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Identification of an Enhancer Sequence within the First Intron Required for Cartilage-specific Transcription of the α2(XI) Collagen Gene

2000; Elsevier BV; Volume: 275; Issue: 17 Linguagem: Inglês

10.1074/jbc.275.17.12712

1083-351X

Ying Liu, Haochuan Li, Kazuhiro Tanaka, Noriyuki Tsumaki, Yoshihiko Yamada,

Cancer-related gene regulation

Type XI collagen, a heterotrimer composed of α1(XI), α2(XI) and α3(XI), is primarily synthesized by chondrocytes in cartilage and is also present in some other tissues. Type XI collagen plays a critical role in collagen fibril formation and skeletal morphogenesis. We investigated a tissue-specific transcriptional enhancer in the first intron of the α2(XI) collagen gene (Col11a2). Transient transfection assays using reporter gene constructs revealed that a 60-base pair (bp) segment within intron 1 increased promoter activity of Col11a2 in rat chondrosarcoma cells but not in either BalB/3T3 cells or undifferentiated ATDC5 cells, suggesting that it contained cell type-specific enhancer activity. In transgenic mice, this 60-bp fragment was also able to target β-galactosidase expression to cartilage including the limbs and axial skeleton, with similar localization specificity as the full-length intron 1 fragment. Competition experiments in gel shift assays using mutated oligonucleotides showed that recombinant Sox9 bound to a 7-bp sequence, CTCAAAG, within the 60-bp segment. Anti-Sox9 antibodies supershifted the complex of the 60-bp segment with recombinant Sox9 or with rat chondrosarcoma cell extracts, confirming the binding of Sox9 to the enhancer. Moreover, a site-specific mutation within the 7-bp segment resulted in essentially complete loss of the enhancer activity in chondrosarcoma cells and transgenic mice. These results suggest that the 7-bp sequence within intron 1 plays a critical role in the cartilage-specific enhancer activity of Col11a2 through Sox9-mediated transcriptional activation. Type XI collagen, a heterotrimer composed of α1(XI), α2(XI) and α3(XI), is primarily synthesized by chondrocytes in cartilage and is also present in some other tissues. Type XI collagen plays a critical role in collagen fibril formation and skeletal morphogenesis. We investigated a tissue-specific transcriptional enhancer in the first intron of the α2(XI) collagen gene (Col11a2). Transient transfection assays using reporter gene constructs revealed that a 60-base pair (bp) segment within intron 1 increased promoter activity of Col11a2 in rat chondrosarcoma cells but not in either BalB/3T3 cells or undifferentiated ATDC5 cells, suggesting that it contained cell type-specific enhancer activity. In transgenic mice, this 60-bp fragment was also able to target β-galactosidase expression to cartilage including the limbs and axial skeleton, with similar localization specificity as the full-length intron 1 fragment. Competition experiments in gel shift assays using mutated oligonucleotides showed that recombinant Sox9 bound to a 7-bp sequence, CTCAAAG, within the 60-bp segment. Anti-Sox9 antibodies supershifted the complex of the 60-bp segment with recombinant Sox9 or with rat chondrosarcoma cell extracts, confirming the binding of Sox9 to the enhancer. Moreover, a site-specific mutation within the 7-bp segment resulted in essentially complete loss of the enhancer activity in chondrosarcoma cells and transgenic mice. These results suggest that the 7-bp sequence within intron 1 plays a critical role in the cartilage-specific enhancer activity of Col11a2 through Sox9-mediated transcriptional activation. kilobase(s) high mobility group base pair(s) rat chondrosarcoma polymerase chain reaction electrophoretic mobility shift assay Cartilage is a highly specialized tissue important for bearing compression loads in joints and also serves as the template for most developing bones. Cartilage contains unique extracellular matrix proteins produced by chondrocytes. The collagen network provides the scaffolding of the cartilage matrix and confers tensile strength important for resisting compression in cartilage. Collagen fibrils in cartilage consist of the major collagen, type II, and minor collagens, type IX and type XI. Type XI collagen, consisting of α1(XI), α2(XI), and α3(XI) chains, co-assembles with type II collagen to form cartilage collagen fibrils, whereas type IX collagen is associated with the surface of these fibrils (1.Mendler M. Eich-Bender S.G. Vaughan L. Winterhalter K.H. Bruckner P. J. Cell Biol. 1989; 108: 191-197Crossref PubMed Scopus (401) Google Scholar, 2.Vaughan L. Mendler M. Huber S. Bruckner P. Winterhalter K.H. Irwin M.I. Mayne R. J. Cell Biol. 1988; 106: 991-997Crossref PubMed Scopus (258) Google Scholar). The α1(XI) and α2(XI) chains are distinct gene products (3.Bernard M. Yoshioka H. Rodriguez E. van der Rest M. Kimura T. Ninomiya Y. Olsen B.R. Ramirez F. J. Biol. Chem. 1988; 263: 17159-17166Abstract Full Text PDF PubMed Google Scholar, 4.Kimura T. Cheah K.S.E. Chan S.D.H. Lui V.C.H. Mattei M.-G. van der Rest M. Ono K. Solomon E. Ninomiya Y. Olsen B.R. J. Biol. Chem. 1989; 264: 13910-13916Abstract Full Text PDF PubMed Google Scholar), whereas the α3(XI) chain is a post-translational variant of the α1(II) chain (5.Furuto D.K. Miller E.J. Arch. Biochem. Biophys. 1983; 226: 604-611Crossref PubMed Scopus (45) Google Scholar). It has been postulated that type IX and type XI collagens regulate the collagen network by determining the diameter of cartilage collagen fibrils and their interactions with other matrix components (6.Keene D.R. Oxford J.T. Morris N.P. J. Histochem. Cytochem. 1995; 43: 967-979Crossref PubMed Scopus (93) Google Scholar, 7.Wu J.J. Eyre D.R. Biochem. Biophys. Res. Commun. 1984; 123: 1033-1039Crossref PubMed Scopus (45) Google Scholar, 8.Smith Jr., G.N. Williams J.M. Brandt K.D. J. Biol. Chem. 1985; 260: 10761-10767Abstract Full Text PDF PubMed Google Scholar, 9.Eikenberry E.F. Mendler M. Burgin R. Winterhalter K.H. Bruckner P. Kuettner K.E. Schleyerbach R. Peyron J.G. Hascall V.C. Articular Cartilage and Osteoarthritis. Raven Press, New York1992: 133-149Google Scholar).Mutations in the type XI collagen genes have been identified and found to cause chondrodysplasia in both humans and mice. For example, mutations in the α2(XI) collagen gene (Col11a2) were identified in patients with Stickler syndrome and otospondylo-megaepiphyseal dysplasia (10.Vikkula M. Mariman E.C.M. Lui V.C.H. Zhidkova N.I. Tiller G.E. Goldring M.B. van Beersum S.E.C. de Waal Malefiit M.C. van den Hoogen F.H.J. Ropers H.-H. Cell. 1995; 80: 431-437Abstract Full Text PDF PubMed Scopus (305) Google Scholar). A null mutation in the α1(XI) gene of cho (chondrodysplasia) mice causes dwarfism with reduced matrix and thickened cartilage collagen fibrils (11.Li Y. Lacerda D.A. Warman M.L. Beier D.R. Yoshioka H. Ninomiya Y. Oxford J.T. Morris N.P. Andrikopoulos K. Ramirez F. Cell. 1995; 80: 423-430Abstract Full Text PDF PubMed Scopus (297) Google Scholar). These findings indicate that type XI collagen plays an important role in skeletal morphogenesis and that expression of type XI collagen is critical for the development of cartilage.Several regulatory regions of the mouse α2(XI) collagen gene have been identified, and their modular arrangements have been proposed (12.Tsumaki N. Kimura T. J. Biol. Chem. 1995; 270: 2372-2378Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 13.Tsumaki N. Kimura T. Tanaka K. Kimura J.H. Ochi T. Yamada Y. J. Biol. Chem. 1998; 273: 22861-22864Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 14.Sugimoto M. Kimura T. Tsumaki N. Matsu Y. Nakata K. Kawabata H. Yasui N. Kitamura Y. Nomura S. Ochi T. Cell Tissue Res. 1998; 292: 325-332Crossref PubMed Scopus (27) Google Scholar). The −742 promoter segment is able to direct transcription ofCol11a2 in most cartilaginous tissues in transgenic mice (15.Tsumaki N. Kimura T. Matsui Y. Nakata K. Ochi T. J. Cell Biol. 1996; 134: 1573-1582Crossref PubMed Scopus (68) Google Scholar, 16.Bridgewater L.C. Lefebvre V. de Crombrugghe B. J. Biol. Chem. 1998; 273: 14998-15006Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 17.Li S.W. Arita M. Kopen G.C. Phinney D.G. Prockop D.J. Matrix Biol. 1998; 17: 213-221Crossref PubMed Scopus (11) Google Scholar) and consists of at least two chondrocyte-specific enhancers containing Sox9 binding sites at ∼−600 and ∼−530 (15.Tsumaki N. Kimura T. Matsui Y. Nakata K. Ochi T. J. Cell Biol. 1996; 134: 1573-1582Crossref PubMed Scopus (68) Google Scholar, 16.Bridgewater L.C. Lefebvre V. de Crombrugghe B. J. Biol. Chem. 1998; 273: 14998-15006Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 17.Li S.W. Arita M. Kopen G.C. Phinney D.G. Prockop D.J. Matrix Biol. 1998; 17: 213-221Crossref PubMed Scopus (11) Google Scholar, 18.Bell D.M. Leung K.K.H. Wheatley S.C. Ng L.J. Zhou S. Ling K.W. Sham M.H. Koopman P. Tam P.P.L. Cheah K.S.E. Nat. Genet. 1997; 16: 174-178Crossref PubMed Scopus (760) Google Scholar, 19.Ng L.J. Wheatley S. Muscat G.E.O. Campbell J.C. Bowles J. Wright E. Bell D.M. Tam P.P.L. Cheah K.S.E. Koopman P. Dev. Biol. 1997; 183: 108-121Crossref PubMed Scopus (556) Google Scholar). Inclusion of a 2.3-kb1segment from the first intron to the −742 promoter construct increased the promoter activity of Col11a2 (15.Tsumaki N. Kimura T. Matsui Y. Nakata K. Ochi T. J. Cell Biol. 1996; 134: 1573-1582Crossref PubMed Scopus (68) Google Scholar). A shorter promoter construct (−453) did not show cartilage-specific expression; however, inclusion of the intron sequence to the construct induced reporter gene expression in cartilage. These results suggest the presence of the enhancer in intron 1. More recently, it was found that the −530 promoter sequence was sufficient for cartilage-specific expression ofCol11a2 and that deletion of a sequence between −530 and −500 abolished reporter gene expression in cartilage, suggesting the presence of regulatory elements within this region required for cartilage-specific expression (13.Tsumaki N. Kimura T. Tanaka K. Kimura J.H. Ochi T. Yamada Y. J. Biol. Chem. 1998; 273: 22861-22864Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 16.Bridgewater L.C. Lefebvre V. de Crombrugghe B. J. Biol. Chem. 1998; 273: 14998-15006Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar).SOX9 was initially identified by positional cloning as associated with the skeletal malformation syndrome campomelic dysplasia (20.Foster J.W. Dominguez-Steglich M.A. Guioli S. Kowk G. Weller P.A. Stevanovic M. Weissenbach J. Mansour S. Young I.D. Goodfellow P.N. et al.Nature. 1994; 372: 525-530Crossref PubMed Scopus (1303) Google Scholar). It contains an HMG-type DNA-binding domain and a transactivation domain (21.Sudbeck P. Schmitz L. Baeuerle P.A. Scherer G. Nat. Genet. 1996; 13: 230-232Crossref PubMed Scopus (184) Google Scholar). The HMG box region of SOX9 binds DNA at the target sequences AACAT and AACAAAG (22.Harley V.R. Jackson D.I. Hextall P.J. Hawkins J.R. Berkovitz G.D. Sockanathan S. Lovell-Badge R. Goodfellow P.N. Science. 1992; 255: 453-456Crossref PubMed Scopus (373) Google Scholar). SOX9 is expressed in primordial cartilage and in other noncartilaginous tissues during development (19.Ng L.J. Wheatley S. Muscat G.E.O. Campbell J.C. Bowles J. Wright E. Bell D.M. Tam P.P.L. Cheah K.S.E. Koopman P. Dev. Biol. 1997; 183: 108-121Crossref PubMed Scopus (556) Google Scholar, 23.Wright E. Hargrave M.R. Christiansen J. Cooper L. Kun J. Evans T. Gangadharan U. Greenfield A. Koopman P. Nat. Genet. 1995; 9: 15-20Crossref PubMed Scopus (550) Google Scholar). It has been shown that SOX-9 regulates Col2a1 transcription through Sox9 binding to the intron enhancer (18.Bell D.M. Leung K.K.H. Wheatley S.C. Ng L.J. Zhou S. Ling K.W. Sham M.H. Koopman P. Tam P.P.L. Cheah K.S.E. Nat. Genet. 1997; 16: 174-178Crossref PubMed Scopus (760) Google Scholar, 24.Lefebvre V. de Crombrugghe B. Matrix Biol. 1998; 16: 529-540Crossref PubMed Google Scholar, 33.Lefebvre V. Huang W. Harley V.R. Goodfellow P.N. de Crombrugghe B. Mol. Cell. Biol. 1997; 17: 2336-2346Crossref PubMed Google Scholar). It has also been shown that the promoter of Col11a2 contains Sox9-binding sites necessary for cartilage-specific expression (16.Bridgewater L.C. Lefebvre V. de Crombrugghe B. J. Biol. Chem. 1998; 273: 14998-15006Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar).In this study, we have characterized the activity of the intron enhancer of Col11a2 by deletion analysis in cell cultures and in transgenic mice and by DNA binding assays. We found that a 60-bp sequence from intron 1 can direct cartilage-specific expression ofCol11a2. Mutation analysis identified a 7-bp sequence within the sequence critical for cell type-specific enhancer activity and Sox9 interactions.DISCUSSIONType XI collagen is an essential structural component in cartilage. Regulation of Col11a2 is mediated by positive and negative regulatory elements. For example, Sox9 binding elements at ∼−600 and ∼−520 promotes cartilage-specific expression ofCol11a2 (16.Bridgewater L.C. Lefebvre V. de Crombrugghe B. J. Biol. Chem. 1998; 273: 14998-15006Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). A neural tissue-specific element (−454 to −500) and a cartilage-specific element (−501 to −530) that converts neuronal tissue-specific expression to cartilage have been identified (13.Tsumaki N. Kimura T. Tanaka K. Kimura J.H. Ochi T. Yamada Y. J. Biol. Chem. 1998; 273: 22861-22864Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). In addition to these promoter elements, the first intron segment enhanced the promoter activity in cartilage and was required for expression of Col11a2 in the notochord (13.Tsumaki N. Kimura T. Tanaka K. Kimura J.H. Ochi T. Yamada Y. J. Biol. Chem. 1998; 273: 22861-22864Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). In this report, we have identified a sequence in the first intron required for cartilage-specific expression of Col11a2.We found that a 300-bp intron segment contained full enhancer activity similar to that of the 2.3-kb intron segment in transfection assays in RCS cells. Transfection analysis using oligonucleotides as competitors identified a 30-bp sequence within the 300-bp segment important for the enhancer activity. This oligonucleotide competition approach has advantages over a conventional method using mutated constructs. It is quick (i.e. no requirement for the creation of mutations in the constructs) and can compare enhancer activity using the same wild type reporter construct without sacrificing the size of the enhancer segment of interest. A disadvantage may be a limitation of the size of oligonucleotides to be used as competitors. Using this approach, we narrowed down an enhancer-containing sequence to 60 bp within the 300-bp segment. The 60-bp sequence enhanced the −453 promoter ofCol11a2 by 10-fold compared with 40-fold enhancement by the 300-bp or 2-kb intron segment. The reduced enhancer activity of the 60-bp sequence may occur because its flanking sequence might be important for stable factor-DNA binding and/or recognized by other factors for the full enhancer activity in a cooperative manner. Consistent with this hypothesis, amplifications of the 60-bp sequence increased enhancer activity (i.e. 2.7, 4.6, 11.5, and 16.0-fold increase by two, four, six, and eight copies, respectively; Table I). These results suggest that multiple factors may be required for the full enhancer activity.Gel shift assays showed that Sox9 protein bound a 7-bp sequence, CTCAAAG (+1334 to +1339), within the 60-bp segment. A substitution mutation in the 7-bp sequence showed little activity in RCS cells (Table I). These results suggest that the 7-bp sequence is critical for enhancer activity through Sox9 binding. When nuclear extracts from RCS cells were used in EMSAs with the 60-bp probe, two complexes were formed. The fast migrating band represents a Sox9-DNA complex because its migration position was similar to that of the complex with the Sox9 protein and the antibodies to Sox9 supershifted the complex. It is likely that the slower migrating band contains another protein factor(s) in addition to Sox9. It was reported that other Sox family proteins (e.g. Sox5 and Sox6) cooperatively work with Sox9 for activation of the enhancer of the Col2a1 gene (34.Lefebvre V. Li P. de Crombrugghe B. EMBO J. 1998; 17: 5718-5733Crossref PubMed Scopus (670) Google Scholar). Our observations are in agreement with these observations. These results indicate that the 7-bp cis-acting element plays a central role in the chondrocyte-specific enhancer activity and strongly suggest that the Sox9 protein is a key mediator for the transcription ofCol11a2 in chondrocyte.The 60-bp sequence was able to direct expression of the −453 promoter of Col11a2 in the cartilage of transgenic mice. The expression patterns of the reporter β-galactosidase gene were similar to those with the construct containing either the 300-bp or the 2.3-kb intron segment (15.Tsumaki N. Kimura T. Matsui Y. Nakata K. Ochi T. J. Cell Biol. 1996; 134: 1573-1582Crossref PubMed Scopus (68) Google Scholar). 2Y. Liu, H. Li, K. Tanaka, N. Tsumaki, and Y. Yamada, unpublished data. A 7-bp substitution mutation in the 60-bp sequence eliminated the enhancer activity, suggesting that this 7-bp sequence is critical for tissue-specific activity, which is in agreement with the transfection analysis. Interestingly, the 60-bp enhancer-containing construct failed to direct β-galactosidase expression in the notochord (data not shown). Because the 2.3-kb intron segment is necessary for notochord-specific expression of Col11a2, the 60-bp sequence likely lacks the information necessary for expression in the notochord. Thus, Co1l1a2 is positively regulated by at least two distinct elements, one for cartilage and another for notochord in the first intron.Col11a2 contains redundant Sox9 sites. Each site may have unique activity in different tissues, and such redundancy may be necessary for the optimum expression of Col11a2 to form collagen fibrils specific to each type of cartilage. Differences in the activity of these sites observed in transfected RCS cells may not be the only regulatory mechanisms of Col11a2 expression in cartilage. Recently, it was reported that sequences adjacent to the Sox9 site are also required for the chondrocyte-specific enhancer activity of Col2a1 and that new members of the Sox family,l-Sox5 and Sox6, form a heterodimer and activate theCol2a1 enhancer co-operatively with Sox9 (34.Lefebvre V. Li P. de Crombrugghe B. EMBO J. 1998; 17: 5718-5733Crossref PubMed Scopus (670) Google Scholar). It is conceivable that protein factors, such as l-Sox5 and Sox6, may also be involved in Col11a2 expression. Levels of Sox9 and these proteins may differ in each cartilage and have preferential utilization of the multi-modular elements containing the Sox9 site forCol11a2 expression. Cartilage is a highly specialized tissue important for bearing compression loads in joints and also serves as the template for most developing bones. Cartilage contains unique extracellular matrix proteins produced by chondrocytes. The collagen network provides the scaffolding of the cartilage matrix and confers tensile strength important for resisting compression in cartilage. Collagen fibrils in cartilage consist of the major collagen, type II, and minor collagens, type IX and type XI. Type XI collagen, consisting of α1(XI), α2(XI), and α3(XI) chains, co-assembles with type II collagen to form cartilage collagen fibrils, whereas type IX collagen is associated with the surface of these fibrils (1.Mendler M. Eich-Bender S.G. Vaughan L. Winterhalter K.H. Bruckner P. J. Cell Biol. 1989; 108: 191-197Crossref PubMed Scopus (401) Google Scholar, 2.Vaughan L. Mendler M. Huber S. Bruckner P. Winterhalter K.H. Irwin M.I. Mayne R. J. Cell Biol. 1988; 106: 991-997Crossref PubMed Scopus (258) Google Scholar). The α1(XI) and α2(XI) chains are distinct gene products (3.Bernard M. Yoshioka H. Rodriguez E. van der Rest M. Kimura T. Ninomiya Y. Olsen B.R. Ramirez F. J. Biol. Chem. 1988; 263: 17159-17166Abstract Full Text PDF PubMed Google Scholar, 4.Kimura T. Cheah K.S.E. Chan S.D.H. Lui V.C.H. Mattei M.-G. van der Rest M. Ono K. Solomon E. Ninomiya Y. Olsen B.R. J. Biol. Chem. 1989; 264: 13910-13916Abstract Full Text PDF PubMed Google Scholar), whereas the α3(XI) chain is a post-translational variant of the α1(II) chain (5.Furuto D.K. Miller E.J. Arch. Biochem. Biophys. 1983; 226: 604-611Crossref PubMed Scopus (45) Google Scholar). It has been postulated that type IX and type XI collagens regulate the collagen network by determining the diameter of cartilage collagen fibrils and their interactions with other matrix components (6.Keene D.R. Oxford J.T. Morris N.P. J. Histochem. Cytochem. 1995; 43: 967-979Crossref PubMed Scopus (93) Google Scholar, 7.Wu J.J. Eyre D.R. Biochem. Biophys. Res. Commun. 1984; 123: 1033-1039Crossref PubMed Scopus (45) Google Scholar, 8.Smith Jr., G.N. Williams J.M. Brandt K.D. J. Biol. Chem. 1985; 260: 10761-10767Abstract Full Text PDF PubMed Google Scholar, 9.Eikenberry E.F. Mendler M. Burgin R. Winterhalter K.H. Bruckner P. Kuettner K.E. Schleyerbach R. Peyron J.G. Hascall V.C. Articular Cartilage and Osteoarthritis. Raven Press, New York1992: 133-149Google Scholar). Mutations in the type XI collagen genes have been identified and found to cause chondrodysplasia in both humans and mice. For example, mutations in the α2(XI) collagen gene (Col11a2) were identified in patients with Stickler syndrome and otospondylo-megaepiphyseal dysplasia (10.Vikkula M. Mariman E.C.M. Lui V.C.H. Zhidkova N.I. Tiller G.E. Goldring M.B. van Beersum S.E.C. de Waal Malefiit M.C. van den Hoogen F.H.J. Ropers H.-H. Cell. 1995; 80: 431-437Abstract Full Text PDF PubMed Scopus (305) Google Scholar). A null mutation in the α1(XI) gene of cho (chondrodysplasia) mice causes dwarfism with reduced matrix and thickened cartilage collagen fibrils (11.Li Y. Lacerda D.A. Warman M.L. Beier D.R. Yoshioka H. Ninomiya Y. Oxford J.T. Morris N.P. Andrikopoulos K. Ramirez F. Cell. 1995; 80: 423-430Abstract Full Text PDF PubMed Scopus (297) Google Scholar). These findings indicate that type XI collagen plays an important role in skeletal morphogenesis and that expression of type XI collagen is critical for the development of cartilage. Several regulatory regions of the mouse α2(XI) collagen gene have been identified, and their modular arrangements have been proposed (12.Tsumaki N. Kimura T. J. Biol. Chem. 1995; 270: 2372-2378Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 13.Tsumaki N. Kimura T. Tanaka K. Kimura J.H. Ochi T. Yamada Y. J. Biol. Chem. 1998; 273: 22861-22864Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 14.Sugimoto M. Kimura T. Tsumaki N. Matsu Y. Nakata K. Kawabata H. Yasui N. Kitamura Y. Nomura S. Ochi T. Cell Tissue Res. 1998; 292: 325-332Crossref PubMed Scopus (27) Google Scholar). The −742 promoter segment is able to direct transcription ofCol11a2 in most cartilaginous tissues in transgenic mice (15.Tsumaki N. Kimura T. Matsui Y. Nakata K. Ochi T. J. Cell Biol. 1996; 134: 1573-1582Crossref PubMed Scopus (68) Google Scholar, 16.Bridgewater L.C. Lefebvre V. de Crombrugghe B. J. Biol. Chem. 1998; 273: 14998-15006Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 17.Li S.W. Arita M. Kopen G.C. Phinney D.G. Prockop D.J. Matrix Biol. 1998; 17: 213-221Crossref PubMed Scopus (11) Google Scholar) and consists of at least two chondrocyte-specific enhancers containing Sox9 binding sites at ∼−600 and ∼−530 (15.Tsumaki N. Kimura T. Matsui Y. Nakata K. Ochi T. J. Cell Biol. 1996; 134: 1573-1582Crossref PubMed Scopus (68) Google Scholar, 16.Bridgewater L.C. Lefebvre V. de Crombrugghe B. J. Biol. Chem. 1998; 273: 14998-15006Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 17.Li S.W. Arita M. Kopen G.C. Phinney D.G. Prockop D.J. Matrix Biol. 1998; 17: 213-221Crossref PubMed Scopus (11) Google Scholar, 18.Bell D.M. Leung K.K.H. Wheatley S.C. Ng L.J. Zhou S. Ling K.W. Sham M.H. Koopman P. Tam P.P.L. Cheah K.S.E. Nat. Genet. 1997; 16: 174-178Crossref PubMed Scopus (760) Google Scholar, 19.Ng L.J. Wheatley S. Muscat G.E.O. Campbell J.C. Bowles J. Wright E. Bell D.M. Tam P.P.L. Cheah K.S.E. Koopman P. Dev. Biol. 1997; 183: 108-121Crossref PubMed Scopus (556) Google Scholar). Inclusion of a 2.3-kb1segment from the first intron to the −742 promoter construct increased the promoter activity of Col11a2 (15.Tsumaki N. Kimura T. Matsui Y. Nakata K. Ochi T. J. Cell Biol. 1996; 134: 1573-1582Crossref PubMed Scopus (68) Google Scholar). A shorter promoter construct (−453) did not show cartilage-specific expression; however, inclusion of the intron sequence to the construct induced reporter gene expression in cartilage. These results suggest the presence of the enhancer in intron 1. More recently, it was found that the −530 promoter sequence was sufficient for cartilage-specific expression ofCol11a2 and that deletion of a sequence between −530 and −500 abolished reporter gene expression in cartilage, suggesting the presence of regulatory elements within this region required for cartilage-specific expression (13.Tsumaki N. Kimura T. Tanaka K. Kimura J.H. Ochi T. Yamada Y. J. Biol. Chem. 1998; 273: 22861-22864Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 16.Bridgewater L.C. Lefebvre V. de Crombrugghe B. J. Biol. Chem. 1998; 273: 14998-15006Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). SOX9 was initially identified by positional cloning as associated with the skeletal malformation syndrome campomelic dysplasia (20.Foster J.W. Dominguez-Steglich M.A. Guioli S. Kowk G. Weller P.A. Stevanovic M. Weissenbach J. Mansour S. Young I.D. Goodfellow P.N. et al.Nature. 1994; 372: 525-530Crossref PubMed Scopus (1303) Google Scholar). It contains an HMG-type DNA-binding domain and a transactivation domain (21.Sudbeck P. Schmitz L. Baeuerle P.A. Scherer G. Nat. Genet. 1996; 13: 230-232Crossref PubMed Scopus (184) Google Scholar). The HMG box region of SOX9 binds DNA at the target sequences AACAT and AACAAAG (22.Harley V.R. Jackson D.I. Hextall P.J. Hawkins J.R. Berkovitz G.D. Sockanathan S. Lovell-Badge R. Goodfellow P.N. Science. 1992; 255: 453-456Crossref PubMed Scopus (373) Google Scholar). SOX9 is expressed in primordial cartilage and in other noncartilaginous tissues during development (19.Ng L.J. Wheatley S. Muscat G.E.O. Campbell J.C. Bowles J. Wright E. Bell D.M. Tam P.P.L. Cheah K.S.E. Koopman P. Dev. Biol. 1997; 183: 108-121Crossref PubMed Scopus (556) Google Scholar, 23.Wright E. Hargrave M.R. Christiansen J. Cooper L. Kun J. Evans T. Gangadharan U. Greenfield A. Koopman P. Nat. Genet. 1995; 9: 15-20Crossref PubMed Scopus (550) Google Scholar). It has been shown that SOX-9 regulates Col2a1 transcription through Sox9 binding to the intron enhancer (18.Bell D.M. Leung K.K.H. Wheatley S.C. Ng L.J. Zhou S. Ling K.W. Sham M.H. Koopman P. Tam P.P.L. Cheah K.S.E. Nat. Genet. 1997; 16: 174-178Crossref PubMed Scopus (760) Google Scholar, 24.Lefebvre V. de Crombrugghe B. Matrix Biol. 1998; 16: 529-540Crossref PubMed Google Scholar, 33.Lefebvre V. Huang W. Harley V.R. Goodfellow P.N. de Crombrugghe B. Mol. Cell. Biol. 1997; 17: 2336-2346Crossref PubMed Google Scholar). It has also been shown that the promoter of Col11a2 contains Sox9-binding sites necessary for cartilage-specific expression (16.Bridgewater L.C. Lefebvre V. de Crombrugghe B. J. Biol. Chem. 1998; 273: 14998-15006Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). In this study, we have characterized the activity of the intron enhancer of Col11a2 by deletion analysis in cell cultures and in transgenic mice and by DNA binding assays. We found that a 60-bp sequence from intron 1 can direct cartilage-specific expression ofCol11a2. Mutation analysis identified a 7-bp sequence within the sequence critical for cell type-specific enhancer activity and Sox9 interactions. DISCUSSIONType XI collagen is an essential structural component in cartilage. Regulation of Col11a2 is mediated by positive and negative regulatory elements. For example, Sox9 binding elements at ∼−600 and ∼−520 promotes cartilage-specific expression ofCol11a2 (16.Bridgewater L.C. Lefebvre V. de Crombrugghe B. J. Biol. Chem. 1998; 273: 14998-15006Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). A neural tissue-specific element (−454 to −500) and a cartilage-specific element (−501 to −530) that converts neuronal tissue-specific expression to cartilage have been identified (13.Tsumaki N. Kimura T. Tanaka K. Kimura J.H. Ochi T. Yamada Y. J. Biol. Chem. 1998; 273: 22861-22864Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). In addition to these promoter elements, the first intron segment enhanced the promoter activity in cartilage and was required for expression of Col11a2 in the notochord (13.Tsumaki N. Kimura T. Tanaka K. Kimura J.H. Ochi T. Yamada Y. J. Biol. Chem. 1998; 273: 22861-22864Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). In this report, we have identified a sequence in the first intron required for cartilage-specific expression of Col11a2.We found that a 300-bp intron segment contained full enhancer activity similar to that of the 2.3-kb intron segment in transfection assays in RCS cells. Transfection analysis using oligonucleotides as competitors identified a 30-bp sequence within the 300-bp segment important for the enhancer activity. This oligonucleotide competition approach has advantages over a conventional method using mutated constructs. It is quick (i.e. no requirement for the creation of mutations in the constructs) and can compare enhancer activity using the same wild type reporter construct without sacrificing the size of the enhancer segment of interest. A disadvantage may be a limitation of the size of oligonucleotides to be used as competitors. Using this approach, we narrowed down an enhancer-containing sequence to 60 bp within the 300-bp segment. The 60-bp sequence enhanced the −453 promoter ofCol11a2 by 10-fold compared with 40-fold enhancement by the 300-bp or 2-kb intron segment. The reduced enhancer activity of the 60-bp sequence may occur because its flanking sequence might be important for stable factor-DNA binding and/or recognized by other factors for the full enhancer activity in a cooperative manner. Consistent with this hypothesis, amplifications of the 60-bp sequence increased enhancer activity (i.e. 2.7, 4.6, 11.5, and 16.0-fold increase by two, four, six, and eight copies, respectively; Table I). These results suggest that multiple factors may be required for the full enhancer activity.Gel shift assays showed that Sox9 protein bound a 7-bp sequence, CTCAAAG (+1334 to +1339), within the 60-bp segment. A substitution mutation in the 7-bp sequence showed little activity in RCS cells (Table I). These results suggest that the 7-bp sequence is critical for enhancer activity through Sox9 binding. When nuclear extracts from RCS cells were used in EMSAs with the 60-bp probe, two complexes were formed. The fast migrating band represents a Sox9-DNA complex because its migration position was similar to that of the complex with the Sox9 protein and the antibodies to Sox9 supershifted the complex. It is likely that the slower migrating band contains another protein factor(s) in addition to Sox9. It was reported that other Sox family proteins (e.g. Sox5 and Sox6) cooperatively work with Sox9 for activation of the enhancer of the Col2a1 gene (34.Lefebvre V. Li P. de Crombrugghe B. EMBO J. 1998; 17: 5718-5733Crossref PubMed Scopus (670) Google Scholar). Our observations are in agreement with these observations. These results indicate that the 7-bp cis-acting element plays a central role in the chondrocyte-specific enhancer activity and strongly suggest that the Sox9 protein is a key mediator for the transcription ofCol11a2 in chondrocyte.The 60-bp sequence was able to direct expression of the −453 promoter of Col11a2 in the cartilage of transgenic mice. The expression patterns of the reporter β-galactosidase gene were similar to those with the construct containing either the 300-bp or the 2.3-kb intron segment (15.Tsumaki N. Kimura T. Matsui Y. Nakata K. Ochi T. J. Cell Biol. 1996; 134: 1573-1582Crossref PubMed Scopus (68) Google Scholar). 2Y. Liu, H. Li, K. Tanaka, N. Tsumaki, and Y. Yamada, unpublished data. A 7-bp substitution mutation in the 60-bp sequence eliminated the enhancer activity, suggesting that this 7-bp sequence is critical for tissue-specific activity, which is in agreement with the transfection analysis. Interestingly, the 60-bp enhancer-containing construct failed to direct β-galactosidase expression in the notochord (data not shown). Because the 2.3-kb intron segment is necessary for notochord-specific expression of Col11a2, the 60-bp sequence likely lacks the information necessary for expression in the notochord. Thus, Co1l1a2 is positively regulated by at least two distinct elements, one for cartilage and another for notochord in the first intron.Col11a2 contains redundant Sox9 sites. Each site may have unique activity in different tissues, and such redundancy may be necessary for the optimum expression of Col11a2 to form collagen fibrils specific to each type of cartilage. Differences in the activity of these sites observed in transfected RCS cells may not be the only regulatory mechanisms of Col11a2 expression in cartilage. Recently, it was reported that sequences adjacent to the Sox9 site are also required for the chondrocyte-specific enhancer activity of Col2a1 and that new members of the Sox family,l-Sox5 and Sox6, form a heterodimer and activate theCol2a1 enhancer co-operatively with Sox9 (34.Lefebvre V. Li P. de Crombrugghe B. EMBO J. 1998; 17: 5718-5733Crossref PubMed Scopus (670) Google Scholar). It is conceivable that protein factors, such as l-Sox5 and Sox6, may also be involved in Col11a2 expression. Levels of Sox9 and these proteins may differ in each cartilage and have preferential utilization of the multi-modular elements containing the Sox9 site forCol11a2 expression. Type XI collagen is an essential structural component in cartilage. Regulation of Col11a2 is mediated by positive and negative regulatory elements. For example, Sox9 binding elements at ∼−600 and ∼−520 promotes cartilage-specific expression ofCol11a2 (16.Bridgewater L.C. Lefebvre V. de Crombrugghe B. J. Biol. Chem. 1998; 273: 14998-15006Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). A neural tissue-specific element (−454 to −500) and a cartilage-specific element (−501 to −530) that converts neuronal tissue-specific expression to cartilage have been identified (13.Tsumaki N. Kimura T. Tanaka K. Kimura J.H. Ochi T. Yamada Y. J. Biol. Chem. 1998; 273: 22861-22864Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). In addition to these promoter elements, the first intron segment enhanced the promoter activity in cartilage and was required for expression of Col11a2 in the notochord (13.Tsumaki N. Kimura T. Tanaka K. Kimura J.H. Ochi T. Yamada Y. J. Biol. Chem. 1998; 273: 22861-22864Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). In this report, we have identified a sequence in the first intron required for cartilage-specific expression of Col11a2. We found that a 300-bp intron segment contained full enhancer activity similar to that of the 2.3-kb intron segment in transfection assays in RCS cells. Transfection analysis using oligonucleotides as competitors identified a 30-bp sequence within the 300-bp segment important for the enhancer activity. This oligonucleotide competition approach has advantages over a conventional method using mutated constructs. It is quick (i.e. no requirement for the creation of mutations in the constructs) and can compare enhancer activity using the same wild type reporter construct without sacrificing the size of the enhancer segment of interest. A disadvantage may be a limitation of the size of oligonucleotides to be used as competitors. Using this approach, we narrowed down an enhancer-containing sequence to 60 bp within the 300-bp segment. The 60-bp sequence enhanced the −453 promoter ofCol11a2 by 10-fold compared with 40-fold enhancement by the 300-bp or 2-kb intron segment. The reduced enhancer activity of the 60-bp sequence may occur because its flanking sequence might be important for stable factor-DNA binding and/or recognized by other factors for the full enhancer activity in a cooperative manner. Consistent with this hypothesis, amplifications of the 60-bp sequence increased enhancer activity (i.e. 2.7, 4.6, 11.5, and 16.0-fold increase by two, four, six, and eight copies, respectively; Table I). These results suggest that multiple factors may be required for the full enhancer activity. Gel shift assays showed that Sox9 protein bound a 7-bp sequence, CTCAAAG (+1334 to +1339), within the 60-bp segment. A substitution mutation in the 7-bp sequence showed little activity in RCS cells (Table I). These results suggest that the 7-bp sequence is critical for enhancer activity through Sox9 binding. When nuclear extracts from RCS cells were used in EMSAs with the 60-bp probe, two complexes were formed. The fast migrating band represents a Sox9-DNA complex because its migration position was similar to that of the complex with the Sox9 protein and the antibodies to Sox9 supershifted the complex. It is likely that the slower migrating band contains another protein factor(s) in addition to Sox9. It was reported that other Sox family proteins (e.g. Sox5 and Sox6) cooperatively work with Sox9 for activation of the enhancer of the Col2a1 gene (34.Lefebvre V. Li P. de Crombrugghe B. EMBO J. 1998; 17: 5718-5733Crossref PubMed Scopus (670) Google Scholar). Our observations are in agreement with these observations. These results indicate that the 7-bp cis-acting element plays a central role in the chondrocyte-specific enhancer activity and strongly suggest that the Sox9 protein is a key mediator for the transcription ofCol11a2 in chondrocyte. The 60-bp sequence was able to direct expression of the −453 promoter of Col11a2 in the cartilage of transgenic mice. The expression patterns of the reporter β-galactosidase gene were similar to those with the construct containing either the 300-bp or the 2.3-kb intron segment (15.Tsumaki N. Kimura T. Matsui Y. Nakata K. Ochi T. J. Cell Biol. 1996; 134: 1573-1582Crossref PubMed Scopus (68) Google Scholar). 2Y. Liu, H. Li, K. Tanaka, N. Tsumaki, and Y. Yamada, unpublished data. A 7-bp substitution mutation in the 60-bp sequence eliminated the enhancer activity, suggesting that this 7-bp sequence is critical for tissue-specific activity, which is in agreement with the transfection analysis. Interestingly, the 60-bp enhancer-containing construct failed to direct β-galactosidase expression in the notochord (data not shown). Because the 2.3-kb intron segment is necessary for notochord-specific expression of Col11a2, the 60-bp sequence likely lacks the information necessary for expression in the notochord. Thus, Co1l1a2 is positively regulated by at least two distinct elements, one for cartilage and another for notochord in the first intron. Col11a2 contains redundant Sox9 sites. Each site may have unique activity in different tissues, and such redundancy may be necessary for the optimum expression of Col11a2 to form collagen fibrils specific to each type of cartilage. Differences in the activity of these sites observed in transfected RCS cells may not be the only regulatory mechanisms of Col11a2 expression in cartilage. Recently, it was reported that sequences adjacent to the Sox9 site are also required for the chondrocyte-specific enhancer activity of Col2a1 and that new members of the Sox family,l-Sox5 and Sox6, form a heterodimer and activate theCol2a1 enhancer co-operatively with Sox9 (34.Lefebvre V. Li P. de Crombrugghe B. EMBO J. 1998; 17: 5718-5733Crossref PubMed Scopus (670) Google Scholar). It is conceivable that protein factors, such as l-Sox5 and Sox6, may also be involved in Col11a2 expression. Levels of Sox9 and these proteins may differ in each cartilage and have preferential utilization of the multi-modular elements containing the Sox9 site forCol11a2 expression. We thank H. Kleinman (NIDCR) for critical reading of the manuscript and N. Iehara for valuable suggestions. We also thank Andrew Cho (NIDCR Gene Targeting Core Facility) for generating transgenic mice.