Vol. March
183,
No.
16,
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1992
Pages
688-693
CHARACTERIZATION OF THE PROMOTJXRREGION OF TIE MURINE FIBROBLAST GRORTEIFACTOR RRCEPTOR 1 GENE Hiroshi
Saito, Haruhiko Kouhara, Soji Kasayama, Tadamitsu Kishimoto and Bunzo Sato’
Department of Internal Medicine III, Osaka University Hospital, Osaka 553, JAPAN
Received
January
27,
1992
Summary: To obtain some clue for the regulatory mechanism by which fibroblast growth factor (FGF) receptor 1 (FGFR1) gene is expressed, we have cloned the promoter region of this gene from genomic library of mouse FGF-responsive The genomic clone isolated here includes the FGFR1 gene from cell lines. position -868 to +697 relative to transcription initiation site. Sequence analysis reveals the presence of various consensus sequences for the binding sites of transcriptional factors such as SP 1, GCF, Ott-I, AP 1 and AP 2, but the absence of TATA and CAAT sequence motif. The transfection of this promoter-CAT constructs into NIH 3T3 cells demonstrates its promoter activity which is at least located between base -106 and +104. 0 1992 Academic
PESS,
Inc.
The basic and acidic role
in
important
fibroblast
growth factors
processes, including
cell
wound healing and promotion of differentiation
(aFGF and bFGF) play
proliferation, of various
cells
(1).
evidence
Recent
reveals that FGF family is also involved in the process of (2, 3). These actions are believed development and progression mediated by their
binding to cell-surfaced
tyrosine
receptors
kinase
identified fms-like
(4-7).
(flg)
related
cancer to be
which form a subclass of
Among these FGF receptors,
from chick embryo was closely gene
receptors
a
angiogenesis,
cDNA first
to a previously
published
(8) or Cek I gene (9). This first FGF receptor is Both aFGF and bFGF were found to be associated
designated as FGFR-1 (6). with FGFR 1 (10).
FGFR 1 is expressed in a variety of cells of mesenchymal, epithelial and neuroectodermal origin (1). However, the molecular mechanism for FGFR 1 to be expressed in target cells is unknown. In addition, the expression of some cell-surfaced up-regulated
receptor mRNAshas been reported to be down-regulated (12) by their corresponding ligand. Actually, our
* To whomcorrespondence should be addressed. 0006-291 X/92 $1.50 Copyright 0 1992 by Academic Press, Inc. All rights of reproductiorl in any form reserved.
688
(11) or previous
Vol.
183,
No.
study
2, 1992
showed
cells (SC-3) the molecular structural this region
that
BIOCHEMICAL
FGFR 1 mRNA levels
are up-regulated mechanism for
analysis
AND
BIOPHYSICAL
RESEARCH
in FGF-responsive
COMMUNICATIONS
mouse
mammary
tumor
in response to bFGF stimuli (13). To clarify regulating the expression of the FGFR 1 gene,
on the promoter
element
of this
gene is prerequisite.
report, we describe the isolation and characterization of FGFR l-encoding gene from the partial genomic
of the library
In promoter of SC-3
cells. HATERIALS AND HEIBODS Screening of a genomic DNA library. Genomic DNA isolated from SC-3 cells as described by Sambrook et al. (14) was digested with Sph I. The digested DNA was electrophoresed in 0.75% agarose gel and subjected to Southernblot analysis (14) using 32P-labeled Pst I-Pst I fragment (480 bp) from 5’noncoding region of SC-3 FGFR-1 cDNA (15) as probe. DNA hybridized with this probe (molecular mass of 3-5 kb) was extracted and inserted into Sph I site of pUC 19 vector. The constructed partial genomic library was transfected into E. coli DHSa, yielding 2x10= independent colonies. After transfer to nitrocellulose filters, this genomic DNA library was screened with the abovecited probe under the high-stringency condition [GxSSC (1xSSC is 0.15 M sodium chloride/l5 mM sodium citrate, 5xDenhardt’s solution PH 7)/ (1xDenhardt’s solution is 0.02% polyvinylpyrrolidone/ 0.02% Ficoll/0.02% bovine serum albumin)/O.l% (w/v) sodium dodecyl sulfate(SDS)/lOO ug/ml denatured salmon sperm DNA at 55’C for 18 h. The positive clones were subjected to second screening. The DNAs isolated from the positive clones were subcloned into pUC 19 and sequenced using the dideoxynucleotide chaintermination method (16). Amplification of FGFR 1 5’-flanking region in NIH 3T3 cells by polymerase chain reaction (PCR). Genomic DNA from NIH 3T3 cells was used as a template for PCR (17) to amplify the FGFR 1 5’-flanking DNA in another murine cell line. Each primer as indicated in Fig. 1 was synthesized. The reaction mixture contained 5 picomoles of each primer, 200 PM each of four deoxynucleoside triphosphates and 1 U of Taq polymerase (Perkin Elmer Cetus) in 50 ~1 of 10 mM Tris hydrochloride (pH 8.3) -50 mM KCl-1.5 mM MgC12-0.01% (w/v) gelatin. Thirty cycles consisting of denaturation at 94-C for 1 min, annealing at 55 ‘C for 1 min and extension at 72 ‘C for 1 min for primer 1 cycles consisting of denaturation at 94 ‘C for 90 s, and 3, and forty annealing at 55 ‘C for 30 s and extension at 75 ‘C for 3 min for primer 2 and 4 were performed to amplify the DNA, respectively. The amplified DNA was inserted into pUC 19 and sequenced as described above. Sl nuclease analysis. To identify the transcription initiation site, mapping by Sl nuclease digestion was carried out as reported previously (18). Total cellular RNA was isolated as described before (12). An aliquot (50 pg) of RNA in 20 1.11of the hybridization buffer containing 80% (v/v) formamide, 40 mM PIPES (pH 6.5), 1 mM EDTA and 0.4 M NaCl was mixed with 380 bp Pst I PstI fragment or 210 bp Pst I - Sma I fragment of SC-3 FGFR 1 genomic DNA which had been end-labeled with T4 polynucleotide kinase and [Y-~*P]ATP. The reaction mixture was heated for 15 min at 80 ‘C, followed by annealing at 55 -C or 60 ‘C for 12 h. After hybridization, 200 pl of the buffer containing 50 mM CH3COONa, 0.25 M NaCl, 4.5 mM ZnSO,, 100 pg of salmon sperm DNA per ml and 900 U of Sl nuclease per ml was added. This mixture was incubated at 37 ‘C for 30 min. The samples were ethanol-precipitated, electrophoresed on 6% polyacrylamide8 M urea gels, and autoradiographed for 15 h at -70 ‘C. Construction and transfection of the FGFR l-CAT chimeric gene. FGFR 1 5’-flanking region was digested with restriction enzymes and inserted into the Sma I site of pSVOOCAT (19). Each fusion gene (10 pg) and pSV- B galactosidase gene (10 vg) were co-transfected into NIH 3T3 cells by calcium phosphate precipitation method (20). After 48 h cultures in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% or 1% fetal calf serum,
689
Vol.
183, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
the cells were harvested and subjected to chloramphenicol acetyltransferase (CAT) activity as described previously (21). Materials. The materials used here were obtained from the following sources : restriction enzymes and Sl nuclease from Toyobo Co., Ltd (Tokyo, Japan) and Takara Co. Ltd (Kyoto, Japan): plasmids from American Tissue Culture Collection (Rockville, MD) and Pharmacia (Uppsala, Sweden); all 32Plabeled nucleotides from AmershamJapan (Tokyo, Japan); culture media from Nissui Pharmaceutical Co., Ltd (Tokyo, Japan). The other reagents were of analytical grade.
RESULTS AND DISCUSSION TO isolate
the
genomic library digested
promoter element of the FGFR1 gene,
prepared from the FGF-responsive SC-3 cells
DNAs with
As shown in Fig.
clone
that
indicated
sequence completely Fig.
1).
to
5’-noncoding
from our laboratory
resistant
out.
hybridization
to protect
region
of
site,
FGFR 1 cDNA
Sl nuclease mapping
position
+l,
analyzed primers
nuclease
with SC-3 or NIH 3T3 mRNA(Fig.
initiation
failed to show the presence of two sites, resolution at the high molecular mass region. initiation
sites.
and the nucleotide
the
+167 to +635 in
the probe from Sl nuclease digestion.
FGFR 1 gene contained two transcription
transcription
a
contained
sites.
2).
The Sl
mapping using the Pst I - Sma I fragment as probe indicated
nuclease
two
The Sph Iconstruct
Using two probes, we were able to observe Sl
fragments after
tRNA failed
which
(15) (from a position initiation
a
sequence of one positive
a DNA fragment
To determine the transcription
was then carried Yeast
1, the nucleotide
we had isolated identical
reported
(22).
a molecular mass of 3-5 kb were used to
genomic library.
previously
we screened
that
The longer
probably due to the One base was inserted
the
probe limited between
The 3’- most residue was designated sequence was aligned
(Fig.
the .promoter region of FGFR1 gene in NIH 3T3 cells.
1).
a
We also Using
four
which had been prepared according to the sequence described in
Fig.
1, two DNA fragments were amplified using NIH 3T3 genomic DNA as a template for PCR. The sequence analysis revealed that the nucleotide sequence from a
position -870 to +104 in NIH 3T3 cells is completely identical with that in SC-3 cells (data not shown). Examination of the murine FGFR 1 gene sequence around the transcription initiation sites reveals notable features. Within
this
However,
region,
neither
the consensus TATA box nor CAAT box was detected.
there
are four consensus sequences for the SP 1 binding site at positions, -246 , +48 , +392 and t416. The other consensus sequences for the transcription factors such as Ott-1, AP 1 and AP 2 were also present in this region. Interestingly, three GCF sites (C/GCGGGGC) which have been proposed to be necessary for epidermal growth factor receptor (EGFR) gene to be downregulated by EGF stimuli (23) were found to exist at positions, -117, +31 and In addition, the nucleotide sequence 3’ downstream from position +636 +120. was not present in the FGFR 1 cDNA, suggesting that an intron is localized in
690
Vol.
183,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
. . -600. . . . . . . . GCA~GCTTCATCAAAACATCGGGATTTCTCATCTCCAAGChCGATCAAAAATTTTTATTTTTTAACAATCTCAATATGATTCACCTCCACTGGACTCAT APl Prirper 1 . . . . . -100. . . . . CCATTAAACAGCCACACATTAACATCTCTCTGGAGGGACCTGGTGGGAAGGGCATTTCAGCGATTT~CTTTCAGGTTCATAGTATACTGTGGAGGCTCT . -600 . . . . . . . . GTTTCACTCTCATTAATTCCTAAGGCAAACAAAACAAAACAAGCAAGCAAGTAAACAAACATCCCCCCCATCCGAAAAAAAAAAAAAACAAAAACCCCT API . . -500. . . . . . . . CTCTCTTCGGTTCCTTCCTCAAAGCTGGTGATTTCTAACGCCT Ott-1 . . . . . . . -400. . . AATTATCTGCCTGGGGAAACTTCAGATCCAAGGAAAGGGTCTGAGGGCGAAAGTCAACCGATCCATCAATAAAAACAGTTCGTATATTGTAAGCTATGT Primer 2 Primer 3 . . . . . . . -300. . . ~GGTCCTATTTATTCCTTTTAGAGACGGGAAAACCCAGATTTAGAGCACGAAAATCATGACCCCAAGGCACAGAGATAACCAAAGACCAAGTTGCCCA AP2 . . . . . . . . -200. . TGCAACCTCAAAA?TATAGCCAGCAeCCCCCCfCCCCACCCTCCACTCCTGTCCCCAAATCGCTGATCTACACACAGTCTGAAATTCGGGCGCC~ SPl AP . . . . . . . -100. . . ~GAAAATTCAAGTTAGACCACACTCTACdCCACTTCTGCTCCCTCCACGCTTCTCCCCGCCTCCACCTTGACAACTACAAGCCCCAGCAGGCAACTG A?2 CCF . . . . . . . .+1 . . AACGGCAGCACCACGAGGTGCCACTACCCCCCGGTGCGAGTCACAGAGCGAGCCCTCGCGCCTCGCCCGCGCACAGCGCTCGCAGCGCTCCTGCGGGTA AA . . . . . . . .+100 . . TTTC~TCTCCCCTGC~CGCCCAACCCCACCCGGAACCCTCGTCCAGCCCCTGCCTCCACAGGACCCGGCCTCCCCAGGCAA~ GCF SPl Prlner 4 CCF . . . . . . . .+200 . . CAGACCTCCGCACTGGACTGAGACTCTCCTTAGCGCATTGCGGCCACCTCGCCTTTCCCGGCCGCGAGCGCGCGCCGCAGCTGGAAAAGCAGCGGAGAC 1 . . . . . . . .t300 . . GACGACTTTTCTCAGCTCCCAGGGGCGCACCACAGCCCTCCTGCAGTCAATGCACGCCGGAGCCCCACGAGGGGTGATCGGAACTCGGGCTGCCAGAAG . . . . . . . .t400 . . CTCAGACCCCCCCACCGCCCCCCCAGCGTACTGGAGAGCGGCGGGCGCA~C~~~~CCGG~~~~~GGC~GC~CG~~~~~~~~~~~CCC~CC~CCCCGCCT SP1 SPI . . . . . . . .+soo . .
.
.
.
.
.
.
.
. . . . . . ~gtaagagtcgcc~gcgcccgcagagccgggtggctgggcgtcccgagcggcccccgccgt---------
.
.+600
.
.
Fig.
1 Nucleotide sequence of the 5’-terminal region of the murine FGFR 1 gene. The nucleotide sequence was aligned as the 3’-residue of two transcription initiation sites (A) was designated a position +1 (see the text and Fig. 2). The potential SP 1, AP 1, AP 2, Ott-1 and GCF binding sites are underlined. The arrow indicates the 5’-end of the murine FGFR 1 cDNA obtained from the expression cDNA library in our laboratory (15). The region identical with PstI-PstI fragment of cDNA which was used for screening of the genomic library is also indicated t----l. The primer pairs (l-3 and 2-4) used for amplification of NIH 3T3 cell DNA (PCR) are indicated ( =) . The lowercase letters denote the possible introgenic sequence.
this region. This notion was further supported by the finding end of this possible introgenic sequence contains GT.
that
the
5’
Next, we attempted to confirm the promoter activity. Various portions of the 5’-flanking region fused with the coding region of CAT were transfected into NIH 3T3 cells and cultured in the serum-supplemented or starved inserted the
condition for 48 h (Fig. 3). When the 5’-flanking region was in a sense orientation, the marked CAT activity was demonstrated in transfected NIH 3T3 cells. The construct fused with CAT gene in an 691
Vol.
183,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
A
-378
-418/+104
P -270
h
-212 -868/+104
-1
+104/-868
+m
L
0 -104
02
-106/+104
03 Fig.
2
Transcription initiation mapping. The Pst I-Pst I fragment (probe 1) SC3 cell RNA at 55 * C or 60 * C (left fragment (probe 2) was also hybridized SC-3 or NIH 3T3 cells (right panel). right panel was obtained by using the
-868/+104
sites
iii
CAT
-1
determined
^'
by
Sl
i
I
nuclease
of the genomic clone was hybridized with panel). The Pst I-Sma I FGFR 1 genomic at 55 ‘C with yeast tRNA, or RNA from The sequence ladder depicted in the probe 2.
Fig.
3 Promoter activities of the murine FGFR 1 gene. Several DNA fragments from the 5’-flanking region of the mouse FGFR 1 gene were used to constract the murine FGFR 1 promoter -CAT chimeric genes. These constructs were transfected into NIH 3T3 cells, followed by culture for 48 h in a high (10%) (panel A) or low (1%) (panel B) serum condition. The SRo promoter in place of the FGFR 1 promoter was also used as a positive control. The arrow indicates the transcription initiation site (+l) and the 5’ to 3’ direction. The transfection efficiency was always adjusted by measuring the activity of B-galactosidase.
orientation failed to show the CAT activity. were also obtained even in the serum-starved condition
opposite
the
DNA sequences responsible
The similar results (Fig. 3B). Moreover, for the basal promoter activity of the murine
FGFR 1 gene were found to be localized at least between -106 and +104. The 5’-flanking sequences of various growth factor receptor genes have been analyzed. Interestingly, the promoter regions of the human EGFR, insulin-like growth factor -1 receptor and nerve growth factor receptor genes (24-26) were observed to lack the TATA as well as CMT box, but to contain the GC box, similar to those found in promoters of cellular housekeeping genes (27). The present study demonstrates that the murine FGFR 1 gene adheres to the general promoter structure identified in growth factor 692
Vol.
183,
No.
receptor
2, 1992
genes.
been found levels
Moreover,
to contain
are
cells (data is directed sequences
BIOCHEMICAL
the promoter
distinct
up-regulated
AND
regulatory
in SC-3 cells
BIOPHYSICAL
region
RESEARCH
COMMUNICATIONS
of the murine
FGFR 1 gene
elements.
Actually,
(13) but down-regulated
not shown) by bFGF stimuli. The current study in our toward identifying the biological role of these for
the expression
has
the FGFR 1 mRNA in
NIH
3T3
laboratory consensus
of the murine FGFR 1 gene. RElXRENCE!3
1. Gospodarowicz, D.J., Neufeld, G., and Schweigerer, L. (1986) Cell. Differ. 19, l-17. 2. Moore, R., Casey, G., Brookes, S., Dixon, M., Peters, G., and Dickson. (1986) EMBO J. 5, 919-924. 3. Taira, M., Yoshida, T., Miyagawa, K., Sakamoto, H., Terada, M., and Sugimura, T. (1987) Proc. Natl. Acad. Sci. USA 84, 2980-2984. 4. Lee, P.L., Johnson, D.E., Cousens. L.S., Fried, V.A., and Williams, L.T. (1989) Science 245, 57-60. 5. Houssaint, E.. Blanquet, P.R., Chambon-Arnaud, P., Gesnel. M.C., Torriglia, Y., Courtois, Y., and Breathnach, R. (1990) Proc. Natl. Acad. Sci. USA 87. 8180-8184. 6. Keegan, K., Johnson, D.E., Williams, L.T., and Hayman, M.J. (1991) Proc. Natl. Acad. Sci. USA 88. 1095-1099. 7. Partanen, J., Makela, T.P., Eerola, E., Korhonen, J., Hirvonen, H., CWelsh, L., and Alitalo, K. (1991) EMBOJ. 10, 1347-1354. 8. Ruta, M., Howk, R., Ricca, G., Drohan, W., Zabelshansky, M., Laureys, G.. Barton, D.E., Francke, U., Schlessinger, J., and Givol, D. (1988) Oncogene 3, 9-15. 9. Pasquale, E.B., and Singer, J.S. (1989) Proc. Natl. Acad. Sci. USA 86, 5449-5453. 10. Dianne, C.A., Crumley, G., Bellot, F., Kaplow, J.M., Searfoss, G., Ruta, M. I Burgess, W.H., and Schlessicger, J. (1990) EMBO J. 9, 2685-2692. 11. Izzo, Jr. N.J., Seidman, C.E., Collins, S., and Colucci, W.S. (1990) Proc. Natl. Acad. Sci. USA 87, 6268-6271. 12. Clark, A.J.L., Ishii, S., Richert, N., Merlino, G.T., and Pastan, I. (1985) Proc. Natl. Acad. Sci. USA 82, 8374-8378. 13. Saito, H., Kasayama. S., Kouhara, H., Matsumoto, K., and Sato, B. (1991) Biochem. Biophys. Res. Commun. 174, 136-141. 14. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular cloning : a laboratory manual. Cold Spring Habor Laboratory, Cold Spring Harbor,N.Y. 15. Kouhara, H., Kasayama, S., Saito, H.. Matsumoto, K., and Sato, B. (1991) Biochem. Biophys. Res. Commun. 176, 31-37. 16. Sanger, F., Nicklen, S., and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 17. Saiki. R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., and Erlich, H.A. (1988) Science 239, 48’7-491. 18. Berk, A.J., and Sharp, P.A. (1977) Cell 12, 721-732. 19. Araki, E., Shimada. F.. Shichiri, M.. Mori, M., and Ebina, Y. (1988) Nucleic Acids Res. 16, 1627. 20. Wigler, M., Pellicer, A., Silverstein, S., and Axel, R. (1978) Cell 14, 725-731. 21. Hirose, T., Koga, M., Saito, H., Kouhara, H., Sumitani, S., Kasayama, S., Matsumoto, K., and Sato, B. (1991) J. Steroid Biochem. Molec. Bio1.38,593-598 22. Kasayama, S., Sumitani, S., Tanaka, A., Yamanishi, H., Nakamura, N., Matsumoto, K., and Sato, B. (1991) J. Cell. Physiol. 148, 260-266. 23. Kageyama, R., and Pastan, I. (1989) Cell 59, 815-825. 24. Ishii, S., Xu, Y.H.. Stratton, R.H.. Roe, B.A., Merlino, G.T., and Pastan. I. (1985) Proc. Natl. Acad. Sci. USA 82, 4920-4924. 25. Cooke, D.W., Bankert, L.A., Roberts, Jr., C.T., Le Roith, D., and Casella, S.J. (1991) Biochem. Biophys. Res. Commun. 177, 1113-1120. 26. Sehgal, A., Patil, N., and Chao. M. (1988) Mol. Cell. Biol. 8, 3160-3167. 693
183,
No.
16,
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1992
Pages
688-693
CHARACTERIZATION OF THE PROMOTJXRREGION OF TIE MURINE FIBROBLAST GRORTEIFACTOR RRCEPTOR 1 GENE Hiroshi
Saito, Haruhiko Kouhara, Soji Kasayama, Tadamitsu Kishimoto and Bunzo Sato’
Department of Internal Medicine III, Osaka University Hospital, Osaka 553, JAPAN
Received
January
27,
1992
Summary: To obtain some clue for the regulatory mechanism by which fibroblast growth factor (FGF) receptor 1 (FGFR1) gene is expressed, we have cloned the promoter region of this gene from genomic library of mouse FGF-responsive The genomic clone isolated here includes the FGFR1 gene from cell lines. position -868 to +697 relative to transcription initiation site. Sequence analysis reveals the presence of various consensus sequences for the binding sites of transcriptional factors such as SP 1, GCF, Ott-I, AP 1 and AP 2, but the absence of TATA and CAAT sequence motif. The transfection of this promoter-CAT constructs into NIH 3T3 cells demonstrates its promoter activity which is at least located between base -106 and +104. 0 1992 Academic
PESS,
Inc.
The basic and acidic role
in
important
fibroblast
growth factors
processes, including
cell
wound healing and promotion of differentiation
(aFGF and bFGF) play
proliferation, of various
cells
(1).
evidence
Recent
reveals that FGF family is also involved in the process of (2, 3). These actions are believed development and progression mediated by their
binding to cell-surfaced
tyrosine
receptors
kinase
identified fms-like
(4-7).
(flg)
related
cancer to be
which form a subclass of
Among these FGF receptors,
from chick embryo was closely gene
receptors
a
angiogenesis,
cDNA first
to a previously
published
(8) or Cek I gene (9). This first FGF receptor is Both aFGF and bFGF were found to be associated
designated as FGFR-1 (6). with FGFR 1 (10).
FGFR 1 is expressed in a variety of cells of mesenchymal, epithelial and neuroectodermal origin (1). However, the molecular mechanism for FGFR 1 to be expressed in target cells is unknown. In addition, the expression of some cell-surfaced up-regulated
receptor mRNAshas been reported to be down-regulated (12) by their corresponding ligand. Actually, our
* To whomcorrespondence should be addressed. 0006-291 X/92 $1.50 Copyright 0 1992 by Academic Press, Inc. All rights of reproductiorl in any form reserved.
688
(11) or previous
Vol.
183,
No.
study
2, 1992
showed
cells (SC-3) the molecular structural this region
that
BIOCHEMICAL
FGFR 1 mRNA levels
are up-regulated mechanism for
analysis
AND
BIOPHYSICAL
RESEARCH
in FGF-responsive
COMMUNICATIONS
mouse
mammary
tumor
in response to bFGF stimuli (13). To clarify regulating the expression of the FGFR 1 gene,
on the promoter
element
of this
gene is prerequisite.
report, we describe the isolation and characterization of FGFR l-encoding gene from the partial genomic
of the library
In promoter of SC-3
cells. HATERIALS AND HEIBODS Screening of a genomic DNA library. Genomic DNA isolated from SC-3 cells as described by Sambrook et al. (14) was digested with Sph I. The digested DNA was electrophoresed in 0.75% agarose gel and subjected to Southernblot analysis (14) using 32P-labeled Pst I-Pst I fragment (480 bp) from 5’noncoding region of SC-3 FGFR-1 cDNA (15) as probe. DNA hybridized with this probe (molecular mass of 3-5 kb) was extracted and inserted into Sph I site of pUC 19 vector. The constructed partial genomic library was transfected into E. coli DHSa, yielding 2x10= independent colonies. After transfer to nitrocellulose filters, this genomic DNA library was screened with the abovecited probe under the high-stringency condition [GxSSC (1xSSC is 0.15 M sodium chloride/l5 mM sodium citrate, 5xDenhardt’s solution PH 7)/ (1xDenhardt’s solution is 0.02% polyvinylpyrrolidone/ 0.02% Ficoll/0.02% bovine serum albumin)/O.l% (w/v) sodium dodecyl sulfate(SDS)/lOO ug/ml denatured salmon sperm DNA at 55’C for 18 h. The positive clones were subjected to second screening. The DNAs isolated from the positive clones were subcloned into pUC 19 and sequenced using the dideoxynucleotide chaintermination method (16). Amplification of FGFR 1 5’-flanking region in NIH 3T3 cells by polymerase chain reaction (PCR). Genomic DNA from NIH 3T3 cells was used as a template for PCR (17) to amplify the FGFR 1 5’-flanking DNA in another murine cell line. Each primer as indicated in Fig. 1 was synthesized. The reaction mixture contained 5 picomoles of each primer, 200 PM each of four deoxynucleoside triphosphates and 1 U of Taq polymerase (Perkin Elmer Cetus) in 50 ~1 of 10 mM Tris hydrochloride (pH 8.3) -50 mM KCl-1.5 mM MgC12-0.01% (w/v) gelatin. Thirty cycles consisting of denaturation at 94-C for 1 min, annealing at 55 ‘C for 1 min and extension at 72 ‘C for 1 min for primer 1 cycles consisting of denaturation at 94 ‘C for 90 s, and 3, and forty annealing at 55 ‘C for 30 s and extension at 75 ‘C for 3 min for primer 2 and 4 were performed to amplify the DNA, respectively. The amplified DNA was inserted into pUC 19 and sequenced as described above. Sl nuclease analysis. To identify the transcription initiation site, mapping by Sl nuclease digestion was carried out as reported previously (18). Total cellular RNA was isolated as described before (12). An aliquot (50 pg) of RNA in 20 1.11of the hybridization buffer containing 80% (v/v) formamide, 40 mM PIPES (pH 6.5), 1 mM EDTA and 0.4 M NaCl was mixed with 380 bp Pst I PstI fragment or 210 bp Pst I - Sma I fragment of SC-3 FGFR 1 genomic DNA which had been end-labeled with T4 polynucleotide kinase and [Y-~*P]ATP. The reaction mixture was heated for 15 min at 80 ‘C, followed by annealing at 55 -C or 60 ‘C for 12 h. After hybridization, 200 pl of the buffer containing 50 mM CH3COONa, 0.25 M NaCl, 4.5 mM ZnSO,, 100 pg of salmon sperm DNA per ml and 900 U of Sl nuclease per ml was added. This mixture was incubated at 37 ‘C for 30 min. The samples were ethanol-precipitated, electrophoresed on 6% polyacrylamide8 M urea gels, and autoradiographed for 15 h at -70 ‘C. Construction and transfection of the FGFR l-CAT chimeric gene. FGFR 1 5’-flanking region was digested with restriction enzymes and inserted into the Sma I site of pSVOOCAT (19). Each fusion gene (10 pg) and pSV- B galactosidase gene (10 vg) were co-transfected into NIH 3T3 cells by calcium phosphate precipitation method (20). After 48 h cultures in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% or 1% fetal calf serum,
689
Vol.
183, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
the cells were harvested and subjected to chloramphenicol acetyltransferase (CAT) activity as described previously (21). Materials. The materials used here were obtained from the following sources : restriction enzymes and Sl nuclease from Toyobo Co., Ltd (Tokyo, Japan) and Takara Co. Ltd (Kyoto, Japan): plasmids from American Tissue Culture Collection (Rockville, MD) and Pharmacia (Uppsala, Sweden); all 32Plabeled nucleotides from AmershamJapan (Tokyo, Japan); culture media from Nissui Pharmaceutical Co., Ltd (Tokyo, Japan). The other reagents were of analytical grade.
RESULTS AND DISCUSSION TO isolate
the
genomic library digested
promoter element of the FGFR1 gene,
prepared from the FGF-responsive SC-3 cells
DNAs with
As shown in Fig.
clone
that
indicated
sequence completely Fig.
1).
to
5’-noncoding
from our laboratory
resistant
out.
hybridization
to protect
region
of
site,
FGFR 1 cDNA
Sl nuclease mapping
position
+l,
analyzed primers
nuclease
with SC-3 or NIH 3T3 mRNA(Fig.
initiation
failed to show the presence of two sites, resolution at the high molecular mass region. initiation
sites.
and the nucleotide
the
+167 to +635 in
the probe from Sl nuclease digestion.
FGFR 1 gene contained two transcription
transcription
a
contained
sites.
2).
The Sl
mapping using the Pst I - Sma I fragment as probe indicated
nuclease
two
The Sph Iconstruct
Using two probes, we were able to observe Sl
fragments after
tRNA failed
which
(15) (from a position initiation
a
sequence of one positive
a DNA fragment
To determine the transcription
was then carried Yeast
1, the nucleotide
we had isolated identical
reported
(22).
a molecular mass of 3-5 kb were used to
genomic library.
previously
we screened
that
The longer
probably due to the One base was inserted
the
probe limited between
The 3’- most residue was designated sequence was aligned
(Fig.
the .promoter region of FGFR1 gene in NIH 3T3 cells.
1).
a
We also Using
four
which had been prepared according to the sequence described in
Fig.
1, two DNA fragments were amplified using NIH 3T3 genomic DNA as a template for PCR. The sequence analysis revealed that the nucleotide sequence from a
position -870 to +104 in NIH 3T3 cells is completely identical with that in SC-3 cells (data not shown). Examination of the murine FGFR 1 gene sequence around the transcription initiation sites reveals notable features. Within
this
However,
region,
neither
the consensus TATA box nor CAAT box was detected.
there
are four consensus sequences for the SP 1 binding site at positions, -246 , +48 , +392 and t416. The other consensus sequences for the transcription factors such as Ott-1, AP 1 and AP 2 were also present in this region. Interestingly, three GCF sites (C/GCGGGGC) which have been proposed to be necessary for epidermal growth factor receptor (EGFR) gene to be downregulated by EGF stimuli (23) were found to exist at positions, -117, +31 and In addition, the nucleotide sequence 3’ downstream from position +636 +120. was not present in the FGFR 1 cDNA, suggesting that an intron is localized in
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. . -600. . . . . . . . GCA~GCTTCATCAAAACATCGGGATTTCTCATCTCCAAGChCGATCAAAAATTTTTATTTTTTAACAATCTCAATATGATTCACCTCCACTGGACTCAT APl Prirper 1 . . . . . -100. . . . . CCATTAAACAGCCACACATTAACATCTCTCTGGAGGGACCTGGTGGGAAGGGCATTTCAGCGATTT~CTTTCAGGTTCATAGTATACTGTGGAGGCTCT . -600 . . . . . . . . GTTTCACTCTCATTAATTCCTAAGGCAAACAAAACAAAACAAGCAAGCAAGTAAACAAACATCCCCCCCATCCGAAAAAAAAAAAAAACAAAAACCCCT API . . -500. . . . . . . . CTCTCTTCGGTTCCTTCCTCAAAGCTGGTGATTTCTAACGCCT Ott-1 . . . . . . . -400. . . AATTATCTGCCTGGGGAAACTTCAGATCCAAGGAAAGGGTCTGAGGGCGAAAGTCAACCGATCCATCAATAAAAACAGTTCGTATATTGTAAGCTATGT Primer 2 Primer 3 . . . . . . . -300. . . ~GGTCCTATTTATTCCTTTTAGAGACGGGAAAACCCAGATTTAGAGCACGAAAATCATGACCCCAAGGCACAGAGATAACCAAAGACCAAGTTGCCCA AP2 . . . . . . . . -200. . TGCAACCTCAAAA?TATAGCCAGCAeCCCCCCfCCCCACCCTCCACTCCTGTCCCCAAATCGCTGATCTACACACAGTCTGAAATTCGGGCGCC~ SPl AP . . . . . . . -100. . . ~GAAAATTCAAGTTAGACCACACTCTACdCCACTTCTGCTCCCTCCACGCTTCTCCCCGCCTCCACCTTGACAACTACAAGCCCCAGCAGGCAACTG A?2 CCF . . . . . . . .+1 . . AACGGCAGCACCACGAGGTGCCACTACCCCCCGGTGCGAGTCACAGAGCGAGCCCTCGCGCCTCGCCCGCGCACAGCGCTCGCAGCGCTCCTGCGGGTA AA . . . . . . . .+100 . . TTTC~TCTCCCCTGC~CGCCCAACCCCACCCGGAACCCTCGTCCAGCCCCTGCCTCCACAGGACCCGGCCTCCCCAGGCAA~ GCF SPl Prlner 4 CCF . . . . . . . .+200 . . CAGACCTCCGCACTGGACTGAGACTCTCCTTAGCGCATTGCGGCCACCTCGCCTTTCCCGGCCGCGAGCGCGCGCCGCAGCTGGAAAAGCAGCGGAGAC 1 . . . . . . . .t300 . . GACGACTTTTCTCAGCTCCCAGGGGCGCACCACAGCCCTCCTGCAGTCAATGCACGCCGGAGCCCCACGAGGGGTGATCGGAACTCGGGCTGCCAGAAG . . . . . . . .t400 . . CTCAGACCCCCCCACCGCCCCCCCAGCGTACTGGAGAGCGGCGGGCGCA~C~~~~CCGG~~~~~GGC~GC~CG~~~~~~~~~~~CCC~CC~CCCCGCCT SP1 SPI . . . . . . . .+soo . .
.
.
.
.
.
.
.
. . . . . . ~gtaagagtcgcc~gcgcccgcagagccgggtggctgggcgtcccgagcggcccccgccgt---------
.
.+600
.
.
Fig.
1 Nucleotide sequence of the 5’-terminal region of the murine FGFR 1 gene. The nucleotide sequence was aligned as the 3’-residue of two transcription initiation sites (A) was designated a position +1 (see the text and Fig. 2). The potential SP 1, AP 1, AP 2, Ott-1 and GCF binding sites are underlined. The arrow indicates the 5’-end of the murine FGFR 1 cDNA obtained from the expression cDNA library in our laboratory (15). The region identical with PstI-PstI fragment of cDNA which was used for screening of the genomic library is also indicated t----l. The primer pairs (l-3 and 2-4) used for amplification of NIH 3T3 cell DNA (PCR) are indicated ( =) . The lowercase letters denote the possible introgenic sequence.
this region. This notion was further supported by the finding end of this possible introgenic sequence contains GT.
that
the
5’
Next, we attempted to confirm the promoter activity. Various portions of the 5’-flanking region fused with the coding region of CAT were transfected into NIH 3T3 cells and cultured in the serum-supplemented or starved inserted the
condition for 48 h (Fig. 3). When the 5’-flanking region was in a sense orientation, the marked CAT activity was demonstrated in transfected NIH 3T3 cells. The construct fused with CAT gene in an 691
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A
-378
-418/+104
P -270
h
-212 -868/+104
-1
+104/-868
+m
L
0 -104
02
-106/+104
03 Fig.
2
Transcription initiation mapping. The Pst I-Pst I fragment (probe 1) SC3 cell RNA at 55 * C or 60 * C (left fragment (probe 2) was also hybridized SC-3 or NIH 3T3 cells (right panel). right panel was obtained by using the
-868/+104
sites
iii
CAT
-1
determined
^'
by
Sl
i
I
nuclease
of the genomic clone was hybridized with panel). The Pst I-Sma I FGFR 1 genomic at 55 ‘C with yeast tRNA, or RNA from The sequence ladder depicted in the probe 2.
Fig.
3 Promoter activities of the murine FGFR 1 gene. Several DNA fragments from the 5’-flanking region of the mouse FGFR 1 gene were used to constract the murine FGFR 1 promoter -CAT chimeric genes. These constructs were transfected into NIH 3T3 cells, followed by culture for 48 h in a high (10%) (panel A) or low (1%) (panel B) serum condition. The SRo promoter in place of the FGFR 1 promoter was also used as a positive control. The arrow indicates the transcription initiation site (+l) and the 5’ to 3’ direction. The transfection efficiency was always adjusted by measuring the activity of B-galactosidase.
orientation failed to show the CAT activity. were also obtained even in the serum-starved condition
opposite
the
DNA sequences responsible
The similar results (Fig. 3B). Moreover, for the basal promoter activity of the murine
FGFR 1 gene were found to be localized at least between -106 and +104. The 5’-flanking sequences of various growth factor receptor genes have been analyzed. Interestingly, the promoter regions of the human EGFR, insulin-like growth factor -1 receptor and nerve growth factor receptor genes (24-26) were observed to lack the TATA as well as CMT box, but to contain the GC box, similar to those found in promoters of cellular housekeeping genes (27). The present study demonstrates that the murine FGFR 1 gene adheres to the general promoter structure identified in growth factor 692
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genes.
been found levels
Moreover,
to contain
are
cells (data is directed sequences
BIOCHEMICAL
the promoter
distinct
up-regulated
AND
regulatory
in SC-3 cells
BIOPHYSICAL
region
RESEARCH
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of the murine
FGFR 1 gene
elements.
Actually,
(13) but down-regulated
not shown) by bFGF stimuli. The current study in our toward identifying the biological role of these for
the expression
has
the FGFR 1 mRNA in
NIH
3T3
laboratory consensus
of the murine FGFR 1 gene. RElXRENCE!3
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