From the Department of Molecular Genetics, the University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
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ABSTRACT |
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To understand the molecular mechanisms by which
mesenchymal cells differentiate into chondrocytes, we have used the
gene for an early and abundant marker of chondrocytes, the mouse
pro-1(II) collagen gene (Col2a1), to delineate a minimal
sequence needed for chondrocyte-specific expression and to identify the
DNA-binding proteins that mediate its activity. We show here that a
48-base pair (bp) Col2a1 intron 1 sequence specifically
targets the activity of a heterologous promoter to chondrocytes in
transgenic mice. Mutagenesis studies of this 48-bp element identified
three separate sites (sites 1-3) that were essential for its
chondrocyte-specific enhancer activity in both transgenic mice and
transient transfections. Mutations in sites 1 and 2 also severely
inhibited the chondrocyte-specific enhancer activity of a 468-bp
Col2a1 intron 1 sequence in vivo. SOX9, an
SRY-related high mobility group (HMG) domain transcription factor, was
previously shown to bind site 3, to bend the 48-bp DNA at this site,
and to strongly activate this 48-bp enhancer as well as larger
Col2a1 enhancer elements. All three sites correspond to
imperfect binding sites for HMG domain proteins and appear to be
involved in the formation of a large chondrocyte-specific complex
between the 48-bp element, Sox9, and other protein(s). Indeed,
mutations in each of the three HMG-like sites of the 48-bp element,
which abolished chondrocyte-specific expression of reporter genes in
transgenic mice and in transiently transfected cells, inhibited
formation of this complex. Overall our results suggest a model whereby
both Sox9 and these other proteins bind to several HMG-like sites in
the Col2a1 gene to cooperatively control its expression in
cartilage.
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INTRODUCTION |
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Acquisition of the chondrocytic phenotype occurs along a major pathway of differentiation of mesenchymal cells (1, 2). With the goal of identifying transcription factors that control chondrocyte-specific gene expression, we used the gene for collagen type II (Col2a1),1 an early and abundant marker of chondrocytes (3-5), to delineate minimal sequences in this gene that control chondrocyte-specific expression in transgenic mice. Elucidation of the transcriptional mechanisms that control the chondrocyte-specific expression of the Col2a1 gene should provide important insights into the molecular specifications of chondrocytes.
We previously identified a 48-bp element in intron 1 of the mouse Col2a1 gene that, when present as four tandem copies, conferred chondrocyte-specific expression both in transgenic mice and in transient expression experiments in tissue culture cells (6). A multimerized 18-bp element located at the 3' end of the 48-bp sequence also acted as a powerful chondrocyte-specific enhancer in transient transfection assays of rat chondrosarcoma (RCS) cells and mouse primary chondrocytes but not of fibroblasts (6).
SOX9 is a member of a family of transcription factors with a DNA-binding domain that shows more than 50% similarity with the high mobility group HMG DNA-binding domain of SRY, the testis-determining factor in mammals (7-12). Recently, heterozygous mutations in human SOX9 have been identified as a cause of campomelic dysplasia (CD), a severe dwarfism syndrome in which essentially all skeletal elements derived from cartilages are affected (13-17). A large proportion of genotypically male (XY) CD patients carrying mutations in SOX9 also show sex reversal. In situ hybridization during mouse embryogenesis showed that Sox9 is expressed in all chondroprogenitor cells; Sox9 expression generally parallels that of Col2a1 even in some non-chondrocytic cells and increases together with that of Col2a1 when frank chondrocyte differentiation takes place (5, 18, 19). The expression of Sox9 in gonadal ridges and later in the Sertoli cells of the testis presumably accounts for the sex reversal in CD patients (20, 21). Overall, the abnormal skeletal manifestations of CD patients and the pattern of expression of Sox9 during embryonic development suggest that Sox9 plays an important role in the pathway of chondrocyte differentiation.
Recent experiments showed that the 48-bp Col2a1 element that confers chondrocyte specificity in transgenic mice is a direct target for Sox9. Indeed, Sox9 was able to bind to a sequence in this element that is essential for chondrocyte-specific enhancer activity, and SOX9 activated this element in cotransfection experiments of nonchondrocytic cells (22). In addition, ectopic expression of SOX9 in transgenic mouse embryos resulted in the activation of the endogenous Col2a1 gene in some but not all areas of ectopic SOX9 expression (23).
Although four tandem copies of the 48-bp Col2a1 sequence and 12 tandem copies of an 18-bp element within this 48-bp sequence both acted as strong chondrocyte-specific enhancers in transient expression experiments, 12 tandem copies of the 18-bp element showed much weaker activity in cartilages of the transgenic mice than did the four tandem copies of the 48-bp enhancer (6). Expression of the reporter gene in embryos harboring the multimerized 18-bp construct was also detected at low levels in skin and brain (6). Therefore, to confer high level chondrocyte-specific reporter gene expression in vivo, the entire 48-bp Col2a1 intronic fragment appeared to be needed.
Although the 18-bp enhancer sequence included the SOX9-binding site of the 48-bp Col2a1 enhancer and was a strong target for SOX9 in transfection experiments (22), the above-mentioned transgenic mice results were consistent with the hypothesis that, to confer chondrocyte specificity in vivo, proteins other than Sox9 might be needed that interact with the 48-bp but not with the 18-bp enhancer sequence. The expression of Sox9 at high levels in Sertoli cells also favors the hypothesis that additional proteins are needed to differentiate the phenotype of chondrocytes from that of Sertoli cells (20, 21). Hence, the purpose of the present study was to further identify sequences in the Col2a1 48-bp enhancer essential for its chondrocyte-specific activity and to determine whether chondrocyte-specific nuclear proteins bound to these sequences. Our results indicate that the 48-bp sequence in Col2a1 contains multiple cis-acting elements essential for chondrocyte-specific expression in vivo and that chondrocytes contain specific nuclear protein(s) in addition to SOX9 that bind to these cis-acting elements and might therefore be involved in chondrocyte-specific enhancer activity.
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MATERIALS AND METHODS |
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Cell Cultures-- All cell types were obtained as described previously and cultured under standard conditions (6, 24).
DNA Constructs--
To generate i(8 × 48)pgloblacZ, eight tandem copies of the 48-bp
enhancer Col2a1 element were cloned upstream of a minimal
human -globin promoter (
44 to +28) in the reporter plasmid placF,
as described previously for other constructs (25).
Transient Expression Experiments--
DNA transfections were
performed by the modified DNA-calcium phosphate coprecipitation method
(26). Monolayers of RCS cells pre-established in 20-cm2
dishes were cotransfected with 7.5 µg of luciferase reporter plasmids
and 2.5 µg of pSV2gal plasmid used as an internal control for
transfection efficiency. Cell extracts were prepared 40-48 h
after the start of transfection, and luciferase and
-galactosidase activities were assayed as described (6).
Generation and Characterization of Transgenic
Mice--
Transgenic mice were generated as described (25). Transgenic
founder embryos were sacrificed at 14.5 days postcoitum (dpc). Southern
blot analysis, staining with X-gal
(5-bromo-4-chloro-3-indolyl--D-galactopyranoside), and
histological analysis were performed as described previously (25).
Synthesis of SOX9 in Vitro and Preparation of Nuclear Extracts-- SOX9 protein was synthesized by in vitro transcription-translation from a previously described SOX9-pcDNA-5'-UT expression vector (22) using the Single-tube Protein System 2 from Novagen, Inc. (Madison, WI). Nuclear extracts from all cell types were prepared as described previously (6) in buffers containing 10 µg/ml leupeptin and pepstatin.
Electrophoretic Mobility Shift Assays (EMSAs)--
The wild-type
and mutant 48-bp Col2a1 double-stranded oligonucleotides
were prepared as described above under "DNA Constructs." Berenil
and distamycin were purchased from Sigma. The OCT and HMG probes were
prepared as described previously (6, 22). All probes were end-labeled
with [-32P]dGTP or [
-32P]dCTP using
the Klenow fragment. Protein-DNA binding reactions were carried out as
described previously (6). Assays with nuclear extracts were performed
with 10 µg of protein and 2 µg of poly(dG-dC)·poly(dG-dC). SOX9
synthesized in vitro was assayed in the presence of 0.1 µg of poly(dG-dC)·poly(dG-dC). Supershift experiments were performed with purified SOX9 antibodies as described previously (22).
Elution of CSEP from Electrophoresis Gels-- RCS cell nuclear extracts were partially purified by passage through a DNA affinity column containing an R2 oligonucleotide containing an 18-bp Col2a1 enhancer sequence (6). The flow-through, which contained CSEP activity, was concentrated 10 times by precipitation with ammonium sulfate (60% saturation) and then subjected to 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Protein elution from gel slices and renaturation were performed as described (6); 10-µl eluates were used in EMSA.
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RESULTS |
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Chondrocyte-specific Targeting by a 48-bp Col2a1 Element in
Transgenic Mice--
We showed previously that a 48-bp sequence of
intron 1 in the Col2a1 gene together with a 309-bp
Col2a1 promoter conferred strict chondrocyte-specific
expression in transgenic mice (6). To investigate further whether the
48-bp Col2a1 sequence by itself contained all the
cis-acting elements needed for chondrocyte-specific enhancer
activity in intact mouse embryos, we generated a transgene i(8 × 48)pgloblacZ in which
eight tandem repeats of the 48-bp enhancer fragment were cloned
upstream of a minimal human -globin promoter (Fig.
1A). Two of the five
transgenic 14.5-dpc founder embryos harboring i(8 × 48)pgloblacZ stained positive for X-gal, a
chromogenic substrate for
-galactosidase, whereas the other three
did not. Moreover, the X-gal-positive embryos harboring
i(8 × 48)pgloblacZ exhibited
cartilage-specific staining similar to that seen at the same
developmental stage (Fig. 1B) in embryos harboring transgene
p309i(4 × 48)Col2a1, in which the same
48-bp fragment was driving a 309-bp Col2a1 promoter (Fig.
6B). X-Gal staining was observed in the cartilages of the
head, scapula, vertebrae, ribs, limbs, and shoulder and pelvic girdles.
Histological analysis showed that X-gal staining was present in
chondrocytes only; no promiscuous X-gal staining was detected in any
nonchondrogenic tissues (examples are shown in Fig. 1, C and
D). Hence, these experiments demonstrated that in transgenic
embryos the sequence of the 48-bp Col2a1 intron 1 element
contained the essential elements required to target the activity of a
minimal heterologous promoter specifically to chondrocytes.
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Formation of a Chondrocyte-specific Complex with the 48-Base Pair Col2a1 Enhancer Element-- In previous EMSA experiments, an 18-bp Col2a1 enhancer probe was used to identify Sox9 and other chondrocyte-enriched proteins in nuclear extracts of primary chondrocytes and RCS cells (6, 22). Since the 48-bp Col2a1 element, which includes the 18-bp sequence, confers a much stricter chondrocyte specificity in transgenic embryos than does the 18-bp element, the 48-bp Col2a1 element was used as a probe in EMSA experiments to (a) identify in chondrocytes unique nuclear factors that would specifically bind to the 48-bp enhancer and (b) locate the DNA-binding sites for these factors.
Formation of a major complex with the 48-bp enhancer probe was observed in reactions using extracts from primary chondrocytes and from two chondrocytic cell lines (MC615 and RCS cells) (Fig. 2). This complex was absent in reactions with extracts from fibroblastic cell lines (10T1/2 and Balb/3T3) and all other nonchondrogenic cell lines tested. The protein (or proteins) forming the major complex was tentatively named CSEP for chondrocyte-specific enhancer-binding proteins.
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Abolition of Chondrocyte-specific Enhancer Activity by Mutations in Three HMG-like Sites-- To test the effect of mutations in the HMG-like sites of the Col2a1 48-bp element on enhancer activity in RCS cells, four tandem repeats of mutant 48-bp elements were cloned into the luciferase vector (Fig. 5). Mutations that strongly decreased the binding of CSEP, i.e. MA1, MA4, and MA6, abolished enhancer activity in RCS cells (Fig. 5). Mutation MA3, which had a much weaker effect on formation of the CSEP·DNA complex in EMSA, had little effect on enhancer activity in RCS cells. Mutation MA7, which increased formation of the CSEP·DNA complex in EMSA, had no significant effect on enhancer activity. Hence, there was a good correlation between formation of the CSEP·48-bp Col2a1 DNA complex and enhancer activity in RCS cells as assayed in transient transfection experiments.
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DISCUSSION |
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In 14.5-dpc transgenic mouse embryos, four tandem repeats of the
48-bp enhancer in combination with a 309-bp Col2a1 promoter targeted the expression of a -galactosidase reporter gene to chondrocytes specifically (6). Transgenic mouse embryos harboring a
309-bp Col2a1 promoter that lacked intron 1 enhancer
sequences showed no
-galactosidase expression in chondrocytes (25).
Now we have shown that when the Col2a1 promoter is replaced
with a minimal
-globin promoter, the multimerized 48-bp intron 1 Col2a1 element is still able to target expression of the
lacZ transgene specifically to chondrocytes in 14.5 dpc
transgenic mouse embryos, indicating that Col2a1 promoter
sequences are dispensable for chondrocyte-specific expression in
vivo and that the 48-bp Col2a1 DNA segment contains all
the information necessary to confer chondrocyte-specific expression in
intact mouse embryos.
In previous transient expression experiments, 12 tandem copies of an 18-bp sub-element of the 48-bp sequence were a more potent chondrocyte-specific enhancer than four tandem copies of the 48-bp element (6). However, in transgenic mice, the 12 tandem copies of the 18-bp sequence were much less effective in conferring a high level and strict chondrocyte specificity in vivo (6). We therefore hypothesized that additional sequences outside the 18-bp segment in the 48-bp element were needed to achieve such high level chondrocyte-specific expression in vivo. Our present results show that mutations in three different sites of the 48-bp element resulted in loss of chondrocyte-specific enhancer activity both in transgenic mouse embryos and in transient expression experiments. Two of these sites, sites 1 and 2, are located outside the 18-bp element (only the 3' part of site 2 is present in the 18-bp sequence), which includes the previously identified Sox9-binding site (site 3) (6, 22). All three mutations disrupt potential binding sites for HMG domain proteins.
When nuclear extracts from chondrocytes were used in EMSAs with the 48-bp probe, no complex formed that had the mobility of the major complex formed between SOX9 synthesized in vitro and the 48-bp probe. However, a prominent, slower migrating complex was formed with nuclear proteins present in primary chondrocytes and chondrocytic cell lines but not in other cell lines (CSEP). Since a limited supershift of the CSEP·DNA complex was observed with SOX9 antibodies, it is likely that Sox9 was present in the complex. However, because SOX9 antibodies supershifted only a fraction of the CSEP·48-bp DNA complex, we hypothesized that the CSEP·48-bp DNA complex also included one or more proteins distinct from Sox9.
Additional lines of evidence further support this hypothesis. First, although Sox9 is present in 10T1/2 cells, no CSEP·48-bp DNA complex was formed with nuclear extracts of these cells. Second, after fractionation of chondrocyte nuclear extracts by SDS-PAGE followed by protein elution and renaturation, a protein or proteins with an apparent Mr of 75,000-95,000 formed a complex with the 48-bp sequence that had the same mobility and the same DNA-binding properties as the CSEP·48-bp DNA complex; this complex was not supershifted by SOX9 antibodies. As Western blot analysis revealed, SOX9 ran as a unique species with an apparent Mr of 68,000 (22). Our data also suggest that proteins present in the CSEP·48-bp complex, which are different from Sox9, are HMG-like proteins. Indeed, a complex with a mobility and DNA-binding properties similar to those of the CSEP·48-bp complex was formed with chondrocyte extracts and an oligonucleotide containing a consensus binding site for HMG domain proteins; this complex was not affected by SOX9 antibodies. Moreover, formation of this complex was competed by the 48-bp probe. In addition, mutations in three HMG-like sites of the 48-bp element decreased formation of the CSEP·DNA complex.
Formation of the CSEP·48-bp DNA complex was decreased by mutations in any one of the three HMG-like sites of the 48-bp enhancer, but it was completely abolished only when the three sites were mutated altogether. Hence, the components of CSEP bound to the three sites of the 48-bp element. Since mutations in any one of these three sites also abolished the chondrocyte-specific enhancer activity of the 48-bp Col2a1 element both in transgenic mice and in DNA transfection experiments, we hypothesize that CSEP has a role in enhancer activity.
In experiments by others (23), a 309-bp intron 1 sequence of the human COL2A1 gene that included the sequence of the 18-bp enhancer at its 5' end was also shown to confer chondrocyte-specific expression in transgenic mouse embryos. Two binding sites for Sox9 were identified in this 309-bp segment, one corresponding to site 3 of Fig. 4 and the other about 50 bp 3' of it. Mutations in either one of these sites inhibited chondrocyte-specific expression of a reporter gene in transgenic embryos either in all or many cartilages (23). In our previous experiments, a single copy Col2a1 468-bp segment that included these two Sox9-binding sites as well as 310 bp 5' of the 48-bp element conferred strict chondrocyte-specific expression in transgenic mice. Now we have shown that a single copy 468-bp fragment with mutations in the two HMG-like sites 1 and 2 located upstream of the two Sox9-binding sites could not confer chondrocyte specificity in transgenic embryos despite the presence of two intact Sox9-binding sites in the fragment. This suggests that several HMG-like binding sites need to be occupied by transcription factors in order to activate the single copy 468-bp transgene at high levels in all cartilages in vivo.
Overall our results suggest a model in which both Sox9 and other proteins present in the CSEP·DNA complex bind to several HMG-like sites in the chondrocyte-specific Col2a1 gene to control its chondrocyte-specific expression. To identify these other proteins and study their function, the cDNAs for these proteins will need to be cloned.
In experiments reported in the accompanying article (38), we have
identified two short chondrocyte-specific enhancer elements within the
promoter of the mouse gene for the 2 subunit of type XI collagen
(Col11a2), a gene that is expressed preferentially in
chondrocytes. Like the Col2a1 48-bp enhancer both elements contain several HMG-like sites and formed a DNA-protein complex with
extracts of RCS cells that was dependent on the sequence of these
sites. Furthermore, these complexes had the same mobility as the
CSEP·48-bp Col2a1 complex and, similarly, appeared to
contain SOX9 and other proteins. Like the Col2a1 enhancer,
the Col11a2 elements were also able to activate reporter
genes in chondrocytes, but not in fibroblasts, and were activated by
forced expression of SOX9 in non-chondrocytic cells. Both
Col11a2 elements also directed transgene expression to
chondrocytes in mouse embryos. On the basis of these similarities, we
speculate that common mechanisms involving both Sox9 and the other
proteins present in CSEP may control the chondrocyte-specific
expression of the Col2a1 and Col11a2 genes and
perhaps a larger genetic program of chondrocyte differentiation.
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ACKNOWLEDGEMENTS |
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We are very grateful to Vincent R. Harley and Peter N. Goodfellow for the generous gift of SOX9 antibodies; James H. Kimura for the gift of RCS cells; and Françoise Coustry, Susin Chen, Lee Ann Garrett, and Xin Zhou for providing nuclear extracts from various cell types. We also thank Janie Finch for editorial assistance.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants R01 AR42909 and P01 AR42919-02 (to B. d. C.). The University of Texas M. D. Anderson Cancer Center Core Sequencing Facility, in which DNA sequencing was performed, is supported by National Institutes of Health Grant CA16672 (NCI).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.
Current address: Dept. of Molecular and Human Genetics, Baylor
College of Medicine, Houston, TX 77030.
§ Recipient of an Arthritis Investigator Award from the Arthritis Foundation.
¶ To whom correspondence should be addressed: Dept. of Molecular Genetics, the University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: 713-792-2590; Fax: 713-794-4295.
1
The abbreviations used are: Col2a1,
pro-1(II) collagen gene; CD, campomelic dysplasia; CSEP,
chondrocyte-specific enhancer-binding protein; dpc, days postcoitum;
geo, a fusion protein with E. coli
-galactosidase and
neomycin resistance activities; bp, base pair(s); HMG, high mobility
group; RCS, rat chondrosarcoma cells; X-gal,
5-bromo-4-chloro-3-indolyl-
-D-galactopyranosides; EMSA, electrophoretic mobility shift assays; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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