are presented as
average -fold difference of luciferase activity versus
control (846Luc alone) vector ± S.D.
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Fig. 5.
Mutations in the SOX/Sry-binding sites D and
E reduce transcriptional activity and transactivation of the
COL9A1 promoter by SOX9 in RCS and NIH/3T3 cells.
A, schematic illustration of the point mutations introduced
into the SOX/Sry binding sites C, D, and E, to generate constructs
CmutLuc, DmutLuc, and EmutLuc. B, mutations in sites D and E
reduce transcriptional activity of the COL9A1 promoter in
RCS cells. The wild-type 846Luc construct or the CmutLuc, DmutLuc, and
EmutLuc constructs (10 µg) were transfected into RCS cells along with
2 µg of the pCMV plasmid. C, mutations in sites D and E
reduce the transactivation by SOX9 in NIH/3T3 cells. The wild-type
846Luc construct or the CmutLuc, DmutLuc, and EmutLuc constructs (10 µg) were co-transfected into NIH/3T3 cells along with 2 µg of
pBluescript or a SOX9 expression vector and 2 µg of the pCMV
plasmid. Transfected cells were incubated for 48 h, and luciferase
and
-galactosidase activities were determined as described under
"Materials and Methods." Data are presented as average percent
luciferase activity of control (846Luc) ± S.D. or as average
-fold difference of luciferase activity versus control
(846Luc, no SOX9) ± S.D.
560 to
357 relative to the transcriptional start site
that is necessary for full promoter activity in RCS cells.
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Fig. 6.
SOX/Sry-binding site-D can
activate a heterologous promoter in RCS cells. A,
schematic illustration of the pGL3promoter constructs in which
fragments encompassing sites C or D were placed in front of the SV40
promoter (see bracketed regions in Fig. 2A).
B, transfection of RCS cells with the pGL3promoter,
pGL3proC, or the pGL3proD constructs. Ten micrograms of either the
pGL3promoter, pGL3proC, or the pGL3proD construct along with 2 µg of
the pCMV plasmid were transfected into RCS cells. Transfected cells
were incubated for 48 h, and luciferase and
-galactosidase
activities were determined as described under "Materials and
Methods." Data are presented as average -fold difference of
luciferase activity versus control (pGL3promoter) ± S.D.
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Fig. 7.
SOX9 specifically interacts with the
SOX/Sry-binding site-D in the human COL9A1 promoter.
A, sequence of the wild-type and mutated SOX/Sry-binding
site-D probes employed in the EMSA experiments. Nucleotides in
bold indicate the SOX/Sry-binding site. B, a
specific DNA-protein complex forms with a probe encompassing the
SOX/Sry-binding site D in NIH/3T3 cells transfected with a SOX9
expression construct. Nuclear extracts were prepared from NIH/3T3 cells
that had either been mock-transfected or transfected with a SOX9
expression vector. The nuclear extracts were subjected to EMSA with
either a labeled SOX/Sry-binding site-D probe (D) or a probe
containing the SOX/Sry-binding sites from the human COL2A1
gene enhancer (IIE). C, DNA-binding competition
analysis of the SOX/Sry-binding site-D. EMSA was performed with RCS
nuclear extract and the addition of 50-fold excess unlabeled human
COL2A1 enhancer probe (IIE), wild-type
SOX/Sry-binding site-D probe (WT), or mutated
SOX/Sry-binding site-D probe (M). Arrow indicates
specific DNA-protein complex that is competed with excess IIE and WT
probes but not with excess M probe. D, antibody supershift
analysis of the SOX-Sry-binding site-D. Nuclear extracts from NIH/3T3
cells transfected with a SOX9 expression construct were subjected to a
antibody supershift assay with anti-SOX9 antibodies. Arrow
indicates super-shifted SOX9-D-site complex. Nuclear
extracts were prepared and EMSAs were performed as described under
"Materials and Methods."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
560 to
357 relative to the transcriptional start site. Further work revealed
that SOX9 can specifically bind to at least one of the two sites
(SOX/Sry-binding site-D) as demonstrated by EMSA competition and
supershift analyses. Our results strengthen the notion that SOX9 is a
master regulatory factor for chondrocyte-specific gene expression.
EF1 has been shown to repress COL2A1 gene expression
through sites in the promoter region in limb-bud mesenchymal cells
before differentiation occurs (3). The zinc-finger transcription factor
A-crystallin binding protein-1 is also a negative regulator of the
COL2A1 gene, acting through sites located in the enhancer
region (4). Certainly, more work is needed to elucidate the complex
network of control mechanisms involved in chondrocyte-specific gene expression.
742 bp and the
transcriptional start site (27). These sites were found to bind
a specific protein complex in RCS nuclear extracts that contained SOX9
and also were able to direct reporter gene expression to cartilaginous
tissues in transgenic mice (27). Other work has implicated a 60-bp
segment within the first intron that is important for
COL11A2 gene expression. This segment was also shown to
interact with SOX9 and promote cartilage-specific expression of
reporter genes in transgenic mice (28). Interestingly, the SOX/Sry
sites located in the COL11A2 proximal promoter region differ
from the sites studied in the present work in that they are much closer
to one another. Furthermore, we found that the first intron of the
COL9A1 gene did not augment the activity of the promoter
region. However, our experiments do not rule out a role for the first
intron in regulating COL9A1 gene expression in
vivo because we have not investigated the activity of the
COL9A1-reporter constructs in the context of transgenic mice.
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FOOTNOTES |
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* This work was partially supported by NIAMS, National Institutes of Health Project Grant AR-39740.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.
Supported by NIAMS, National Institutes of Health Training Grant
AR07583-08.
§ To whom correspondence should be addressed: Thomas Jefferson University, Division of Rheumatology, 233 S. 10th St., Rm. 511 BLSB, Philadelphia, PA 19107-5541. Tel.: 215-503-1011; Fax: 215-923-4649; E-mail: david.g.stokes@mail.tju.edu.
Published, JBC Papers in Press, October 23, 2002, DOI 10.1074/jbc.M208049200
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ABBREVIATIONS |
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The abbreviations used are: SOX, Sry-type HMG box; EMSA, electrophoretic mobility-shift assay; RCS, rat chondrosarcoma; HTB, human chondrosarcoma.
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