Article1 October 1998free access A new long form of Sox5 (L-Sox5), Sox6 and Sox9 are coexpressed in chondrogenesis and cooperatively activate the type II collagen gene Véronique Lefebvre Corresponding Author Véronique Lefebvre Department of Molecular Genetics, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 11, Houston, TX, 77030 USA Search for more papers by this author Ping Li Ping Li Department of Molecular Genetics, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 11, Houston, TX, 77030 USA Search for more papers by this author Benoit de Crombrugghe Corresponding Author Benoit de Crombrugghe Department of Molecular Genetics, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 11, Houston, TX, 77030 USA Search for more papers by this author Véronique Lefebvre Corresponding Author Véronique Lefebvre Department of Molecular Genetics, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 11, Houston, TX, 77030 USA Search for more papers by this author Ping Li Ping Li Department of Molecular Genetics, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 11, Houston, TX, 77030 USA Search for more papers by this author Benoit de Crombrugghe Corresponding Author Benoit de Crombrugghe Department of Molecular Genetics, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 11, Houston, TX, 77030 USA Search for more papers by this author Author Information Véronique Lefebvre 1, Ping Li1 and Benoit de Crombrugghe 1 1Department of Molecular Genetics, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 11, Houston, TX, 77030 USA *Corresponding authors. E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (1998)17:5718-5733https://doi.org/10.1093/emboj/17.19.5718 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Transcripts for a new form of Sox5, called L-Sox5, and Sox6 are coexpressed with Sox9 in all chondrogenic sites of mouse embryos. A coiled-coil domain located in the N-terminal part of L-Sox5, and absent in Sox5, showed >90% identity with a similar domain in Sox6 and mediated homodimerization and heterodimerization with Sox6. Dimerization of L-Sox5/Sox6 greatly increased efficiency of binding of the two Sox proteins to DNA containing adjacent HMG sites. L-Sox5, Sox6 and Sox9 cooperatively activated expression of the chondrocyte differentiation marker Col2a1 in 10T1/2 and MC615 cells. A 48 bp chondrocyte-specific enhancer in this gene, which contains several HMG-like sites that are necessary for enhancer activity, bound the three Sox proteins and was cooperatively activated by the three Sox proteins in non-chondrogenic cells. Our data suggest that L-Sox5/Sox6 and Sox9, which belong to two different classes of Sox transcription factors, cooperate with each other in expression of Col2a1 and possibly other genes of the chondrocytic program. Introduction Sox (Sry-type HMG box) proteins, which form a subfamily of DNA-binding proteins with a high-mobility-group (HMG) domain, have critical functions in a number of developmental processes, including sex determination, neurogenesis and skeleton formation (Laudet et al., 1993; Pevny and Lovell-Badge, 1997; Southard-Smith et al., 1998). Individual members of the Sox family show >50% identity in their HMG domain to Sry, the testis-determining factor (Wright et al., 1993). An essential role for SOX9 in skeleton formation was demonstrated with the identification of mutations in SOX9 in patients with campomelic dysplasia (Foster et al., 1994; Wagner et al., 1994; Kwok et al., 1995; Meyer et al., 1997). This disease is characterized by severe malformations of essentially all cartilage-derived structures and is also often associated with XY sex reversal (Houston et al., 1983; Mansour et al., 1995). Cartilage formation is a complex and essential process in vertebrates. Cartilages are obligatory templates for the formation of endochondral bones during development and also constitute permanent skeletal structures in the respiratory tract, in articular joints and other organs. Chondrocytes differentiate following condensation of mesenchymal cells in different locations of the embryo, including the frontonasal mass, branchial arches, sclerotomes and limb buds. Typically, chondrocytes express a set of genes encoding cartilage-specific extracellular matrix components such as collagen II (encoded by the Col2a1 gene), collagens IX and XI, and aggrecan. In growth plates, chondrocytes undergo further differentiation and hypertrophy, producing a matrix in which type X collagen is abundant and calcification occurs. Apoptosis follows and cartilage is replaced by bone. Our understanding of chondrogenesis at the molecular level is still limited. Several cytokines, including bone morphogenetic proteins, Indian Hedgehog, parathyroid hormone-related peptide and fibroblast growth factors, are involved in either skeletal patterning or discrete steps of the chondrogenic pathway, and several transcription factors, such as Hox and Pax family members, are involved in patterning of skeletal primordia (Cancedda et al., 1995; Erlebacher et al., 1995; Hall and Miyake, 1995). However, less is known about the transcription factors that control the determinative switch for chondrocyte differentiation and the activation of marker genes at each step of the chondrogenic cascade. Our laboratory has used genes for specific cartilage matrix components to identify transcription factors that control gene expression in chondrocytes. We have shown that a multimerized 48 bp sequence in the first intron of Col2a1 is sufficient to confer chondrocyte-specific expression both in transgenic mice and in transient transfection of cultured cells (Lefebvre et al., 1996). SOX9 binds to the enhancer and activates Col2a1 constructs in transient transfections of non-chondrocytic cells (Lefebvre et al., 1997) and in transgenic mice (Bell et al., 1997). SOX9 also activates the Col2a1 gene when ectopically expressed in some non-cartilaginous sites in transgenic mice (Bell et al., 1997). Moreover, Sox9 is expressed along with Col2a1 during chondrogenesis in mouse embryos (Wright et al., 1995; Ng et al., 1997; Zhao et al., 1997). Therefore, direct activation of COL2A1 is believed to be an important function of SOX9 in chondrogenesis (Lefebvre and de Crombrugghe, 1998). Several lines of evidence suggest that other transcription factors in addition to SOX9 may be needed to specify the high-level expression of COL2A1 in chondrocytes. Sox9 is expressed in cells that do not express Col2a1, such as those in genital ridges and specific areas of the embryonic heart (Ng et al., 1997; Zhao et al., 1997). Sox9 is highly expressed in the Sertoli cells of the testis and is involved in male gonad differentiation (Kent et al., 1996; Morais da Silva et al., 1996). The target genes of Sox9 in these cells and in chondrocytes are not clearly defined, but the phenotypes of these two cell types are so different that it is likely that Sox9 contributes to their differentiation by controlling expression of different genes. Different functions of Sox9 in these cells must be specified by differential expression of other factors. Ectopic expression of SOX9 in transfected cells, in which Col2a1 was silent, did not result in Col2a1 activation (V.Lefebvre and B.de Crombrugghe, unpublished data), and ectopic expression of SOX9 in transgenic mice led to activation of Col2a1 only in a subset of tissues within the domain of ectopic expression of SOX9 (Bell et al., 1997). We therefore hypothesized that other factors either activate or derepress SOX9 or cooperate with SOX9 in COL2A1-expressing cells. We recently reported that the 48 bp enhancer of Col2a1 formed a large and abundant complex with nuclear proteins from chondrocytes but not from other cells (Zhou et al., 1998). These proteins were designated CSEPs, for chondrocyte-specific enhancer-binding proteins. They included Sox9 and unidentified protein(s). CSEPs appeared to contact the 48 bp DNA at several sites homologous to a consensus for HMG-domain proteins. Mutagenesis demonstrated a good correlation between binding of CSEPs to DNA and enhancer activity in chondrocytes, both in transient transfection experiments and in transgenic mice. In addition, we showed that two chondrocyte-specific enhancer elements located in the Col11a2 promoter contained HMG-like sites that were essential for enhancer activity and formation of an enhancer–CSEP-like complex (Bridgewater et al., 1998). These results suggested that other HMG-domain proteins cooperate with Sox9 to generate Col2a1 and Col11a2 enhancer activity and presumably gene expression in chondrocytes. We show here that in addition to Sox9, CSEPs are composed of a new long form of Sox5 (L-Sox5), and of Sox6, which both are members of a Sox subclass different from that of Sox9. L-Sox5 and Sox6 harbor a coiled-coil domain that mediates protein dimerization and efficient binding to adjacent HMG DNA sites. The three Sox genes are coexpressed in chondrogenesis and cooperate in Col2a1 activation. Our data strongly suggest that L-Sox5, Sox6 and Sox9 together contribute to control Col2a1, and perhaps other important genes of the chondrocyte phenotype. Results A long form of Sox5 (L-Sox5), Sox6 and Sox9 form complexes with the 48-bp Col2a1 enhancer The CSEP proteins that form a chondrocyte-specific complex with the 48 bp Col2a1 enhancer were previously shown to include a protein or proteins with an apparent Mr of 75–95 kDa (Zhou et al., 1998). These proteins exhibited DNA-binding properties of HMG-domain proteins, including binding to several HMG-like sites in the Col2a1 48 bp enhancer, binding to a probe containing a consensus binding site for HMG-domain proteins (1HMG probe), binding to the minor groove of DNA and binding to DNA in the presence of poly(dG–dC) but not poly(dI–dC) (Zhou et al., 1998). We also obtained evidence that CSEP bound with high affinity to a tandem dimer of the 1HMG probe (2HMG probe) both in EMSA and in Southwestern blots (data not shown). On the basis of these results, the 2HMG probe was chosen to clone cDNAs for CSEPs by the Southwestern screening approach. cDNA expression libraries were made from primary chondrocytes of newborn mouse ribs. Several clones that showed stronger binding to the 2HMG probe in the presence of poly(dG–dC) than poly(dI–dC) encoded sequences of Sox5 or Sox6. Interestingly, whereas the previously reported transcript for Sox5 had a length of 2 kb and encoded a 43 kDa protein (Denny et al., 1992), the Sox5 cDNA that was reconstituted from overlapping clones was 3.9 kb long and encoded a 75 kDa protein corresponding to Sox5 with an additional N-terminal sequence (data submitted to DDBJ/EMBL/GenBank; see later, in Figure 4). This long form of Sox5 was designated L-Sox5. Sox6 cDNA clones encoded Sox6 isoforms identical or very similar to those described for testis (data submitted to DDBJ/EMBL/GenBank; see later, in Figure 4). Figure 1.Comparison of Sox5, L-Sox5 and Sox6 cDNAs and polypeptides. (A) Schematic comparison of Sox6A, Sox6B and Sox6C polypeptides. Three forms of Sox6 differed in segments S1 and S2. Sox6A and Sox6B are schematized according to the sequences reported by Connor et al. (1995) and Takamatsu et al. (1995), respectively. Sox6C, whose cDNA sequencing has not been completed, is schematized based on the assumption that it is identical to Sox6A and Sox6B outside of segments S1 and S2. Two coiled-coil domains (1st cc and 2d cc) and the HMG DNA-binding domain, are indicated. S1A and S1B segments are compared at the nucleotide level and, in parentheses, at the amino acid level. (B) Comparison of Sox5 and L-Sox5 cDNAs. Sequences available for Sox5 and L-Sox5 cDNAs are presented as blocks. Shaded areas indicate coding sequences, between the first in-frame ATG codon and the next stop codon. Sox5 and L-Sox5 cDNAs are identical in the segment delineated by the two dotted lines, but they differ totally on either sides of this segment. Numbers refer to nucleotide positions relative to the 5′ end. The Sox5 cDNA representation follows the sequence published by Denny et al. (1992). L-Sox5 cDNA is schematized according to a 3881 bp sequence obtained from overlapping clones. Both cDNAs must lack 5′ and/or 3′ untranslated sequences, as RNA transcript lengths were estimated to be ∼2 kb for Sox5 and ∼6.3 kb for L-Sox5. (C) Alignment of L-Sox5 and Sox6A amino acid sequences. The sequence of L-Sox5 (upper sequence), derived from its cDNA sequence, is compared with that of Sox6A (lower sequence) (Connor et al., 1995). Numbers refer to amino acid positions in the proteins. Sequence alignment was generated using the GAP program from the Genetics Computer Group (GCG, Madison, WI). Dots were introduced within sequences to maximize alignment. Vertical lines denote amino acid identities. Double and single dots between sequences indicate amino acid changes with high and low degrees of conservation, respectively. The methionine translation initiation codon of the short form of Sox5 is circled in the L-Sox5 sequence (residue 288). Boxes outline two potential coiled-coil domains and the HMG DNA-binding domain. The sequence of Sox6A spanning residues 330–379 was omitted because it has no counterpart in L-Sox5. (D) Schematic comparison of Sox5, L-Sox5 and Sox6A polypeptides. Each protein is represented as a block between its N- and C-termini. HMG, HMG DNA-binding domain; cc, potential coiled-coil domain. Hatched boxes represent regions of >10 residues in Sox6A that do not exist in L-Sox5. Dotted lines link regions of similarity between the proteins. Numbers refer to the position of the residues marking domain limits. The predicted molecular weight of each protein is given in parentheses. (E) Delineation of potential coiled-coil domains in L-Sox5 and Sox6A. The amino acid sequences of L-Sox5 and Sox6A were analyzed with the Coilscan program from GCG using an unweighted matrix at a window size of 28 residues. The probability of each residue participating in coiled-coil formation is plotted against its position in the sequences. A score of 1.0 indicates a maximum probability. The first potential coiled coil involves residues 158–240 in L-Sox5 and residues 181–262 in Sox6A; the second potential coiled coil spans residues 364–403 in L-Sox5 and 488–515 in Sox6A. Download figure Download PowerPoint Antibodies were generated against the C-termini of Sox5, Sox6 and Sox9, and affinity purified. In Western blotting, each antibody species specifically recognized its Sox protein target in extracts of fibroblasts transfected with Sox expression plasmids (Figure 1A). In EMSA, each antibody preparation specifically supershifted complexes formed between DNA and its target (Figure 1B). In Western blots of RCS cell extracts, the antibodies strongly reacted with proteins at the level of L-Sox5, Sox6 and Sox9 (Figure 1C), indicating that each of these Sox proteins was indeed made by these cells. The same reactions were seen with extracts of primary chondrocytes and chondrocytic MC615 cells (data not shown), but not with extracts of BALB/3T3 (Figure 1C) or 10T1/2 fibroblasts (see control lanes in Figure 1B). Note that Sox5 antibodies showed no reaction in chondrocyte samples at the level of the 43-kDa short form of Sox5. In extracts of adult mouse testis, Sox5 antibodies recognized Sox5 but not L-Sox5 (Denny et al., 1992). It appeared, therefore, that only L-Sox5 is made in chondrocytes, not Sox5, whereas only Sox5 is made in the testis. These protein data are consistent with RNA data, which show that chondrocytes express only the transcript for L-Sox5 and testis only the transcript for short Sox5 (see Figure 2A). In EMSA, L-Sox5 and Sox6 made in transfected fibroblasts formed complexes with the 48 bp Col2a1 enhancer probe that migrated at the level of the CSEP–DNA complex (Figure 1D). Sox5 did not bind to the 48 bp element. As shown previously (Zhou et al., 1998), SOX9 formed two complexes with the enhancer, a major one migrating faster than the CSEP–DNA complex and a minor one migrating at the level of the CSEP–DNA complex (Figure 1D). Sox5, Sox6 and Sox9 antibodies were each able to partially supershift the CSEP–48-bp-Col2a1 enhancer complex formed with RCS cell nuclear extracts (Figure 1E), indicating that each of these Sox proteins contributed to complex formation. A virtually complete supershift of the CSEP–DNA complex was obtained by incubating EMSA reactions with both Sox5 and Sox6 antibodies. No further supershift was visible when the three Sox protein antibodies were included in the reactions. The same results were obtained with primary chondrocyte extracts (data not shown). L-Sox5 and Sox6 therefore appeared to be predominant CSEP components. Figure 2.L-Sox5, Sox6 and Sox9 are present in chondrocytes and form the CSEP–Col2a1-48-bp enhancer complex. (A) Antibodies (AB) against Sox5, Sox6 and SOX9 specifically recognized their target in Western blots. Samples were extracts of 10T1/2 fibroblasts transfected with expression plasmids encoding no protein (−), L-Sox5, Sox6A or SOX9. The Mr of protein standards (×103) is indicated. Reaction with antibodies is seen at the level expected for each Sox protein. (B) Antibodies against Sox5, Sox6 and Sox9 specifically supershifted the complex of their target with DNA. L-Sox5 and Sox6 protein samples were cell extracts from 10T1/2 fibroblasts transfected with Sox expression plasmids. The SOX9 sample was the product of in vitro transcription/translation with SOX9 plasmid. Samples were used in EMSA with the 2HMG probe. Two microliters of SOX crude antiserums were included, as indicated. A preimmune serum was used as a control (first lane for each protein). None of the complexes seen in the figure was seen in samples containing no SOX protein (data not shown). SOX9 synthesized in vitro formed two distinct complexes with the 2HMG probe. The lower and upper complexes probably involved one and two molecules of SOX9 per molecule of DNA, respectively. (C) Antibodies against Sox5, Sox6 and Sox9 identified their target in Western blots of nuclear extracts of RCS cells but not BALB/3T3 fibroblasts. An intense reaction with antibodies was seen at the level of each Sox protein. (D) L-Sox5, Sox6 and SOX9 bound to the 48 bp probe. 10T1/2 fibroblasts were transfected with expression plasmids for no protein (−), L-Sox5, Sox6, SOX9 or Sox5. Extracts of these cells and nuclear extracts of RCS cells were used in EMSA with the 48 bp Col2a1 probe. L-Sox5 and Sox6 formed a complex with the probe that ran with a mobility similar to that of the CSEP–DNA complex. As shown previously (Zhou et al., 1998), SOX9 formed two complexes with the Col2a1 enhancer probe, a minor one that migrated at the level of the CSEP–DNA complex (arrow), and a major one that migrated faster (arrowhead). (E) Antibodies against Sox5, Sox6 and Sox9 supershifted the CSEP–48-bp-Col2a1 complex. Two or four microliters of SOX antiserums (AB) were included in EMSA of RCS nuclear extracts with the 48 bp Col2a1 probe, as indicated. Sox5 preimmune serum was used in the control reaction (first lane). None of the preimmune serums supershifted any protein–DNA complex (data not shown). As described previously (Zhou et al., 1998), Sox9 present in RCS extracts did not form a fast migrating complex with DNA (as seen in D). (F) EMSA with wild-type and mutant 48 bp Col2a1 probes. The upper strand of the wild-type 48 bp element (wt) is shown from 5′ to 3′. Sites 1–4 are 7 bp HMG-like binding sites. Nucleotides corresponding to those of the heptamer consensus HMG site C[A/T]TTG[A/T][A/T] are underlined. Mutated sites are spelt out, whereas wild-type nucleotides are indicated by dots. Sites 1–3 and mutants mA1, mA3, mA4, mA6 and mA8 were described previously (Zhou et al., 1998). Note that site 2 in mA3 harbors the same five consensus nucleotides as wild-type site 2, whereas, in other mutations, sites 1–4 retained only three or four consensus nucleotides. Binding of CSEP to wild-type and mutant 48 bp probes was tested using RCS nuclear extracts. Binding of L-Sox5, Sox6 and SOX9 was tested using extracts of 10T1/2 cells transfected with either one of the Sox protein expression plasmids. All probes had the same radioactivity. Arrow, migration level of CSEP. Arrowhead, fast-migrating complex of SOX9 with DNA. Download figure Download PowerPoint Figure 3.Long transcripts of Sox6 and Sox5 are expressed at high levels in chondrocytes. (A) Northern blot with total RNA. Samples were as follows: Pr. Ch., primary chondrocytes; 2d p. Ch., chondrocytes at the second passage in culture; RCS cells; newborn mouse brain and adult mouse testis. Hybridization was performed with probes that recognized both the long and short transcripts of either Sox5 or Sox6. Staining of 28S and 18S rRNA is shown as the reference for RNA loading. The sizes of Sox5 and Sox6 transcripts were calculated by comparison with the migration of RNA standards. (B) Northern blot with total RNA. Samples were as follows: Pr. Ch., primary chondrocytes; Pr. sk. fibr., primary skin fibroblasts from newborn mice; RCS cells; MC615 mouse immortalized chondrocytic cells at an early passage; 10T1/2 and BALB/3T3 mouse embryo fibroblasts; ROS rat osteosarcoma cells; C2C12 mouse myoblastic cells; EL4 mouse lymphoma cells; HeLa human carcinoma cells; COS monkey kidney cells; and MCTs, mouse immortalized chondrocytes. Hybridization was performed with probes for the long and short transcripts of either Sox5 or Sox6. Only signals at the level of the long transcripts are shown. No significant hybridization was seen at the level of the short transcripts in any sample (data not shown). Hybridization with an 18S rRNA probe is shown as the control for RNA loading. RNA samples were the same as in Lefebvre et al. (1997). (C) Same Northern blot as in (B), but with total RNA from newborn mouse tissues. Download figure Download PowerPoint Altogether these results indicated that three different Sox proteins, L-Sox5, Sox6 and Sox9, were present in chondrocytes and bound to the 48 bp Col2a1 element. L-Sox5 is a long product of the Sox5 gene that has not been identified previously. L-Sox5, Sox6 and Sox9 contact several HMG-like sites in the 48 bp Col2a1 enhancer The Col2a1 48 bp enhancer contains four HMG-like sites, each containing 5 or 6 bp of the 7 bp consensus HMG site (Figure 1F). Previously we showed that mutations that disrupted any one of sites 1–3 inhibited enhancer activity in chondrocytes in transgenic mice and partially inhibited binding of CSEP to the enhancer (mutants mA1, mA4 and mA6 in Figure 1F; Zhou et al., 1998). A mutation that disrupted site 4 also impaired binding of CSEP to the enhancer (mutant mA9 in Figure 1F). The effect of this mutation on enhancer activity has not been tested. A mutation that preserved all five HMG consensus nucleotides of site 2 did not affect the binding of CSEP (mA3 in Figure 1F) and only weakly inhibited enhancer activity in RCS cells (Zhou et al., 1998). A mutation that disrupted three sites completely inhibited binding of CSEP (mA8 in Figure 1F; Zhou et al., 1998). We also reported previously that subfragments of the 48 bp element containing either one of the four HMG-like sites alone were unable to bind CSEP or to compete with the 48 bp element for binding of CSEP (Lefebvre et al., 1996, 1997; Zhou et al., 1998; our data, not shown). These results indicated that the four sites of the 48 bp element cooperatively contributed to formation of the CSEP–enhancer complex. As described previously (Lefebvre et al., 1997; Zhou et al., 1998), the faster-migrating SOX9–DNA complex was inhibited by mutation of site 3 but not by mutation of other sites (Figure 1F). In contrast, the slower-migrating SOX9–DNA complex was partially inhibited by mutations disrupting any of the four sites and was completely inhibited by mutation of several sites. These results are consistent with the notion that the faster-migrating complex was formed by binding of one molecule of SOX9 to site 3, whereas the slower-migrating complex was formed by cooperative binding of two or more SOX9 molecules to several sites on each DNA molecule. Consistent with the results obtained with CSEP, binding of L-Sox5 or Sox6 to the 48 bp enhancer was partially inhibited by mutations that disrupted any one of the four HMG-like sites and completely inhibited by a mutation of several sites (Figure 1F). Mutation mA1 slightly but reproducibly inhibited binding of L-Sox5 and Sox6 to DNA. As seen with CSEP, L-Sox5 and Sox6 were unable to bind to DNA probes harboring only one of the Col2a1 HMG-like sites (data not shown). Taken together, these data indicated that L-Sox5 and Sox6 were able to contact cooperatively all four HMG-like sites of the Col2a1 48 bp enhancer. In chondrocyte nuclear extracts, the faster-migrating complex of Sox9 with the 48 bp enhancer was not seen (Zhou et al., 1998; compare also extracts of RCS cells and extracts of SOX9-transfected 10T1/2 cells in Figure 1D), but antibody supershift experiments indicated that Sox9 was present in the slower migrating CSEP complex. This result strongly suggested that, in chondrocytes, Sox9 bound the 48 bp element not as a single molecule but in cooperativity with other Sox9, L-Sox5 or Sox6 molecules. These data suggest a model whereby the four HMG-like sites of the 48 bp element and the three Sox proteins could participate in vivo in the formation of a large protein–enhancer complex that would activate expression of Col2a1 in chondrocytes. Consistent with this model in which mutation of any of the HMG-like sites would dismantle the protein–enhancer complex, we have observed that disruption of any of sites 1, 2 or 3 resulted in abolition of the activity of the enhancer in chondrocytes of transgenic mice (Zhou et al., 1998). A search for other HMG consensus and HMG-like sites (with six nucleotides of the heptamer consensus) in 1 kb of Col2a1 promoter sequence and in a 468 bp element of the first intron (+1878/+2345), which acts as a strong chondrocyte-specific enhancer in transgenic mice (Zhou et al., 1995), revealed the presence of a few scattered sites, but no cluster of two or more binding sites for Sox9, L-Sox5 and Sox6 was found that resembled that of the 48 bp element (data not shown). Long transcripts of the Sox6 and Sox5 genes are expressed in chondrocytes Sox5 and Sox6 were previously shown to be highly expressed in the testis of adult mice (Denny et al., 1992; Connor et al., 1995; Takamatsu et al., 1995). The transcripts were ∼2.0 and ∼3.2 kb long, respectively. Traces of longer transcripts (∼10 kb) were described for Sox6 in immature mouse testis (Takamatsu et al., 1995) and several tissues of adult mice (Connor et al., 1995), and also for SOX5 in human fetal brain (Wunderle et al., 1996). In Northern blots of total RNA, the short transcripts of Sox6 (3.2 kb) and Sox5 (2 kb) were abundant in adult mouse testis, as expected (Figure 2A). These short transcripts were not expressed (or were expressed at a very low level in the case of Sox6) in chondrocytes or in any cell line or newborn mouse tissue examined (Figure 2A; data not shown). A 6.3 kb Sox5 transcript and a 7.7 kb Sox6 transcript were found in similar relative abundance in primary chondrocytes from ribs of newborn mice, as well as in RCS and early-passage MC615 cells (Figure 2A and B). These three chondrocyte cells were previously shown to be well differentiated, expressing Col2a1 at a high level in parallel with Sox9 (Lefebvre et al., 1997). When rib chondrocytes were allowed to dedifferentiate by two passages in monolayer culture, Sox5 and Sox6 expression sharply declined (Figure 2A), as did Col2a1 and Sox9 expression (Lefebvre et al., 1997). Transcripts of Sox5, Sox6 and Sox9 were found in some non-chondrocytic cell types, but in contrast to chondrocytes, none of the non-chondrocytic cells coexpressed the three Sox genes or expressed Col2a1 at high levels (Figure 2B; Lefebvre et al., 1997). The long transcripts of Sox5 and Sox6 were present in the brain and some other non-cartilaginous tissues of newborn mice (Figure 2A and C). However, no non-cartilaginous tissue, besides brain and testis, was found to coexpress Sox9, Sox5 or and Sox6 (Figure 2A and C; Lefebvre et al., 1997). In testis, Sox9 expression is restricted to the somatic Sertoli cells (Kent et al., 1996; Morais da Silva et al., 1996); Sox5 is expressed as a short transcript in post-meiotic germ cells, mostly in round spermatids (Denny et al., 1992); and, since expression of Sox6 correlates with that of the protamine gene (Takamatsu et al., 1995), a marker of spermatid differentiation, it is possible that Sox6 is expressed in testis in the same cells and at the same time as Sox5. The three Sox proteins, therefore, do not appear to be expressed in the same cells in testis. In conclusion, chondrocytes, and perhaps some brain cells, appear to express together long transcripts for Sox5 and Sox6 and transcripts for Sox9. Expression of these transcripts correlates with expression of Col2a1 in chondrocytes. The long transcripts of Sox5 and Sox6 are coexpressed with Sox9 and Col2a1 during chondrogenesis in mouse embryos Previous whole-mount in situ experiments showed that Sox6 was expressed in the developing nervous system of mouse embryos; expression was high in early-stage embryos, but disappeared by embryonic day 12.5 (Connor et al., 1995). To obtain information on Sox6 and Sox5 expression during chondrogene
