From the Department of Molecular Biology and Pharmacology, Washington University Medical School, St. Louis, Missouri 63110
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ABSTRACT |
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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 these cis-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.
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INTRODUCTION |
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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)1 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 (1, 2). To date, 15 FGF ligands have been described (3, 4). 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 (5). 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 (6). 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 (3, 7). Recently, both gain-of-function and loss-of-function mutations in the FGFRs have revealed unique roles for these receptors during development (8-13). Specifically, point mutations in FGFR3 have been genetically linked to achondroplasia, thanatophoric dysplasia, and hypochondroplasia (8); 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 (14). 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 (15, 16). 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 (10, 12), as well as deafness due to defects in the development of the organ of Corti (10). 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.
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EXPERIMENTAL PROCEDURES |
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Genomic Locus Isolation--
phage isolates encompassing the
5' end of the FGFR3 gene were previously obtained using standard
techniques (17). Identification of the locus was confirmed by
dideoxy-terminator sequencing. The insert was cloned as two
NotI fragments (p113.1.2 and p113.3A1214) and subsequently
used to generate both luciferase and
-galactosidase reporter
constructs as described below.
Luciferase Reporter Construction--
To generate
p(2951/
27)FR3-luc (c1), p113.1.2 (a NotI
phage subclone encompassing the 5' end of the FGFR3 gene) was digested with BssHII and blunted. A 3-kilobase pair fragment was then
excised with SalI and cloned directionally into pGL2-Basic
(Promega Corp.) between XhoI and a blunted NheI
site. p(
1537/
27)FR3-luc (c3) and p(
220/
27)FR3-luc
(c4) were subsequently generated by deletion of either a
NcoI/Asp718 fragment or
SacI/Asp718 fragment, respectively, from
c1 (see Fig. 1A for map positions). The
BamHI/BglII fragment from c1 was
inserted into the BglII site of pGL2-Basic to generate p(
2311/
27)FR3-luc (c2). p(
1537/
220)FR3-luc
(c5) was constructed by deleting the
SacI-BssHII fragment from c3.
p(
175/
27)FR3-luc (c6), p(
126/
27)FR3-luc
(c7), and p(
79/
27)FR3-luc (c8) constructs were assembled by polymerase chain reaction using one of three specific
sense oligonucleotides (
175 (DM64), 5'-CGG GGT ACC CTC CGC CCC AGC
TGG GCT CC-3';
126, 5'-CGG GGT ACC CCT GAC CAC GCC TCT TCG GA-3';
79, 5'-CGG GGT ACC GGA AGG GGA GTG TTC GGG GC-3') and a common
antisense oligonucleotide (DM67, 5'-GGA AGA TCT GGC TCC AGA GCG GGG GCC
GC-3'). Amplified products were digested with Asp718 and
BssHII and cloned into pGL2-Basic restricted with the same
endonucleases.
Heterologous Promoter Plasmids--
pRSV(i)-luc and p(i)RSV-luc
were generated by cloning the intron (i) into both the
HindIII and XhoI sites of pRSV-luc (18), respectively. The intron was subdivided into overlapping 60-bp fragments by polymerase chain reaction amplification. The
oligonucleotides used for polymerase chain reaction amplification were
as follows: (sense), 5'-GTC GAC GCG TGT GAG TTG GGC TCT AG-3';
(antisense), 5'-CTC GAG ACC TCC GAG TGC CAG G-3';
(sense), 5'-GTC
GAC GCG TGT GTG AAG GAC TCG CC-3';
(antisense), 5'-CTC GAG ACC CCC
TGA CTG CTT C-3';
(sense), 5'-GTC GAC GCG TTC CGG GCG GGC TCA G-3';
(antisense), 5'-CTC GAG TAC GAA CCG CTC CAC-3';
(sense), 5'-GTC GAC GCG TGA GAT ATG CGG GAA G-3';
(antisense), 5'-CTC GAG CCC GCG
CCT TGC CC-3';
(sense), 5'-GTC GAC GCG TAG TGC GGC GAG GCC G-3';
(antisense), 5'-CTC GAG CAG TAG CCG CAA ACT T-3';
(sense), 5'-GTC GAC GCG TCT TCC CGC TCT GGA A-3';
(antisense), 5'-CTC GAG
GTT GCC GCA GAG CC-3';
(sense), 5'-GTC GAC GCG TAC CTC CGT CCT GGG
AG-3';
(antisense), 5'-CTC GAG CGC GGT TCC TCC CTC C-3';
(sense), 5'-GTC GAC GCG TCA GGG AGG GAA GGA GG-3';
(antisense), 5'-CTC GAG CTG GCC TGG CGC CGG-3'; o (sense), 5'-GTC GAC GCG TGC TGG
GAG GAG GCG G-3'; o (antisense), 5'-CTC GAG CCG CCC GCC GAC TCC-3';
(sense), 5'-GTC GAC GCG TCC CGG GAG AGA GCT A-3';
(antisense),
5'-CTC GAG CTC GCT CCT GAA CC-3';
(sense), 5'-GTC GAC GCG TGG CGG
CCG GGG GTC GG-3'; and
(antisense), 5'-CTC GAG CTA CAG GAG GAG
AGC-3'.
Expression Plasmids and Other DNA Reagents--
The expression
plasmid pPACSp1 (19) was a generous gift of R. Tjian, and
the pPACUSp3 (20) expression plasmid was provided by G. Suske. The Sp1-responsive, dihydrofolate reductase-luciferase reporter
was kindly provided by J. Azizkhan. The FGFR3 (14) and mouse rpL32 (21)
RNase protection probes were as described previously. The RNase
protection probe for -galactosidase was generated by subcloning the
EcoRV-ClaI fragment of
-galactosidase into
pBluescript KS (Stratagene, Inc.).
RNase Protection Analysis--
Gene expression was determined by
RNase protection analysis (22) with the following modifications. Each
RNA sample was hybridized with a mixture of 5 × 105
cpm FGFR3, 5 × 105 cpm -gal, and 0.5 × 105 cpm rpL32 RNA probes. Single-stranded RNA probes, as
well as duplex RNA hybrids, were purified with RNAzol B (Teltest, Inc.) as described by the manufacturer. The expected sizes for the protected fragments are as follows: FGFR3, 430 nt;
-gal, 288 nt; and rpL32, 179 nt.
Cell Culture and Transfection--
NBT-II, CFK2, and RCJ (clone
3.1C5.18) cell lines were obtained from J. P. Thiery, J. Henderson, and J. Aubin, respectively. FGFR expression was determined
as described (23). All cells were transfected in triplicate using a
modified calcium phosphate precipitant (15). During each experiment, 5 µg of each DNA construct (double banded on a CsCl2
gradient) was co-precipitated with 0.5 µg of CScyto-gal (24, 25).
Sixty hours post-transfection, adherent cells were washed with 1 ml of
1 × phosphate-buffered saline, and subsequently assayed for
luciferase activity as described (26).
-Galactosidase activity was
determined with the Galacto-Light Plus system, as described by the
manufacturer (Tropix, Inc.). To normalize for transfection
efficiencies, luciferase values from each transfection were divided by
the corresponding
-galactosidase activities.
Nuclear Extracts and Electrophoretic Mobility Shift Assays
(EMSAs)--
Nuclear extracts were prepared according to the
protocol of Dignam et al. (27). Protein concentrations of
the extracts were determined with a modified Bradford assay (Bio-Rad,
Inc.). Finally, aliquots of the nuclear extracts were transferred to
silanized tubes, snap-frozen in liquid nitrogen, and stored at
80 °C.
Transgenic Mouse Constructs and Founder Generation--
Various
lengths of suspected 5' cis-regulatory sequences, all
terminating at the FGFR3 ATG codon (+612), were fused in-frame to a
nuclear localized form of the bacterial -galactosidase gene at the
initiation methionine. Constructs, double banded on CsCl2 gradients, were digested to remove all vector sequence, purified with
Qiaex II resin (Qiagen, Inc.), and resuspended in injection buffer (10 mM Tris·HCl (pH 7.5), 0.2 mM EDTA) at a
concentration of 5 ng/µl. Prior to injection, samples were filtered
through a 0.2-µm membrane. DNA was injected into the male pronucleus
of FVB/N embryos, and founder animals were identified as described previously (30). Founder animals were bred to wild type FVB/N mice, and
the resulting offspring were analyzed for expression of the transgene.
With the exception of p(
2951/+609)FR3nlacZ construct, multiple
independent transgenic lines were analyzed to control for insertion
site effects on transgene expression.
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RESULTS |
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Identification of a CpG Island at the 5' End of the FGFR3 Gene-- Genomic DNA encompassing the 5' end of the FGFR3 gene was isolated using a cDNA-derived DNA probe. Three kilobases of DNA extending 5' from the end of the published FGFR3 cDNA and the complete 5'-untranslated region (UTR) were sequenced. Analysis of genomic sequences encompassing the 5' end of the FGFR3 gene identified a CpG island (Fig. 1A), whereas comparisons with the cDNA sequence (GenBank accession no. M81342) identified a 376-bp intron within the 5'-UTR sequences. The intron/exon organization of the 5' end of the FGFR3 gene as well as the donor and acceptor splice sites are indicated in Fig. 1 (A and B). To determine the start site of transcription, two fragments (p222a2 and p222b3) corresponding to genomic DNA that overlaps the 5' end of the published FGFR3 cDNA were used to synthesize RNase protection probes (Fig. 1A). Both probes mapped the major start site of transcription to the first cytosine in the sequence, 5'-CACTGT-3', denoted as +1 in Fig. 1 (B and C). The 5' proximal sequence corresponding to the CpG island, as well as the start site of transcription, is shown in Fig. 1B.
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Basal Promoter Activity of the FGFR3 CpG Island--
To
demonstrate the transcriptional potential of the sequences near the 5'
end of the FGFR3 gene, various fragments of proximal sequence were
cloned upstream of the firefly luciferase reporter gene (luc). A 5'
deletion series with a fixed 3' end at the 27 position was generated
through restriction endonuclease digestion. The constructs were
transfected into a series of different cell lines that either express
(NBT-II and CFK2 cell lines) or fail to express (NIH/3T3 and RCJ cell
lines) the endogenous FGFR3 gene. All deletion constructs tested were
capable of inducing a 20-40-fold increase in luciferase activity from
a promoter-less vector (pGL2-Basic) (Fig.
2A). Nevertheless, this
activity, at least in transient transfection assays, is independent of
cellular background and fails to mimic the expression pattern of the
endogenous gene.
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Localization of Enhancer Elements in FGFR3 Intron I--
The
transcriptional activity of basal promoters is often regulated by
enhancer elements found at some distance from the start site of
transcription (37). To screen for potential enhancer-like elements,
constructs were generated that contained the sequences encoding the
5'-UTR and the first intron. Addition of the sequences between 26 and
+609 to the
2951/
27 promoter fragment resulted in an 8-fold
increase in reporter gene expression in NBT-II cells (Fig.
3A, compare c9 to
c1), as well as in NIH/3T3 and CFK2 cell lines (data not
shown). Furthermore, this enhancer-like activity requires only the
minimal promoter elements, as evidenced by its ability to
trans-activate promoter elements between
220 and
27 to
the same extent as larger segments of cis-regulatory
sequence (Fig. 3A; compare c9, c10,
and c11 to c1, c3, and c4,
respectively). Sequences between
27 and +609 were also capable of
enhancing the transcriptional activity of the
175/
27,
126/
27,
and
79/
27 promoter fragments (data not shown).
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Mapping Enhancer Activity to Polypurine Stretches in Intron
I--
Although the intron preferentially enhances transcription from
the 3' position, manipulation of the intron sequences, when in the 3'
position, could affect the efficiency of mRNA splicing, mRNA
stability, and/or proper translational initiation of the luciferase
reporter. To avoid these potential problems and still identify the
cis-acting enhancer elements found in intron I, the transcriptional potential of a series of overlapping 60-bp fragments derived from the intron was assessed in the 5' position, relative to
the RSV minimal promoter. Three overlapping fragments, +310/+368, +339/+395, and +369/+427 (henceforth designated ,
, and o,
respectively), were capable of enhancing reporter activity in both CFK2
(Fig. 5B) and NBT-II cells
(data not shown), whereas all other fragments showed little or no
stimulatory activity in both cell lines. To further refine this
analysis, subdivision of the
fragment into three overlapping
30-mers, designated A, B, and C in
Fig. 5A, was conducted. Although all three fragments could
enhance the transcriptional activity of the RSV minimal promoter, only
fragment B (+354 to +384) could promote transcriptional activity that
was comparable to either the complete intron or to the
fragment (Fig. 5C).
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Sp1-like Molecules Bind Polypurine Enhancer Elements--
To
determine the potential role of Sp1 in regulating the expression of
FGFR3, EMSAs were utilized to characterize the trans-acting factors that bind to both the and B fragments. Three independent DNA-protein complexes are observed when nuclear extracts, prepared from
the NBT-II cells, are incubated with the
fragment from intron I
(Fig. 6A, lane 2).
Although the slowest migrating complex is competed away by a
nonspecific competitor (
fragment in Fig. 6A, lanes
3-5), two specific DNA-protein complexes were identified (Fig.
6A, lanes 7-10). These complexes were disrupted
through competition with as little as 10-fold molar excess of unlabeled probe (Fig. 6A, lanes 8-10). Significant
competition for the binding of nuclear factors is also observed with
the B fragment (Fig. 6A, lanes 11-14) and C
fragments (data not shown) of
, whereas the A fragment requires
5-10-fold higher concentrations to compete effectively (data not
shown).
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Sp1 Family Members Regulate Transcription of the FGFR3 Promoter in
SL2 Cells--
To confirm the trans-activation of the
promoter sequences by members of the Sp1 family of transcription
factors, the Drosophila SL2 cell line was utilized. SL2
cells lack Sp1-like activity and have been used by others to directly
assess the transcriptional role of Sp1 and Sp1-like family members (19,
33, 34, 44). To determine if Sp1 could regulate the transcriptional
potential of the FGFR3 promoter, p(2951/
27)FR3-luc (c1)
and p(
2951/+609)FR3-luc (c9) constructs were
co-transfected with an expression vector for Sp1. Expression of Sp1
resulted in a 13- and 25-fold increase in the transcriptional activity
of
2951/
27 and
2951/+609 promoter fragments, respectively (Fig.
7A). This regulatory activity
is comparable to the 11-fold stimulation seen with the Sp1-responsive dihydrofolate reductase promoter (33) (Fig. 7A). To examine whether Sp1 or Sp3 could regulate expression through the A, B, and C
fragments various RSV-luc reporter constructs were transfected into SL2
cells along with expression vectors for either Sp1 or Sp3. Both Sp1 and
Sp3 weakly stimulated the activity of pA-RSV-luc and pC-RSV-luc,
whereas pB-RSV-luc was stimulated 15-fold and 12-fold by Sp1 and Sp3,
respectively. These data, and the ability of Sp1 consensus competitors
to compete for binding of nuclear proteins to the intron enhancer
elements, demonstrate that members of the Sp1 family of transcription
factors can promote transcription of the FGFR3 promoter.
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Minimal cis-Regulatory Sequences Confer Tissue-specific Expression
in Vivo--
The reporter constructs containing various FGFR3 promoter
fragments direct transcription in all four cell lines tested,
regardless of whether the endogenous FGFR3 gene is expressed (Fig.
2A). However, FGFR3 is expressed in a very cell-specific
pattern in vivo (14). To examine the tissue-specific
expression pattern of the defined promoter sequences, various reporter
constructs were used to generate transgenic animals. RNase protection
assays were used to simultaneously monitor the expression of the
endogenous FGFR3 gene, the transgene reporter (-gal), and a
ubiquitously expressed ribosomal RNA molecule (rpL32). The endogenous
gene is detected to some extent in all tissues examined (Fig.
8). However, the reporter gene is only expressed in a subset of these tissues in all lines examined. Comparisons of reporter gene expression between independent transgenic lines derived with increasing lengths of 5' cis-regulatory
sequences demonstrated that the elements found between
220 and +609
are sufficient to direct the tissue-specific expression of a reporter gene in lung, liver, small intestine, kidney, and skin (Fig. 8, compare
220/+609 to
15 (kilobase pairs)/+609). Furthermore, 3 of the 13 lines examined also expressed
-gal mRNA in the brain. However,
the inconsistent nature of this expression suggests that it results
from insertion site effects. The ability of these proximal sequences to
promote the tissue-specific expression of the reporter gene in
vivo demonstrates that much of the tissue-specific regulation of
the FGFR3 gene is controlled by the regulatory elements found between
220 and +609.
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DISCUSSION |
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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 (44-46). In such instances, Sp1 is capable of stabilizing transcriptional initiation complexes approximately 50 bp downstream from an Sp1 binding site (45). 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. (47) 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. (47) 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 (48, 49).
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 (50, 51). 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 Johnson et al. (42), 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,3 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 (50, 52, 53). Through these studies, it has been shown that Sp1-Sp1 protein interactions can induce looping of the intervening cis-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 (54, 55). 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 (56) 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
(57-59) or by recruiting methyl binding transcriptional repressor
proteins (60, 61). 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant CA60673 and a grant from Monsanto/Searle, Inc.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. E-mail:
dornitz{at}pharmdec.wustl.edu.
1
The 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); -gal,
-galactosidase; EMSA,
electrophoretic mobility shift assay; InR, initiator region.
2 D. G. McEwen, M. Naski, D. Towler, and D. M. Ornitz, manuscript in preparation.
3 D. G. McEwen and D. M. Ornitz, unpublished observations.
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REFERENCES |
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