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Regulation of the Fibroblast Growth Factor Receptor 3 Promoter and Intron I Enhancer by Sp1 Family Transcription Factors

1998; Elsevier BV; Volume: 273; Issue: 9 Linguagem: Inglês

10.1074/jbc.273.9.5349

1083-351X

Donald G. McEwen, David M. Ornitz,

Epigenetics and DNA Methylation

Fibroblast growth factor receptor 3 (FGFR3) has a complex spatial and temporal pattern of expression and is essential for the normal development of a diverse set of tissues. Recently, mutations have been identified in FGFR3 that result in constitutive tyrosine kinase activity and cause a number of different human skeletal disorders. To examine the regulatory mechanisms governing FGFR3 expression, the promoter for the FGFR3 gene was identified and characterized. It resides in a CpG island, which encompasses the 5′ end of the FGFR3 gene and lacks classical cis-regulatory motifs. As little as 100 base pairs of sequence 5′ to the initiation site can confer a 20–40-fold increase in transcriptional activity upon a promoter-less vector. The transcriptional activity of thesecis-regulatory sequences is further stimulated by elements found within the first intron. Mapping of the enhancer activity found within intron I identified two purine-rich sequence motifs between +340 and +395. Electrophoretic mobility shift assays demonstrated that sequences within this region bind members of the Sp1 family of transcription factors. In a background lacking Sp1-like activity, we demonstrate that Sp1 can enhance transcription of the minimal promoter (which contains five classical Sp1 sites), whereas both Sp1 and Sp3 can enhance transcription through the elements found in intron I. Although these transcription factors are ubiquitously expressed, we demonstrate that the sequences between −220 and +609 of the FGFR3 gene are sufficient to promote the tissue-specific expression of a reporter gene in transgenic mice. Fibroblast growth factor receptor 3 (FGFR3) has a complex spatial and temporal pattern of expression and is essential for the normal development of a diverse set of tissues. Recently, mutations have been identified in FGFR3 that result in constitutive tyrosine kinase activity and cause a number of different human skeletal disorders. To examine the regulatory mechanisms governing FGFR3 expression, the promoter for the FGFR3 gene was identified and characterized. It resides in a CpG island, which encompasses the 5′ end of the FGFR3 gene and lacks classical cis-regulatory motifs. As little as 100 base pairs of sequence 5′ to the initiation site can confer a 20–40-fold increase in transcriptional activity upon a promoter-less vector. The transcriptional activity of thesecis-regulatory sequences is further stimulated by elements found within the first intron. Mapping of the enhancer activity found within intron I identified two purine-rich sequence motifs between +340 and +395. Electrophoretic mobility shift assays demonstrated that sequences within this region bind members of the Sp1 family of transcription factors. In a background lacking Sp1-like activity, we demonstrate that Sp1 can enhance transcription of the minimal promoter (which contains five classical Sp1 sites), whereas both Sp1 and Sp3 can enhance transcription through the elements found in intron I. Although these transcription factors are ubiquitously expressed, we demonstrate that the sequences between −220 and +609 of the FGFR3 gene are sufficient to promote the tissue-specific expression of a reporter gene in transgenic mice. Cellular differentiation requires the proper interpretation of external stimuli. Although many types of stimuli influence cell fate, a target cell must express the appropriate complement of receptors to perceive, interpret, and respond to environmental signals. The fibroblast growth factors (FGFs) 1The abbreviations used are: FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; HSPG, heparan sulfate proteoglycan; luc, luciferase; i, intron; UTR, untranslated region; RSV, Rous sarcoma virus; EGFR, epidermal growth factor; bp, base pair(s); nt, nucleotide(s); β-gal, β-galactosidase; EMSA, electrophoretic mobility shift assay; InR, initiator region.1The abbreviations used are: FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; HSPG, heparan sulfate proteoglycan; luc, luciferase; i, intron; UTR, untranslated region; RSV, Rous sarcoma virus; EGFR, epidermal growth factor; bp, base pair(s); nt, nucleotide(s); β-gal, β-galactosidase; EMSA, electrophoretic mobility shift assay; InR, initiator region. are small molecular mass polypeptides (18–27 kDa), which have been implicated in many developmentally regulated events such as mesoderm induction, angiogenesis, chondrogenesis, malignant transformation, and neuronal differentiation (1Basilico C. Moscatelli D. Adv. Cancer Res. 1992; 59: 115-165Google Scholar, 2Wilkie A.O.M. Morriss-Kay G.M. Jones E.Y. Heath J.K. Curr. Biol. 1995; 5: 500-507Google Scholar). To date, 15 FGF ligands have been described (3Coulier F. Pontarotti P. Roubin R. Hartung H. Goldfarb M. Birnbaum D. J. Mol. Evol. 1997; 44: 43-56Google Scholar,4McWhirter J.R. Goulding M. Weiner J.A. Chun J. Murre C. Development. 1997; 124: 3221-3232Google Scholar). These FGFs all have unique patterns of expression as well as a high affinity for heparin and heparan sulfate proteoglycans (HSPGs). HSPGs have been shown to regulate the biological activity of many FGFs and serve as an essential cofactor required for FGF-induced receptor autophosphorylation (5Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Google Scholar). Because HSPGs are a major component of the extracellular matrix, they have been implicated in limiting the diffusion of FGFs from their site of production (6Flaumenhaft R. Moscatelli D. Rifkin D.B. J. Cell Biol. 1990; 111: 1651-1659Google Scholar). Thus, the spatial and temporal regulation of expression of FGFs provides one mechanism through which FGF-mediated signaling can be regulated during development.The fibroblast growth factor receptor (FGFR) family consists of four genes, each of which encodes a membrane-spanning tyrosine kinase receptor (3Coulier F. Pontarotti P. Roubin R. Hartung H. Goldfarb M. Birnbaum D. J. Mol. Evol. 1997; 44: 43-56Google Scholar, 7Johnson D.E. Williams L.T. Adv. Cancer Res. 1993; 60: 1-41Google Scholar). Recently, both gain-of-function and loss-of-function mutations in the FGFRs have revealed unique roles for these receptors during development (8Muenke M. Schell U. Trends Genet. 1995; 11: 308-313Google Scholar, 9Skaer H. Curr. Biol. 1997; 7: R238-R241Google Scholar, 10Colvin J.S. Bohne B.A. Harding G.W. McEwen D.G. Ornitz D.M. Nat. Genet. 1996; 12: 390-397Google Scholar, 11Deng C.X. Wynshaw-Boris A. Shen M.M. Daugherty C. Ornitz D.M. Leder P. Genes Dev. 1994; 8: 3045-3057Google Scholar, 12Deng C. Wynshaw-Boris A. Zhou F. Kuo A. Leder P. Cell. 1996; 84: 911-921Google Scholar, 13Yamaguchi T.P. Harpal K. Henkemeyer M. Rossant J. Genes Dev. 1994; 8: 3032-3044Google Scholar). Specifically, point mutations in FGFR3 have been genetically linked to achondroplasia, thanatophoric dysplasia, and hypochondroplasia (8Muenke M. Schell U. Trends Genet. 1995; 11: 308-313Google Scholar); all are diseases where bones fail to grow to normal lengths. These skeletal disorders result from defects in the epiphyseal growth plate, a place where FGFR3 is known to be highly expressed (14Peters K. Ornitz D.M. Werner S. Williams L. Dev. Biol. 1993; 155: 423-430Google Scholar). When the mutations corresponding to those of achondroplasia (G380R) and thanatophoric dysplasia (R248C and K650E) are introduced into the murine FGFR3 cDNA, ligand-independent activation of the receptor is observed (15Naski M.C. Wang Q. Xu J. Ornitz D.M. Nat. Genet. 1996; 13: 233-237Google Scholar, 16Webster M.K. Donoghue D.J. EMBO J. 1996; 15: 520-527Google Scholar). The constitutive activity of this receptor is thought to disrupt normal development by initiating intracellular signals in the absence of ligand. In contrast, loss-of-function alleles of FGFR3 lead to the overgrowth of long bones (10Colvin J.S. Bohne B.A. Harding G.W. McEwen D.G. Ornitz D.M. Nat. Genet. 1996; 12: 390-397Google Scholar, 12Deng C. Wynshaw-Boris A. Zhou F. Kuo A. Leder P. Cell. 1996; 84: 911-921Google Scholar), as well as deafness due to defects in the development of the organ of Corti (10Colvin J.S. Bohne B.A. Harding G.W. McEwen D.G. Ornitz D.M. Nat. Genet. 1996; 12: 390-397Google Scholar). Although redundancy in the FGFR family may compensate for the loss of FGFR3 activity in some tissues, these results demonstrate that both the regulation of FGFR3 expression and kinase-mediated signaling activity are required for proper development.To understand the mechanisms that regulate the expression of FGFR3, we have identified and characterized the FGFR3 promoter both in vitro and in vivo. Here we demonstrate that sequences derived from the CpG island found at the 5′ end of the murine FGFR3 gene are capable of promoting transcription in transient transfection assays. Furthermore, the activity of these sequences can be further stimulated by sequences found within the first intron. Localization of the intron enhancer element identified binding sites for the Sp1 family of transcription factors. Characterization of the trans-acting factors demonstrated that Sp1 and Sp3 could promote transcriptional activity through these elements in the Drosophila SL2 cell line. Although Sp1 and Sp3 transcription factors are ubiquitously expressed, the defined minimal promoter and intron enhancer are sufficient to promote the tissue-specific expression of a reporter gene in transgenic mice.DISCUSSIONThe promoter for the FGFR3 gene resides in a CpG island that lacks the classical CAAT box and TATA box motifs found in many eukaryotic promoters. Sequence analysis of the FGFR3 promoter revealed a number of transcription factor binding sites, including five classical Sp1 sites, within the first 200 bp 5′ of the transcription start site. The positioning of the basal transcriptional machinery in a TATA-less promoter can occur independent of InR sequences when Sp1 binding sites are present (44Dennig J. Hagen G. Beato M. Suske G. J. Biol. Chem. 1995; 270: 12737-12744Google Scholar, 45Kollmar R. Sukow K.A. Sponagle S.K. Farnham P.J. J. Biol. Chem. 1994; 269: 2252-2257Google Scholar, 46Blake M.C. Jambou R.C. Swick A.G. Kahn J.W. Azizkhan J.C. Mol. Cell. Biol. 1990; 10: 6632-6641Google Scholar). In such instances, Sp1 is capable of stabilizing transcriptional initiation complexes approximately 50 bp downstream from an Sp1 binding site (45Kollmar R. Sukow K.A. Sponagle S.K. Farnham P.J. J. Biol. Chem. 1994; 269: 2252-2257Google Scholar). Mapping of the start site of transcription was achieved through RNase protection, and it was shown that transcriptional initiation occurs 22 bp 5′ from the end of the longest published mouse FGFR3 cDNA (GenBank accession no. M81342) and 57 bp 3′ of the most proximal Sp1 binding site. Our start site differs by only two nucleotides from that previously described by Perez-Castro et al. (47Perez-Castro A.V. Wilson J. Altherr M.R. Genomics. 1997; 41: 10-16Google Scholar) and both start sites are positioned such that Sp1 could facilitate organization of the transcription initiation complex.Through comparison to the published mouse FGFR3 cDNA, it was also determined that sequences encoding for the 5′-UTR are divided by a 376-bp intron. Our placement of the 5′ splice donor site 26 bp 5′ to that determined by Perez-Castro et al. (47Perez-Castro A.V. Wilson J. Altherr M.R. Genomics. 1997; 41: 10-16Google Scholar) is consistent with the 5′-UTR sequences of the published mouse FGFR3 cDNA. Alternative splicing or the use of a cryptic splice donor site could account for the differences between these two studies. The utilization of alternative splice donor and splice acceptor sites is known to occur in many of the FGFR genes (48Eisemann A. Ahn J.A. Graziani G. Tronick S.T. Ron D. Oncogene. 1991; 6: 1195-1202Google Scholar, 49Shi E. Kan M. Xu J. Wang F. Hou J. McKeehan W.L. Mol. Cell. Biol. 1993; 13: 3907-3918Google Scholar).cis-Regulatory sequences found within the CpG island were analyzed for transcriptional activity. Luciferase reporter constructs were transfected into four different cell lines, and the activity of the reporter gene examined. Constructs with as little as 100 bp (−126/−27) of 5′ cis-regulatory sequence still brought about a 20–40-fold increase in transcriptional activity. Within the CpG-rich sequence found between −126 and −27, there are two classical Sp1 binding sites. Deletions that remove the distal-most Sp1 site result in a 34–43% reduction in transcriptional activity, depending upon the cell line examined. Neighboring Sp1 sites frequently act synergistically (50Courey A.J. Holtzman D.A. Jackson S.P. Tjian R. Cell. 1989; 59: 827-836Google Scholar, 51Pascal E. Tjian R. Genes Dev. 1991; 5: 1646-1656Google Scholar). However, the data presented here, in conjunction with the known requirement of Sp1 for the formation of a transcription initiation complex in a TATA-less promoter, suggests that the sequence context of the FGFR3 promoter simply provides for additive effects mediated by Sp1. Although the transcriptional activity is dependent upon the 5′ proximal sequence, this activity is independent of cellular background in that it fails to mimic the expression profile of the endogenous FGFR3 gene. Finally, it should also be noted that binding sites for transcription factors not yet identified may also regulate FGFR3 promoter function through the −126/−27 promoter fragment. A detailed linker scanning analysis will be needed to identify such sites.In an attempt to find transcriptional enhancers, it was determined that addition of the FGFR3 UTR and intervening intron sequences could promote an 8–10-fold increase in transcriptional activity. To rule out the role of initiator region (InR) effects on the efficiency of transcriptional initiation, constructs that contained the endogenous initiation site were compared with the previously defined promoter constructs. These experiments showed that addition of the −26 to +10 FGFR3 sequence failed to affect transcriptional activity. To localize this enhancer activity, additional constructs were analyzed. Constructs that contained the 5′-UTR sequences but lacked intron I failed to result in significant transcriptional enhancement, whereas placement of the intron alone 3′ relative to the FGFR3 promoter sequences afforded the same transcriptional enhancement seen with the UTR/intron combination. These results demonstrated that the enhancer-like activity resides in intron I.Mapping of the intron enhancer activity to sequences between +340 and +395 identified two polypurine direct repeat sequence motifs. The sequence and organization of these motifs is similar to a motif previously identified in the EGFR promoter. From the studies of Johnsonet al. (42Johnson A.C. Jinno Y. Merlino G.T. Mol. Cell. Biol. 1988; 8: 4174-4184Google Scholar), it was determined that this site was capable of enhancing the transcription of the EGFR promoter in vitro. Through their studies, they also showed that these elements were sensitive to S1 nuclease and bound the Sp1 transcription factor. Although this site in the FGFR3 promoter is not sensitive to S1 nuclease, 3D. G. McEwen and D. M. Ornitz, unpublished observations. it does interact with Sp1-like DNA binding activity as shown through gel shift analysis. The specificity of this interaction was demonstrated through competition with the classical Sp1 (GC box) binding site and a non-classical Sp1 binding site derived from the promoter for the human EGFR. The classical Sp1 element is unrelated to the polypurine stretch; however, it was capable of competing for the DNA binding activity found in all but one of the resulting DNA-protein complexes. Furthermore, transfection studies in SL2 cells demonstrated that Sp1 could promote transcriptional activity through either the basal promoter alone or the promoter/enhancer combination whereas co-transfection studies demonstrated that both Sp1 and Sp3 can enhance transcription through the intron enhancer element. Together, these data suggest that binding sites for members of the Sp1 family of transcription factors, both proximal and distal to the start site of transcription, can work together to enhance transcription of the FGFR3 gene.The ability of proximal and distal Sp1 binding sites to synergistically regulate transcription has been observed by others (50Courey A.J. Holtzman D.A. Jackson S.P. Tjian R. Cell. 1989; 59: 827-836Google Scholar, 52Su W. Jackson S. Tjian R. Echols H. Genes Dev. 1991; 5: 820-826Google Scholar, 53Mastrangelo I.A. Courey A.J. Wall J.S. Jackson S.P. Hough P.V. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5670-5674Google Scholar). Through these studies, it has been shown that Sp1-Sp1 protein interactions can induce looping of the interveningcis-regulatory sequences. These proximal-distal interactions are hypothesized to regulate gene transcription by increasing the local concentration of Sp1 glutamine-rich activation domains near the start site. Such a model would explain the synergistic ability of the intron enhancer to regulate the transcriptional activity of the FGFR3 basal promoter.Although Sp3 has usually been shown to serve a negative regulatory role by competing for Sp1 binding sites, at least two other studies have demonstrated that SP3 can promote transcriptional activity (54Majello B. De Luca P. Lania L. J. Biol. Chem. 1997; 272: 4021-4026Google Scholar, 55Liang Y. Robinson D.F. Dennig J. Suske G. Fahl W.E. J. Biol. Chem. 1996; 271: 11792-11797Google Scholar). This transcription-promoting ability of Sp3 in our experiments may reflect the sequence-specific context of the binding site, as evidenced by the ability of Sp3 to transactivate the B fragment. Additional experiments will be required to demonstrate the in vivo role of Sp3 in FGFR3 promoter regulation.Transient transfection assays demonstrated that promoter activity resides in the CpG island found at the 5′ end of the FGFR3 gene, whereas an enhancer element was located in the first intron. However, this activity failed to parallel the cell-type specific expression of the endogenous FGFR3 gene. To assess whether or not the minimal promoter sequences defined above were capable of promoting cell-type specific expression in vivo, various lengths of regulatory sequences were used to generate transgenic animals. Surprisingly, analysis of the transgene expression in 13 independent transgenic lines demonstrated that the sequences between −220 and +609 provide the proper regulatory elements required for the expression of a reporter gene in a subset of the tissues that normally express the endogenous FGFR3 gene, whereas these same elements fail to limit the cell-specific expression pattern of the endogenous FGFR3 gene in vitro. These data suggest that other undefined mechanisms exist to regulate the expression of the FGFR3 minimal promoter in vivo.Due to the increased mutability associated with 5-methylcytosine, the conservation of the CpG island at the 5′ end of the FGFR3 gene suggests that it plays some important regulatory role in vivo. One possible way in which these sequences might regulate gene expression in a tissue-specific manner is through the methylation of any of the 83 CpG dinucleotides found within the −220 to +609 region. The establishment of methylation patterns during development (56Li E. Bestor T.H. Jaenisch R. Cell. 1992; 69: 915-926Google Scholar) is required for embryo viability, and has been shown to regulate the transcriptional activity of many genes by either directly interfering with the binding of transcription factors to their DNA cognates (57Watt F. Molloy P.L. Genes Dev. 1988; 2: 1136-1143Google Scholar, 58Iguchi-Ariga S.M. Schaffner W. Genes Dev. 1989; 3: 612-619Google Scholar, 59Iannello R.C. Young J. Sumarsono S. Tymms M.J. Dahl H.H. Gould J. Hedger M. Kola I. Mol. Cell. Biol. 1997; 17: 612-619Google Scholar) or by recruiting methyl binding transcriptional repressor proteins (60Nan X. Campoy F.J. Bird A. Cell. 1997; 88: 471-481Google Scholar, 61Boyes J. Bird A. Cell. 1991; 64: 1123-1134Google Scholar). Unlike the hypomethylated state of most CpG islands, preliminary studies3 in which we examined the methylation status of the FGFR3 CpG island in numerous tissues, as well as the transcriptional activity of in vitro methylated reporter constructs, suggested that methylation may be a contributing factor to the tissue specificity exhibited by the FGFR3 minimal promoter in vivo. Cellular differentiation requires the proper interpretation of external stimuli. Although many types of stimuli influence cell fate, a target cell must express the appropriate complement of receptors to perceive, interpret, and respond to environmental signals. The fibroblast growth factors (FGFs) 1The abbreviations used are: FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; HSPG, heparan sulfate proteoglycan; luc, luciferase; i, intron; UTR, untranslated region; RSV, Rous sarcoma virus; EGFR, epidermal growth factor; bp, base pair(s); nt, nucleotide(s); β-gal, β-galactosidase; EMSA, electrophoretic mobility shift assay; InR, initiator region.1The abbreviations used are: FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; HSPG, heparan sulfate proteoglycan; luc, luciferase; i, intron; UTR, untranslated region; RSV, Rous sarcoma virus; EGFR, epidermal growth factor; bp, base pair(s); nt, nucleotide(s); β-gal, β-galactosidase; EMSA, electrophoretic mobility shift assay; InR, initiator region. are small molecular mass polypeptides (18–27 kDa), which have been implicated in many developmentally regulated events such as mesoderm induction, angiogenesis, chondrogenesis, malignant transformation, and neuronal differentiation (1Basilico C. Moscatelli D. Adv. Cancer Res. 1992; 59: 115-165Google Scholar, 2Wilkie A.O.M. Morriss-Kay G.M. Jones E.Y. Heath J.K. Curr. Biol. 1995; 5: 500-507Google Scholar). To date, 15 FGF ligands have been described (3Coulier F. Pontarotti P. Roubin R. Hartung H. Goldfarb M. Birnbaum D. J. Mol. Evol. 1997; 44: 43-56Google Scholar,4McWhirter J.R. Goulding M. Weiner J.A. Chun J. Murre C. Development. 1997; 124: 3221-3232Google Scholar). These FGFs all have unique patterns of expression as well as a high affinity for heparin and heparan sulfate proteoglycans (HSPGs). HSPGs have been shown to regulate the biological activity of many FGFs and serve as an essential cofactor required for FGF-induced receptor autophosphorylation (5Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Google Scholar). Because HSPGs are a major component of the extracellular matrix, they have been implicated in limiting the diffusion of FGFs from their site of production (6Flaumenhaft R. Moscatelli D. Rifkin D.B. J. Cell Biol. 1990; 111: 1651-1659Google Scholar). Thus, the spatial and temporal regulation of expression of FGFs provides one mechanism through which FGF-mediated signaling can be regulated during development. The fibroblast growth factor receptor (FGFR) family consists of four genes, each of which encodes a membrane-spanning tyrosine kinase receptor (3Coulier F. Pontarotti P. Roubin R. Hartung H. Goldfarb M. Birnbaum D. J. Mol. Evol. 1997; 44: 43-56Google Scholar, 7Johnson D.E. Williams L.T. Adv. Cancer Res. 1993; 60: 1-41Google Scholar). Recently, both gain-of-function and loss-of-function mutations in the FGFRs have revealed unique roles for these receptors during development (8Muenke M. Schell U. Trends Genet. 1995; 11: 308-313Google Scholar, 9Skaer H. Curr. Biol. 1997; 7: R238-R241Google Scholar, 10Colvin J.S. Bohne B.A. Harding G.W. McEwen D.G. Ornitz D.M. Nat. Genet. 1996; 12: 390-397Google Scholar, 11Deng C.X. Wynshaw-Boris A. Shen M.M. Daugherty C. Ornitz D.M. Leder P. Genes Dev. 1994; 8: 3045-3057Google Scholar, 12Deng C. Wynshaw-Boris A. Zhou F. Kuo A. Leder P. Cell. 1996; 84: 911-921Google Scholar, 13Yamaguchi T.P. Harpal K. Henkemeyer M. Rossant J. Genes Dev. 1994; 8: 3032-3044Google Scholar). Specifically, point mutations in FGFR3 have been genetically linked to achondroplasia, thanatophoric dysplasia, and hypochondroplasia (8Muenke M. Schell U. Trends Genet. 1995; 11: 308-313Google Scholar); all are diseases where bones fail to grow to normal lengths. These skeletal disorders result from defects in the epiphyseal growth plate, a place where FGFR3 is known to be highly expressed (14Peters K. Ornitz D.M. Werner S. Williams L. Dev. Biol. 1993; 155: 423-430Google Scholar). When the mutations corresponding to those of achondroplasia (G380R) and thanatophoric dysplasia (R248C and K650E) are introduced into the murine FGFR3 cDNA, ligand-independent activation of the receptor is observed (15Naski M.C. Wang Q. Xu J. Ornitz D.M. Nat. Genet. 1996; 13: 233-237Google Scholar, 16Webster M.K. Donoghue D.J. EMBO J. 1996; 15: 520-527Google Scholar). The constitutive activity of this receptor is thought to disrupt normal development by initiating intracellular signals in the absence of ligand. In contrast, loss-of-function alleles of FGFR3 lead to the overgrowth of long bones (10Colvin J.S. Bohne B.A. Harding G.W. McEwen D.G. Ornitz D.M. Nat. Genet. 1996; 12: 390-397Google Scholar, 12Deng C. Wynshaw-Boris A. Zhou F. Kuo A. Leder P. Cell. 1996; 84: 911-921Google Scholar), as well as deafness due to defects in the development of the organ of Corti (10Colvin J.S. Bohne B.A. Harding G.W. McEwen D.G. Ornitz D.M. Nat. Genet. 1996; 12: 390-397Google Scholar). Although redundancy in the FGFR family may compensate for the loss of FGFR3 activity in some tissues, these results demonstrate that both the regulation of FGFR3 expression and kinase-mediated signaling activity are required for proper development. To understand the mechanisms that regulate the expression of FGFR3, we have identified and characterized the FGFR3 promoter both in vitro and in vivo. Here we demonstrate that sequences derived from the CpG island found at the 5′ end of the murine FGFR3 gene are capable of promoting transcription in transient transfection assays. Furthermore, the activity of these sequences can be further stimulated by sequences found within the first intron. Localization of the intron enhancer element identified binding sites for the Sp1 family of transcription factors. Characterization of the trans-acting factors demonstrated that Sp1 and Sp3 could promote transcriptional activity through these elements in the Drosophila SL2 cell line. Although Sp1 and Sp3 transcription factors are ubiquitously expressed, the defined minimal promoter and intron enhancer are sufficient to promote the tissue-specific expression of a reporter gene in transgenic mice. DISCUSSIONThe promoter for the FGFR3 gene resides in a CpG island that lacks the classical CAAT box and TATA box motifs found in many eukaryotic promoters. Sequence analysis of the FGFR3 promoter revealed a number of transcription factor binding sites, including five classical Sp1 sites, within the first 200 bp 5′ of the transcription start site. The positioning of the basal transcriptional machinery in a TATA-less promoter can occur independent of InR sequences when Sp1 binding sites are present (44Dennig J. Hagen G. Beato M. Suske G. J. Biol. Chem. 1995; 270: 12737-12744Google Scholar, 45Kollmar R. Sukow K.A. Sponagle S.K. Farnham P.J. J. Biol. Chem. 1994; 269: 2252-2257Google Scholar, 46Blake M.C. Jambou R.C. Swick A.G. Kahn J.W. Azizkhan J.C. Mol. Cell. Biol. 1990; 10: 6632-6641Google Scholar). In such instances, Sp1 is capable of stabilizing transcriptional initiation complexes approximately 50 bp downstream from an Sp1 binding site (45Kollmar R. Sukow K.A. Sponagle S.K. Farnham P.J. J. Biol. Chem. 1994; 269: 2252-2257Google Scholar). Mapping of the start site of transcription was achieved through RNase protection, and it was shown that transcriptional initiation occurs 22 bp 5′ from the end of the longest published mouse FGFR3 cDNA (GenBank accession no. M81342) and 57 bp 3′ of the most proximal Sp1 binding site. Our start site differs by only two nucleotides from that previously described by Perez-Castro et al. (47Perez-Castro A.V. Wilson J. Altherr M.R. Genomics. 1997; 41: 10-16Google Scholar) and both start sites are positioned such that Sp1 could facilitate organization of the transcription initiation complex.Through comparison to the published mouse FGFR3 cDNA, it was also determined that sequences encoding for the 5′-UTR are divided by a 376-bp intron. Our placement of the 5′ splice donor site 26 bp 5′ to that determined by Perez-Castro et al. (47Perez-Castro A.V. Wilson J. Altherr M.R. Genomics. 1997; 41: 10-16Google Scholar) is consistent with the 5′-UTR sequences of the published mouse FGFR3 cDNA. Alternative splicing or the use of a cryptic splice donor site could account for the differences between these two studies. The utilization of alternative splice donor and splice acceptor sites is known to occur in many of the FGFR genes (48Eisemann A. Ahn J.A. Graziani G. Tronick S.T. Ron D. Oncogene. 1991; 6: 1195-1202Google Scholar, 49Shi E. Kan M. Xu J. Wang F. Hou J. McKeehan W.L. Mol. Cell. Biol. 1993; 13: 3907-3918Google Scholar).cis-Regulatory sequences found within the CpG island were analyzed for transcriptional activity. Luciferase reporter constructs were transfected into four different cell lines, and the activity of the reporter gene examined. Constructs with as little as 100 bp (−126/−27) of 5′ cis-regulatory sequence still brought about a 20–40-fold increase in transcriptional activity. Within the CpG-rich sequence found between −126 and −27, there are two classical Sp1 binding sites. Deletions that remove the distal-most Sp1 site result in a 34–43% reduction in transcriptional activity, depending upon the cell line examined. Neighboring Sp1 sites frequently act synergistically (50Courey A.J. Holtzman D.A. Jackson S.P. Tjian R. Cell. 1989; 59: 827-836Google Scholar, 51Pascal E. Tjian R. Genes Dev. 1991; 5: 1646-1656Google Scholar). However, the data presented here, in conjunction with the known requirement of Sp1 for the formation of a transcription initiation complex in a TATA-less promoter, suggests that the sequence context of the FGFR3 promoter simply provides for additive effects mediated by Sp1. Although the transcriptional activity is dependent upon the 5′ proximal sequence, this activity is independent of cellular background in that it fails to mimic the expression profile of the endogenous FGFR3 gene. Finally, it should also be noted that binding sites for transcription factors not yet identified may also regulate FGFR3 promoter function through the −126/−27 promoter fragment. A detailed linker scanning analysis will be needed to identify such sites.In an attempt to find transcriptional enhancers, it was determined that addition of the FGFR3 UTR and intervening intron sequences could promote an 8–10-fold increase in transcriptional activity. To rule out the role of initiator region (InR) effects on the efficiency of transcriptional initiation, constructs that contained the endogenous initiation site were compared with the previously defined promoter constructs. These experiments showed that addition of the −26 to +10 FGFR3 sequence failed to affect transcriptional activity. To localize this enhancer activity, additional constructs were analyzed. Constructs that contained the 5′-UTR sequences but lacked intron I failed to result in significant transcriptional enhancement, whereas placement of the intron alone 3′ relative to the FGFR3 promoter sequences afforded the same transcriptional enhancement seen with the UTR/intron combination. These results demonstrated that the enhancer-like activity resides in intron I.Mapping of the intron enhancer activity to sequences between +340 and +395 identified two polypurine direct repeat sequence motifs. The sequence and organization of these motifs is similar to a motif previously identified in the EGFR promoter. From the studies of Johnsonet al. (42Johnson A.C. Jinno Y. Merlino G.T. Mol. Cell. Biol. 1988; 8: 4174-4184Google Scholar), it was determined that this site was capable of enhancing the transcription of the EGFR promoter in vitro. Through their studies, they also showed that these elements were sensitive to S1 nuclease and bound the Sp1 transcription factor. Although this site in the FGFR3 promoter is not sensitive to S1 nuclease, 3D. G. McEwen and D. M. Ornitz, unpublished observations. it does interact with Sp1-like DNA binding activity as shown through gel shift analysis. The specificity of this interaction was demonstrated through competition with the classical Sp1 (GC box) binding site and a non-classical Sp1 binding site derived from the promoter for the human EGFR. The classical Sp1 element is unrelated to the polypurine stretch; however, it was capable of competing for the DNA binding activity found in all but one of the resulting DNA-protein complexes. Furthermore, transfection studies in SL2 cells demonstrated that Sp1 could promote transcriptional activity through either the basal promoter alone or the promoter/enhancer combination whereas co-transfection studies demonstrated that both Sp1 and Sp3 can enhance transcription through the intron enhancer element. Together, these data suggest that binding sites for members of the Sp1 family of transcription factors, both proximal and distal to the start site of transcription, can work together to enhance transcription of the FGFR3 gene.The ability of proximal and distal Sp1 binding sites to synergistically regulate transcription has been observed by others (50Courey A.J. Holtzman D.A. Jackson S.P. Tjian R. Cell. 1989; 59: 827-836Google Scholar, 52Su W. Jackson S. Tjian R. Echols H. Genes Dev. 1991; 5: 820-826Google Scholar, 53Mastrangelo I.A. Courey A.J. Wall J.S. Jackson S.P. Hough P.V. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5670-5674Google Scholar). Through these studies, it has been shown that Sp1-Sp1 protein interactions can induce looping of the interveningcis-regulatory sequences. These proximal-distal interactions are hypothesized to regulate gene transcription by increasing the local concentration of Sp1 glutamine-rich activation domains near the start site. Such a model would explain the synergistic ability of the intron enhancer to regulate the transcriptional activity of the FGFR3 basal promoter.Although Sp3 has usually been shown to serve a negative regulatory role by competing for Sp1 binding sites, at least two other studies have demonstrated that SP3 can promote transcriptional activity (54Majello B. De Luca P. Lania L. J. Biol. Chem. 1997; 272: 4021-4026Google Scholar, 55Liang Y. Robinson D.F. Dennig J. Suske G. Fahl W.E. J. Biol. Chem. 1996; 271: 11792-11797Google Scholar). This transcription-promoting ability of Sp3 in our experiments may reflect the sequence-specific context of the binding site, as evidenced by the ability of Sp3 to transactivate the B fragment. Additional experiments will be required to demonstrate the in vivo role of Sp3 in FGFR3 promoter regulation.Transient transfection assays demonstrated that promoter activity resides in the CpG island found at the 5′ end of the FGFR3 gene, whereas an enhancer element was located in the first intron. However, this activity failed to parallel the cell-type specific expression of the endogenous FGFR3 gene. To assess whether or not the minimal promoter sequences defined above were capable of promoting cell-type specific expression in vivo, various lengths of regulatory sequences were used to generate transgenic animals. Surprisingly, analysis of the transgene expression in 13 independent transgenic lines demonstrated that the sequences between −220 and +609 provide the proper regulatory elements required for the expression of a reporter gene in a subset of the tissues that normally express the endogenous FGFR3 gene, whereas these same elements fail to limit the cell-specific expression pattern of the endogenous FGFR3 gene in vitro. These data suggest that other undefined mechanisms exist to regulate the expression of the FGFR3 minimal promoter in vivo.Due to the increased mutability associated with 5-methylcytosine, the conservation of the CpG island at the 5′ end of the FGFR3 gene suggests that it plays some important regulatory role in vivo. One possible way in which these sequences might regulate gene expression in a tissue-specific manner is through the methylation of any of the 83 CpG dinucleotides found within the −220 to +609 region. The establishment of methylation patterns during development (56Li E. Bestor T.H. Jaenisch R. Cell. 1992; 69: 915-926Google Scholar) is required for embryo viability, and has been shown to regulate the transcriptional activity of many genes by either directly interfering with the binding of transcription factors to their DNA cognates (57Watt F. Molloy P.L. Genes Dev. 1988; 2: 1136-1143Google Scholar, 58Iguchi-Ariga S.M. Schaffner W. Genes Dev. 1989; 3: 612-619Google Scholar, 59Iannello R.C. Young J. Sumarsono S. Tymms M.J. Dahl H.H. Gould J. Hedger M. Kola I. Mol. Cell. Biol. 1997; 17: 612-619Google Scholar) or by recruiting methyl binding transcriptional repressor proteins (60Nan X. Campoy F.J. Bird A. Cell. 1997; 88: 471-481Google Scholar, 61Boyes J. Bird A. Cell. 1991; 64: 1123-1134Google Scholar). Unlike the hypomethylated state of most CpG islands, preliminary studies3 in which we examined the methylation status of the FGFR3 CpG island in numerous tissues, as well as the transcriptional activity of in vitro methylated reporter constructs, suggested that methylation may be a contributing factor to the tissue specificity exhibited by the FGFR3 minimal promoter in vivo. The promoter for the FGFR3 gene resides in a CpG island that lacks the classical CAAT box and TATA box motifs found in many eukaryotic promoters. Sequence analysis of the FGFR3 promoter revealed a number of transcription factor binding sites, including five classical Sp1 sites, within the first 200 bp 5′ of the transcription start site. The positioning of the basal transcriptional machinery in a TATA-less promoter can occur independent of InR sequences when Sp1 binding sites are present (44Dennig J. Hagen G. Beato M. Suske G. J. Biol. Chem. 1995; 270: 12737-12744Google Scholar, 45Kollmar R. Sukow K.A. Sponagle S.K. Farnham P.J. J. Biol. Chem. 1994; 269: 2252-2257Google Scholar, 46Blake M.C. Jambou R.C. Swick A.G. Kahn J.W. Azizkhan J.C. Mol. Cell. Biol. 1990; 10: 6632-6641Google Scholar). In such instances, Sp1 is capable of stabilizing transcriptional initiation complexes approximately 50 bp downstream from an Sp1 binding site (45Kollmar R. Sukow K.A. Sponagle S.K. Farnham P.J. J. Biol. Chem. 1994; 269: 2252-2257Google Scholar). Mapping of the start site of transcription was achieved through RNase protection, and it was shown that transcriptional initiation occurs 22 bp 5′ from the end of the longest published mouse FGFR3 cDNA (GenBank accession no. M81342) and 57 bp 3′ of the most proximal Sp1 binding site. Our start site differs by only two nucleotides from that previously described by Perez-Castro et al. (47Perez-Castro A.V. Wilson J. Altherr M.R. Genomics. 1997; 41: 10-16Google Scholar) and both start sites are positioned such that Sp1 could facilitate organization of the transcription initiation complex. Through comparison to the published mouse FGFR3 cDNA, it was also determined that sequences encoding for the 5′-UTR are divided by a 376-bp intron. Our placement of the 5′ splice donor site 26 bp 5′ to that determined by Perez-Castro et al. (47Perez-Castro A.V. Wilson J. Altherr M.R. Genomics. 1997; 41: 10-16Google Scholar) is consistent with the 5′-UTR sequences of the published mouse FGFR3 cDNA. Alternative splicing or the use of a cryptic splice donor site could account for the differences between these two studies. The utilization of alternative splice donor and splice acceptor sites is known to occur in many of the FGFR genes (48Eisemann A. Ahn J.A. Graziani G. Tronick S.T. Ron D. Oncogene. 1991; 6: 1195-1202Google Scholar, 49Shi E. Kan M. Xu J. Wang F. Hou J. McKeehan W.L. Mol. Cell. Biol. 1993; 13: 3907-3918Google Scholar). cis-Regulatory sequences found within the CpG island were analyzed for transcriptional activity. Luciferase reporter constructs were transfected into four different cell lines, and the activity of the reporter gene examined. Constructs with as little as 100 bp (−126/−27) of 5′ cis-regulatory sequence still brought about a 20–40-fold increase in transcriptional activity. Within the CpG-rich sequence found between −126 and −27, there are two classical Sp1 binding sites. Deletions that remove the distal-most Sp1 site result in a 34–43% reduction in transcriptional activity, depending upon the cell line examined. Neighboring Sp1 sites frequently act synergistically (50Courey A.J. Holtzman D.A. Jackson S.P. Tjian R. Cell. 1989; 59: 827-836Google Scholar, 51Pascal E. Tjian R. Genes Dev. 1991; 5: 1646-1656Google Scholar). However, the data presented here, in conjunction with the known requirement of Sp1 for the formation of a transcription initiation complex in a TATA-less promoter, suggests that the sequence context of the FGFR3 promoter simply provides for additive effects mediated by Sp1. Although the transcriptional activity is dependent upon the 5′ proximal sequence, this activity is independent of cellular background in that it fails to mimic the expression profile of the endogenous FGFR3 gene. Finally, it should also be noted that binding sites for transcription factors not yet identified may also regulate FGFR3 promoter function through the −126/−27 promoter fragment. A detailed linker scanning analysis will be needed to identify such sites. In an attempt to find transcriptional enhancers, it was determined that addition of the FGFR3 UTR and intervening intron sequences could promote an 8–10-fold increase in transcriptional activity. To rule out the role of initiator region (InR) effects on the efficiency of transcriptional initiation, constructs that contained the endogenous initiation site were compared with the previously defined promoter constructs. These experiments showed that addition of the −26 to +10 FGFR3 sequence failed to affect transcriptional activity. To localize this enhancer activity, additional constructs were analyzed. Constructs that contained the 5′-UTR sequences but lacked intron I failed to result in significant transcriptional enhancement, whereas placement of the intron alone 3′ relative to the FGFR3 promoter sequences afforded the same transcriptional enhancement seen with the UTR/intron combination. These results demonstrated that the enhancer-like activity resides in intron I. Mapping of the intron enhancer activity to sequences between +340 and +395 identified two polypurine direct repeat sequence motifs. The sequence and organization of these motifs is similar to a motif previously identified in the EGFR promoter. From the studies of Johnsonet al. (42Johnson A.C. Jinno Y. Merlino G.T. Mol. Cell. Biol. 1988; 8: 4174-4184Google Scholar), it was determined that this site was capable of enhancing the transcription of the EGFR promoter in vitro. Through their studies, they also showed that these elements were sensitive to S1 nuclease and bound the Sp1 transcription factor. Although this site in the FGFR3 promoter is not sensitive to S1 nuclease, 3D. G. McEwen and D. M. Ornitz, unpublished observations. it does interact with Sp1-like DNA binding activity as shown through gel shift analysis. The specificity of this interaction was demonstrated through competition with the classical Sp1 (GC box) binding site and a non-classical Sp1 binding site derived from the promoter for the human EGFR. The classical Sp1 element is unrelated to the polypurine stretch; however, it was capable of competing for the DNA binding activity found in all but one of the resulting DNA-protein complexes. Furthermore, transfection studies in SL2 cells demonstrated that Sp1 could promote transcriptional activity through either the basal promoter alone or the promoter/enhancer combination whereas co-transfection studies demonstrated that both Sp1 and Sp3 can enhance transcription through the intron enhancer element. Together, these data suggest that binding sites for members of the Sp1 family of transcription factors, both proximal and distal to the start site of transcription, can work together to enhance transcription of the FGFR3 gene. The ability of proximal and distal Sp1 binding sites to synergistically regulate transcription has been observed by others (50Courey A.J. Holtzman D.A. Jackson S.P. Tjian R. Cell. 1989; 59: 827-836Google Scholar, 52Su W. Jackson S. Tjian R. Echols H. Genes Dev. 1991; 5: 820-826Google Scholar, 53Mastrangelo I.A. Courey A.J. Wall J.S. Jackson S.P. Hough P.V. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5670-5674Google Scholar). Through these studies, it has been shown that Sp1-Sp1 protein interactions can induce looping of the interveningcis-regulatory sequences. These proximal-distal interactions are hypothesized to regulate gene transcription by increasing the local concentration of Sp1 glutamine-rich activation domains near the start site. Such a model would explain the synergistic ability of the intron enhancer to regulate the transcriptional activity of the FGFR3 basal promoter. Although Sp3 has usually been shown to serve a negative regulatory role by competing for Sp1 binding sites, at least two other studies have demonstrated that SP3 can promote transcriptional activity (54Majello B. De Luca P. Lania L. J. Biol. Chem. 1997; 272: 4021-4026Google Scholar, 55Liang Y. Robinson D.F. Dennig J. Suske G. Fahl W.E. J. Biol. Chem. 1996; 271: 11792-11797Google Scholar). This transcription-promoting ability of Sp3 in our experiments may reflect the sequence-specific context of the binding site, as evidenced by the ability of Sp3 to transactivate the B fragment. Additional experiments will be required to demonstrate the in vivo role of Sp3 in FGFR3 promoter regulation. Transient transfection assays demonstrated that promoter activity resides in the CpG island found at the 5′ end of the FGFR3 gene, whereas an enhancer element was located in the first intron. However, this activity failed to parallel the cell-type specific expression of the endogenous FGFR3 gene. To assess whether or not the minimal promoter sequences defined above were capable of promoting cell-type specific expression in vivo, various lengths of regulatory sequences were used to generate transgenic animals. Surprisingly, analysis of the transgene expression in 13 independent transgenic lines demonstrated that the sequences between −220 and +609 provide the proper regulatory elements required for the expression of a reporter gene in a subset of the tissues that normally express the endogenous FGFR3 gene, whereas these same elements fail to limit the cell-specific expression pattern of the endogenous FGFR3 gene in vitro. These data suggest that other undefined mechanisms exist to regulate the expression of the FGFR3 minimal promoter in vivo. Due to the increased mutability associated with 5-methylcytosine, the conservation of the CpG island at the 5′ end of the FGFR3 gene suggests that it plays some important regulatory role in vivo. One possible way in which these sequences might regulate gene expression in a tissue-specific manner is through the methylation of any of the 83 CpG dinucleotides found within the −220 to +609 region. The establishment of methylation patterns during development (56Li E. Bestor T.H. Jaenisch R. Cell. 1992; 69: 915-926Google Scholar) is required for embryo viability, and has been shown to regulate the transcriptional activity of many genes by either directly interfering with the binding of transcription factors to their DNA cognates (57Watt F. Molloy P.L. Genes Dev. 1988; 2: 1136-1143Google Scholar, 58Iguchi-Ariga S.M. Schaffner W. Genes Dev. 1989; 3: 612-619Google Scholar, 59Iannello R.C. Young J. Sumarsono S. Tymms M.J. Dahl H.H. Gould J. Hedger M. Kola I. Mol. Cell. Biol. 1997; 17: 612-619Google Scholar) or by recruiting methyl binding transcriptional repressor proteins (60Nan X. Campoy F.J. Bird A. Cell. 1997; 88: 471-481Google Scholar, 61Boyes J. Bird A. Cell. 1991; 64: 1123-1134Google Scholar). Unlike the hypomethylated state of most CpG islands, preliminary studies3 in which we examined the methylation status of the FGFR3 CpG island in numerous tissues, as well as the transcriptional activity of in vitro methylated reporter constructs, suggested that methylation may be a contributing factor to the tissue specificity exhibited by the FGFR3 minimal promoter in vivo. We thank I. Boime, J. Gordon, and D. Towler for their insightful discussions. We also thank J. Azizkhan for help in establishing the SL2 cell culture system and C. Neville for help in assembling this manuscript.