From the Department of Pediatrics and Pathology, Washington University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
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Fibroblast growth factor-8 (FGF8) plays a
critical role in vertebrate development and is expressed normally in
temporally and spatially restricted regions of the vertebrate embryo.
We now report on the identification of regions of Fgf8
important for its transcriptional regulation in murine ES cell-derived
embryoid bodies. Stable transfection of ES cells, using a human growth hormone reporter gene, was employed to identify regions of the Fgf8 gene with promoter/enhancer activity. A 2-kilobase 5'
region of Fgf8 was shown to contain promoter activity. A
0.8-kilobase fragment derived from the large intron of Fgf8
was found to enhance human growth hormone expressed from the
Fgf8 promoter 3-4-fold in an orientation dependent manner.
The intronic fragment contains DNA-binding sites for the AP2, Pbx1, and
Engrailed transcription factors. Gel shift and Western blot experiments
documented the presence of these transcription factors in nuclear
extracts from ES cell embryoid bodies. In vitro mutagenesis
of the Engrailed or Pbx1 site demonstrated that these sites modulate
the activity of the intronic fragment. In addition, in
vitro mutagenesis of both Engrailed and Pbx1 sites indicated that
other unidentified sites are responsible for the transcriptional
enhancement observed with the intronic fragment.
The fibroblast growth factors
(FGFs),1 a family of secreted
proteins encoded by at least 18 genes in mammals, are mitogens and
morphogens for a variety of cell types and tissues (reviewed in Ref.
1). Despite the discrete FGF expression patterns, the molecular
mechanisms controlling the regulation of these genes are poorly
understood. Human FGF1 spans over 120 kb, contains three
protein coding exons, and at least four upstream untranslated exons
(2). Multiple FGF1 transcripts are generated by alternate promoter usage and tissue-specific splicing (3, 4). Murine Fgf3 contains three distinct promoters and two separate
polyadenylation sites that generate different mRNA, but all of them
encode the same protein (5-8). The Fgf4 gene has an
enhancer element in the 3'-noncoding region of exon 3, both in mice and
humans (9-12).
Fgf8 is primarily an embryonic growth factor that is
expressed in several regions of vertebrate embryos: i.e.
epiblast, primitive streak, surface ectoderm of branchial arches,
apical ectodermal ridge of the limb bud, isthmus of midbrain-hindbrain
junction, and forebrain/nasal placode (13-17). Fgf8 is also
weakly expressed in adult gonads, primarily in germ cells (18, 19).
This discrete temporal and spatial expression pattern of
Fgf8 is important for induction and patterning of the embryo
during gastrulation, limb development, and midbrain-hindbrain formation
(20-23). FGF8 is pathologically expressed in some human
breast cancer cell lines (24). Examining how Fgf8 expression
is regulated could provide an understanding of both physiological and
pathological actions of this growth factor.
Currently, little is known about the Fgf8 promoter. Its
5'-flanking region is very GC-rich and it is located 5 kb from the 3'
end of Npm3, which encodes a ubiquitously expressed member of the nucleoplasmin/nucleophosmin family (25). The transcriptional start site(s) for Fgf8 are unknown, but are inferred to be
within 300 bp upstream of the start codon. In the 5'-region of
Fgf8, there is no TATA box. Prior work demonstrated that the
2-kb 5'-region of Fgf8 did not direct expression of a
reporter transgene in a Fgf8-like expression
pattern.2 We therefore
postulated that DNA elements cis to Fgf8, but not included
in this 2 kb of upstream DNA sequence, might be important for
Fgf8 expression. Because of the presence of the
Npm3 gene 5 kb upstream of Fgf8 (25), we searched
for DNA sequences downstream of the beginning of Fgf8 that
were conserved between mice and humans. Small regions in the proximal
portion of the large intron are conserved between murine
Fgf8 and human FGF8 (26), despite the lack of
coding function for this region. This large intron lies between the
last exon of the cluster of first exons (exon 1D) and exon 2 (18).
There is no conservation between murine Fgf8 and human
FGF8 in the remainder of the large intron, and in other
introns (26).
Characterization of cis-regulatory elements in Fgf8 through
the usual transient transfection methods is complicated since Fgf8 is not normally expressed in established cell lines,
and hence these cell lines may lack appropriate transcription factors that regulate Fgf8 expression. Because transgenic approaches
are expensive, we sought a relevant in vitro system to
analyze potential Fgf8 regulatory regions. Prior work
demonstrated Fgf8 expression in murine ES cells
differentiated into embryoid bodies (27). In this paper we use a stable
transfection human growth hormone (hGH) reporter assay in ES cell
embryoid bodies to analyze Fgf8 DNA sequences for promoter
and enhancer activity. We find that the 2-kb 5'-region of
Fgf8 contains promoter activity, and that a 0.8-kb fragment
of the large intron enhances transcription of the reporter gene. We
show co-localization of expression of the endogenous Fgf8
gene and the reporter hGH gene in embryoid bodies derived from ES cells
transfected with the Fgf8-hGH reporter construct. DNA
sequence analysis, in conjunction with electrophoretic mobility shift
assays (EMSA), identify multiple transcription factors that bind to the
Fgf8 promoter and enhancer. In vitro mutagenesis
experiments demonstrate that although the Engrailed and Pbx1-binding
sites modulate the enhancer activity of the intronic fragment, other sites are responsible for most of the transcriptional enhancement seen
with the intronic fragment.
Plasmid Constructions--
The 1.8-kb
XhoI-XbaI fragment containing neomycin gene was
isolated from pPNT vector (28), made blunt with Klenow fragment, and
after addition of EcoRI linkers, inserted into the
EcoRI site of the promoter-less plasmid, p Mutagenesis--
Mutations in the binding sites for
transcription factors AP2, Pbx1 or Engrailed were introduced into the
plasmid, pGEM-7Zf(+), containing the 0.8-kb Fgf8 intronic
fragment subcloned into the SphI site, using the Quick
Change Site-directed Mutagenesis kit (Stratagene). The sequences of the
oligonucleotides used for mutagenesis were as follows (lowercase
letters denote mutant substitutions): forward primer for AP2-binding
site, 5'-GAAAGAGGATATGTCGaaAGGGCCCACAAAAGG-3'; reverse primer
for AP2 binding site, 5'-CCTTTTGTGGGCCCTttCGACATATCCTCTTTC-3'; forward
primer for Pbx1-binding site, 5'-GGGGCTGAGAACTGAccGATTTCTACAACATTC-3'; reverse primer for Pbx1-binding site,
5'-GAATGTTGTAGAAATCggTCAGTTCTCAGCCCC-3'; forward primer for
Engrailed-binding site
5'-GGAGAAGGAAAACTCTAggAAAATGTAATAAAATGTAATAAAAAGG3'; reverse
primer for Engrailed-binding site
5'-CCTTTTTATTACATTTTccTAGAGTTTTCCTTCTCC-3'. Oligonucleotides were
synthesized by Biosynthesis, Inc. (Lewisville, TX). Following
introduction of the AP2, Pbx1, or Engrailed mutation into the intronic
fragment of Fgf8 subcloned into the SphI site of
the pGEM-7Zf(+) vector, the mutated enhancer was subcloned into p DNA Sequencing--
The plasmids above were sequenced by the
thermocycle technique, using the fmol kit (Promega), and a
combination of plasmid and Fgf8 sequence-specific primers
(26). Sequences were read with the IBI-Kodak gel reader and MacVector
v5.0 software (IBI-Kodak).
Cell Culture--
CCE ES cells (29) were routinely cultured on
mitomycin C-treated STO feeder cells in culture medium consisting of
high glucose Dulbecco's modified Eagle's medium supplemented with
10% fetal calf serum, 10% newborn calf serum, 0.1 mM
2-mercaptoethanol, 0.1 mM non-essential amino acids, 2 mM L-glutamine, and 50 units/ml penicillin/streptomycin with addition of leukemia inhibitory factor at
a concentration of 1,000 units/ml (30). The cells were incubated in
humidified incubators at 37 °C and 5% CO2.
Transfection--
ES cells (1 × 10 7) were
electroporated with 25 µg of SalI-linearized targeting
plasmid (240 V, 500 microfarads, electrode distance 0.4 cm) as
described by Hogan et al. (55) and the cells were then
seeded into 100-mm plates. After 24 h, 250 µg/ml active G418 was
added and the cells cultured for 7-9 days. Antibiotic-resistant ES
clones were pooled and expanded at 125 µg/ml G418. The cells were
passaged once through leukemia inhibitory factor-containing media to
remove feeder cells. Then ES cells were differentiated in suspension
culture using the method of Doetschman et al. (30). Conditioned cell culture medium was changed every other day, and was
harvested at day 9 and assayed for hGH using a commercially available
radioimmunoassay (Nichols Institute Diagnostics}. Embryoid bodies
were washed twice with phosphate-buffered saline. Then they were
resuspended in 500 µl of ice-cold 0.25 M Tris-HCl, pH 7.5, and lysed by three freeze-thaw cycles (31). Total protein was
quantitated in supernatant fraction to normalize hGH levels to number
of embryoid bodies.
Gel Shift Analysis--
For gel shift assays, nuclear extracts
from day 9 embryoid bodies were prepared as described by Andrews and
Faller (32). Oligonucleotides, 21-44 bases in length containing
binding sites for transcription factors in the middle of sequence, were
designed based on known murine Fgf8 gene sequence and they
were made by Biosynthesis, Inc. (Lewisville, TX). Double-stranded
oligonucleotides were prepared (33) and radiolabeled with
[
Binding reaction mixtures (20 µl) contained 50,000 cpm of DNA probe,
10 µg of nuclear extract in 10 mM Tris-HCl, pH 7.5, buffer with 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 5% glycerol, and 1 µg of poly(dI-dC) to inhibit
nonspecific binding of the labeled probe to nuclear extract proteins
(34). For competition assays, a 5-100-fold molar excess of specific or
nonspecific oligonucleotides were included in each binding reaction.
Mutant oligonucleotides were bought from Santa Cruz Biotechnology, Inc.
if available or custom made by Biosynthesis (Engrailed mutant,
corresponding to mutant primer above). All mutants contain 2-bp
substitutions in the middle of the binding motif for the transcription
factor. Following 20-min incubation at room temperature, the binding
reactions were fractionated on 4% polyacrylamide gels run in 45 mM Tris borate buffer, pH 8.0, containing 1 mM
EDTA for 2 h at 200 V. The gels were subsequently dried and autoradiographed.
Protein Quantitation and Western Blot Assays--
Protein in
nuclear extracts and in the conditioned media after lysis of embryoid
bodies was quantitated with the Bradford assay kit (Bio-Rad). Western
blots on nuclear extracts were performed as described previously (35),
using a primary antibody to En1 and En2 (36), or a primary antibody to
Pbx1 (Santa Cruz Biotechnology, Inc.).
In Situ Hybridization--
Day 9 embryoid bodies were washed in
phosphate-buffered saline, fixed for 20 min in 4% paraformaldehyde in
phosphate-buffered saline, rinsed with phosphate-buffered saline
containing 10% sucrose, and frozen in OCT cryopreservation solution
(Tissue-Tek). 10-µm sections were subjected to in situ
hybridization with sense or antisense Fgf8 or hGH
riboprobes. Fgf8 probes (765 nucleotides, derived from the
FGF8b cDNA) were prepared as described previously (13). hGH sense
or antisense probes were prepared by in vitro transcription
of EcoRI-linearized pGEM vector (Promega) containing a
240-bp fragment of hGH exon V, kindly provided by D. Ornitz. Riboprobes
were labeled with [ Fgf8 Sequence--
We were interested in understanding the
regulation of Fgf8, a developmentally regulated FGF with
expression in discrete regions of the vertebrate embryo, but only
minimal expression in the gonads of the adult organism (13-19). We
cloned the murine Fgf8 gene in prior work (Fig.
1A) (18), and now report the
sequence of the 1462 bp upstream of the translation start codon
(GenBank accession number AF065607). Analysis of the sequence revealed
DNA-binding sites for the transcription factors Msx2 (562-568 bp),
CREB/ATF (814-819 bp), Egr1/Krox24 (971-979 bp and 1060-1069 bp),
AP2 (1068-1075 bp), and Sp1 (1072-1077) (Fig. 1B).
As we prepared to test two different 5'-regions for promoter activity,
we learned of the inability of this region to direct reporter transgene
expression in a Fgf8-like pattern in mouse embryos.2 We therefore sought to identify additional
Fgf8 sequence elements that might be important for
regulating Fgf8 expression. We previously cloned the human
FGF8 gene (26). The homology between the mouse and human
genes is very high in coding regions (85-90% at nucleic acid level),
but not in noncoding regions. The mouse and human noncoding sequences
were analyzed for short stretches (30-60 bp) of complete identity, as
we hypothesized that noncoding sequences conserved in mice and humans
might be important in gene regulation. We identified a 767-bp region of
the proximal portion of the large intron of Fgf8 that had
several well conserved stretches of DNA sequence when compared with the
corresponding region of human FGF8 (Fig. 1B).
This sequence is now reported in GenBank (accession number AF065608).
Analysis of this sequence revealed DNA-binding sites for transcription
factors AP2 (69-77 bp), Sp1 (293-300 bp), Pbx1 (305-312 bp), and
Engrailed (494-503 bp) (Fig. 1B).
Reporter Gene Analysis of Fgf8 Gene Elements--
To analyze
possible Fgf8 promoter/enhancer elements in
vitro, we sought to identify a cell line that expresses
Fgf8. Mouse ES cells made to differentiate into embryoid
bodies express Fgf8 by the eighth day of this process
(27).3 We therefore
constructed reporter plasmids containing the hGH gene with various
Fgf8 5'-regions ± intron region (Fig. 1C).
The larger 5'-region runs from EcoRV to the second
SmaI site, which is 6 bp upstream of the translation start
site (Fig. 1, A and B). The shorter 5'-region
runs from the EcoRV site to the first SmaI site,
and lacks the binding sites for AP2 and Sp1.
Following stable transfection into ES cells, the clones from each
transfection were pooled and allowed to differentiate into embryoid
bodies. The conditioned media from these embryoid bodies at day 9 were
analyzed for hGH protein by radioimmunoassay, and the results were
normalized to total protein present in the embryoid bodies. The shorter
5'-region had promoter activity in this assay (Fig.
2). However, the larger 5'-region had
significantly more promoter activity than the shorter 5'-region (Fig.
2), implicating Fgf8 DNA sequences between the two
SmaI sites in the 5'-region in the regulation of
Fgf8 expression. When the intronic fragment was placed
downstream of the hGH reporter gene in the forward orientation (Fig.
1C, constructs 5 and 7), very high level
expression was obtained (3-4-fold increase) (Fig. 2). In contrast,
placing the intronic fragment in the reverse orientation (Fig.
1C, constructs 6 and 8) did not
increase reporter gene expression above that observed without the
intronic fragment (Fig. 2). This intronic fragment by itself did not
have promoter activity, as hGH reporter gene expression in construct 4 was no higher than hGH reporter gene expression in the construct
lacking any Fgf8 sequence (Fig. 2).
Temporal and Spatial Analysis of Reporter Constructs--
With the
identification of potential DNA elements in Fgf8 that
stimulate reporter gene expression, we sought to determine if these
same elements directed expression of the reporter gene in a fashion
similar to the endogenous Fgf8 gene. Previously, it was
shown that steady state levels of the endogenous Fgf8 RNA could be detected by ribonuclease protection assays at days 10 and 18, but not days 0, 4, or 6 (27). We have subsequently demonstrated expression of Fgf8 at day 8 of development of embryoid
bodies {data not shown}. We therefore did a temporal analysis of
the hGH reporter gene expression from our highest expressing construct in Fig. 2, construct 5. As for the endogenous Fgf8 gene, we
did not detect reporter gene expression at day 0 and day 1 of
development of embryoid bodies (Fig.
3A). We detected low level
reporter hGH expression at day 3, that increased to full expression at
day 8 (Fig. 2). This reporter hGH expression pattern is similar to the
endogenous Fgf8 expression pattern seen in embryoid bodies (27), although expression levels of the reporter gene seem higher compared with the endogenous Fgf8 gene (see below).
We compared the spatial pattern of the hGH reporter gene expression
with the endogenous Fgf8 gene by in situ
hybridization at day 9 of development of embryoid bodies, using
constructs 3 and 5 (Fig. 1C). There was co-localization of
endogenous Fgf8 expression with reporter hGH gene expression
in the less eosinophilic cells of the embryoid bodies, compared with
expression in the more eosinophilic cells in construct 5 (Fig.
3B). In contrast, the reporter hGH gene was expressed in
both the less eosinophilic and the more eosinophilic cells in construct
3 (Fig. 3C). Although there was co-localization of the
reporter hGH gene with the endogenous Fgf8 gene in construct
5, the reporter gene was expressed at higher levels (note that
Fgf8 probe is 3 times the length of hGH probe), possibly due
to higher reporter gene copy number in the transfected cells. Despite
the differences in expression levels, the time course and
co-localization experiments (Fig. 3) demonstrate that the reporter gene
containing the intronic fragment has expression similar to the
endogenous Fgf8 gene.
Gel Shift and Western Blot Analyses--
To determine if the
identified DNA sequences in Fgf8 actually were occupied by
transcription factors present in nuclei of day 9 embryoid bodies, we
prepared nuclear extracts and performed EMSA. Specific gel shifts were
obtained when nuclear extracts from day 9 embryoid bodies were assayed
with radiolabeled oligonucleotides corresponding to the Msx2-,
CREB/ATF-, and Egr1/Sp1/AP2-binding sites in the 5'-region of
Fgf8 (Fig. 4). The gel shifts
were competed with non-radiolabeled authentic oligonucleotide, but not
mutant oligonucleotide (when available).
Similarly, specific gel shifts were obtained when the nuclear extracts
of day 9 embryoid bodies were assayed with radiolabeled oligonucleotides corresponding to the AP2, Sp1/Pbx1, and Engrailed DNA-binding sites in Fgf8 (Fig.
5). The AP2 and Sp1/Pbx1 sites were
competed with non-radiolabeled authentic, but not mutant, oligonucleotide (Fig. 5). The Engrailed site was fully competed by
authentic non-radiolabeled oligonucleotide, and partially competed by
an Engrailed mutant oligonucleotide, suggesting the possibility of
additional factors binding at adjacent sites in the oligonucleotide, or
some ability of a transcription factor in day 9 embryoid bodies to bind
to the mutated Engrailed site.
We were unsuccessful in our attempts to supershift complexes seen in
EMSA assays using antisera, and thereby confirm the identity of the
proteins binding to these sites in Fgf8. We therefore
performed Western blots to demonstrate that En1, En2, and Pbx1 were
present in day 9 embryoid extracts (Fig.
6).
In Vitro Mutagenesis--
To determine whether the AP2, Pbx1,
and/or Engrailed DNA-binding sites in the intron of Fgf8
were responsible for the enhancement of reporter hGH gene expression
seen in construct 5 compared with construct 3 (Fig. 1C), we
created two base mutations in these binding sites by polymerase chain
reaction methods. The mutated constructs were compared with the wild
type construct 5 in the ES cell stable transfection assay. Although
proteins bound to the AP2 site in the intron (Fig. 1B) in a
specific fashion (Fig. 5), mutation in this AP2-binding site did not
significantly affect expression of the reporter gene (Fig.
7). In contrast, a mutation in the
Engrailed-binding site of the intron (Fig. 1B) reduced hGH
expression to 45% of the wild type construct (Fig. 7). A mutation in
the Pbx1 DNA-binding site in the Fgf8 intron (Fig.
1B) reduced expression to 75% of the wild type construct
(Fig. 7). Surprisingly, a double Engrailed/Pbx1 mutant gave wild type
expression of the reporter gene (Fig. 7). As the intron increases
expression of the reporter transgene from 26 ng of hGH/mg of protein to
89 ng of hGH/mg of protein (Fig. 2, constructs 3 and 5), the mutation in the Engrailed-binding site removes 75% of the enhancing activity of
the intron. The mutation in the Pbx1-binding site removes 35% of the
enhancing activity of the intron. Mutations in both Engrailed- and
Pbx1-binding sites do not alter the ability of the intronic fragment to
increase reporter gene expression, and indicate that additional site(s)
are responsible for the transcriptional enhancement seen with the
intronic fragment.
In this paper, we identified promoter activity in the 2-kb
5'-region of Fgf8, spanning the region between the
EcoRV and SmaI sites (Figs. 1 and 2). We also
demonstrated that deletion of the 0.2-kb
SmaI-SmaI fragment in this 5'-region of
Fgf8 results in 44% reduction of hGH production (Figs.
1C and 2), reflecting a significant decrease in
Fgf8 promoter activity. Sequence analysis of this region of
Fgf8 identified binding sites for AP2 and Sp1 at the
SmaI site, which are destroyed when the 0.2-kb
SmaI-SmaI fragment is removed from longer
construct. Specific gel shifts were observed in EMSA with this region
of Fgf8 (Fig. 4). These results suggest that the AP2/Sp1
DNA-binding site in the Fgf8 5'-region is important for
transcriptional activity of Fgf8 in mouse ES cells.
Our data further indicate that a Fgf8 intronic fragment in
the proximal portion of the large intron regulates Fgf8
expression in mouse ES cells (Figs. 1-3). Specific gel shifts were
observed for the AP2-, Pbx1-, and Engrailed-binding sites of this
intronic fragment, when analyzed with nuclear extracts of day 9 embryoid bodies (Fig. 5). Pbx1, En1, and En2 are present in day 9 embryoid bodies as determined by Western blotting (Fig. 6). Although,
mutation of the AP2-binding site in the intronic fragment abolished
competition in the gel shift assay (Fig. 5), the mutation had no effect
on the Fgf8 intronic enhancing activity (Fig. 7). In
contrast, mutation of the Engrailed-binding site in the intronic
fragment markedly affected the ability of this region to compete with
the authentic binding site, but did not abolish competition (Fig. 5).
The same Engrailed mutation decreased the enhancing activity of the
Fgf8 intron by 75% in mouse ES cells (Fig. 7). A mutation
in the Pbx1-binding site that completely abolished competition in a gel
shift assay (Fig. 5) decreased the enhancing activity of the
Fgf8 intron by 35% in mouse ES cells (Fig. 7). In contrast
to the decrease in expression of the reporter gene seen with either the
Pbx- or Engrailed-binding sites, the Pbx1/Engrailed double mutant
showed a wild type level of expression (Fig. 7).
To explain our data, we propose the following model. First, we
hypothesize that there are activating sequences, presently unidentified, in the 0.8-kb intronic fragment that increase
Fgf8 expression. Second, the Pbx and En proteins modulate
the activity of the intronic activator sequence(s). If only Pbx
proteins, or only Engrailed proteins, are present, they inhibit the
activity of the intronic fragment and decrease Fgf8
expression. If both Engrailed and Pbx proteins are present, then they
interact with their binding sites and relieve the inhibition of each
other. The mechanism of this interaction, based on prior studies (see below), may well involve heterodimerization of Pbx and Engrailed proteins versus homodimerization.
The Engrailed proteins are members of a homeodomain-containing protein
family (37), encoded by two mammalian genes, En1 and
En2 (38). Both genes are expressed in posterior portions of
the midbrain-hindbrain junction and spinal cord in the mouse embryo
(38). Mice lacking En1 die at birth with severe
midbrain-hindbrain deletion, and rib and limb defects (39), while mice
lacking En2 survive with subtler cerebellar defects (40).
En2 can substitute for En1 in midbrain-hindbrain
function (41). En1 is essential for ventral limb patterning,
as loss of Engrailed function results in a dorsal-ventral patterning
defect of the limb (42). En1 was shown responsible for
limiting the ventral aspect of the apical ectodermal ridge expression
of Fgf8 (42). Additionally, En1 is expressed
prior to Fgf8 in the midbrain-hindbrain junction (isthmus)
(15, 43, 44), and may be responsible for localizing the expression of
Fgf8 to the cells of the isthmus (45).
The studies in the limb suggest that Engrailed represses
Fgf8 expression in this location (42). Our results in ES
cells suggest that Engrailed inhibits Fgf8 expression in the
absence of Pbx1. Engrailed proteins can heterodimerize with members of the Pbx family of homeodomain proteins (46, 47). Extradenticle, a
Drosophila homolog of the vertebrate Pbx proteins, has been shown to heterodimerize with homeodomain proteins and convert a
transcriptional repressor homeodomain protein (as a monomer or
homodimer) to a transcriptional activator (48). We identified single
Engrailed- and Pbx-binding sites in the Fgf8 intron that inhibit expression of Fgf8 in mouse ES cells, but that
together can allow full Fgf8 expression. We also demonstrate
binding of these elements by nuclear proteins present in mouse ES cell
embryoid bodies, and identify both Pbx and Engrailed proteins in day 9 mouse ES cell embryoid bodies by Western blotting. Our data support the
model that Engrailed heterodimerized with Pbx might activate transcription, while Engrailed or Pbx proteins alone might repress transcription (48).
An interesting finding in our work is the weak competition of the
Engrailed mutant site observed in EMSA, but not seen for any of the
other sites (Figs. 4 and 5). This weak competition may occur for two
reasons. First, the mutated site may still be bound by the
transcription factor, albeit in a much weaker fashion. Second, other
unidentified binding site(s) in this region may exist that is/are not
affected by the mutation. Evidence for the second possibility exists,
since one of the gel shifts observed is competed equally well by the
Engrailed mutant and wild type probes (open arrow, Fig.
5).
This report is the first example of transcriptional regulation by
intron sequences in the FGF family of genes, but numerous examples of
intronic regulation of transcription exist for other genes (49-52).
Traditionally, enhancer sequences are thought to be position and
orientation independent. The Fgf8 intron's ability to
enhance transcription shows orientation dependence, activating only in
the forward orientation (Figs. 1C and 2). Hprt
intronic sequences that regulate transcription in ES cells also show
similar orientation and position dependence (53).
Our use of stable transfection of ES cells, and the analysis of
reporter genes in embryoid bodies derived from these transfected ES
cells, allows one to analyze transcriptional control elements of
developmentally regulated genes in an appropriate physiological context
in vitro. Similar analyses can be performed in P19 cells following differentiation in vitro (54).
In summary, we have identified important DNA-binding sites in the
Fgf8 gene that regulate in vitro expression of a
reporter gene in embryoid bodies following transfection into ES cells. Future work will seek to identify the proteins that bind to these elements, and to generate transgenic and/or chimeric mice to test whether the identified Fgf8 regulatory elements are
necessary and sufficient for Fgf8 expression in
vivo.
INTRODUCTION
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Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
GH (Nichols
Institute Diagnostics). The 1.8- or 2-kb
EcoRV-SmaI fragment containing the 5'-region of
murine Fgf8 gene (Fig. 1), after addition of
BamHI linkers, was inserted in the forward orientation into
the BamHI site of p
GH with neomycin gene subcloned
previously. The 0.8-kb PstI-XhoI intronic
fragment from the Fgf8 gene (Fig. 1) was made blunt with T4
DNA polymerase, and subcloned in forward and reverse orientation into
the SphI site of p
GH vector downstream from the hGH gene, after adding SphI linkers. Promoter sequences and the
neomycin gene were then added to the constructs as described above.
GH,
at the SphI site as described above.
-32P]ATP, using polynucleotide kinase. The sense
(plus) strand sequences for the oligonucleotides in the 5'-region of
Fgf8 were as follows: Msx2,
5'-TTTGCCCCTGCTAATTGCTCAGAAGCA-3'; CREB/ATF,
5'-GCCCAAGGGGACGTCAGCACCGGGGC-3'; Egr1,
5'-CCACACCAGCCGCCCCCGCCCCTCCCGCGG-3'; and Egr1/AP2/Sp1, 5'-GCCCCGCCCCCGCCCCGGGCGGCGACCCGGC-3'. The sense (plus) strand sequences for the oligonucleotides in the intron were as follows: AP2,
5'-GAAAGAGGATATGTCGCCAGGGCCCACAAAAGG-3'; Sp1/Pbx1,
5'-GGGGCTGAGAACTGATTGATTTCTACAACATTC-3'; Engrailed,
5'-GGAGAAGGAAAACTCTATTAAAATGTAATAAAATGTAATAAAAAGG-3'.
-33P]UTP (ICN or Amersham) and SP6,
T3, or T7 RNA polymerases (Promega) in in vitro
transcription reactions as described (13). Hybridization, washing,
exposure to emulsion, counterstaining, and photography were as
described (13).
RESULTS
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Fig. 1.
Fgf8-hGH reporter constructs.
The murine Fgf8 gene is represented in A. An
upstream repetitive element that separates Npm3 from
Fgf8 is indicated by , noncoding Fgf8 exons by
, exons coding for all isoforms by
, and alternatively spliced
exons by
. The promoters, S and L, and the
downstream enhancer, E, are shown in B. They are
directly under their locations in the Fgf8 gene depicted in
A. The DNA-binding sites in Fgf8 for AP2,
CREB/ATF, Egr1, Engrailed (En), Msx2, Pbx1, and Sp1 are
indicated in B. In C, the reporter constructs
used in the experiments are indicated. Arrows indicate the
orientation (5'
3') of the hGH and enhancer elements relative to
the promoters.
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Fig. 2.
Intronic region of Fgf8
enhances transcription from hGH reporter gene. The reporter
constructs, numbered as shown in Fig. 1C, were analyzed in
mouse ES cell embryoid bodies. The results are presented as nanograms
of hGH (determined by radioimmunoassay) normalized to the amount of
protein (in mg) of the sample. The error bars represent the
standard error of the mean, based on four separate experiments with
duplicate samples in each experiment.
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Fig. 3.
Fgf8-hGH reporter construct mimics endogenous
Fgf8 in mouse ES cell embryoid bodies.
A, time course of reporter gene activity versus
day of differentiation of embryoid bodies in mouse ES cells transfected
with construct 5. The results are presented as in Fig. 2, and the
error bars represent the standard error of the mean, based
on two separate experiments with duplicate samples in each experiment.
Prior studies have demonstrated Fgf8 steady-state RNA
detectable in 8-day embryoid bodies (27). B, in
situ hybridization pattern of day 9 ES cell embryoid bodies
transfected with construct 5. The left figure is a bright
field photomicrograph, the center figure is a dark field
photomicrograph of the left figure, hybridized to antisense
Fgf8 cDNA probe (AS Fgf8), and the
right figure is an adjacent section to the left figure,
hybridized to antisense hGH probe (AS hGH). Expression of
both the endogenous Fgf8 gene and the Fgf8-hGH
reporter genes are primarily in lighter staining cells of the embryoid
body. C, in situ hybridization pattern of day 9 ES cell embryoid bodies transfected with construct 3. Panels are the
same as in B. The endogenous Fgf8 gene is
expressed in lighter staining cells as in B, but the
reporter hGH is expressed in both lighter and darker staining cells at
similar levels. In panels B and C,
bars in the upper right corner of the
photomicrographs represent 10 µm. The exposure was carried out for 7 days in panel B, and for 3 days in panel C.
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Fig. 4.
Identification of transcription
factor-binding sites in Fgf8 promoter. EMSAs were
performed on nuclear extracts from day 9 ES cell embryoid bodies, using
radiolabeled oligonucleotides corresponding to the Msx2-, CREB/ATF-,
and Egr1/Sp1/AP2-binding sites in the Fgf8 promoter region
(Fig. 1B). Solid arrows at left of
gels indicate specific gel shifts that are competed with
non-radiolabeled authentic oligonucleotide (self), but not with
non-radiolabeled oligonucleotide mutated at the binding site indicated.
Dashes (- -) in the competitor row refer to no competitor,
while numbers refer to the fold excess of non-radiolabeled
oligonucleotide used as competitor. Zero (0) in the extract
refers to no added extract, where plus (+) refers to lanes
with nuclear extract added.
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Fig. 5.
Identification of transcription
factor-binding sites in Fgf8 enhancer. EMSAs were
performed as described in the legend to Fig. 4, with radiolabeled
oligonucleotides corresponding to the AP2, Sp1/Pbx1, and Engrailed (En)
DNA-binding sites in the intronic enhancer of Fgf8 (Fig.
1B). The gels are labeled as in Fig. 4. Open
arrows at the left of the gels indicate specific gel
shifts that are competed equally well with authentic and mutated
oligonucleotides.
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Fig. 6.
En1, En2, and Pbx1 transcription factors
present in day 9 embryoid bodies. Western blots of nuclear
extracts, probed with antisera against Engrailed proteins (left
panel) or Pbx1 (right panel). The 55-kDa En1 and 41-kDa
En2 proteins are indicated in the left blot, while the
53-kDa Pbx1 protein is indicated in the right blot. The
protein molecular weight sizes are indicated in kilodaltons between the
blots.
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Fig. 7.
Mutation in Engrailed- and Pbx1-binding sites
of intronic enhancer inhibit Fgf8-hGH reporter gene
activity. Construct 5 in Fig. 2 was in vitro
mutagenized to disrupt the AP2-, Engrailed (En)-, and/or
Pbx1-binding sites. The resulting mutated constructs were compared with
wild type Fgf8-hGH reporter genes, including the intron
(wt), or lacking the intron (no intron), in day 9 ES cell embryoid bodies, as in Fig. 2. The results are presented as
nanograms of hGH (determined by radioimmunoassay) normalized to the
amount of protein (in mg) of the sample. The error bars
represent the standard error of the mean, based on a minimum of two
separate experiments with duplicate samples in each experiment.
DISCUSSION
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ACKNOWLEDGEMENTS |
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We thank A. Joyner for the gift of Engrailed antisera, G. Martin for communication of unpublished results, M. Bielinska for technical advice regarding ES cells, and D. Wilson and L. Muglia for critical review of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant R01 CA 70106 (to C. A. M.) and by a MOD Basil O'Connor Starter Scholar Award (to C. A. M.).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: Washington University
School of Medicine, Dept. of Pediatrics, Campus Box 8116, One
Children's Place, St. Louis, MO 63110. Tel.: 314-454-2547; Fax:
314-454-2685; E-mail: macarthur{at}kids.wustl.edu.
2 G. Martin, personal communication.
3 J. Gemel, C. Jacobsen, and C. A. MacArthur, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: FGF, fibroblast growth factor; kb, kilobase(s); bp, base pair(s); hGH, human growth hormone; EMSA, electrophoretic mobility shift assay.
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REFERENCES |
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