From the Departments of Biology and
§ Pharmacology and the ¶ Institute for Biomolecular
Science, University of South Florida, Tampa, Florida 33620
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ABSTRACT |
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Bmp2, a highly conserved member of
the transforming growth factor- Bone morphogenetic proteins
(BMPs)1 are developmentally
critical growth factors of the transforming growth factor- The evolutionary conservation of the Bmp2 and
Bmp4 genes and their Drosophila homolog
dpp is remarkable. Conservation exists at both the
functional and sequence levels (8, 12-14). Both BMP2 in mouse and DPP
in Drosophila have pleiotropic functions and are expressed
in a highly tissue- and stage-specific manner. Multiple promoters and
alternative splicing produce three major and several minor
dpp transcripts (15). Our work in murine cells and the
Drosophila studies indicate that the multiple core promoters are closely involved in Bmp2 and dpp
tissue-specific regulation. It is likely that the regulation of this
essential growth factor in mammals equals the complexity of
dpp regulation in Drosophila.
Many Bmp2-expressing tissues develop abnormally in
vitamin A-deficient embryos or after exposure to the potent teratogen
retinoic acid (RA). These include the heart and cardiovasculature,
limbs, central nervous system, craniofacial structures, and
vertebrae (see Refs. 3 and 16 and references therein). The first
indication that the Bmp2 gene was regulated by RA was the
discovery that it was strongly induced in F9 embryonal carcinoma cells
stimulated to differentiate with RA (17). Subsequently, the
Bmp2 gene was found to be induced by RA in the developing
chick limb (18). Since retinoid signaling may contribute to the normal
pattern of embryonic Bmp2 expression and since the aberrant
induction of Bmp2 by excess RA may cause some RA-associated
deformities, elucidating the genetic regulatory elements controlling
the RA inducibility of Bmp2 will increase our understanding
of normal development and teratogenesis.
F9 cells, a widely used model of cellular differentiation and early
embryonic development, are an excellent biochemical system for
investigating RA-inducible genes. F9 embryonal carcinoma cells differentiate rapidly and synchronously into primitive endoderm upon
treatment with RA and into parietal endoderm upon treatment with RA and
cAMP analogs (19). This model system has been used to identify retinoic
acid response elements (RAREs) controlling the expression of several
important developmental genes, such as Hoxa1 (20), laminin
B1 (21), and now Bmp2.
Here we describe the first genetic regulatory elements controlling the
RA-regulated induction of the Bmp2 gene in embryonic cells.
RA-regulated gene expression is mediated by nuclear receptors, which
act as retinoid-dependent transcription factors (22). Encoded by six different genes, RAR F9 Cell Culture and Differentiation--
F9 embryonal carcinoma
cells were grown in Dulbecco's modified Eagle's medium supplemented
with 10% heat-inactivated calf serum and 2 mM glutamine.
The cells were induced to differentiate into primitive endoderm by
adding RA alone and into parietal endoderm by adding RA, 250 µM dibutyryl cAMP, and 500 µM theophylline (RACT).
Library Screen--
Using two sets of primers
(5'-GAATTCCGGACTCAGGAGTG-3' and 5'-CTCGAGACAGTCCAGCTGCG-3'
(GenBankTM accession number
L25602)2;
5'-AAACAGTAGTTTCCAGCAGC-3' and 5'-TCTGATTCACTAACCTGGTG-3' polymerase chain reaction was performed on amplified aliquots of a genomic library
in Plasmids--
Plasmids were constructed as follows. All
nucleotide positions are indicated with respect to the translation
start site. For pCAT5'NN6.8 ( Nuclear Run-on Assays--
F9 cells were untreated or treated
72 h with 0.5 µM RA, dibutyryl cAMP, and
theophylline as described above. Nuclei were isolated and nuclear
run-on assays were performed as described previously (25, 28). Plasmids
36B4 (29), pGEM3Z (Promega), and pBMP2-452 were used for hybridization.
Sequencing--
Sequencing was performed manually (25), by the
Molecular Biology Core Facility at the H. Lee Moffitt Cancer Center
(Tampa, FL) or by the DNA Sequencing Laboratory at the
Interdisciplinary Center for Biotech Research (Gainesville, FL), using
primers from the vector and internal sequences. Analysis of the
RA-responsive upstream sequence for putative transcription factor
binding sites was performed using
TFSEARCH3 versus
the TFMATRIX transcription factor binding site profile data base (30)
and by visual inspection. Promoterscan
II4 was used to located
putative promoter sequences (31).
Primer Extension--
Primer extension was performed as
described previously (25). The primer 5'-GTGGGAAGCGCAGCGGCGGC-3',
corresponding to the complement of the sequence extending from Ribonuclease Protection Assays--
Ribonuclease protection
assays were performed as described by Zinn et al. (32) with
the following modifications. 32P-Labeled RNA probes were
made from pBMP2-H linearized with BglII and transcribed with
T3 RNA polymerase and pBMP2-NX linearized with NotI and
transcribed with T7 RNA polymerase. 106 cpm of each probe
were hybridized to 10 µg of RNA overnight at 45 °C in 80%
deionized formamide, 40 mM PIPES, pH 8.5, 400 mM NaCl, and 1 mM EDTA. After treatment with
ribonuclease A, the product was electrophoresed on a sequencing gel.
F9 Cell Transfections and CAT Assays--
All methods were
essentially as described by Vasios et al. (21). F9 cells
were cultured for 48 h without drugs or in the presence of CT, RA,
or RACT, transfected by calcium phosphate precipitation, and then
cultured an additional 24 or 48 h with drugs. All cells were
cotransfected with the reporter gene and with p Yeast Transformations and Electrophoretic Mobility Shift Assays--
Electrophoretic
mobility shift assays were performed essentially as described by
Ausubel et al. (25). DNA probe was made by gel-purifying a
145-bp Sau3AI fragment ( Bmp2 Transcription in F9 Cells Increases in Response to RACT
Treatment--
RNA abundance may be regulated by alterations in
transcription rate and in message stability. Previous experiments using
the transcriptional inhibitors actinomycin D and
5,6-dichloro-1- Location of the Transcription Start Site--
Two Bmp2
promoters have been described in osteoblast cells (36, 37). We used
ribonuclease protection assays to determine if these transcription
start sites or others were RA-inducible in F9 cells (Fig.
1B). An antisense RNA probe that extended from nucleotide
To confirm the transcription start site, primer extension was performed
utilizing reverse transcriptase and a primer complementary to base
pairs Regulation of Bmp2 Promoter Activity by RA in F9 Cells--
CAT
assays were used to detect RA- or RACT-dependent increases
in CAT reporter activity driven by Bmp2 genomic DNA in F9
cells. The reporter constructs containing sequence flanking the
Bmp2 gene are shown in Fig. 2.
Since two transcriptional start sites have been described (37), all
nucleotide positions are indicated relative to the translational start
site (Fig. 2). Several large regions of the Bmp2 upstream
flanking region were inserted upstream of the Herpes simplex virus TK
minimal promoter in pBLCAT2. These fragments, extending from
Since developmental regulation of the dpp gene is mediated
by the core promoter (38), we hypothesized that the RA responsiveness of the Bmp2 gene was similarly controlled. If so, then the
TK minimal promoter in pBLCAT2 might have interfered with RA-induced transcription. Therefore, Bmp2 sequences were inserted into
pBLCAT3, which lacks a minimal promoter. A 1,709-bp fragment,
containing nucleotides A Bmp2 RARE Drives RA-dependent
In addition to the naturally occurring all-trans-RA, which
activates only RARs, and 9-cis-RA, which activates both RARs
and RXRs, several synthetic receptor-selective retinoids are available. TTNPB is often used to demonstrate RAR selectivity in mammalian cells
because, unlike all-trans-RA, it cannot be converted to 9-cis-RA. LG100268 is an RXR-selective retinoid (41). We
treated RAR
Having proven that this sequence could drive the yeast
Sequencing and Analysis of the Upstream Region of the Bmp2
Gene--
We sequenced base pairs
The sequence was also scanned for putative regulatory protein binding
sites. A putative Sp1 site was identified between Approximately 200 genes have been shown to be RA-responsive in one
cell or another. Some genes are regulated directly by RA-bound receptors, e.g. Hoxa1 (20), while others are
secondarily regulated by other transcription factors modulated in
RA-treated cells, e.g. Fgf4 (47). Since both
retinoid deficiencies and overdoses can cause embryonic malformations
via the aberrant expression of key proteins controlling
differentiation, proliferation, apoptosis, and morphogenesis, it is
important to understand which genes are directly regulated. We now
present evidence that the gene encoding the essential growth and
differentiation factor, BMP2, is a direct target of RA.
Bmp2 is transcriptionally induced by RA in F9 embryonal
carcinoma cells (Fig. 1A). Several pieces of evidence
suggest that F9 cells utilize a Bmp2 promoter initiating
transcription at nucleotide In mouse osteoblasts, distal and proximal transcription start sites
were observed at nucleotides We have shown that this promoter and 1,709 base pairs of flanking
region drive RA-dependent reporter gene expression in F9 cells. RA, which induces primitive endoderm differentiation, and RA and
CT (dibutyryl cyclic AMP and theophylline), which induce parietal
endoderm differentiation, caused equal activation of the CAT reporter
gene (Fig. 3B). The endogenous Bmp2 RNA is
undetectable in undifferentiated cells and is induced modestly in
RA-treated cells. In contrast, although CT does not induce
differentiation and has no effect on Bmp2 mRNA
abundance, the combination of RA and CT induces the message abundance
strikingly. Thus, sequence outside of Our demonstration that a 57-bp fragment of Bmp2 genomic DNA
can drive the expression of a Known retinoic acid-responsive elements are highly polymorphic and
conform loosely to the form of two repeated half-sites separated by
nonconserved "spacer" DNA: RG(G/T)TCAN5RG(G/T)TCA (22). Although the most frequent forms are direct repeats separated by
5 base pairs (N), some RAREs consist of inverted repeats, much wider
spacing, and diverse arrangements of half-sites. Since the 57-bp
Bmp2 RARE lacks identity to any previously described RAREs, point mutational analyses will be necessary to identify the precise sequences bound by retinoid receptors. Considering that the numerous combinations of the six retinoid receptors and their various isozymes have distinct ligand- and DNA-binding specificities and that over 200 genes are known to be modulated in retinoid-treated cells (48), many
more types of functional RAREs are likely to be found.
gene family, is crucial for normal
development. Retinoic acid, combined with cAMP analogs, sharply induces
the Bmp2 mRNA during the differentiation of F9
embryonal carcinoma cells into parietal endoderm. Retinoic acid (RA)
also induces the Bmp2 gene in chick limb buds. Since normal
Bmp2 expression may require an endogenous retinoid signal
and aberrant Bmp2 expression may cause some aspects of
RA-induced teratogenesis, we studied the mechanism underlying the
induction of Bmp2. Measurements of the Bmp2
mRNA half-life and nuclear run-on assays indicated that RA
stimulated the transcription rate of the Bmp2 gene. The
results of ribonuclease protection and primer extension assays
indicated that Bmp2 transcription started 2,127 nucleotides
upstream of the translation start site in F9 cells. To identify genetic
elements controlling this transcription rate increase, upstream and
downstream genomic sequences flanking the Bmp2 gene were
screened using chloramphenicol acetyltransferase reporter genes in F9
cells and
-galactosidase reporter genes in Saccharomyces
cerevisiae that were cotransformed with retinoic acid receptor
and retinoid X receptor expression plasmids. RA-dependent transcriptional activation was detected between base pairs
2,373 and
2,316 relative to the translation start site. We also identified a
required Sp1 binding site between
2,308 and
2,298. The data indicate that Bmp2 is directly regulated by retinoic
acid-bound receptors and Sp1.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
family that were first described as having osteogenic activity in rats (1-3).
Bmp2 and Bmp4 transcripts are widely expressed in
vertebrate embryonic structures undergoing induction and morphogenesis
(4-6). BMP signaling is involved in key embryonic processes such as
epithelio-mesenchymal interactions (5), interdigital apoptosis in
the developing limb (7), and dorsal-ventral axis specification (8).
Mice having null mutations in the Bmp2 or Bmp4
(9, 10) or the Bmp receptor IA (11) genes die during early
embryogenesis. The phenotypes of these mutants prove that BMP signaling
is required for numerous extraembryonic and embryonic developmental processes.
, -
, and -
and RXR
,
-
, and -
, the receptors can act as homodimers and heterodimers, often with unique DNA binding and transactivation specificities (see
Refs. 23 and 24). RARs bind to and are activated by
all-trans-RA and 9-cis-RA, while the RXRs are
activated only by 9-cis-RA. Our experiments in yeast suggest
that, like Hoxa1 and RAR
, ligands that bind both RARs and
RXRs synergistically activate the Bmp2 promoter. The work
also suggests that, as in Drosophila, multiple transcription
start sites are utilized in different mammalian tissues.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
DASH II. Polymerase chain reaction-positive aliquots were then
screened (25) with 32P-labeled probe from the full-length
Bmp2 cDNA (pBMP2-452, kindly provided by David Israel)
or a Bmp2 subclone (pBMP2-68, nucleotides 6,483-6,724 with
respect to the translation start site) to isolate two overlapping
bacteriophage that contain the entire Bmp2 gene and
extensive 5'- and 3'-flanking sequences (16.5 kilobases total).
mBMP2-4771 contains the 5'-flanking region of Bmp2
(nucleotides
8,583 to +3,360 relative to the translation start site).
mBMP2-1 contains the remainder of the transcribed region and the
3'-flanking region (nucleotides 2,979-15,700; see Fig. 2).
8,583 to
2,320) and pCAT5'NN6.3
(
2,287 to +3,360), NotI fragments extending from
NotI sites in
DASH II to each of two genomic
NotI sites from
mBMP2-4771 were filled with Klenow fragment and ligated into the filled XbaI site in front of
the pBLCAT2 Herpes simplex virus thymidine kinase (TK) minimal promoter (26). For pCAT5'XX4.5 (
3,367 to +1,206), a XbaI fragment
was ligated into the XbaI site of pBLCAT2 in front of the
pBLCAT2 TK promoter. For pCAT3'BB3.4 (9,318-12,700), a
BamHI fragment from bacteriophage
mBMP2-1 was filled and
ligated into the SmaI site of pBLCAT2 downstream of the
chloramphenicol acetyltransferase (CAT) coding region. For pCAT3'BN3.0
(12,700-15,700), a fragment downstream of the Bmp2 gene was
obtained by digesting bacteriophage
mBMP2-1 at the genomic
BamHI site and the
DASH II NotI site. The
ends were filled and ligated into the SmaI site of pBLCAT2 downstream of the CAT coding region. For pCAT4.5X (
3,367 to
1,658), a XbaI-XhoI fragment was ligated into the
XbaI and XhoI sites of the promoterless vector,
pBLCAT3 (26). For pCAT4.5X
Not (
3,367 to
2,320;
2,287 to
1,658), two NotI sites at
2,320 and
2,288 in pCAT4.5X
were digested to remove a 32-bp fragment and religated. For
pCAT5'NB6.3B (
2,288 to
1,537), a NotI-BglII
fragment was ligated into XbaI-BglII-digested
pBLCAT3 after filling the NotI and XbaI ends. For
pBMP2-H (
2,231 to
1,232), a BamHI fragment was ligated
into the BamHI site of pBSIISK+ (Stratagene). For pBMP2-NX
(
2,289 to
1,658), a NotI-XhoI fragment was
ligated into the NotI and XhoI sites of pBSIISK+.
For p
ss-BMP2 (
3,367 to
1,658), a
SalI-XhoI fragment from pCAT4.5X was inserted
into the XhoI site preceding the cyc1 promoter in
the yeast reporter plasmid p
ss (27). For p
ss-SN1.05 (
3,367 to
2,316), a NotI-XhoI fragment in p
ss-BMP2
was removed and religated after filling the ends. For p
ss-BN.88
(
3,195 to
2,316), a BglII fragment from pGL-XN1.05 was
inserted into the XhoI site of p
ss after partially
filling the ends. For pGL-XN1.05 (
3,364 to
2,316), a
XbaI-XhoI fragment from pCAT4.5X was inserted
into the NheI and XhoI site of pGL2 (Promega) to
make pGL-XX1.7, and then a NotI-XhoI fragment in
pGL-XX1.7 was removed and religated after filling the ends. For
p
ss-BB.83 (
3,195 to
2,369), a BglII-BamHI fragment from pCAT4.5X was inserted into the XhoI site of
p
ss after partially filling the ends. For p
ss-BB.06 (
2,373 to
2,316), a BamHI-BglII fragment from pGL-XN1.05
was inserted into the XhoI site of p
ss after partially
filling the ends. For p
ss-SB.18 (
3,367 to
3,191), a
BglII-XhoI fragment in p
ss-BMP2 was removed and religated after filling the ends.
2,064
to
2,045, was labeled and hybridized to 29.7 µg of RNA and extended
with avian myeloblastosis virus reverse transcriptase (Life
Technologies, Inc.).
AclacZ (21), which
contains the
-galactosidase coding region driven by the constitutive
-actin promoter. Cell extracts were normalized for transfection
efficiency as determined by
-galactosidase expression. Equivalent
amounts of extract were incubated at 37 °C for 7 h with 250 mM Tris, pH 7.8, 5.3 mM acetyl coenzyme A, and
32.4 µM 14C-chloramphenicol (51.5 µCi/µmol; NEN Life Science Products). After separation by thin
layer chromatography (Whatman No. 4410221), chloramphenicol acetylation
was quantified with a Molecular Dynamics PhosphorImager or a Beckman
60001C liquid scintillation counter.
-Galactosidase Assays--
The
p
ss
-galactosidase reporter vector (URA3) and the
retinoid receptor expression vectors p2HG-RAR
(HIS3),
pG1-RAR
(TRP1), and pG1-RXR
(TRP1) have
been described (27, 33). The reporter vector and various receptor
expression vectors were used to transform the yeast strain BJ5409
(his3, leu2, trp1, ura3) using the lithium acetate method
(34). Double or triple transformants were selected by plating on
synthetic medium lacking the appropriate nutrients. For
-galactosidase assays, yeast cells were grown in selective medium in
the presence and absence of retinoids for 24 h
(all-trans-RA, Sigma; TTNPB and 9-cis-RA,
Hoffman-La Roche; LG100268, Ligand Pharmaceuticals). The cells
were lysed, and
-galactosidase activity was assayed by
o-phenylphosphogalactopyranoside hydrolysis at 30 °C
(25). Normalized
-galactosidase values were determined as follows:
(A420/A600) × 1,000/min
of reaction time.
2,372 to
2,227) from pCAT4.5X
containing a Sp1 consensus sequence. The ends were filled in with
Klenow fragment in the presence of [32P]dCTP and
[32P]dGTP. Binding reactions contained 1 unit of rhSP1
(Promega), 60,000 cpm (2 ng) of probe, 10 mM HEPES, pH 7.9, 40 mM KCl, 6 mM MgCl2, 0.1% Triton
X-100, 0.1 mM dithiothreitol, 0.25 mg/ml acetylated bovine
serum albumin (New England BioLabs), 2% Ficoll, and 0.05 mg/ml
sonicated salmon sperm DNA (Sigma). Samples were incubated for 30 min
at room temperature and then loaded onto a 5% polyacrylamide gel (74:1
acrylamide:bisacrylamide, 5% glycerol (w/v)). Electrophoresis was
performed at 300 V for 45 min in 0.5× TBE at 4 °C. The gel was
dried under vacuum onto filter paper (Whatman) and exposed overnight to
x-ray film (Eastman Kodak Co.).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-D-ribofuranosylbenzimidazole showed that
Bmp2 mRNA stability does not change with RA treatment (35). This observation suggested, but did not prove, that RA increased
Bmp2 transcription rates. To test this hypothesis, we isolated nuclei from untreated cells or cells treated with RACT for
72 h and performed nuclear run-on assays (Fig.
1A). Bmp2 gene transcription increased 3.7-fold in RACT-treated cells relative to
untreated cells. In contrast, the transcription of 36B4, a constitutively expressed ribosomal protein, did not vary. This directly
demonstrated transcriptional induction of the Bmp2 gene by
RA.
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Fig. 1.
Identification of the RA-inducible Bmp2
promoter. F9 cells were grown for 72 (A) or
96 h (B and C) in the presence
(RACT) or absence (Stem) of 0.5 (A) or
1 µM (B and C) RA and CT.
A, nuclear run-on assays. 32P-Labeled probes
generated from transcripts initiated in these cells at the time of RNA
extraction were hybridized to identical nitrocellulose strips spotted
with a vector control (pGEM3Z) or plasmids containing the cDNA
encoding a constitutive ribosomal protein (36B4) or BMP2 (pBMP2-452).
B, ribonuclease protection assays. Antisense probes
extending from nucleotide 1,541 to
1,233 and nucleotide
2,287 to
1,663 were hybridized to F9 cell RNA or to yeast tRNA, and
ribonuclease protection assays were performed. The molecular weight
marker (M) was pBR322 digested with MspI. Visible
fragments are 622, 527, 404, 307, 242, 238, 217, 201, 190, 180, 160,
147, 123, and 110 bp in length. The positions of the undigested probe
and the protected fragment are indicated by the thin and
thick arrows, respectively. The open
arrow indicates the predicted size of a fragment generated
by the proximal promoter used in osteoblast cells (36). C,
primer extension assays. An antisense oligonucleotide
corresponding to nucleotides
2,065 to
2,046 was hybridized to F9
cell RNA or to yeast tRNA, and primer extension was performed. One band
was visible in the RACT lane only. A sequence generated from this
oligonucleotide and pBMP2-NX is shown to the left of the
primer extension lanes.
1,541 to
1,233 relative to the translation initiation site was
generated from pBMP2-H digested with BglII. The entire Bmp2 probe was protected by RNA isolated from RACT-treated
cells (Fig. 1B, thick arrow),
indicating that Bmp2 transcription initiated upstream of
nucleotide
1,541 in F9 cells. No protected fragments were observed in
the reactions containing RNA from untreated cells, yeast tRNA (Fig.
1B), or a sense probe extending from
2,230 to
1,233
(data not shown). The open arrow indicates the
predicted location of a fragment generated by a transcript originating
at the proximal promoter (nucleotide
1,344) described by Feng
et al. (36). The absence of a fragment at this
location indicates that, in contrast to osteoblast cells, this promoter
is not used in F9 cells. Using an antisense probe that extended from
2,287 to
1,663 generated from pBMP2-NX, we observed a fragment of
approximately 493 nucleotides. This suggests that transcription starts
at approximately
2,156, near the distal promoter used in osteoblasts.
2,064 to
2,045 relative to the translation start site (Fig.
1C). An extended product was detected only in the reaction containing RNA from cells treated with 1 µM RA and CT for
96 h and not in the reactions containing RNA from untreated cells
or yeast tRNA. Comparison with the genomic sequence designated a start
site at nucleotide
2,127. No other RACT-dependent
extended products were observed, suggesting utilization of one major
transcriptional start site in F9 cells. The differences in mobility
between RNA and DNA molecular weight markers explain the small
discrepancy in start site position as determined by primer extension or
RNase protection assays.
8,583 to
2,320 (pCAT5'NN6.8),
3,367 to +1,206 (pCATXX4.5), and
2,287 to
+3,360 (pCAT5'NN6.3), failed to drive CAT expression in F9 cells
treated for 96 h with 1 µM RA or 1 µM
RA and CT (Fig. 3A). A
fragment that included the 3'-end of the transcribed region
(9,316-12,700; pCAT3'BB3.4) did not affect CAT activity. In contrast,
a fragment distal to the 3'-end (12,700-15,700; pCAT3'BN3.0) caused a
3.5-4-fold CAT activation relative to the pBLCAT2 vector alone (Fig.
3A). Since activation occurred in cells treated with CT, RA,
or RACT, this sequence must contain a non-RA-dependent
regulatory element. Considering the highly tissue- and stage-specific
expression of Bmp2, many regulatory elements are likely to
control Bmp2 expression.
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Fig. 2.
Bmp2 gene structure and CAT reporter
constructs. A schematic representation of the Bmp2
genomic sequence is shown. The sequence is numbered with respect to the
translation start site (36). Filled boxes
represent exons (36). D (distal) indicates transcription
initiation from the RA-dependent promoter in F9 cells,
whereas P (proximal) indicates the additional transcription
initiation site utilized in osteoblasts (37). The bars
below indicate the location and relative sizes of the
sequences cloned into CAT reporter vectors pBLCAT2 and pBLCAT3. For
pCAT5'NN6.8, pCAT5'XX4.5, and pCAT5'NN6.3, fragments were inserted
5' of the TK promoter in pBLCAT2. For pCAT3'BB3.4 and pCAT3'BN3.0,
fragments were inserted 3' of the CAT coding region. For
pCAT5'NB6.3B, pCAT4.5X, and pCAT4.5X Not, fragments were inserted
5' of the CAT coding region in the promoterless reporter vector
pBLCAT3.
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Fig. 3.
Regulation of Bmp2 reporter
activity in F9 cells. F9 cells were grown for 48 h in the
absence of drug (Stem) or the presence of 1 µM
RA (RA) or 250 µM dibutyryl cAMP and 500 µM theophylline (CT) or all three drugs
(RACT). Cells were then transfected with the plasmids
indicated under each group of bars and p AclacZ, which
contains a constitutive promoter driving
-galactosidase. Reporter
constructs containing the TK promoter are shown in A;
constructs without the TK promoter are shown in B. 48 h
later, cell extracts were made.
-Galactosidase assays were performed
to normalize for transfection efficiency. Amounts of extract containing
equivalent
-galactosidase activity were then used for CAT assays.
Each bar shows the average of 3-6 experiments and the S.E.
measurement.
3,367 to
1,658 (pCAT4.5X), induced CAT
activity 2.8-fold in cells treated for 96 h with 1 µM RA or 1 µM RA and CT relative to the
activity observed in CT-treated cells (Fig. 3B). This
fragment included 1,240 base pairs upstream of the transcription start
site at
2,127. Finally, a fragment containing only 161 nucleotides
upstream of the transcriptional start site (
2,288 to
1,537;
pCAT5'NB6.3B) failed to induce CAT activity (Fig. 3B). These
results are consistent with the presence of elements required for the
RA response and promoter activity between nucleotides
3,367 and
2,288. As will be discussed below, we used a yeast reporter system to
further delineate this RARE.
-Galactosidase
Expression in Yeast--
It is difficult to distinguish genes
regulated directly by retinoid-activated receptors from those
indirectly activated by other transcription factors induced by RA in
mammalian cells. To avoid the complications associated with endogenous
receptors and other transcription factors in F9 cells, we
co-transformed yeast with mammalian receptor expression vectors and
reporter genes driven by Bmp2 genomic sequences. Although
yeast do not normally express retinoid receptors, yeast transformed
with receptor genes synthesize functional receptors. These can
stimulate the RA-dependent expression of reporter genes
controlled by mammalian RAREs (33, 39, 40). We inserted a fragment
containing base pairs
3,367 to
1,658 of the Bmp2 gene in
front of the cyc1 promoter and the
-galactosidase coding
region of the yeast vector, p
SS (27). The yeast strain BJ5409 was
transformed with this plasmid and various combinations of RAR
,
RAR
or RXR
yeast expression vectors. Treatment of yeast
expressing RAR
or RAR
and RXR
with 1 µM
all-trans-RA or 9-cis-RA induced
-galactosidase activity 1.7- and 2.3-fold, respectively, relative to
untreated yeast (Fig. 4A).
Yeast transfected with RAR
or RAR
alone and treated with 9-cis-RA also induced
-galactosidase activity 1.6-fold,
indicating that the RAR homodimers could activate Bmp2
nearly as efficiently as the RAR/RXR heterodimer (Fig. 4B).
In contrast, yeast expressing RXR alone or yeast lacking receptors
failed to express
-galactosidase in response to RA treatment (Fig.
4B). These experiments indicate that the RA responsiveness
of this Bmp2 sequence in yeast requires activation of RAR
homodimers or RAR/RXR heterodimers.
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Fig. 4.
Regulation of Bmp2 reporter
activity in yeast. Yeast strain BJ5409 was transformed with
expression vectors encoding RAR or RAR
alone or with RXR
as
indicated under each bar (A and B) or
with RAR
and RXR
(C). These yeast were subsequently
transformed with the empty reporter plasmid p
SS or p
SS-BMP2 as
indicated under each bar (A) or with p
SS-BMP2
(B and C). Cultures were treated with the
indicated retinoids at 1 µM.
-Galactosidase activities
and S.E. measurements are shown. n = 3.
- and RXR
-expressing yeast with 1 µM
TTNPB, LG100268, or 9-cis-RA or the combination of TTNPB and
LG100268. Like all-trans-RA, TTNPB activated transcription
slightly (1.5-fold) but less effectively than 9-cis-RA
(2.1-fold, Fig. 4C). Interestingly, the RXR agonist induced
activity as effectively as the panagonist 9-cis-RA, which can activate both the RARs and the RXRs. Combined exposure to these
retinoids stimulated activity by 3.6-fold (Fig. 4C). The synergistic activation of several RA-responsive genes by
simultaneous ligand binding of each receptor subunit within a
heterodimer has also been observed in mammalian cells (42, 43). These
results are the first to demonstrate that the developmentally crucial Bmp2 gene is activated directly by retinoid-bound receptors.
-galactosidase reporter gene in a ligand- and
receptor-dependent manner, we localized this element more
precisely using a series of deletion constructs (Fig.
5). Deletions of 3'-flanking sequences up
to position
2,316 and 5'-flanking sequences up to
2,373 do not alter the induction by 9-cis-RA (bars
1-4). The reporter constructs containing only base pairs
3,195 to
2,369 or
3,367 to
3,191 were not induced by
9-cis-RA (bars 5-6). These results
indicate that a 57-bp Bmp2 promoter sequence located between
2,373 and
2,316 bp contains a RARE that is necessary and sufficient
to induce RA-mediated transcription.
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Fig. 5.
Deletion analysis of RA-responsive Bmp2
sequences in yeast. Yeast strain BJ5409 was transformed with
expression vectors encoding RAR and RAR
. These yeasts were
subsequently transformed with various portions of Bmp2
genomic DNA driving the
-galactosidase gene as indicated.
Transcriptional induction by 1 µM 9-cis-RA is
presented by -fold induction as indicated in the histogram.
"1-Fold" induction indicates no difference in
-galactosidase
activity between untreated and RA-treated cells. n = 3. Bars show S.E.
3,367 to
1,658 of the
Bmp2 gene to identify consensus sequences for other known
regulatory proteins. Sequences consistent with a TATA-containing
promoter sequence and a transcription start site at nucleotide
2,127
are depicted in Fig. 6. As shown in Fig.
1C, the primer extension assay confirmed the activity of
this promoter in RACT-treated F9 cells. These features are consistent
with a promoter at this site.
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Fig. 6.
Nucleotide sequence of the upstream region of
the Bmp2 gene. The sequence is numbered relative to
the translational start site. The start site used in RA-treated F9
cells is indicated by an arrow. The sequence sufficient for
RA-dependent activation in yeast is underlined.
The Sp1 consensus sequence and a putative TATA box are in
boldface type. The sequence complementary to the primer
extension oligonucleotide is shown by a heavy
underline.
2,308 and
2,298
(Fig. 6). Since others have demonstrated the importance of Sp1 sites
for RA responsiveness (44-46), we deleted a 32-bp fragment containing
the site (Fig. 2). This deletion failed to alter the magnitude of RA
inducibility in either yeast (data not shown) or F9 cells (Fig.
7A). However, in F9 cells,
both the basal and the induced transcription activity of the CAT
reporter gene declined by 30% (Fig. 7A). We also
demonstrated that recombinant Sp1 protein bound this sequence (Fig.
7B). These observations suggest that Sp1 influences the
transcription activity of the Bmp2 gene but does not play a
role in RA responsiveness.
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Fig. 7.
Sp1 protein binds a transcription activating
sequence. A, F9 cells were transfected with the
reporter gene CAT driven by bp 3,367 to
1,658 (pCAT4.5X) or the
same sequence lacking a 32-bp fragment containing an Sp1 consensus
sequence (pCAT4.5X
Not) as described in the legend to Fig. 2. Cells
were extracted 48 or 24 h after transfection, resulting in 96 or
72 h of total exposure to drugs. The activity of these reporter
genes was induced 4.2- and 3.9-fold by RA. However, both the induced
and basal activity of the fragment lacking the Sp1 site was reduced
approximately 30%. Bars show the range; n = 2. B, a 145-bp fragment containing the Sp1 site (
2,372 to
2,227) was end-labeled with 32P-dCTP and
32P-dGTP and bound to recombinant human Sp1.
Lane 1 indicates the migration of free probe
(open arrow). Lane 2 shows
the retardation caused by binding of the DNA fragment to the
unglycosylated (95-kDa) and glycosylated (105-kDa) forms of Sp1
(closed arrows).
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
2,127 relative to the translation
initiation site. First, ribonuclease protection assays indicate that
the longest Bmp2 transcript initiates near this site (Fig.
1B). Second, this is the end of an RA-inducible primer
extension product (Fig. 1C). Third, the predicted size of a
transcript starting at
2,127 is consistent with the mRNA size of
3.8 kilobases (17, 36). Finally, sequences resembling a TATA-containing
promoter are near nucleotide
2,127 (31).
2,127 and
1,344 (36, 37). We did not
observe a ribonuclease protection fragment corresponding to the
proximal start site in untreated or in RACT-treated F9 cells (Fig.
2B). Thus, the proximal promoter does not mediate the
RA-induced transcription of Bmp2 in F9 cells. The existence of multiple Bmp2 transcription start sites in different cell
types is not surprising, because the expression of the Bmp2
gene is highly dynamic. In addition, dpp, the gene encoding
the Drosophila homolog of Bmp2, has three major
and several minor transcripts produced from several promoters (15).
Like the dpp transcript, the Bmp2 transcript has
an unusually long 5'-untranslated region of 1,125 nucleotides that
might contain as yet uncharacterized regulatory elements. Since
Bmp2 and dpp are pivotal developmental genes,
their spatial and temporal expression must be tightly regulated. We
have demonstrated here that tissue-specific promoters are one mechanism
involved in this tight regulation.
3,367 to
1,658 must contain
the elements responsible for the synergistic activity of RA and cAMP.
-galactosidase reporter gene in yeast
transformed with retinoid receptors strongly suggests that this gene is
directly regulated by receptor binding. The RAR/RXR heterodimer in the
presence of ligands that activate both subunits activated the
Bmp2-driven reporter most efficiently (Fig. 4C). Similar synergy has been observed for many genes in mammalian cells,
including Hoxa1 and the RAR
gene (42, 43). A requirement for specific receptor combinations and specific ligand activities may
mediate the tight regulation of developmentally crucial genes such as
Bmp2.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. M.A. Glozak for critical reading
of this manuscript and Drs. D. Israel for murine Bmp2
cDNA probes, E. W. Jones and C. A. Woodford for yeast
strain BJ5409, and M. L. Privalsky for retinoid receptor
expression vectors and reporter vector pSS. We also thank S. M. Smith, E. C. Schuetz, S. Shiflett, and Dr. A. C. Cannons for
technical assistance.
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FOOTNOTES |
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* This work has been supported in part by the Molecular Biology Core Facility at the H. Lee Moffitt Cancer Center and Research Institute; NICHD, National Institutes of Health, Grant R29 HD31117 (to M. B. R.); and a postdoctoral fellowship from the American Heart Association, Florida affiliate (to L. C. H.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF074942.
To whom correspondence should be addressed: Dept. of Biology,
BSF119, University of South Florida, 4202 E. Fowler Ave., Tampa, FL
33620. Tel.: 813-974-2623; Fax: 813-974-1614; E-mail:
rogers{at}chuma.cas.usf.edu.
The abbreviations used are: BMP, bone morphogenetic protein; CAT, chloramphenicol acetyltransferase; CT, dibutyryl cyclic AMP and theophylline; RA, all-trans-retinoic acid; RACT, all-trans-retinoic acid, dibutyryl cyclic AMP, and theophylline; RAR, retinoic acid receptor; RARE, retinoic acid-responsive element; RXR, retinoid X receptor; TK, thymidine kinase; bp, base pair(s); PIPES, 1,4-piperazinediethanesulfonic acid; TTNPB, (E)-4-[2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1-propenyl]benzoic acid.
2 D. Israel, personal communication,
3 TFSEARCH is available on the World Wide Web at http://pdap1.trc.rwcp.or.jp/research/db/TFSEARCH.html.
4 Promoterscan II is available on the World Wide Web at http://biosci.cbs.umn.edu/software/promoterscan.htm.
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REFERENCES |
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