(Received for publication, July 21, 1995)
From the
We show that a new rat chondrosarcoma (RCS) cell line
established in long-term culture from the Swarm tumor displayed a
stable differentiated chondrocyte-like phenotype. Indeed, these cells
produced the collagen types II, IX, and XI and alcian blue-stainable
cartilage-specific proteoglycans, but no type I or type III collagen.
To functionally characterize their chondrocytic nature, the cells were
stably transfected with a type II collagen/geo chimeric gene which
confers essentially perfect chondrocyte-specific expression in
transgenic mice. RCS cells expressed both
-galactosidase and G418
resistance, in comparison with similarly transfected 10T1/2 and NIH/3T3
fibroblasts which did not. These cells were then used to perform a
systematic deletion analysis of the first intron of the mouse type II
collagen gene (Col2a1) using transient expression experiments to
determine which segments stimulated expression of a luciferase reporter
gene in RCS cells but not in 10T1/2 fibroblasts. Cloning of two tandem
copies of a 156-base pair (bp) intron 1 fragment (+2188 to
+2343) in a construction containing a 314-bp Col2a1 promoter
caused an almost 200-fold increase in promoter activity in RCS cells
but no increase in 10T1/2 cells. DNase I footprint analysis over this
156-bp fragment revealed two adjacent protected regions, FP1 and FP2,
located in the 3`-half of this segment, but no differences were seen
with nuclear extracts of RCS cells and 10T1/2 fibroblasts. Deletion of
FP2 to leave a 119-bp segment decreased enhancer activity by
severalfold, but RCS cell specificity was maintained. Further deletions
indicated that sequences both in the 5` part of the 119-bp fragment and
in FP1 were needed simultaneously for RCS cell-specific enhancer
activity. A series of deletions in the promoter region of the mouse
Col2a1 gene progressively reduced activity when these promoters were
tested by themselves in transient expression experiments. However,
these promoter deletions were all activated to a similar level in RCS
cells by a 231-bp intron 1 fragment that included the 156-bp enhancer.
The RCS cell-specific activity persisted even if the Col2a1 promoter
was replaced by a minimal adenovirus major late promoter. This 231-bp
intron 1 fragment also had strong enhancing activity in transiently
transfected mouse primary chondrocytes. Our experiments establish the
usefulness of RCS cells as an experimental system for studies of the
control of chondrocyte-specific genes, provide an extensive delineation
of segments in the Col2a1 first intron involved in chondrocyte-specific
activity, and show that promoter sequences are dispensable for
chondrocyte specificity.
The differentiation of mesenchymal cells into chondrocytes results in the synthesis and secretion of a series of proteins characteristic of the extracellular matrix of cartilages. These include types II, IX, and XI collagens, the proteoglycan aggrecan, link protein, and cartilage matrix protein, the expression of which is part of a genetic program of differentiation specific for chondrocytes(1, 2) . Earlier studies on the biosynthesis of these cartilage components have used mainly primary chondrocytes, but the phenotypic instability of these cells, which varies with culture conditions, has made experiments on chondrocyte differentiation difficult(3, 4, 5, 6) . A small number of chondrocyte cell lines that maintain at least part of the chondrocyte phenotype during culture have been isolated recently(7, 8, 9, 10, 11, 12) . Such stable chondrocytic cell lines are essential for achieving further understanding of the genetic program of chondrocyte differentiation.
Because the gene for type II collagen is expressed at very high levels in chondrocytes(2, 13, 14) , it is an excellent candidate for study of chondrocyte differentiation. Humans and mice with mutations in type II collagen often show severe cartilage defects and skeletal malformations (15, 16, 17) . During embryonic development, the gene becomes active in cartilage anlages as early as the time of mesenchymal condensation preceding cartilage formation. Hence, type II collagen can be considered an early and abundantly expressed marker of chondrocyte differentiation. Although the gene for type II collagen is also expressed transiently in some extrachondrogenic sites during embryonic development(14) , this occurs at much lower levels, and the significance of this extrachondrocytic expression is not understood, in contrast to the richly documented role played by this molecule in cartilage function.
Our long-term goal is to identify
and characterize the DNA-binding proteins involved in the
chondrocyte-specific activity of the type II collagen gene. In this
study we sought to better delineate sequences within the mouse type II
collagen gene (Col2a1) ()that can confer
chondrocyte-specific expression to reporter genes. Previous transient
expression experiments using primary chick chondrocytes had identified
a 620-bp chondrocyte-specific enhancer in the first intron of the rat
Col2a1 gene(3) . Subsequently, similar experiments showed that
a 380-bp segment within this 620-bp fragment activated expression of a
reporter gene 11-fold in chondrocytes and that a 260-bp subfragment
activated expression 6-fold(4) . A point mutation in this
260-bp fragment decreased this activity by about two-thirds. In other
experiments, two silencer elements identified in the rat Col2a1
promoter were proposed to participate in inactivating the gene in
nonchondrocytic cells (18) .
In the present study we first examined the expression of a series of molecular markers to characterize the chondrocytic phenotype of a new rat chondrosarcoma (RCS) cell line derived from the Swarm chondrosarcoma tumor after long-term culture. The cells were further characterized by comparing them with 10T1/2 and NIH/3T3 fibroblasts after stable transfection with a mouse Col2a1 chimeric DNA construction and by establishing that the Col2a1-derived transgene was selectively expressed in chondrosarcoma cells. We then used these cells in transient expression experiments to delineate minimal sequences in the first intron of the mouse Col2a1 gene that were needed for chondrocyte-specific enhancer activity and to determine active sequences in the promoter of this gene.
Permanent
transfections were performed by the modified DNA-calcium phosphate
coprecipitation method described by Chen and Okayama(25) . 5
10
cells were plated in 50-cm
tissue
culture dishes and transfected with 1 µg of pPGK-HYG alone or
together with 20 µg of pCol2a1-
geo. Hygromycin-resistant cells
were selected by treatment for 10 days with 200 µg/ml hygromycin B.
Southern analysis was performed on the pools of hygromycin-resistant
cells to verify integration of the Col2a1 transgene. This was done by
using the 3-kb Escherichia coli
-galactosidase gene (lacZ) as a probe. Selection of cells expressing the neomycin
resistance gene was performed by treatment with G418 at 300 or 500
µg/ml as indicated.
-Galactosidase activities were measured
with a chemiluminescent assay kit (Tropix, Bedford, MA), and protein
was assayed with the Bradford reagent (Bio-Rad).
Figure 1: Morphology of the RCS cells and staining with alcian blue. A, nonconfluent culture of RCS cells 24 h after plating. B, confluent culture 3 days after plating. C, alcian blue staining of a confluent culture. The extracellular matrix synthesized by the RCS cells is produced as a thick viscous layer of material that easily detached during the staining procedure. This might account for the nonuniform alcian blue staining. A and B are dark-field photographs. C is a bright-field photograph. Bars in A, B, and C correspond to 100 µm.
Figure 2:
Northern blot analysis of RCS cell RNAs.
Total RNA was prepared from the following cell cultures: MPC,
mouse rib primary chondrocytes maintained in culture for 2 days (A) or 3 days (B, C, and D); RCS, rat chondrosarcoma cells; RPC, rat articular
primary chondrocytes cultured for 3 days; ROS, rat
osteosarcoma cells (ROS 17/2.8). Northern blots were performed with 10
µg of RNA per sample. Membranes were hybridized with DNA probes for
various extracellular matrix protein transcripts, and autoradiographs
were taken for different periods of time as follows: A, mouse
pro-1(II) collagen (Col2a1), 10 or 60 min as indicated; B, mouse pro-
2(IX) collagen (Col9a2), 16 h; C, mouse
1(X) collagen (Col10a1), 4 days; mouse
pro-
1(I) collagen (Col1a1), 22 h; D, rat
osteopontin (OP) and mouse matrix Gla protein (MGP),
2 h 30 min; mouse bone Gla protein (BGP), 6 days. Staining of
28 S rRNA with ethidium bromide (A and B) or
hybridization with the human glyceraldehyde-3-phosphate dehydrogenase
probe (GAPDH, C and D) are shown as
references for the amount of RNA loaded for each
sample.
Figure 3:
Analysis of collagens produced by RCS
cells. Confluent cultures of 10T1/2 fibroblasts, RCS cells, and mouse
primary chondrocytes (MPC, 20 h after release from cartilage)
were labeled with [H]proline and further
processed for the analysis of collagens as described under
``Materials and Methods.'' Aliquots from the culture media
and from the cell and matrix layers of each culture were treated with
pepsin only (C and M, respectively) or with pepsin
followed by bacterial collagenase (C* and M*,
respectively). SDS-PAGE were run without (A) or with
dithiothreitol (B). Numbers indicate the molecular
mass of protein standards which were run alongside on the same gels.
Collagen chains produced by 10T1/2 cells were separated into different
bands as follows: a,
1(III) trimer; b,
1(V); c,
1(I) and probably
2(V); d,
2(I); j,
1(I),
1(III), and probably
2(V).
Collagen chains produced by RCS cells and mouse primary chondrocytes
were as follows: e, likely trimers of type IX collagen chains
undigested or partially digested by pepsin; f,
1(XI); g,
2(XI); h,
1(II) and probably
3(XI); k, l, and m, likely type IX collagen chains
undigested or partially digested by pepsin.
Altogether, these results indicated that the RCS cells express a number of characteristic chondrocyte differentiation markers, including type II, IX, and XI collagens, and cartilage-specific proteoglycans. The absence of type X collagen RNA suggests that they were frozen in a state before transition into hypertrophic chondrocytes. Importantly, the complete absence of type I collagen indicates that these cells, unlike other chondrocytic cell lines previously described(7, 8, 9, 10, 11) , do not express a partial mesenchymal or fibroblastic phenotype.
Figure 4:
Expression of a Col2a1-geo
transgene permanently transfected in RCS, 10T1/2, and NIH/3T3 cells. A, schematic of the Col2a1-
geo construct. See
``Materials and Methods'' for details. B, expression
of neomycin resistance. A pool of cell clones that had stably
integrated the Col2a1-
geo construct was obtained as described
under ``Materials and Methods'' for each of the RCS (squares), 10T1/2 (circles), and NIH/3T3 (triangles) cell lines. Expression of the
geo gene in
these cell pools was determined by measuring the growth of the cells
during 9 days of culture in the presence of G418. 3
10
cells of each pool were plated in 50-cm
dishes, 500
µg/ml of G418 was added to the medium, and the number of cells
excluding trypan blue was estimated at 3-day intervals in a
hemocytometer after release of the cells from plastic with
trypsin/EDTA. Three similar independent experiments were carried out
with essentially the same results, i.e. only the stably
transfected RCS cells survived in the presence of G418. C,
expression of
-galactosidase. Pools of either RCS, 10T1/2, or
NIH/3T3 cell clones were obtained that had stably integrated either a
pPGK-HYG construct only or both the pPGK-HYG and the Col2a1-
geo
constructs (see ``Materials and Methods'').
-Galactosidase was assayed after selection with hygromycin (+HYG) and, also, for RCS cells transfected with both
pPGK-HYG and Col2a1-
geo constructs, after selection with
hygromycin, followed by 1 week of selection with 500 µg/ml G418 (+HYG/+G418).
-Galactosidase activities are
presented in relative chemiluminescent units per µg of protein. The
activity of
-galactosidase in cells transfected with pPGK-HYG
alone corresponds to the endogenous level of
-galactosidase
activity in each cell type; these endogenous
-galactosidase levels
were higher in RCS cells than in 10T1/2 and NIH/3T3 fibroblasts. Only
the RCS cells stably transfected with the Col2a1-
geo construct
contained levels of
-galactosidase above the endogenous levels. *,
-galactosidase assays could not be performed since these cells did
not survive in the presence of G418. Similar results were obtained in
two independent experiments.
After a first selection
of the cells with hygromycin, a Southern hybridization with the DNA of
the pooled colonies showed a signal of about equal intensity for the
three cell types, indicating that the Col2a1 transgene had been stably
integrated (data not shown). To determine whether the Col2a1 transgene
was expressed, the pooled colonies were first tested for resistance to
neomycin. As shown in Fig. 4B, RCS cells continued to
grow in the presence of G418, whereas 10T1/2 and NIH/3T3 fibroblasts
rapidly died. In control experiments, RCS cells that did not contain
the Col2a1-geo transgene died within a few days in G418 selection
medium (data not shown). Results similar to those in Fig. 4B were also obtained when a lower G418 concentration (350 instead of
500 µg/ml) was used (data not shown).
We then tested expression
of -galactosidase in RCS, 10T1/2, and NIH/3T3 cells stably
transfected with pPGK-HYG alone or with both pPGK-HYG and
Col2a1-
geo (Fig. 4C). After selection with
hygromycin, no difference in
-galactosidase activity was observed
whether 10T1/2 and NIH/3T3 fibroblasts were transfected with one or
with both plasmids. RCS cells contained endogenous
-galactosidase
activity that was considerably higher than that of 10T1/2 and NIH/3T3
cells. However, the RCS cell
-galactosidase activity was much
higher in cells containing the Col2a1-
geo construct than in
control cells. After G418 selection, these levels increased by an order
of magnitude in RCS cells transfected with the Col2a1-
geo
construct. This increase was probably due to the G418 selection which
eliminated cells that were transfected by the hygromycin vector only
and probably also the cells that expressed levels of
geo below the
G418 threshold. These functional experiments indicated that RCS cells
were able to support the activity of a stably transfected
chondrocyte-specific chimeric transgene whereas in 10T1/2 and NIH/3T3
cells the transgene was inactive. These results are consistent with the
notion that, unlike 10T1/2 and NIH/3T3 fibroblasts, RCS cells contain
the transcription factors needed for the activity of a
chondrocyte-specific transgene.
Figure 5:
Delineation of a 231-bp Col2a1 intron 1
fragment showing enhancer activity specifically in RCS cells in
transient transfection experiments. The pP1L plasmid was obtained by
cloning an 886-bp fragment of the mouse Col2a1 promoter (box with
grid) in the SmaI site located just upstream of the
luciferase gene (box labeled LUC) in the pALUC
vector. The pI1P1L to pI10P1L plasmids and the pI91P1L plasmid contain
different fragments of the first intron of the mouse Col2a1 gene (boxes with stripes) cloned in the BglII site of the
pP1L vector located
800 bp upstream of the promoter insertion
site. The sizes of the promoter and intron fragments are indicated
inside their respective boxes, and the position of their first and last
nucleotides relative to the Col2a1 transcription start site is
indicated at the bottom left and right sides of the
boxes. In the pI9`P1L and pI10P1L constructs, two copies of the
intron fragments were cloned head to tail, as indicated between brackets (2
). All these DNA constructs were transiently
transfected into 10T1/2, C
C
, and RCS cells.
Luciferase activities were measured in cell extracts and normalized for
transfection efficiency (see ``Materials and Methods'').
Transcriptional activation of the promoter induced by intron fragments
is given relative to the activity obtained with the pP1L construct
(considered as 1) and is presented as the average ±
S.E. of all transfection assays performed, with the number of assays
indicated in parentheses.
The results of the luciferase
assays are presented as multiples of the luciferase values obtained
with the promoter alone (Fig. 5). Activity of the promoter
fragment alone was about 5 to 15 times higher in 10T1/2 cells and about
30 times higher in CC
myoblasts than in RCS
cells. A 3-kb intron 1 fragment (+306 to +3325) produced a
modest 3-fold increase in RCS cells, but a 5- to 7-fold decrease in
10T1/2 and C
C
cells. This intron fragment is
similar to the one that was used in conjunction with a 3-kb mouse
Col2a1 promoter segment in stable transfections of Fig. 4and
also in transgenic mice.
In contrast to the modest increase
in activity seen in transiently transfected RCS cells, mice harboring
the latter construction showed strong expression of the transgene that
was completely chondrocyte-specific. This difference could reflect
differences between transient expression in tissue culture cells and
stable integration in transgenic mice or differences in the placement
of the intron sequences, which were located in their natural position
3` of the transcription start site in the construction used for
transgenic mice. In other experiments we have shown that when a 300-bp
promoter instead of a 3-kb promoter was used, no change occurred in
chondrocyte-specific expression in transgenic mice.
The
5`-half of the 3-kb intron fragment (+306 to +1880) showed no
enhancing activity, whereas the 3`-half (+1637 to +3251)
showed 10 times more activity than the 3-kb fragment in RCS cells (Fig. 5). It is possible that the 5`-half of the 3-kb fragment
contained inhibitory sequences that decreased the activity of the 3-kb
intronic enhancer. Further fragmentation of this active segment showed
that a 953-bp fragment (+1394 to +2346) was an active
enhancer in RCS cells but that a 908-bp fragment (+2344 to
+3251) located 3` of this segment was much less active. Further
work was focused on smaller fragments. A 546-bp fragment (+1880 to
+2425) showed a 14-fold increase specifically in chondrosarcoma
cells but smaller fragments were clearly less active. However, we found
that when two copies of a 465-bp fragment (+1880 to +2344)
were cloned tandemly a 44-fold increase in activity occurred in
chondrosarcoma cells whereas practically no increase occurred in the
other two cell types. Similarly, two tandem copies of a 231-bp fragment
(+2113 to +2343) produced a 100-fold increase in activity in
RCS cells, but much less in 10T1/2 and C
C
cells. It is possible that several modular elements in the first
intron contribute to the strength of the chondrosarcoma cell-specific
enhancer and that, when smaller fragments are generated, duplication of
one or more of these modular elements is necessary to compensate for
the loss of other active elements.
Figure 6:
Protein binding and nucleotide sequence
analysis of the 231-bp Col2a1 enhancer. In A, a DNase I
protection assay was performed on the coding strand of the Col2a1
231-bp enhancer fragment in the absence of nuclear extracts (no
N.E.) or after preincubation with nuclear extracts from 10T1/2 or
RCS cells, as indicated at the top of the lanes. The first
lane shows a G + A sequencing of the same DNA strand. Two
regions protected by nuclear proteins are bracketed and
labeled FP1 and FP2. In B, the coding strand
of the 231-bp enhancer is aligned with analogous regions in the human
and rat type II collagen genes. Only 96 bp of the corresponding rat
sequence are available from the GenBank data base. Only
bases in the human and rat sequences that differ from the mouse
sequence are shown; identical bases are indicated by a dot.
Gaps, marked by a dash, have been added to maximize alignment.
A decamer sequence in the rat gene, that was postulated to have a role
in chondrocyte-specific expression, is shown in italics. The
regions footprinted in DNase I protection assays are underlined in the mouse sequence and labeled FP1 and FP2.
The numbers +2113 to +2343 above the mouse
sequence indicate the distance in nucleotides from the mouse Col2a1
transcription start site. Repeated DNA sequencings have indicated that
nucleotide 2280 in the mouse sequence is a C residue rather than a T
residue as reported earlier (23) .
Figure 7:
Further deletions of the RCS-specific
231-bp Col2a1 enhancer. In A, the pP1L and pI9`P1L plasmids
are as described in Fig. 3. Plasmids pI11P1L, pI12P1L, and
pI13P1L were obtained by cloning 2 (2) or 4 (4
) tandem
copies of small Col2a1 intron sequences in the BglII site of
pP1L. In B, plasmid pP2L was obtained by cloning a 443-bp
fragment of the mouse Col2a1 promoter (box with grid) in the SmaI site located just upstream of the luciferase gene (box labeled LUC) in the pA
LUC vector. Plasmids
pI9`P2L and pI14P2L to pI17P2L contain two copies (2
) of
different fragments of the Col2a1 first intron (boxes with
stripes) cloned head to tail in the BglII site of the
pP2L vector. In A and B, the sizes of the promoter
and intron fragments are indicated inside their respective boxes, and
the position of their first and last nucleotides relative to the Col2a1
transcription start site is indicated at the bottom left and right sides of the boxes. All these DNA constructions were
transiently transfected in RCS cells (A and B) and
10T1/2 cells (B). Luciferase activities were measured in cell
extracts and normalized for transfection efficiency (see
``Materials and Methods''). Transcriptional activation of the
promoter induced by intron fragments is given relative to the activity
obtained with the corresponding promoter constructs (considered as 1) and is presented as the average ± S.E. of all
transfection assays performed, with the number of assays indicated in parentheses.
In subsequent experiments, a shorter 433-bp (-314 to +119) promoter fragment was used to replace the 886-bp (-767 to +119) promoter. The activity of this promoter was reduced about 5 to 10 times compared to the 886-bp promoter in both 10T1/2 and RCS cells (data not shown), despite the fact that this shorter promoter removed two potential silencer elements that were identified in the rat gene(18) . Sequence comparison of the rat, human, and mouse genes failed to show conservation of these silencer elements(33) . When two tandem copies of the 231-bp fragment (+2113 to +2343) were cloned upstream of this promoter, an 88-fold activation was seen in transient expression experiments using RCS cells and no activation using 10T1/2 cells (Fig. 7B). Additional deletions of the 231-bp enhancer fragment were tested in constructions containing two tandem copies of these subfragments. A 5` deletion producing a 156-bp fragment resulted in even greater activity than the 231-bp fragment in RCS cells (Fig. 7B). When 37 bp at the 3` end of this 156-bp fragment were deleted, which removed most of FP2, the remaining 119-bp fragment was still active in RCS cells albeit at a severalfold lower level. A further 30-bp deletion of 3` sequences in this 119-bp fragment, which removed FP2 and the 3` part of FP1, including the previously described decamer sequence, leaving an 89-bp fragment (+2188 to +2276), resulted in complete loss of activity in RCS cells. Similar results were obtained with a construction that deleted both 5` and 3` sequences (+2232 to +2285).
Hence, the RCS-specific enhancer was located in a 156-bp fragment in which both 5` and 3` sequences appeared to be required simultaneously for activity since neither the 3` 97-bp segment, containing FP1 and FP2, nor the 5` 89-bp segment was able to show enhancer activity by themselves. Deletion of the 3`-DNA corresponding to most of FP2 in the 156-bp fragment significantly decreased the activity of the enhancer, but RCS specificity was maintained indicating that the elements necessary for chondrocyte enhancer activity were contained in a 119-bp segment.
Figure 8:
Transcriptional activity of various
deletions of the Col2a1 promoter and RCS-specific promoter activation
induced by the 231-bp Col2a1 enhancer. Plasmids pP2L and pI9`P2L are as
described in Fig. 5. Plasmid pP3L was constructed by cloning a
321-bp promoter fragment of the Col2a1 gene into the Asp-718 and SpeI sites of the pLuc4 vector. Plasmids pP4L and pP5L were
obtained by cloning, respectively, a 165-bp and a 95-bp promoter
fragment (boxes with grid) of the Col2a1 gene into the HindIII site of the pALuc vector. The sizes of the
promoter fragments are indicated inside their respective boxes, and the
position of their first and last nucleotides relative to the Col2a1
transcription start site is indicated at the bottom left and right sides of the boxes. Plasmids pI9`P3L, pI9`P4L, and
pI9`P5L contain 2 (2
) tandem copies of the 231-bp Col2a1
enhancer (boxes with stripes) inserted in the SmaI
site of the corresponding promoter-luciferase vectors. These DNA
constructs were transiently transfected in RCS and 10T1/2 cells.
Luciferase activities are presented as the average with standard
deviation of three independent transfection
assays.
To determine whether any sequences in the -89 Col2a1 promoter were needed for chondrosarcoma cell specificity, the Col2a1 promoter was replaced by a short segment of the adenovirus major late promoter (-46 to +10) (Fig. 9). In the absence of enhancer, this promoter was essentially inactive in either RCS or 10T1/2 cells. Addition of the 231-bp enhancer increased promoter activity in RCS cells to levels similar to those observed with constructions in which Col2a1 promoter fragments were present. This enhancement was much less in 10T1/2 cells (Fig. 9). This experiment showed that no specific element in the Col2a1 promoter was required to produce selective activation by the 231-bp enhancer in RCS cells.
Figure 9:
RCS-specific activation of a minimal
adenovirus major late promoter by the 231-bp Col2a1 enhancer. Plasmid
padMLPL contains a 56-bp fragment of the adenovirus major late promoter
(-46 to +10) (box with grid) inserted into the KpnI and HindIII sites of the pALuc
vector. Plasmid pI9`adMLPL was made by cloning two copies (2
) of
the 231-bp Col2a1 enhancer (box with stripes) in the BglII site of padMLPL. The plasmids were transiently
transfected in RCS and 10T1/2 cells. Luciferase activities are
presented as the average ± S.D. of three independent
assays.
Our experiments demonstrated that the presence
of two tandem copies of a 156-bp segment of intron 1 in a construction
containing a 314-bp Col2a1 promoter segment provided an almost 200-fold
promoter activation in RCS cells but had no effect on promoter activity
in 10T1/2 cells. By comparison, previous transient expression
experiments from another laboratory using primary chondrocytes
indicated that a 260-bp minimal active enhancer sequence of the intron
1 of the rat Col2a1 gene provided a much more modest 6-fold
cell-specific enhancement in activity(4) . This rat DNA segment
contained sequences that are in part homologous to the mouse 156-bp
sequence. In transient expression experiments in mouse primary
chondrocytes, a construction containing a 231-bp intron fragment, which
included the 156-bp fragment, showed a high level of expression of the
reporter gene which paralleled its activity in RCS cells. In separate
experiments separate from our laboratory, a fragment of 182 bp, which
included the 156-bp fragment, conferred essentially perfect
chondrocyte-specific expression of a lacZ reporter gene in
transgenic mice.
In the 3` part of the 156-bp segment, we identified two adjacent DNase I footprints, FP1 and FP2, but these same footprints were found with nuclear extracts from chondrosarcoma cells and 10T1/2 fibroblastic cells. The 5` footprint (FP1) included part of a decamer sequence that had previously been postulated to be involved in chondrocyte-specific expression of the rat Col2a1 gene(4) . In nuclear extracts of primary chick chondrocytes, Wang et al.(4) identified a protein binding to this decamer sequence that was enriched in chondrocytes, although it did not appear to be completely chondrocyte-specific. Removal of most of the FP2 sequence decreased the activity of the enhancer severalfold, but the remaining 119-bp DNA was still RCS cell-specific. Since the FP2 sequence in the mouse gene is not present at an analogous location in the rat and human sequences, we speculate that in these species a similar sequence present at some other location can substitute for this GC-rich sequence. Removal of the sequence of FP1, leaving an 89-bp fragment at the 5` end of the 156-bp segment, abolished the activity of the enhancer. Importantly, a 97-bp fragment containing the 3` part of the 156-bp segment including both FP1 and FP2 was also completely inactive. Hence, our deletion analysis indicates that sequences both in the 5` and in the 3` portions of this 119-bp fragment are needed together for RCS cell-specific enhancer activity. The 5`-DNA segment of the 119-bp fragment contains an inverted repeat sequence which is completely conserved in the mouse and human genes. The sequence of this inverted repeat contains 11 bp in one arm of the repeat and 10 bp out of 11 that are conserved in the other arm. We propose a model whereby proteins that bind to the 3` part of the 119-bp segment and proteins that bind to the 5` part of this segment cooperate with each other either in DNA binding or in transcriptional activation to provide RCS cell-specific enhancing activity.
In summary, our results establish the unique usefulness of a new RCS cell line as a tissue culture system to study chondrocyte-specific regulatory elements of the Col2a1 gene and presumably other chondrocyte-specific genes. Our experiments indicate that both 5` and 3` sequences in a 119-bp intron 1 segment are needed for RCS cell-specific enhancer activity. We also demonstrate that the Col2a1 promoter sequences are dispensable for this cell-specific enhancer activity. Further work is needed to better characterize the binding sites within the 119-bp segment for proteins involved in the chondrocyte-specific expression of the Col2a1 gene and to isolate cDNA clones for these proteins.