During the cartilage-to-bone transition,
participating chondrocytes eventually undergo hypertrophy and are
replaced by bone and marrow. Type X collagen is synthesized by
chondrocytes specifically when they become hypertrophic, and this
specificity is primarily regulated at the level of transcription.
Previously, we demonstrated that a proximal promoter region from
nucleotide
562 to +86 contained cis-acting elements that
directed high level expression of a reporter gene in a cell-specific
manner (Long, F., and Linsenmayer, T. F. (1995) J. Biol. Chem. 270, 31310-31314). In the present study, we have
further dissected this region by generating a series of constructs and
examining their expression in hypertrophic versus nonhypertrophic chondrocytes. Several positive and negative elements have been delineated within the proximal promoter region to mediate the
regulation of transcription in hypertrophic chondrocytes. Most notably,
a sequence from nucleotide
139 to +5 was sufficient to direct high
level expression in this cell type. Electrophoresis mobility shift
assay and supershift experiments identified within this sequence two
10-base pair noncanonical binding sites for Sp1 proteins. Mutations
within the Sp1 binding sites either diminished or abolished the
expression driven by the sequence from
139 to +5. These results
indicate that the Sp1 proteins mediate the cell-specific expression of
type X collagen.
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INTRODUCTION |
During endochondral bone formation, a notable event is the
hypertrophy of chondrocytes prior to their removal and replacement by
either bony tissue or marrow (1). A major product of hypertrophic chondrocytes is collagen type X (2-5). The chains of this homotrimeric molecule are encoded by a single gene, which becomes transcriptionally active following chondrocyte hypertrophy (6). This event occurs concomitant with a precipitous decrease in the production of certain other extracellular matrix molecules such as collagen types II and IX
(6, 7), proteoglycan Lb (8), and cartilage matrix protein (9).
The tissue-specific expression of type X collagen, and several
functional studies, suggest its potential importance for the development of endochondral bones. Individuals with Schmid metaphyseal chondrodysplasia have mutations in the type X collagen gene (10) that
disrupt the assembly of the chains into a triple-helical molecule (11).
In a transgenic study, major abnormalities in the cartilage growth
plate were observed in mice carrying a chicken type X transgene with
deletions (12). However, other investigators, using a gene knock-out
technique (13, 14), reported only subtle phenotypic changes in a type X
collagen-null mouse (14). Furthermore, type X collagen was shown to be
associated with preexisting types II/XI/IX collagen fibrils by
immunoelectron microscopy studies (15-17), and this was experimentally
demonstrated in in situ diffusion studies (18). The
diffusion studies also suggest that type X collagen may affect the
biosynthesis of cartilage proteoglycans and alter the biochemical and
physical properties of hypertrophic cartilage matrix (19).
The expression of type X collagen is controlled primarily at the level
of transcription, as demonstrated by both in situ
hybridization (6) and nuclear run-off experiments (20). We observed
previously in transfection experiments, that a proximal promoter region
that extended from 562 bp1
upstream to 86 bp downstream of the transcription start site conferred
cell specificity for the expression of the gene (21).
In the present study, we have explored further the proximal promoter
region and its neighboring sequences for their involvement in the
tissue-specific regulation of the type X collagen gene. In transient
transfection experiments with constructs containing serial deletions of
the proximal promoter region, we found multiple regulatory elements
that appear to contribute to the expression in hypertrophic
chondrocytes. A detailed analysis focused on a sequence from
135 to
+5, which conferred high level expression in this cell type, revealed
two noncanonical Sp1 binding sites. The binding of Sp1 proteins to
these sequences appears to be essential for high level expression in
hypertrophic chondrocytes .
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MATERIALS AND METHODS |
Generation of CAT Constructs--
The constructs pCAT I and pCAT
II were as described previously (21). Constructs pCAT III through pCAT
XII were generated as follows. The genomic inserts were obtained by PCR
from the plasmid pFL2, which contained a 1.5-kilobase pair genomic
fragment spanning the transcription start site of the type X collagen
gene (21). To each corresponding genomic fragment, a HindIII
site was added at the 5' end and a SalI site at the 3' end.
The resultant fragments were purified with the QIAquick PCR
Purification Kit (QIAGEN Inc.) and subjected to HindIII and
SalI digestion. The digested products were cloned into the
HindIII and SalI sites of the vector pCAT-Basic
(Promega). The resultant plasmids were sequenced (Life Technologies,
Inc.) to confirm their identities. To generate constructs pCAT XIII,
XIV, and XV, mutations were introduced into the sequence by PCR using
primers containing these mutations. The resultant DNA was then cloned
into pCAT-Basic as described above.
Cell Culture and Transfections--
Primary cultures of
hypertrophic chondrocytes and nonhypertrophic chondrocytes were
prepared from tibia and sterna of 14-day chick embryos, respectively,
as described previously (21).
The transient transfections were done as described previously (21).
Briefly, 1 µg of pSV
-gal (Promega) was used as an internal control
and cotransfected with 4 µg of constructs in 25 µg of Lipofectin
(Life Technologies, Inc.). Cells were transfected for 5-8 h, allowed
to recover in complete medium (Dulbecco's modified Eagle's medium
from Life Technologies, Inc. supplemented with 10% calf serum from
HyClone Laboratories) for 60 h, and the cells were harvested. In
each experiment, transfections of each construct were done at least in
triplicate.
-Galactosidase activity was measured according to
Sambrook et al. (22) and expressed as the optical density of
the substrate reaction at the wavelength of 420 nm (OD420).
CAT (pg) was determined by enzyme-linked immunosorbent assay using an
anti-CAT antibody (Boehringer Mannheim). Normalized levels of CAT
expression were calculated as CAT protein to
-galactosidase activity
(i.e. CAT/
-galactosidase). The mean value of
CAT/
-galactosidase ratios from multiple transfections for each
construct was taken as its CAT expression level in the experiment.
Results presented in the paper are from two or more separate
experiments.
Electrophoretic Mobility Shift Assay (EMSA)--
Nuclear
extracts were prepared from monolayer cell cultures at different salt
concentrations (0.8-1.6 M KCl) according to Abmayr and
Workman (23), and protein concentrations were determined by a protein
assay kit (Bio-Rad). Double-stranded probes were generated either by
PCR or by annealing complementary synthetic oligonucleotides, according
to Mueller et al. (24). These were then labeled with
32P by a forward reaction with T4 kinase (Life
Technologies, Inc.) and the products purified by Nensorb 20 cartridges
(NEN Life Science Products). EMSA was performed according to Chodosh
(25). Nuclear extracts of 7-14 µg and approximately 1-3 × 104 cpm of probes were used in each reaction. For
competition assays, cold competitors were added in 200-400 molar
excess. The nucleotide sequence of the competitor for Sp1 binding
(Santa Cruz Biotechnology, Inc.) is as follows (shown for a single
strand): 5'-ATTCGATCGGGGCGGGGCGAGC-3'. The sequence of the
mutant form (Sp1') is 5'-ATTCGATCGGTTCGGGGCGAGC-3'.
For supershift experiments, binding was performed as described above,
and subsequently 2 µl of Sp1 antibody (1 µg/µl rabbit polyclonal
antibody, Santa Cruz Biotechnology, Inc.) was added to the reaction. An
additional 20-min incubation at 30 °C followed prior to gel
analyses.
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RESULTS |
Multiple cis-Acting Elements Mediating Expression in Hypertrophic
Chondrocytes--
Previously, we showed that the proximal promoter
region, comprising 562 bp upstream and 86 bp downstream of the
transcription start site (pCAT I, see Fig.
1), directs high level expression of a
reporter gene in a cell-specific manner (21). To delineate the
cis-acting elements within this region that are responsible for the regulation, we have now generated a series of constructs with
nested deletions (see Fig. 1) and tested them by transient transfections. In nonhypertrophic chondrocytes, all constructs examined
directed only a low level of expression. Specifically, the expression
levels by these constructs in this type X-negative cell type were less
than 30% of the expression elicited by pCAT I in hypertrophic
chondrocytes (Table I). Examination of
these constructs in hypertrophic chondrocytes, however, revealed a
number of potential cis-acting elements, both positive and
negative.

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Fig. 1.
A diagram of constructs used for
transfections. Shown for each construct is the insert cloned in
CAT expression vector. Also shown is a partial organizational map of
the type X collagen gene. The transcription start site is denoted by
+1.
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Table I
Minimal expression of CAT constructs in nonhypertrophic chondrocytes
(n = 3)
Expression is expressed as percent of the expression level of pCAT I in
hypertrophic chondrocytes.
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Effects of the First Exon--
The first exon of the type X
collagen gene appeared to contain a positive regulatory activity for
transcription. The 86-bp transcribed sequence (nt +1 to +85, see Fig.
1) present in pCAT I represents a large portion of the first exon (26).
The deletion construct pCAT III, however, contains only 5 bp of the
transcribed region and thus lacks the bulk of the first exon (see Fig.
1). This construct elicited expression significantly lower than that by
pCAT I (see Fig. 2), indicating the
presence of a positive regulatory activity in the sequence from nt +6
to + 85. Consistent with this, another construct that lacked the
sequence, pCAT X, also directed considerably lower expression than
pCAT XI, which contained it.

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Fig. 2.
Relative expression of CAT constructs in
hypertrophic chondrocytes. Expression by pCAT I is termed 100%.
Results are expressed as mean ± S.D. of triplicates in two or
more separate experiments.
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Effects of an Upstream Sequence and the First
Intron--
Previously, we reported that the 2.6-kilobase pair
upstream sequence included in pCAT II did not affect cell specificity
to any substantial degree. By comparing pCAT II and pCAT I within hypertrophic chondrocytes themselves, however, we found that expression by the former was consistently lower than that by the latter (see Fig.
2), suggesting that the 2.6-kilobase pair upstream sequence may
actually repress expression.
The first intron of several collagen genes has been shown to contain
regulatory activities responsible for tissue-specific expression (see
"Discussion"). To test whether this is true for the type X collagen
gene, we examined the expression by pCAT XII, a construct that
contained the entire first intron in addition to the proximal promoter
region (see Fig. 1). Our results showed that the expression level by
this construct is similar to that by pCAT I (see Fig. 2), indicating
that the first intron does not have any detectable effect on
expression.
Effects of the Sequence from nt
562 to
140--
To determine
whether other regulatory elements might be present in the proximal
promoter region, serial deletions were made from the 5'-end inward
within the pCAT III insert (i.e. sequence from nt
562 to
+5). This procedure resulted in the constructs pCAT IV through pCAT X
(see Fig. 1). From the experiments using this set of constructs, a
number of potentially interesting features of the promoter were
uncovered.
A positive activity was found from nt
562 to
493, as the expression
level elicited by pCAT IV was lower than that by pCAT III (see Fig. 2).
Likewise, a second positive activity was located within the sequence
from nt
492 to
413, as shown by the lower expression level by pCAT
V compared with that by pCAT IV.
Negative regulatory activities were also found in the proximal promoter
region. These were present from nt
412 to
292 and from nt
291 to
140, as demonstrated by an increase of expression by progressively
shorter constructs, starting from pCAT V to pCAT VI to pCAT VII (see
Fig. 2).
Activation by Sequence from nt
139 to
55 in Hypertrophic
Chondrocytes--
We next further dissected the sequence from
139 to
55, which appeared to contain a potent activity for high level
expression in hypertrophic chondrocytes. Whereas pCAT X (containing the
sequence from nt
54 to +5, see Fig. 1) elicited only a negligible
level of expression, inclusion of the sequence from nt
139 to
55 in pCAT VII resulted in an 8-10-fold increase in CAT expression (Fig. 2).
Further analyses of the region from nt
139 to
55 revealed that
sequences from nt
139 to
115, and from nt
89 to
55 are important for the high level expression. This is indicated by the
results that the expression level dropped about 2-fold from pCAT VII to
pCAT VIII and 3-fold from pCAT IX to pCAT X. Thus, by performing
transient transfection experiments in hypertrophic chondrocytes, we
have identified multiple regions with regulatory activities in the type
X collagen gene. These results are summarized in Table
II.
Identification of Core Sequences for Binding of Nuclear
Proteins--
To begin to examine the potential involvement of nuclear
proteins in the regulation of expression in hypertrophic chondrocytes, EMSA was carried out using double-stranded DNA from nt
139 to
55 as
a probe. We chose to examine this region because the transfections have
shown that a construct containing this relatively short sequence, pCAT
VII, elicited high level expression (about 70% of that by pCAT I) in
hypertrophic chondrocytes (see above). Nuclear extracts prepared at
different salt concentrations (0.8-1.6 M KCl) were examined, and all gave similar results (Fig.
3 and data not shown). Two major bands
specific to the probe were detected (see Fig. 3B). To
further dissect this region, a panel of oligonucleotides designed to
cover the full length of the probe were used as competitors in EMSA
(Fig. 3A). These experiments showed that DNA from nt
89 to
55 (Com4) competed for binding just as efficiently as the full-length
cold probe (Com1), and that DNA from nt
139 to
115 (Com2) also
competed for binding but to a somewhat lesser extent. DNA from nt
114
to
90 (Com3), from nt
120 to
94 (Com5), and from nt
99 to
75
(Com6), however, did not compete (see Fig. 3B). These
results localized nucleotides important for the binding to the regions
from nt
139 to
121 and from nt
75 to
55. Furthermore, both Com2
and Com4, when themselves used as probes in EMSA, exhibited two major
bands (see Fig. 4, A and
B) with mobilities similar to those produced by the
full-length probe (Fig. 3B), confirming that they contribute
to the binding of nuclear factors. These results are consistent with
the finding from the transfection studies that nucleotides from
nt
139 to
115 and from nt
89 to
55 are important for the high
level expression conferred by the sequence from nt
139 to
55
(described above).

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Fig. 3.
EMSA and competition assays. The
sequence from 139 to 55 was used as a probe, and its various
regions were as competitors (Com1 through Com6) (A).
Abbreviations in B are as follows: N, no nuclear
extract; NC, no competitor; C1-C6, Com1-6;
F, free probe. The arrows indicate the two major
shifted bands.
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Fig. 4.
EMSA and mutation analyses. Com2
(A) and Com4 (B) were used as probes. Sequences
containing mutations (mutant 1 through mutant 5) were analyzed for
their ability to compete with corresponding probes. Dotted
lines indicate non-mutated nucleotides. Nucleotides in bold
face constitute the core sequences (CS1 and CS2, see
"Results"). Also as competitors are the cold probes (C2,
Com2; C4, Com4). NC, no competitor;
M1-M5, mutant 1-5; F, free probe. The
arrows point to the two major bands detected.
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Sequence comparisons between the sequences from nt
139 to
121 and
from nt
75 to
55 revealed two 10-bp nucleotide stretches of high
similarity. These nucleotide stretches, located from
130 to
121 and
from
74 to
65, are 5'-CCCCACCCCT-3' and 5'-TGGGGAGGAG-3', respectively. We term them "core sequences"
1 (CS1) and 2 (CS2), respectively (see Fig. 4, A and
B). To determine whether the core sequences are important
for binding of nuclear factors, oligonucleotides containing mutations
within these sequences were examined for competition in EMSA. As shown
for probe Com2 in Fig. 4A, a mutation within CS1 (mutant 1)
abolished its competition. Similarly for probe Com4, mutations within
CS2 (mutants 2 and 3) abolished competition, whereas mutations in other
regions had no effect (mutants 4 and 5) (Fig. 4B). These
data indicate that CS1 and CS2 are essential for the sequence from
139 to
55 to bind nuclear proteins from hypertrophic
chondrocytes.
To determine whether Com2 and Com4 bind the same proteins, these
sequences were used to compete with each other in EMSA. Shown in Fig.
4A, Com4 competed with the probe Com2 and the competition was abolished by the mutation within CS2 (mutant 2). Likewise, Com2
competed with the probe Com4, and the competition was eliminated by a
mutation within CS1 (mutant 1) (Fig. 4B). Thus, CS1 and CS2 appear to be involved in binding the same proteins.
Identification of the Core Sequences as Sp1 Binding Sites--
A
computer search identified 70% similarity between the core sequences
and the consensus binding site for Sp1 and its related proteins. To
determine whether it is Sp1 family proteins that are responsible for
the binding to the core sequences in hypertrophic chondrocytes, we
first performed additional competition assays using EMSA. In these
assays, double-stranded oligonucleotides containing either a wild-type
or a mutant Sp1 binding site were used as competitors for binding. As
shown in Fig. 5A, in reactions using either Com2 or Com4 as a probe, the wild-type Sp1 oligonucleotide (lanes 4 and 10) disrupted formation of the two
major complexes (arrows), whereas the mutant oligonucleotide
Sp1', with a two-nucleotide alteration (see "Materials and Methods"
for sequences), did not (lanes 5 and 11). These
results indicate that the Sp1 binding sequence specifically competes
with both Com2 and Com4 for the binding.

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Fig. 5.
Binding of Sp1 to Com2 and Com4. Either
Com2 or Com4 (see Fig. 4) was used as a probe in hypertrophic
chondrocytes (A) and nonhypertrophic chondrocytes
(B). The arrows point to the two major complexes.
Comp., competitor; N, no nuclear extracts; NC, no competitor; C2, Com2; C4, Com4;
Sp1, Sp1 binding sequence; Sp1', mutant Sp1
binding sequence; Ab, Sp1 antibody; F, free
probe.
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To determine whether the core sequences bind Sp1, we performed
supershift experiments using an antibody against this protein. As shown
in Fig. 5A (lanes 6 and 12), the
bottom band of the two main complexes (arrows) detected by
Com2 and Com4 was supershifted by the Sp1 antibody. The upper band,
however, does not appear to be shifted by the antibody. Thus, the lower
band contains Sp1, whereas the upper band may contain a related family
member. Overall, together with the competition data above, these
results establish that both CS1 and CS2 bind Sp1.
A similar observation was also made with nuclear extracts from
nonhypertrophic chondrocytes (Fig. 5B). Specifically, probes Com2 (lane 13) and Com4 (lane 18) both detected
two major complexes that were competed out by the Sp1 sequence
(lanes 15 and 20) but not by the mutant Sp1'
oligonucleotide (lanes 16 and 21). Of the two
complexes, the lower but not the upper band was also supershifted by
the Sp1 antibody (lanes 17 and 22). Therefore, as
in hypertrophic chondrocytes, Sp1 and its related proteins are also
present and bind to the core sequences in nonhypertrophic chondrocytes.
However, the ratio of the two complexes appears different between
hypertrophic and nonhypertrophic chondrocytes. For instance, the bottom
band formed by Com4 predominates over the upper band in nonhypertrophic chondrocytes (lane 18) whereas the two appear similar in
intensity in hypertrophic chondrocytes (lane 8). These
differences presumably reflect differential expression of the Sp1
family members in the different cell types, which could be important
for cell-specific regulation of the type X collagen gene (see
"Discussion").
Effects of Mutations in Sp1 Binding Sites on Expression--
To
determine whether Sp1 family binding is functionally important for
regulating expression of the type X collagen gene, we performed
transfection experiments with mutant constructs. The mutations, in
either CS1 or CS2 or both (Fig.
6A), were introduced into the
construct pCAT VII and the effects of these alterations were examined
for changes in the expression levels. As shown in Fig. 6B, a
mutated CS1 (pCAT XIII) reduced expression by approximately 2-fold
(compared with pCAT VII), and mutations in both CS1 and CS2 (pCAT XV)
completely abolished expression (i.e. reduced it to the
level of pCAT X). Likewise, a mutated CS2 alone (pCAT XIV) also
eliminated expression. These data, when taken together with the results
that these mutations abolish binding (see above), demonstrate that the
binding of these Sp1 family proteins at both CS1 and CS2 are important
for high level expression in hypertrophic chondrocytes. Furthermore,
the binding of Sp1 proteins at CS2 appears to be indispensable.

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Fig. 6.
Effects of mutations within core sequences on
CAT expression. Nucleotides were mutated within either one or both
of the sequences (A). Dotted lines indicate
non-mutated nucleotides. Also shown is pCAT X, lacking the upstream
region altogether. Shown in B are the effects of these
mutations on expression. The expression by the wild type sequence (pCAT
VII) is termed 100%. Results are expressed as mean ± S.D. of six
transfections.
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DISCUSSION |
In the present study, we have uncovered a number of potential
cis-acting elements involved in the transcriptional control of the chicken type X collagen gene. Most notably, an 85-bp sequence from nt
139 to
55 contained a potent positive regulatory activity and this sequence, in conjunction with a short fragment containing the
TATA box (nt
54 to +5), was sufficient to direct high level expression in a cell-specific manner. Detailed analysis of this region
revealed two noncanonical Sp1 binding sites, whose binding to Sp1
family proteins appeared to be critical for high level expression of
the gene in hypertrophic chondrocytes.
The present study suggests that the Sp1 family proteins mediate the
expression of type X collagen in a cell-specific manner. Similar
cell-specific regulation by these factors has also been reported for
other matrix genes, including those of collagens and elastin (27-30).
Although the exact mechanism for this is unclear at present, several
scenarios can be postulated. One is based on information showing that
not all members of the family produce the same regulation. For
instance, it is known that Sp3 functions as an inhibitor, whereas Sp1
is an activator (29, 31). Thus it is conceivable that, in chondrocytes
at different stages of maturation, regulation is achieved by the
relative level or availability of these factors. This is of particular
interest in light of our gel shift data, which showed differential
relative intensities for the bands of Sp1 family proteins in
hypertrophic versus nonhypertrophic chondrocytes (Fig. 5).
Furthermore, since Sp1 is a phosphoprotein (32-35), its
phosphorylation status in different cell types could influence its
regulatory function in a cell-specific manner.
Alternatively, other protein-DNA interactions in the promoter may
influence the function of Sp1 proteins in nonhypertrophic chondrocytes.
In support of this possibility, our preliminary data showed that when
an 85-bp fragment (
139 to
55) containing the Sp1 sites and their
neighboring sequence was used as an EMSA probe, three additional bands
exclusive to nonhypertrophic chondrocytes were detected, in addition to
the two major Sp1 complexes (data not shown). Two of the three bands
appear to represent higher order Sp1 complexes. The third band,
however, seems to be a separate entity, since its formation occurs more
efficiently in the absence of Sp1 binding. These observations, when
coupled with the result that this 85-bp sequence conferred cell
specificity in transfections, suggest a possible inhibitory mechanism
in the nonhypertrophic chondrocytes.
Nonetheless, Sp1 appears to be critical for the high level expression
of type X collagen in hypertrophic chondrocytes. Nucleotide comparisons
of the type X collagen gene across several species revealed that the
Sp1 binding site CS2 identified in this study is greatly conserved
throughout evolution (see Table III). For instance, the promoter for the human gene contains an element that is
90% identical with the chicken CS2, despite the fact that the overall
similarity between the two species from nt
100 to
1 is only about
60%.
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Table III
Conservation of a Sp1 binding site (CS2) across species
Bold letters denote nucleotides conserved with those in the chicken.
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Of the two Sp1 binding sites, CS2 appears to have a higher affinity.
This is suggested both by the competition analyses in which Com4
(containing CS2) competed better than Com2 (containing CS1) (Fig.
3B), and by the mutation study where probe 2 (with a mutated
CS1) bound Sp1 more efficiently than probe 1 (with a mutated CS2) (data
not shown). This differential binding affinity is consistent with the
greater reduction of expression in transfections by the mutation in CS2
versus that in CS1 (see Fig. 6B). The
mechanism(s) by which CS1 and CS2 combinatorially regulate expression
of the gene remain to be investigated. Nonetheless, CS2 appears to play a pivotal role for activation of the gene. Activation by CS1 is not
only synergistic with that by CS2 but also seems to be dependent upon
the presence of CS2.
The present study also revealed several other potential regulatory
regions in the promoter of the type X collagen gene. For instance, the
sequence from nt
562 to
413 appeared to have a positive regulatory
activity. Conversely, the fragment from nt
3163 to
563 and that
from nt
412 to
140 seemed to repress expression in hypertrophic
chondrocytes. Further studies, however, are required to elucidate the
regulation in these regions. Taken together, the results from this
study suggest that multiple cis-acting regulatory elements
exist within the proximal promoter region of the chicken type X
collagen gene and that Sp1 family proteins mediate signals promoting
the cell-specific expression of the type X collagen gene.