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A Combination of cis-Acting Elements Is Required to Activate the Pro-α1(I) Collagen Promoter in Tendon Fibroblasts of Transgenic Mice

2002; Elsevier BV; Volume: 277; Issue: 21 Linguagem: Inglês

10.1074/jbc.m200125200

1083-351X

Catherine Terraz, Gaëlle Brideau, Pierre Ronco, Jérôme Rossert,

Dupuytren's Contracture and Treatments

The genes encoding the two type I collagen chains are selectively activated in few cell types, including fibroblasts and osteoblasts. By generating transgenic mice, we have previously shown that the activity of the mouse pro-α1(I) promoter was controlled by separate cell-specific cis-acting elements. In particular, a sequence located between −3.2 and −2.3 kb was needed to induce expression of the reporter gene at high levels in tendon fibroblasts. In the present work, by using the same transgenic approach, we have identified two short elements in this sequence, named tendon-specific element (TSE) 1 and TSE2, that were necessary to direct reporter gene expression selectively in tendon fibroblasts. Gel shift assays showed that TSE1 and TSE2 bound proteins specifically present in nuclear extracts from tendon fibroblasts and that the sequence of TSE2 binding a tendon-specific protein corresponded to an E-box. Analysis of transgenic mice further indicated that TSE1 and TSE2 needed to cooperate not only with each other but also with other cis-acting elements of the proximal promoter to activate reporter gene expression in tendon fibroblasts. Similarly, it pointed out that the so-called osteoblast-specific element had to interact with downstream sequences to drive reporter gene expression in osteoblasts of transgenic mice. Thus, expression of the mouse pro-α1(I) collagen gene in tendon fibroblasts appears to be the result of a unique combination of different cis-acting elements, including TSE1 and TSE2. The genes encoding the two type I collagen chains are selectively activated in few cell types, including fibroblasts and osteoblasts. By generating transgenic mice, we have previously shown that the activity of the mouse pro-α1(I) promoter was controlled by separate cell-specific cis-acting elements. In particular, a sequence located between −3.2 and −2.3 kb was needed to induce expression of the reporter gene at high levels in tendon fibroblasts. In the present work, by using the same transgenic approach, we have identified two short elements in this sequence, named tendon-specific element (TSE) 1 and TSE2, that were necessary to direct reporter gene expression selectively in tendon fibroblasts. Gel shift assays showed that TSE1 and TSE2 bound proteins specifically present in nuclear extracts from tendon fibroblasts and that the sequence of TSE2 binding a tendon-specific protein corresponded to an E-box. Analysis of transgenic mice further indicated that TSE1 and TSE2 needed to cooperate not only with each other but also with other cis-acting elements of the proximal promoter to activate reporter gene expression in tendon fibroblasts. Similarly, it pointed out that the so-called osteoblast-specific element had to interact with downstream sequences to drive reporter gene expression in osteoblasts of transgenic mice. Thus, expression of the mouse pro-α1(I) collagen gene in tendon fibroblasts appears to be the result of a unique combination of different cis-acting elements, including TSE1 and TSE2. Type I collagen is a fibrillar collagen composed of two α1 chains and one α2 chain coiled around each other in a triple helix. It is a major protein of mammalian bodies and an essential component of most extracellular matrices. In the extracellular space, type I collagen molecules self-assemble into highly organized fibrils and then into fibers, which largely contribute to the high tensile strength of the framework supporting body structures (reviewed in Ref.1Van der Rest M. Garrone R. FASEB J. 1991; 5: 2814-2823Crossref PubMed Scopus (969) Google Scholar). Evidence for a role of type I collagen in providing mechanical strength to tissues comes from pathophysiological analyses of genetic diseases resulting from mutations in one of the genes encoding type I collagen, such as osteogenesis imperfecta and Ehlers-Danlos syndromes type VIIA and VIIB (2Kuivaniemi H. Tromp G. Prockop D.J. Hum. Mutat. 1997; 9: 300-315Crossref PubMed Scopus (280) Google Scholar). The hallmark of osteogenesis imperfecta is brittle bones, but the disease can also involve other tissues rich in type I collagen, such as ligaments, tendons, fascia, sclerae, and teeth (3Prockop D.J. Kivirikko K.I. N. Engl. J. Med. 1984; 311: 376-386Crossref PubMed Scopus (388) Google Scholar). Ehlers-Danlos syndromes are characterized by skin hyperextensibility, vascular fragility, and increased ligament elasticity responsible for joint hypermobility (4Byers P.H. Duvic M. Atkinson M. Robinow M. Smith L.T. Krane S.M. Greally M.T. Ludman M. Matalon R. Pauker S. et al.Am. J. Med. Genet. 1997; 72: 94-105Crossref PubMed Scopus (93) Google Scholar).Type I collagen is synthesized by a discrete subset of cells of mesenchymal origin, which contrasts with its wide distribution throughout the body. These cells are mostly fibroblasts, osteoblasts, and odontoblasts. Studies using transgenic mice harboring various fragments of the mouse and rat pro-α1(I) collagen promoters have shown that a modular arrangement of separate cell-specific cis-acting elements was responsible for the expression of the pro-α1(I) collagen gene in different subsets of type I collagen-producing cells (reviewed in Ref. 5Rossert J. de Combrugghe B. Principles of Bone Biology. Academic Press, San Diego, CA2002Google Scholar). Analyses of transgenic mice showed that the mouse pro-α1(I) promoter contained at least three different cell-specific regulatory elements. An element located within 900 bp of the proximal promoter induced lacZ and luciferase reporter gene expression in some skin fibroblasts. A second element located between −2.3 and −0.9 kb conferred high-level expression of these two reporter genes in osteoblasts and odontoblasts. A third element located between −3.2 and −2.3 kb was responsible for high-level expression of the lacZ gene in tendon and fascia fibroblasts (6Rossert J. Eberspaecher H. de Crombrugghe B. J. Cell Biol. 1995; 129: 1421-1432Crossref PubMed Scopus (209) Google Scholar). Similarly, in the rat pro-α1(I) promoter, a 13-bp sequence located between −1683 and −1670 bp was necessary to drive chloramphenicol acetyltransferase reporter gene expression in bones, whereas an upstream sequence, located between −1.7 and −3.5 kb, was required to obtain high-level expression of the reporter gene in tendon fibroblasts (7Dodig M. Kronenberg M.S. Bedalov A. Kream B.E. Gronowicz G. Clark S.H. Mack K. Liu Y.H. Maxon R. Pan Z.Z. et al.J. Biol. Chem. 1996; 271: 16422-16429Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 8Bogdanovic Z. Bedalov A. Krebsbach P.H. Pavlin D. Woody C.O. Clark S.H. Thomas H.F. Rowe D.W. Kream B.E. Lichtler A.C. J. Bone Miner. Res. 1994; 9: 285-292Crossref PubMed Scopus (91) Google Scholar). The human pro-α1(I) collagen promoter probably displays such a modular organization because the first 2.3 kb of this promoter induced growth hormone reporter gene expression in bones, tendons, and fascia of transgenic mice, but not in other type I collagen-containing tissues, such as perichondrium and skeletal muscles (9Liska D.J. Reed M.J. Sage E.H. Bornstein P. J. Cell Biol. 1994; 125: 695-704Crossref PubMed Scopus (59) Google Scholar, 10Slack J.L. Liska D.J. Bornstein P. Mol. Cell. Biol. 1991; 11: 2066-2074Crossref PubMed Google Scholar).Analysis of the rat and mouse pro-α1(I) promoters led to the precise identification of an osteoblast-specific element (7Dodig M. Kronenberg M.S. Bedalov A. Kream B.E. Gronowicz G. Clark S.H. Mack K. Liu Y.H. Maxon R. Pan Z.Z. et al.J. Biol. Chem. 1996; 271: 16422-16429Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 11Rossert J.A. Chen S.S. Eberspaecher H. Smith C.N. de Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1027-1031Crossref PubMed Scopus (90) Google Scholar). Introduction of short mutations or deletions in this cis-acting element abolished the expression of the reporter gene in osteoblastic cells of transgenic mice (7Dodig M. Kronenberg M.S. Bedalov A. Kream B.E. Gronowicz G. Clark S.H. Mack K. Liu Y.H. Maxon R. Pan Z.Z. et al.J. Biol. Chem. 1996; 271: 16422-16429Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), whereas transgenic mice harboring the mouse osteoblast-specific element multimerized four times and cloned upstream of a minimal promoter and the lacZ gene displayed X-gal 1The abbreviations used are: X-gal5-bromo-4-chloro-3-indolyl-β-d-galactopyranosideTSEtendon-specific element 1The abbreviations used are: X-gal5-bromo-4-chloro-3-indolyl-β-d-galactopyranosideTSEtendon-specific element staining selectively in osteoblasts (11Rossert J.A. Chen S.S. Eberspaecher H. Smith C.N. de Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1027-1031Crossref PubMed Scopus (90) Google Scholar). In contrast, no fibroblast-specific element has been precisely identified thus far in either the pro-α1(I) or pro-α2(I) collagen genes (reviewed in Ref. 5Rossert J. de Combrugghe B. Principles of Bone Biology. Academic Press, San Diego, CA2002Google Scholar).We report the identification of two short cis-acting sequences of the mouse pro-α1(I) promoter that are necessary for reporter gene expression at high levels specifically in tendon fibroblasts of transgenic embryos and bind nuclear proteins selectively present in tendon fibroblasts. We also show that these two elements, as well as the osteoblast-specific element, need to cooperate with other elements of the proximal promoter to drive expression of the lacZ reporter gene in tendons and ossification centers, respectively. This suggests that type I collagen expression in tendon fibroblasts and osteoblasts is finely tuned by unique combinations of cis-acting elements and complex interactions between trans-acting factors.DISCUSSIONMolecular mechanisms governing type I collagen gene expression are still quite elusive; in particular, the cis-acting elements that induce expression of the pro-α1(I) collagen gene in fibroblastic cells are unknown (reviewed in Ref. 5Rossert J. de Combrugghe B. Principles of Bone Biology. Academic Press, San Diego, CA2002Google Scholar). Previous studies performed using transgenic mice harboring various segments of the mouse pro-α1(I) promoter have shown that the −3.2 to −2.3 kb segment contains cis-acting elements that are necessary to induce high-level expression of the lacZ reporter gene in tendon fibroblasts (6Rossert J. Eberspaecher H. de Crombrugghe B. J. Cell Biol. 1995; 129: 1421-1432Crossref PubMed Scopus (209) Google Scholar). To identify these cis-acting elements, we have generated transgenic embryos harboring different subsegments of the −3.2 to −2.3 kb fragment cloned upstream of a 2.3-kb segment of the pro-α1(I) proximal promoter and the lacZ reporter gene. The lacZ gene allows precise identification of the cell types in which the transgene is active. The use of the 2.3-kb segment of the pro-α1(I) proximal promoter permits easy identification of embryos that express the lacZ gene because this sequence induces high-level expression of the reporter gene in ossification centers (6Rossert J. Eberspaecher H. de Crombrugghe B. J. Cell Biol. 1995; 129: 1421-1432Crossref PubMed Scopus (209) Google Scholar). Foster mothers were sacrificed at 15.5 days of gestation because at this time the expression of the lacZ gene can be easily detected in ossification centers and developing tendons (6Rossert J. Eberspaecher H. de Crombrugghe B. J. Cell Biol. 1995; 129: 1421-1432Crossref PubMed Scopus (209) Google Scholar). At least three embryos expressing the lacZ gene were obtained for each construct to eliminate a site of integration effect. Our deletion analysis demonstrated that two short cis-acting elements located between −3.2 and −2.3 kb were necessary to induce high-level expression of the reporter gene specifically in tendon fibroblasts of transgenic mice. These elements, which we have named TSE1 and TSE2, are located about 2.8 and 2.3 kb upstream of the transcription start site, respectively. Mouse embryos harboring a transgene containing both TSE1 and TSE2 cloned upstream of a 2.3-kb segment of the pro-α1(I) proximal promoter consistently co-expressed the reporter gene in tendon fibroblasts and ossification centers. In contrast, embryos harboring a transgene containing either TSE1 alone or TSE2 alone, cloned upstream of 2.3 kb of the pro-α1(I) proximal promoter, expressed the reporter gene in ossification centers at high levels, but no staining of tendons could be detected in whole-mount embryos or in histological sections. Comparison of the sequences of TSE1 and TSE2 and the sequences of the rat, bovine, and human pro-α1(I) promoters showed that the most 3′ part of TSE1 and the whole TSE2 have highly similar counterparts, as seen for the osteoblast-specific element (11Rossert J.A. Chen S.S. Eberspaecher H. Smith C.N. de Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1027-1031Crossref PubMed Scopus (90) Google Scholar). Gel shift experiments showed that both TSE1 and TSE2 can bind proteins that are present in nuclear extracts from tendon fibroblasts, but not proteins from other type I collagen-producing cells or from epithelial cells. Competition experiments performed using double-stranded oligonucleotides harboring 5-bp deletions within the TSE2 sequence showed that the tendon-specific nuclear proteins binding TSE2 actually bound a consensus E-box (CACGTG) located at −2325 bp. E-boxes are known to bind basic helix-loop-helix transcription factors, and it is very tempting to speculate that the E-box located within TSE2 binds a basic helix-loop-helix transcription factor named scleraxis. Schweitzer et al. (19Schweitzer R. Chyung J. Murtaugh L. Brent A. Rose V. Olsen E. Lassar A. Tabin C. Development. 2001; 126: 3855-3866Google Scholar) have recently shown that in chick embryos, the expression of scleraxis becomes rapidly specific to the developing tendons and ligaments and that in the developing mouse limb, scleraxis transcripts are selectively found in tendons and their progenitors. Furthermore, they have also shown that induction of excess of scleraxis-positive mesenchymal cells did not result in the production of extra tendons (19Schweitzer R. Chyung J. Murtaugh L. Brent A. Rose V. Olsen E. Lassar A. Tabin C. Development. 2001; 126: 3855-3866Google Scholar). This last finding is fully consistent with the fact that activation of the pro-α1(I) proximal promoter in tendon fibroblasts required the presence of TSE2 and also of TSE1. Analysis of TSE1 using electrophoretic mobility shift assays in the presence of competitors also led to the identification of a short sequence containing a GAACT motif that bound a tendon-specific nuclear protein. Computer analysis of this sequence did not enable us to identify a potential DNA-binding protein that could interact with it. Nevertheless, the role of this sequence has been confirmed by generating transgenic harboring an 18-bp deletion that encompasses the GAACT motif within TSE1. Whereas E15.5 transgenic embryos expressed the lacZ reporter gene at high levels in ossification centers, no X-gal staining could be detected in tendons.Interestingly, when TSE1 was multimerized four times and cloned upstream of four copies of the osteoblast-specific element and 220 pb of the pro-α1(I) proximal promoter, the lacZ reporter gene was expressed in ossification centers but not in tendons, and no staining of tendon fibroblasts could be detected by histological analysis. This shows that unlike the osteoblast-specific element, multimerization of TSE1 cannot overcome the absence of other cis-acting elements (11Rossert J.A. Chen S.S. Eberspaecher H. Smith C.N. de Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1027-1031Crossref PubMed Scopus (90) Google Scholar). Moreover, generation of transgenic mice harboring a deletion between −1537 and −220 bp (i.e. between the osteoblast-specific element and the minimal promoter) showed that TSE1 and TSE2 need to cooperate with elements located within this sequence to induce expression of the lacZ gene in tendon fibroblasts of transgenic mice. Similarly, the absence of staining of ossification centers in embryos harboring this deleted construct showed that the osteoblast-specific element also needs to cooperate with downstream elements. The need for such cooperativity had previously been masked by the systematic usage of four copies of the osteoblast-specific element (11Rossert J.A. Chen S.S. Eberspaecher H. Smith C.N. de Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1027-1031Crossref PubMed Scopus (90) Google Scholar).The existence of cis-acting elements located between −1537 and −220 bp that are able to induce expression of reporter genes in tendons and ossification centers is consistent with data obtained from other groups with the human, rat, and mouse pro-α1(I) promoters (9Liska D.J. Reed M.J. Sage E.H. Bornstein P. J. Cell Biol. 1994; 125: 695-704Crossref PubMed Scopus (59) Google Scholar,10Slack J.L. Liska D.J. Bornstein P. Mol. Cell. Biol. 1991; 11: 2066-2074Crossref PubMed Google Scholar, 18Houglum K. Buck M. Alcorn J. Contreras S. Bornstein P. Chojkier M. J. Clin. Invest. 1995; 96: 2269-2276Crossref PubMed Scopus (47) Google Scholar, 20Bedalov A. Salvatori R. Dodig M. Kronenberg M. Kapural B. Bogdanovic Z. Kream B. Woody C. Clark S. Mack K. Rowe D. Lichtler A. J Bone Miner. Res. 1995; 10: 1443-1451Crossref PubMed Scopus (42) Google Scholar, 21Kern B. Shen J. Starbuck M. Karsenty G. J. Biol. Chem. 2001; 276: 7101-7107Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Slack et al. (10Slack J.L. Liska D.J. Bornstein P. Mol. Cell. Biol. 1991; 11: 2066-2074Crossref PubMed Google Scholar) and Liska et al. (9Liska D.J. Reed M.J. Sage E.H. Bornstein P. J. Cell Biol. 1994; 125: 695-704Crossref PubMed Scopus (59) Google Scholar) have shown that a transgene containing 2.3 kb of the human pro-α1(I) collagen promoter cloned upstream of the growth hormone gene drove expression of this reporter gene in ossification centers and also in tendons. Bedalov et al. (20Bedalov A. Salvatori R. Dodig M. Kronenberg M. Kapural B. Bogdanovic Z. Kream B. Woody C. Clark S. Mack K. Rowe D. Lichtler A. J Bone Miner. Res. 1995; 10: 1443-1451Crossref PubMed Scopus (42) Google Scholar) have shown that a sequence of the rat promoter located between −1670 and −944 is responsible for reporter gene expression in tendons at low levels. Similarly, Houglum et al. (18Houglum K. Buck M. Alcorn J. Contreras S. Bornstein P. Chojkier M. J. Clin. Invest. 1995; 96: 2269-2276Crossref PubMed Scopus (47) Google Scholar) have shown that transgenic mice harboring a construct containing 1626 bp of the mouse pro-α1(I) collagen promoter cloned upstream of lacZ expressed the reporter gene in tendons at low levels. More recently, Kern et al. (21Kern B. Shen J. Starbuck M. Karsenty G. J. Biol. Chem. 2001; 276: 7101-7107Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar) have shown the existence of a second osteoblast-specific element in the pro-α1(I) promoter. They showed that four copies of a sequence of the mouse pro-α1(I) collagen promoter extending from −1347 to −1338 bp cloned upstream of 220 bp of the pro-α1(I) proximal promoter drove expression of the luciferase reporter gene in developing bones (21Kern B. Shen J. Starbuck M. Karsenty G. J. Biol. Chem. 2001; 276: 7101-7107Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Thus, expression of the lacZ gene in tendon fibroblasts and also in ossification centers appears to be the result of a unique combination of different cis-acting elements. Whereas some of these elements bind tissue-specific transcription factors, others may bind factors that are ubiquitously expressed or expressed in both osteoblasts and tendon fibroblasts.Tissue-specific expression induced by a unique combination of different transcription factors has already been demonstrated for different genes, such as the albumin gene, the fibroblast growth factor 4 gene, and the atrial natriuretic peptide gene (22Gualdi R. Bossard P. Zheng M. Hamada Y. Coleman J.R. Zaret K.S. Genes Dev. 1996; 10: 1670-1682Crossref PubMed Scopus (462) Google Scholar, 23Ambrosetti D.C. Schöler H. Dailey L. Basilico C. J. Biol. Chem. 2000; 275: 23387-23397Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 24Shiojima I. Komuro I. Oka T. Hiroi Y. Mizuno T. Takimoto E. Monzen K. Aikawa R. Akazawa H. Yamazaki T. et al.J. Biol. Chem. 1999; 274: 8231-8239Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). For example, the albumin gene requires the recruitment by hepatocyte nuclear factor-3α of several cis-activators and their corresponding DNA-binding proteins to be expressed in the liver (22Gualdi R. Bossard P. Zheng M. Hamada Y. Coleman J.R. Zaret K.S. Genes Dev. 1996; 10: 1670-1682Crossref PubMed Scopus (462) Google Scholar). Similarly, Sox2 and Oct3 bind to adjacent sites and synergistically drive fibroblast growth factor-4 gene expression (23Ambrosetti D.C. Schöler H. Dailey L. Basilico C. J. Biol. Chem. 2000; 275: 23387-23397Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Finally, the transcription factor Nkx-2.5 recruits other DNA-binding proteins such as GATA-4 by direct protein-protein interactions to activate the atrial natriuretic peptide gene (24Shiojima I. Komuro I. Oka T. Hiroi Y. Mizuno T. Takimoto E. Monzen K. Aikawa R. Akazawa H. Yamazaki T. et al.J. Biol. Chem. 1999; 274: 8231-8239Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Similarly, type I collagen expression in fibroblasts and osteoblasts may also be the result of finely tuned regulation under the control of unique combinations of cis-acting elements and complex interactions between trans-acting factors. We speculate that their identification might help to better define the molecular mechanisms responsible for the differentiation of mesenchymal cells into fibroblasts. Type I collagen is a fibrillar collagen composed of two α1 chains and one α2 chain coiled around each other in a triple helix. It is a major protein of mammalian bodies and an essential component of most extracellular matrices. In the extracellular space, type I collagen molecules self-assemble into highly organized fibrils and then into fibers, which largely contribute to the high tensile strength of the framework supporting body structures (reviewed in Ref.1Van der Rest M. Garrone R. FASEB J. 1991; 5: 2814-2823Crossref PubMed Scopus (969) Google Scholar). Evidence for a role of type I collagen in providing mechanical strength to tissues comes from pathophysiological analyses of genetic diseases resulting from mutations in one of the genes encoding type I collagen, such as osteogenesis imperfecta and Ehlers-Danlos syndromes type VIIA and VIIB (2Kuivaniemi H. Tromp G. Prockop D.J. Hum. Mutat. 1997; 9: 300-315Crossref PubMed Scopus (280) Google Scholar). The hallmark of osteogenesis imperfecta is brittle bones, but the disease can also involve other tissues rich in type I collagen, such as ligaments, tendons, fascia, sclerae, and teeth (3Prockop D.J. Kivirikko K.I. N. Engl. J. Med. 1984; 311: 376-386Crossref PubMed Scopus (388) Google Scholar). Ehlers-Danlos syndromes are characterized by skin hyperextensibility, vascular fragility, and increased ligament elasticity responsible for joint hypermobility (4Byers P.H. Duvic M. Atkinson M. Robinow M. Smith L.T. Krane S.M. Greally M.T. Ludman M. Matalon R. Pauker S. et al.Am. J. Med. Genet. 1997; 72: 94-105Crossref PubMed Scopus (93) Google Scholar). Type I collagen is synthesized by a discrete subset of cells of mesenchymal origin, which contrasts with its wide distribution throughout the body. These cells are mostly fibroblasts, osteoblasts, and odontoblasts. Studies using transgenic mice harboring various fragments of the mouse and rat pro-α1(I) collagen promoters have shown that a modular arrangement of separate cell-specific cis-acting elements was responsible for the expression of the pro-α1(I) collagen gene in different subsets of type I collagen-producing cells (reviewed in Ref. 5Rossert J. de Combrugghe B. Principles of Bone Biology. Academic Press, San Diego, CA2002Google Scholar). Analyses of transgenic mice showed that the mouse pro-α1(I) promoter contained at least three different cell-specific regulatory elements. An element located within 900 bp of the proximal promoter induced lacZ and luciferase reporter gene expression in some skin fibroblasts. A second element located between −2.3 and −0.9 kb conferred high-level expression of these two reporter genes in osteoblasts and odontoblasts. A third element located between −3.2 and −2.3 kb was responsible for high-level expression of the lacZ gene in tendon and fascia fibroblasts (6Rossert J. Eberspaecher H. de Crombrugghe B. J. Cell Biol. 1995; 129: 1421-1432Crossref PubMed Scopus (209) Google Scholar). Similarly, in the rat pro-α1(I) promoter, a 13-bp sequence located between −1683 and −1670 bp was necessary to drive chloramphenicol acetyltransferase reporter gene expression in bones, whereas an upstream sequence, located between −1.7 and −3.5 kb, was required to obtain high-level expression of the reporter gene in tendon fibroblasts (7Dodig M. Kronenberg M.S. Bedalov A. Kream B.E. Gronowicz G. Clark S.H. Mack K. Liu Y.H. Maxon R. Pan Z.Z. et al.J. Biol. Chem. 1996; 271: 16422-16429Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 8Bogdanovic Z. Bedalov A. Krebsbach P.H. Pavlin D. Woody C.O. Clark S.H. Thomas H.F. Rowe D.W. Kream B.E. Lichtler A.C. J. Bone Miner. Res. 1994; 9: 285-292Crossref PubMed Scopus (91) Google Scholar). The human pro-α1(I) collagen promoter probably displays such a modular organization because the first 2.3 kb of this promoter induced growth hormone reporter gene expression in bones, tendons, and fascia of transgenic mice, but not in other type I collagen-containing tissues, such as perichondrium and skeletal muscles (9Liska D.J. Reed M.J. Sage E.H. Bornstein P. J. Cell Biol. 1994; 125: 695-704Crossref PubMed Scopus (59) Google Scholar, 10Slack J.L. Liska D.J. Bornstein P. Mol. Cell. Biol. 1991; 11: 2066-2074Crossref PubMed Google Scholar). Analysis of the rat and mouse pro-α1(I) promoters led to the precise identification of an osteoblast-specific element (7Dodig M. Kronenberg M.S. Bedalov A. Kream B.E. Gronowicz G. Clark S.H. Mack K. Liu Y.H. Maxon R. Pan Z.Z. et al.J. Biol. Chem. 1996; 271: 16422-16429Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 11Rossert J.A. Chen S.S. Eberspaecher H. Smith C.N. de Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1027-1031Crossref PubMed Scopus (90) Google Scholar). Introduction of short mutations or deletions in this cis-acting element abolished the expression of the reporter gene in osteoblastic cells of transgenic mice (7Dodig M. Kronenberg M.S. Bedalov A. Kream B.E. Gronowicz G. Clark S.H. Mack K. Liu Y.H. Maxon R. Pan Z.Z. et al.J. Biol. Chem. 1996; 271: 16422-16429Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), whereas transgenic mice harboring the mouse osteoblast-specific element multimerized four times and cloned upstream of a minimal promoter and the lacZ gene displayed X-gal 1The abbreviations used are: X-gal5-bromo-4-chloro-3-indolyl-β-d-galactopyranosideTSEtendon-specific element 1The abbreviations used are: X-gal5-bromo-4-chloro-3-indolyl-β-d-galactopyranosideTSEtendon-specific element staining selectively in osteoblasts (11Rossert J.A. Chen S.S. Eberspaecher H. Smith C.N. de Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1027-1031Crossref PubMed Scopus (90) Google Scholar). In contrast, no fibroblast-specific element has been precisely identified thus far in either the pro-α1(I) or pro-α2(I) collagen genes (reviewed in Ref. 5Rossert J. de Combrugghe B. Principles of Bone Biology. Academic Press, San Diego, CA2002Google Scholar). 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside tendon-specific element 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside tendon-specific element We report the identification of two short cis-acting sequences of the mouse pro-α1(I) promoter that are necessary for reporter gene expression at high levels specifically in tendon fibroblasts of transgenic embryos and bind nuclear proteins selectively present in tendon fibroblasts. We also show that these two elements, as well as the osteoblast-specific element, need to cooperate with other elements of the proximal promoter to drive expression of the lacZ reporter gene in tendons and ossification centers, respectively. This suggests that type I collagen expression in tendon fibroblasts and osteoblasts is finely tuned by unique combinations of cis-acting elements and complex interactions between trans-acting factors. DISCUSSIONMolecular mechanisms governing type I collagen gene expression are still quite elusive; in particular, the cis-acting elements that induce expression of the pro-α1(I) collagen gene in fibroblastic cells are unknown (reviewed in Ref. 5Rossert J. de Combrugghe B. Principles of Bone Biology. Academic Press, San Diego, CA2002Google Scholar). Previous studies performed using transgenic mice harboring various segments of the mouse pro-α1(I) promoter have shown that the −3.2 to −2.3 kb segment contains cis-acting elements that are necessary to induce high-level expression of the lacZ reporter gene in tendon fibroblasts (6Rossert J. Eberspaecher H. de Crombrugghe B. J. Cell Biol. 1995; 129: 1421-1432Crossref PubMed Scopus (209) Google Scholar). To identify these cis-acting elements, we have generated transgenic embryos harboring different subsegments of the −3.2 to −2.3 kb fragment cloned upstream of a 2.3-kb segment of the pro-α1(I) proximal promoter and the lacZ reporter gene. The lacZ gene allows precise identification of the cell types in which the transgene is active. The use of the 2.3-kb segment of the pro-α1(I) proximal promoter permits easy identification of embryos that express the lacZ gene because this sequence induces high-level expression of the reporter gene in ossification centers (6Rossert J. Eberspaecher H. de Crombrugghe B. J. Cell Biol. 1995; 129: 1421-1432Crossref PubMed Scopus (209) Google Scholar). Foster mothers were sacrificed at 15.5 days of gestation because at this time the expression of the lacZ gene can be easily detected in ossification centers and developing tendons (6Rossert J. Eberspaecher H. de Crombrugghe B. J. Cell Biol. 1995; 129: 1421-1432Crossref PubMed Scopus (209) Google Scholar). At least three embryos expressing the lacZ gene were obtained for each construct to eliminate a site of integration effect. Our deletion analysis demonstrated that two short cis-acting elements located between −3.2 and −2.3 kb were necessary to induce high-level expression of the reporter gene specifically in tendon fibroblasts of transgenic mice. These elements, which we have named TSE1 and TSE2, are located about 2.8 and 2.3 kb upstream of the transcription start site, respectively. Mouse embryos harboring a transgene containing both TSE1 and TSE2 cloned upstream of a 2.3-kb segment of the pro-α1(I) proximal promoter consistently co-expressed the reporter gene in tendon fibroblasts and ossification centers. In contrast, embryos harboring a transgene containing either TSE1 alone or TSE2 alone, cloned upstream of 2.3 kb of the pro-α1(I) proximal promoter, expressed the reporter gene in ossification centers at high levels, but no staining of tendons could be detected in whole-mount embryos or in histological sections. Comparison of the sequences of TSE1 and TSE2 and the sequences of the rat, bovine, and human pro-α1(I) promoters showed that the most 3′ part of TSE1 and the whole TSE2 have highly similar counterparts, as seen for the osteoblast-specific element (11Rossert J.A. Chen S.S. Eberspaecher H. Smith C.N. de Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1027-1031Crossref PubMed Scopus (90) Google Scholar). Gel shift experiments showed that both TSE1 and TSE2 can bind proteins that are present in nuclear extracts from tendon fibroblasts, but not proteins from other type I collagen-producing cells or from epithelial cells. Competition experiments performed using double-stranded oligonucleotides harboring 5-bp deletions within the TSE2 sequence showed that the tendon-specific nuclear proteins binding TSE2 actually bound a consensus E-box (CACGTG) located at −2325 bp. E-boxes are known to bind basic helix-loop-helix transcription factors, and it is very tempting to speculate that the E-box located within TSE2 binds a basic helix-loop-helix transcription factor named scleraxis. Schweitzer et al. (19Schweitzer R. Chyung J. Murtaugh L. Brent A. Rose V. Olsen E. Lassar A. Tabin C. Development. 2001; 126: 3855-3866Google Scholar) have recently shown that in chick embryos, the expression of scleraxis becomes rapidly specific to the developing tendons and ligaments and that in the developing mouse limb, scleraxis transcripts are selectively found in tendons and their progenitors. Furthermore, they have also shown that induction of excess of scleraxis-positive mesenchymal cells did not result in the production of extra tendons (19Schweitzer R. Chyung J. Murtaugh L. Brent A. Rose V. Olsen E. Lassar A. Tabin C. Development. 2001; 126: 3855-3866Google Scholar). This last finding is fully consistent with the fact that activation of the pro-α1(I) proximal promoter in tendon fibroblasts required the presence of TSE2 and also of TSE1. Analysis of TSE1 using electrophoretic mobility shift assays in the presence of competitors also led to the identification of a short sequence containing a GAACT motif that bound a tendon-specific nuclear protein. Computer analysis of this sequence did not enable us to identify a potential DNA-binding protein that could interact with it. Nevertheless, the role of this sequence has been confirmed by generating transgenic harboring an 18-bp deletion that encompasses the GAACT motif within TSE1. Whereas E15.5 transgenic embryos expressed the lacZ reporter gene at high levels in ossification centers, no X-gal staining could be detected in tendons.Interestingly, when TSE1 was multimerized four times and cloned upstream of four copies of the osteoblast-specific element and 220 pb of the pro-α1(I) proximal promoter, the lacZ reporter gene was expressed in ossification centers but not in tendons, and no staining of tendon fibroblasts could be detected by histological analysis. This shows that unlike the osteoblast-specific element, multimerization of TSE1 cannot overcome the absence of other cis-acting elements (11Rossert J.A. Chen S.S. Eberspaecher H. Smith C.N. de Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1027-1031Crossref PubMed Scopus (90) Google Scholar). Moreover, generation of transgenic mice harboring a deletion between −1537 and −220 bp (i.e. between the osteoblast-specific element and the minimal promoter) showed that TSE1 and TSE2 need to cooperate with elements located within this sequence to induce expression of the lacZ gene in tendon fibroblasts of transgenic mice. Similarly, the absence of staining of ossification centers in embryos harboring this deleted construct showed that the osteoblast-specific element also needs to cooperate with downstream elements. The need for such cooperativity had previously been masked by the systematic usage of four copies of the osteoblast-specific element (11Rossert J.A. Chen S.S. Eberspaecher H. Smith C.N. de Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1027-1031Crossref PubMed Scopus (90) Google Scholar).The existence of cis-acting elements located between −1537 and −220 bp that are able to induce expression of reporter genes in tendons and ossification centers is consistent with data obtained from other groups with the human, rat, and mouse pro-α1(I) promoters (9Liska D.J. Reed M.J. Sage E.H. Bornstein P. J. Cell Biol. 1994; 125: 695-704Crossref PubMed Scopus (59) Google Scholar,10Slack J.L. Liska D.J. Bornstein P. Mol. Cell. Biol. 1991; 11: 2066-2074Crossref PubMed Google Scholar, 18Houglum K. Buck M. Alcorn J. Contreras S. Bornstein P. Chojkier M. J. Clin. Invest. 1995; 96: 2269-2276Crossref PubMed Scopus (47) Google Scholar, 20Bedalov A. Salvatori R. Dodig M. Kronenberg M. Kapural B. Bogdanovic Z. Kream B. Woody C. Clark S. Mack K. Rowe D. Lichtler A. J Bone Miner. Res. 1995; 10: 1443-1451Crossref PubMed Scopus (42) Google Scholar, 21Kern B. Shen J. Starbuck M. Karsenty G. J. Biol. Chem. 2001; 276: 7101-7107Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Slack et al. (10Slack J.L. Liska D.J. Bornstein P. Mol. Cell. Biol. 1991; 11: 2066-2074Crossref PubMed Google Scholar) and Liska et al. (9Liska D.J. Reed M.J. Sage E.H. Bornstein P. J. Cell Biol. 1994; 125: 695-704Crossref PubMed Scopus (59) Google Scholar) have shown that a transgene containing 2.3 kb of the human pro-α1(I) collagen promoter cloned upstream of the growth hormone gene drove expression of this reporter gene in ossification centers and also in tendons. Bedalov et al. (20Bedalov A. Salvatori R. Dodig M. Kronenberg M. Kapural B. Bogdanovic Z. Kream B. Woody C. Clark S. Mack K. Rowe D. Lichtler A. J Bone Miner. Res. 1995; 10: 1443-1451Crossref PubMed Scopus (42) Google Scholar) have shown that a sequence of the rat promoter located between −1670 and −944 is responsible for reporter gene expression in tendons at low levels. Similarly, Houglum et al. (18Houglum K. Buck M. Alcorn J. Contreras S. Bornstein P. Chojkier M. J. Clin. Invest. 1995; 96: 2269-2276Crossref PubMed Scopus (47) Google Scholar) have shown that transgenic mice harboring a construct containing 1626 bp of the mouse pro-α1(I) collagen promoter cloned upstream of lacZ expressed the reporter gene in tendons at low levels. More recently, Kern et al. (21Kern B. Shen J. Starbuck M. Karsenty G. J. Biol. Chem. 2001; 276: 7101-7107Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar) have shown the existence of a second osteoblast-specific element in the pro-α1(I) promoter. They showed that four copies of a sequence of the mouse pro-α1(I) collagen promoter extending from −1347 to −1338 bp cloned upstream of 220 bp of the pro-α1(I) proximal promoter drove expression of the luciferase reporter gene in developing bones (21Kern B. Shen J. Starbuck M. Karsenty G. J. Biol. Chem. 2001; 276: 7101-7107Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Thus, expression of the lacZ gene in tendon fibroblasts and also in ossification centers appears to be the result of a unique combination of different cis-acting elements. Whereas some of these elements bind tissue-specific transcription factors, others may bind factors that are ubiquitously expressed or expressed in both osteoblasts and tendon fibroblasts.Tissue-specific expression induced by a unique combination of different transcription factors has already been demonstrated for different genes, such as the albumin gene, the fibroblast growth factor 4 gene, and the atrial natriuretic peptide gene (22Gualdi R. Bossard P. Zheng M. Hamada Y. Coleman J.R. Zaret K.S. Genes Dev. 1996; 10: 1670-1682Crossref PubMed Scopus (462) Google Scholar, 23Ambrosetti D.C. Schöler H. Dailey L. Basilico C. J. Biol. Chem. 2000; 275: 23387-23397Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 24Shiojima I. Komuro I. Oka T. Hiroi Y. Mizuno T. Takimoto E. Monzen K. Aikawa R. Akazawa H. Yamazaki T. et al.J. Biol. Chem. 1999; 274: 8231-8239Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). For example, the albumin gene requires the recruitment by hepatocyte nuclear factor-3α of several cis-activators and their corresponding DNA-binding proteins to be expressed in the liver (22Gualdi R. Bossard P. Zheng M. Hamada Y. Coleman J.R. Zaret K.S. Genes Dev. 1996; 10: 1670-1682Crossref PubMed Scopus (462) Google Scholar). Similarly, Sox2 and Oct3 bind to adjacent sites and synergistically drive fibroblast growth factor-4 gene expression (23Ambrosetti D.C. Schöler H. Dailey L. Basilico C. J. Biol. Chem. 2000; 275: 23387-23397Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Finally, the transcription factor Nkx-2.5 recruits other DNA-binding proteins such as GATA-4 by direct protein-protein interactions to activate the atrial natriuretic peptide gene (24Shiojima I. Komuro I. Oka T. Hiroi Y. Mizuno T. Takimoto E. Monzen K. Aikawa R. Akazawa H. Yamazaki T. et al.J. Biol. Chem. 1999; 274: 8231-8239Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Similarly, type I collagen expression in fibroblasts and osteoblasts may also be the result of finely tuned regulation under the control of unique combinations of cis-acting elements and complex interactions between trans-acting factors. We speculate that their identification might help to better define the molecular mechanisms responsible for the differentiation of mesenchymal cells into fibroblasts. Molecular mechanisms governing type I collagen gene expression are still quite elusive; in particular, the cis-acting elements that induce expression of the pro-α1(I) collagen gene in fibroblastic cells are unknown (reviewed in Ref. 5Rossert J. de Combrugghe B. Principles of Bone Biology. Academic Press, San Diego, CA2002Google Scholar). Previous studies performed using transgenic mice harboring various segments of the mouse pro-α1(I) promoter have shown that the −3.2 to −2.3 kb segment contains cis-acting elements that are necessary to induce high-level expression of the lacZ reporter gene in tendon fibroblasts (6Rossert J. Eberspaecher H. de Crombrugghe B. J. Cell Biol. 1995; 129: 1421-1432Crossref PubMed Scopus (209) Google Scholar). To identify these cis-acting elements, we have generated transgenic embryos harboring different subsegments of the −3.2 to −2.3 kb fragment cloned upstream of a 2.3-kb segment of the pro-α1(I) proximal promoter and the lacZ reporter gene. The lacZ gene allows precise identification of the cell types in which the transgene is active. The use of the 2.3-kb segment of the pro-α1(I) proximal promoter permits easy identification of embryos that express the lacZ gene because this sequence induces high-level expression of the reporter gene in ossification centers (6Rossert J. Eberspaecher H. de Crombrugghe B. J. Cell Biol. 1995; 129: 1421-1432Crossref PubMed Scopus (209) Google Scholar). Foster mothers were sacrificed at 15.5 days of gestation because at this time the expression of the lacZ gene can be easily detected in ossification centers and developing tendons (6Rossert J. Eberspaecher H. de Crombrugghe B. J. Cell Biol. 1995; 129: 1421-1432Crossref PubMed Scopus (209) Google Scholar). At least three embryos expressing the lacZ gene were obtained for each construct to eliminate a site of integration effect. Our deletion analysis demonstrated that two short cis-acting elements located between −3.2 and −2.3 kb were necessary to induce high-level expression of the reporter gene specifically in tendon fibroblasts of transgenic mice. These elements, which we have named TSE1 and TSE2, are located about 2.8 and 2.3 kb upstream of the transcription start site, respectively. Mouse embryos harboring a transgene containing both TSE1 and TSE2 cloned upstream of a 2.3-kb segment of the pro-α1(I) proximal promoter consistently co-expressed the reporter gene in tendon fibroblasts and ossification centers. In contrast, embryos harboring a transgene containing either TSE1 alone or TSE2 alone, cloned upstream of 2.3 kb of the pro-α1(I) proximal promoter, expressed the reporter gene in ossification centers at high levels, but no staining of tendons could be detected in whole-mount embryos or in histological sections. Comparison of the sequences of TSE1 and TSE2 and the sequences of the rat, bovine, and human pro-α1(I) promoters showed that the most 3′ part of TSE1 and the whole TSE2 have highly similar counterparts, as seen for the osteoblast-specific element (11Rossert J.A. Chen S.S. Eberspaecher H. Smith C.N. de Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1027-1031Crossref PubMed Scopus (90) Google Scholar). Gel shift experiments showed that both TSE1 and TSE2 can bind proteins that are present in nuclear extracts from tendon fibroblasts, but not proteins from other type I collagen-producing cells or from epithelial cells. Competition experiments performed using double-stranded oligonucleotides harboring 5-bp deletions within the TSE2 sequence showed that the tendon-specific nuclear proteins binding TSE2 actually bound a consensus E-box (CACGTG) located at −2325 bp. E-boxes are known to bind basic helix-loop-helix transcription factors, and it is very tempting to speculate that the E-box located within TSE2 binds a basic helix-loop-helix transcription factor named scleraxis. Schweitzer et al. (19Schweitzer R. Chyung J. Murtaugh L. Brent A. Rose V. Olsen E. Lassar A. Tabin C. Development. 2001; 126: 3855-3866Google Scholar) have recently shown that in chick embryos, the expression of scleraxis becomes rapidly specific to the developing tendons and ligaments and that in the developing mouse limb, scleraxis transcripts are selectively found in tendons and their progenitors. Furthermore, they have also shown that induction of excess of scleraxis-positive mesenchymal cells did not result in the production of extra tendons (19Schweitzer R. Chyung J. Murtaugh L. Brent A. Rose V. Olsen E. Lassar A. Tabin C. Development. 2001; 126: 3855-3866Google Scholar). This last finding is fully consistent with the fact that activation of the pro-α1(I) proximal promoter in tendon fibroblasts required the presence of TSE2 and also of TSE1. Analysis of TSE1 using electrophoretic mobility shift assays in the presence of competitors also led to the identification of a short sequence containing a GAACT motif that bound a tendon-specific nuclear protein. Computer analysis of this sequence did not enable us to identify a potential DNA-binding protein that could interact with it. Nevertheless, the role of this sequence has been confirmed by generating transgenic harboring an 18-bp deletion that encompasses the GAACT motif within TSE1. Whereas E15.5 transgenic embryos expressed the lacZ reporter gene at high levels in ossification centers, no X-gal staining could be detected in tendons. Interestingly, when TSE1 was multimerized four times and cloned upstream of four copies of the osteoblast-specific element and 220 pb of the pro-α1(I) proximal promoter, the lacZ reporter gene was expressed in ossification centers but not in tendons, and no staining of tendon fibroblasts could be detected by histological analysis. This shows that unlike the osteoblast-specific element, multimerization of TSE1 cannot overcome the absence of other cis-acting elements (11Rossert J.A. Chen S.S. Eberspaecher H. Smith C.N. de Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1027-1031Crossref PubMed Scopus (90) Google Scholar). Moreover, generation of transgenic mice harboring a deletion between −1537 and −220 bp (i.e. between the osteoblast-specific element and the minimal promoter) showed that TSE1 and TSE2 need to cooperate with elements located within this sequence to induce expression of the lacZ gene in tendon fibroblasts of transgenic mice. Similarly, the absence of staining of ossification centers in embryos harboring this deleted construct showed that the osteoblast-specific element also needs to cooperate with downstream elements. The need for such cooperativity had previously been masked by the systematic usage of four copies of the osteoblast-specific element (11Rossert J.A. Chen S.S. Eberspaecher H. Smith C.N. de Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1027-1031Crossref PubMed Scopus (90) Google Scholar). The existence of cis-acting elements located between −1537 and −220 bp that are able to induce expression of reporter genes in tendons and ossification centers is consistent with data obtained from other groups with the human, rat, and mouse pro-α1(I) promoters (9Liska D.J. Reed M.J. Sage E.H. Bornstein P. J. Cell Biol. 1994; 125: 695-704Crossref PubMed Scopus (59) Google Scholar,10Slack J.L. Liska D.J. Bornstein P. Mol. Cell. Biol. 1991; 11: 2066-2074Crossref PubMed Google Scholar, 18Houglum K. Buck M. Alcorn J. Contreras S. Bornstein P. Chojkier M. J. Clin. Invest. 1995; 96: 2269-2276Crossref PubMed Scopus (47) Google Scholar, 20Bedalov A. Salvatori R. Dodig M. Kronenberg M. Kapural B. Bogdanovic Z. Kream B. Woody C. Clark S. Mack K. Rowe D. Lichtler A. J Bone Miner. Res. 1995; 10: 1443-1451Crossref PubMed Scopus (42) Google Scholar, 21Kern B. Shen J. Starbuck M. Karsenty G. J. Biol. Chem. 2001; 276: 7101-7107Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Slack et al. (10Slack J.L. Liska D.J. Bornstein P. Mol. Cell. Biol. 1991; 11: 2066-2074Crossref PubMed Google Scholar) and Liska et al. (9Liska D.J. Reed M.J. Sage E.H. Bornstein P. J. Cell Biol. 1994; 125: 695-704Crossref PubMed Scopus (59) Google Scholar) have shown that a transgene containing 2.3 kb of the human pro-α1(I) collagen promoter cloned upstream of the growth hormone gene drove expression of this reporter gene in ossification centers and also in tendons. Bedalov et al. (20Bedalov A. Salvatori R. Dodig M. Kronenberg M. Kapural B. Bogdanovic Z. Kream B. Woody C. Clark S. Mack K. Rowe D. Lichtler A. J Bone Miner. Res. 1995; 10: 1443-1451Crossref PubMed Scopus (42) Google Scholar) have shown that a sequence of the rat promoter located between −1670 and −944 is responsible for reporter gene expression in tendons at low levels. Similarly, Houglum et al. (18Houglum K. Buck M. Alcorn J. Contreras S. Bornstein P. Chojkier M. J. Clin. Invest. 1995; 96: 2269-2276Crossref PubMed Scopus (47) Google Scholar) have shown that transgenic mice harboring a construct containing 1626 bp of the mouse pro-α1(I) collagen promoter cloned upstream of lacZ expressed the reporter gene in tendons at low levels. More recently, Kern et al. (21Kern B. Shen J. Starbuck M. Karsenty G. J. Biol. Chem. 2001; 276: 7101-7107Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar) have shown the existence of a second osteoblast-specific element in the pro-α1(I) promoter. They showed that four copies of a sequence of the mouse pro-α1(I) collagen promoter extending from −1347 to −1338 bp cloned upstream of 220 bp of the pro-α1(I) proximal promoter drove expression of the luciferase reporter gene in developing bones (21Kern B. Shen J. Starbuck M. Karsenty G. J. Biol. Chem. 2001; 276: 7101-7107Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Thus, expression of the lacZ gene in tendon fibroblasts and also in ossification centers appears to be the result of a unique combination of different cis-acting elements. Whereas some of these elements bind tissue-specific transcription factors, others may bind factors that are ubiquitously expressed or expressed in both osteoblasts and tendon fibroblasts. Tissue-specific expression induced by a unique combination of different transcription factors has already been demonstrated for different genes, such as the albumin gene, the fibroblast growth factor 4 gene, and the atrial natriuretic peptide gene (22Gualdi R. Bossard P. Zheng M. Hamada Y. Coleman J.R. Zaret K.S. Genes Dev. 1996; 10: 1670-1682Crossref PubMed Scopus (462) Google Scholar, 23Ambrosetti D.C. Schöler H. Dailey L. Basilico C. J. Biol. Chem. 2000; 275: 23387-23397Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 24Shiojima I. Komuro I. Oka T. Hiroi Y. Mizuno T. Takimoto E. Monzen K. Aikawa R. Akazawa H. Yamazaki T. et al.J. Biol. Chem. 1999; 274: 8231-8239Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). For example, the albumin gene requires the recruitment by hepatocyte nuclear factor-3α of several cis-activators and their corresponding DNA-binding proteins to be expressed in the liver (22Gualdi R. Bossard P. Zheng M. Hamada Y. Coleman J.R. Zaret K.S. Genes Dev. 1996; 10: 1670-1682Crossref PubMed Scopus (462) Google Scholar). Similarly, Sox2 and Oct3 bind to adjacent sites and synergistically drive fibroblast growth factor-4 gene expression (23Ambrosetti D.C. Schöler H. Dailey L. Basilico C. J. Biol. Chem. 2000; 275: 23387-23397Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Finally, the transcription factor Nkx-2.5 recruits other DNA-binding proteins such as GATA-4 by direct protein-protein interactions to activate the atrial natriuretic peptide gene (24Shiojima I. Komuro I. Oka T. Hiroi Y. Mizuno T. Takimoto E. Monzen K. Aikawa R. Akazawa H. Yamazaki T. et al.J. Biol. Chem. 1999; 274: 8231-8239Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Similarly, type I collagen expression in fibroblasts and osteoblasts may also be the result of finely tuned regulation under the control of unique combinations of cis-acting elements and complex interactions between trans-acting factors. We speculate that their identification might help to better define the molecular mechanisms responsible for the differentiation of mesenchymal cells into fibroblasts. We thank F. Ruggiero for the generous gift of FV6M cells and M. Delauche for expert technical assistance. We are also grateful to J. Chambaz and C. Lasne for welcoming and helping us in the IFR 58 transgenic facility.