DNA Sequences Downstream from the Vitamin D Response Element of the Rat Osteocalcin Gene Are Required for Ligand-Dependent Transactivation
W. Bruce Sneddon,
Cesar E. Bogado1,
M. Susan Kiernan2 and
Marie B. Demay
Endocrine Unit Massachusetts General Hospital Harvard
Medical School Boston, Massachusetts 02114
 |
ABSTRACT
|
---|
The sequences in the rat osteocalcin gene that lie
3' to the vitamin D response element (VDRE) have been shown to augment
transcriptional activation by 1,25-dihydroxyvitamin
D3
[1,25-(OH)2D3]. These
DNA sequences, however, are unable to bind the VDR or mediate
1,25-(OH)2D3
responsiveness independently of the VDRE. To further characterize this
region, the functional properties of a series of mutant
oligonucleotides were examined in transiently transfected ROS 17/2.8
cells. When these mutant oligonucleotides were expressed upstream of
the heterologous herpes simplex virus thymidine kinase promoter, the
bases between -420 and -414 of the rat osteocalcin gene were
identified as critical for maximal transactivation by
1,25-(OH)2D3.
Furthermore, mutation of these sequences in the context of the native
osteocalcin promoter and enhancer totally abolished the ability of the
VDRE to mediate
1,25-(OH)2D3
responsiveness. These bases, which are essential for the
1,25-(OH)2D3
responsiveness of the rat osteocalcin gene, are also present in a
similar position, relative to the VDRE, in the human osteocalcin gene.
To explore whether these sequences could enhance transactivation by
other inducible transcription factors, they were examined for their
ability to synergize with the chick vitellogenin estrogen response
element and the rat somatostatin cAMP response element. When placed
upstream to the herpes simplex virus thymidine kinase promoter and
transfected into ROS 17/2.8 cells, these sequences were able to enhance
transcriptional responsiveness to 17ß-estradiol and forskolin,
respectively, demonstrating that they also contribute to
transactivation by other inducible transcription factors.
 |
INTRODUCTION
|
---|
Vitamin D, through its biologically active metabolite,
1,25-dihydroxyvitamin D3
[1,25-(OH)2D3], interacts with target genes
by binding to a nuclear steroid hormone receptor (reviewed in Ref.1),
the vitamin D receptor (VDR), which heterodimerizes with the retinoid X
receptor (RXR) and binds to up-regulatory vitamin D response elements
(VDRE) on target genes (2, 3). As a consequence, presumably through
stabilization of the transcription preinitiation complex,
transcriptional activation occurs.
The VDRE of the rat osteocalcin gene has been characterized as an
imperfect direct hexameric repeat separated by three bases
(GGGTGAATGAGGACA) (4). Initial studies directed
at characterizing this VDRE revealed that the inclusion of bases 3' to
the VDRE resulted in a 3-fold increase in
1,25-(OH)2D3 responsiveness in transient gene
expression assays (5). Interactions between VDREs and binding sites for
other transcription factors have previously been reported. When placed
upstream to the minimal adenovirus E1b promoter, the mouse osteopontin
VDRE is able to synergize with SP-1, nuclear factor-1, octamer binding
protein-1, and activating protein-1 binding sites (6) to augment the
transcriptional response to 1,25-(OH)2D3.
Although none of these binding sites is found in close proximity 3' to
any of the known, naturally occurring VDREs (5, 7, 8, 9, 10, 11, 12), this
observation supports the hypothesis that the level of
1,25-(OH)2D3 responsiveness of the rat
osteocalcin gene may be affected by transcription factors that interact
with sequences flanking the VDRE.
The classical model for steroid-activated transcription is the
hormone-bound receptor binding to its response element, leading to
transactivation. Recent information indicates that this process is much
more complex (13, 14, 15, 16, 17). Gene promoters are thought to integrate multiple
signals to modulate transcriptional responses. We have identified a
sequence 3' to the VDRE of the rat osteocalcin gene that is absolutely
required for VDR-mediated transactivation of the rat osteocalcin gene,
suggesting that multiple factors contribute to transcriptional
activation by the VDR.
 |
RESULTS
|
---|
In our initial studies aimed at characterizing the VDRE of the rat
osteocalcin gene, we observed that an oligonucleotide containing the
DNA sequences from -458 to -403 (1D3) was able to confer
6-fold 1,25-(OH)2D3 responsiveness to the
herpes simplex virus thymidine kinase (HSV-tk) promoter in transient
gene expression assays in ROS 17/2.8 cells (5). Further studies
revealed that the minimal VDRE was contained in the bases from -456 to
-442 (4) and that the DNA sequences from -458 to -427
(10D3) were only able to confer 2-fold
1,25-(OH)2D3 responsiveness in transient gene
expression assays (4). The sequences from -428 to -403 did not bind
the VDR, nor could they confer 1,25-(OH)2D3
responsiveness independently of the VDRE (5), thereby excluding the
possibility that they contained a second VDRE. To explore the
hypothesis that this region of the osteocalcin gene contained specific
DNA sequences responsible for enhanced transactivation by
1,25-(OH)2D3, a series of oligonucleotides
representing 3' deletions of 1D3 was synthesized and
ligated into pUTKAT3 (18). Their ability to mediate transcriptional
activation by 1,25-(OH)2D3 was assessed after
transfection into ROS 17/2.8 cells (Fig. 1
). The fusion
gene, 14D3-tkCAT (CAT = chloramphenicol
acetyltransferase; Table 1
), which contained seven
fewer bases than 1D3-tkCAT, retained a 6.0 ± 0.3-fold
induction of CAT activity in response to 10-8
M 1,25-(OH)2D3 (Fig. 1
). In
contrast, 13D3-tkCAT (Table 1
), which contained eight fewer
3' bases than 14D3-tkCAT, conferred a 2.4 ± 0.1-fold
responsiveness to 1,25-(OH)2D3 (Fig. 1
). There
was no difference in basal expression between 14D3-tkCAT
and 13D3-tkCAT (data not shown). The human (h) homologs of
14D3-tkCAT and 13D3-tkCAT (Table 3
) were also
examined for their ability to mediate ligand-dependent transactivation
in ROS 17/2.8 cells. h14D3-tkCAT was able to respond
2.6-fold to 1,25-(OH)2D3, whereas the
responsiveness of h13D3-tkCAT was only 1.2-fold.

View larger version (12K):
[in this window]
[in a new window]
|
Figure 1. Relative Stimulation of CAT Activity in Response to
10 nM 1,25-(OH)2D3
ROS 17/2.8 cells were transiently transfected with the indicated fusion
genes. Relative stimulation of CAT activity in response to 10
nM 1,25-(OH)2D3 represents the
mean ± SEM of three independent experiments using at
least two different preparations of each plasmid. Each transfection was
performed in triplicate and normalized for transfection efficiency with
RSV-luc cotransfection.
|
|
View this table:
[in this window]
[in a new window]
|
Table 1. Double-Stranded Oligonucleotides Containing
the Sense Sequences Indicated, with GATC Overhangs, were subcloned into
the BamHI site of pUTKAT3
|
|
View this table:
[in this window]
[in a new window]
|
Table 3. Double-Stranded Oligonucleotides with the
Sense Sequences Shown Were Employed in Gel Retardation Analysis (Fig 7 )
|
|
Because deletion of eight bases at the 3' end of the 14D3
oligonucleotide (13D3) reduced transcriptional activation
by the VDRE, a series of 14D3 oligonucleotides with
mutations in the region 3' to the VDRE was tested for their ability to
confer 1,25-(OH)2D3 responsiveness to the
HSV-tk promoter when transfected into ROS 17/2.8 cells (Table 2
and Fig. 2
). All fusion genes contained
one copy of the oligonucleotide in the correct orientation. Mutations
in the region corresponding to -420 to -414 of the rat osteocalcin
gene, like 13D3-tkCAT, resulted in a 3-fold reduction in
1,25-(OH)2D3 responsiveness in ROS 17/2.8
cells. These sequences were, therefore, critical for the increased
1,25-(OH)2D3 responsiveness observed with
14D3-tkCAT.
View this table:
[in this window]
[in a new window]
|
Table 2. Double-Stranded Mutant 14D3
Oligonucleotides Containing the Sense Sequences Indicated, with GATC
Overhangs, Were Subcloned into the BamHI Site of
pUTKAT3
|
|

View larger version (16K):
[in this window]
[in a new window]
|
Figure 2. The Sequences Required for the Increased
1,25-(OH)2D3 Responsiveness Are Located between
-420 and -414 of the Rat Osteocalcin Gene
A series of mutant 14D3 oligonucleotides was ligated to
pUTKAT3 and transiently transfected into ROS 17/2.8 cells. The mutant
oligonucleotides are shown in Table 2 . Relative stimulation of CAT
activity in response to 10 nM
1,25-(OH)2D3 represents the mean ±
SEM of four independent experiments using at least two
different preparations of each plasmid. Each transfection was performed
in triplicate and normalized for transfection efficiency with RSV-luc
cotransfection.
|
|
To assess whether these same DNA sequences augmented transcriptional
activation by 1,25-(OH)2D3 in osteosarcoma
cells, which do not express the endogenous osteocalcin gene, two fusion
genes were expressed in UMR-106 cells. 14D3-tkCAT and a
mutant with decreased responsiveness (M5-14D3-tkCAT) were
examined for their ability to confer
1,25-(OH)2D3 responsiveness to a heterologous
viral promoter. When 14D3-tkCAT was expressed in UMR-106
cells (Fig. 3
), a 2.5 ± 0.4-fold induction of CAT
activity was observed in response to 10-8 M
1,25-(OH)2D3, whereas M5-14D3-tkCAT
only conferred a 1.3 ± 0.2-fold response (Fig. 3
).

View larger version (12K):
[in this window]
[in a new window]
|
Figure 3. The Sequences between -420 and -414 of the Rat
Osteocalcin Gene Augment 1,25-(OH)2D3
Responsiveness in UMR-106 Cells
UMR-106 cells were transiently transfected with plasmids containing
either the wild-type 14D3 or mutant M5 oligonucleotide
fused to pUTKAT3. Relative stimulation of CAT activity in response to
10 nM 1,25-(OH)2D3 represents the
mean ± SEM of three independent experiments using at
least two different preparations of each plasmid. Each transfection was
performed in triplicate and normalized for transfection efficiency with
RSV-luc cotransfection.
|
|
To demonstrate that the sequences that augment transcriptional
responsiveness to 1,25-(OH)2D3 in the context
of a heterologous viral promoter do so with the native osteocalcin
promoter as well, a composite M3 and M4 mutation was introduced by
site-directed mutagenesis of an osteocalcin-CAT fusion gene containing
the sequences from -522 to -8 of the rat osteocalcin gene. The
wild-type (OC-CAT) and the mutant plasmid (M3,M4-OC-CAT) were
transfected into ROS 17/2.8 cells to examine their ability to mediate
transactivation by 1,25-(OH)2D3 (Fig. 4
). The M3-M4 composite mutation abolished
1,25-(OH)2D3 responsiveness, demonstrating that
these sequences are essential for 1,25-(OH)2D3
induction of rat osteocalcin gene expression in the context of the
native promoter. The combined M1-M2 mutation, which separately had no
effect on the transcriptional responsiveness to
1,25-(OH)2D3 in the context of a heterologous
viral promoter, also abrogated 1,25-(OH)2D3
responsiveness (Fig. 4
), indicating that sequences 5' to the GGTTTGG
are involved in transcriptional activation by
1,25-(OH)2D3. Neither the M3-M4 nor the M1-M2
mutation had an effect on the basal expression of CAT activity (data
not shown).

View larger version (12K):
[in this window]
[in a new window]
|
Figure 4. The Bases from -420 to -414 Are Essential for the
1,25-(OH)2D3 Responsiveness of the Rat
Osteocalcin Gene
A composite mutation containing the base changes in M3 and M4 was
introduced into the 5'-flanking region of the rat osteocalcin gene by
site-directed mutagenesis to yield M3,M4-OC-CAT. The same procedure was
employed to introduce a composite mutation containing the base changes
in M1 and M2 to yield M1,M2-OC-CAT. ROS 17/2.8 cells were transiently
transfected with the mutant and wild-type plasmids, and their ability
to induce CAT activity in response to 10 nM
1,25-(OH)2D3 was assessed. Relative stimulation
of CAT activity represents the mean ± SEM of three
independent experiments using at least two different preparations of
each plasmid. Each transfection was performed in triplicate and
normalized for transfection efficiency with RSV-luc cotransfection.
|
|
Experiments were undertaken to address whether the sequences
shown to augment 1,25-(OH)2D3 responsiveness
could modulate transactivation by other transcription factors. The
estrogen response element from the chick vitellogenin gene (19) was
substituted for the VDRE in both 14D3-tkCAT and
9D3-tkCAT (Fig. 5
). These fusion genes were
transfected into ROS 17/2.8 cells and assayed for transcriptional
induction by 10-8 M 17ß-estradiol. In cells
transfected with 14ERE-tkCAT (ERE = estrogen response element),
17ß-estradiol induced CAT reporter activity 3.1 ± 0.4-fold
(Fig. 5
). In contrast, cells transfected with 9ERE-tkCAT only displayed
a 1.4 ± 0.4-fold response to 17ß-estradiol (Fig. 5
). To
determine whether the augmented transcriptional responsiveness mediated
by these sequences extended beyond the nuclear receptor superfamily,
the cAMP response element from the rat somatostatin gene (20) was
substituted for the VDRE in both 14D3-tkCAT and
9D3-tkCAT (Fig. 6
). These fusion genes were
transfected into ROS 17/2.8 cells and assayed for responsiveness to
10-6 M forskolin. In cells transfected with
14CRE-tkCAT (CRE = cAMP response element), forskolin induced CAT
activity 3.3 ± 0.4-fold (Fig. 6
), whereas 9CRE-tkCAT mediated a
1.3 ± 0.1-fold responsiveness to forskolin. These data
demonstrate that the augmented transcriptional responsiveness mediated
by the sequence we have identified is not limited to its interactions
with a VDRE.

View larger version (17K):
[in this window]
[in a new window]
|
Figure 5. The Sequences between -420 and -414 Augment
Transcriptional Responsiveness Mediated by an ERE
The ERE from the chick vitellogenin II gene was substituted for the
VDRE in the 14D3 and 9D3 oligonucleotides to
generate 14-ERE and 9-ERE. Double stranded oligonucleotides containing
the sense sequence shown, with GATC overhangs, were ligated into the
BamHI site of pUTKAT3 and transiently transfected into
ROS 17/2.8 cells. The arrows indicate the ERE. Relative
stimulation of CAT activity in response to 10 nM
17ß-estradiol represents the mean ± SEM of three
independent experiments using at least two different preparations of
each plasmid. Each transfection was performed in triplicate and
normalized for transfection efficiency with RSV-luc cotransfection.
|
|

View larger version (17K):
[in this window]
[in a new window]
|
Figure 6. The Sequences between -420 and -414 Augment
Transcriptional Responsiveness Mediated by a CRE
The rat somatostatin cAMP response element was substituted for the VDRE
in the 14D3 and 9D3 oligonucleotides to
generate 14-CRE and 9-CRE. Double stranded oligonucleotides containing
the sense sequences shown, with GATC overhangs, were ligated into
BamHI site of pUTKAT3 and transiently transfected into
ROS 17/2.8 cells. The CRE is indicated by the line. Relative
stimulation of CAT activity in response to 1 µM forskolin
represents the mean ± SEM of seven independent
experiments performed using at least two different preparations of each
plasmid. Each transfection was performed in triplicate and normalized
for transfection efficiency with RSV-luc cotransfection.
|
|
To examine the protein/DNA interactions in the region of the rat
osteocalcin gene 3' to the VDRE, gel retardation analysis was
performed. A double stranded oligonucleotide, -D14D3
(14D3 lacking the VDRE), representing the rat osteocalcin
sequence between -434 and -410 was employed as a probe (Table 3
). Gel retardation analysis was performed using
nuclear extracts from ROS 17/2.8 (Fig. 7A
) and UMR-106
(Fig. 7B
) cells. Two major retarded bands (see arrows in
Fig. 7
) were observed, all of which were competed for by excess
unlabeled -D14D3, wild-type 14D3, and the
human homolog of 14D3 (h14D3). Double stranded
oligonucleotide competitors, containing mutations that abolished
1,25-(OH)2D3 responsiveness in the context of
the native osteocalcin promoter (-DM3,M4 and -DM1,M2 Table 3
),
failed to compete for two high mol wt protein-DNA complexes present in
nuclear extracts from either ROS 17/2.8 or UMR-106 cells (Fig. 7
, A and
B).

View larger version (52K):
[in this window]
[in a new window]
|
Figure 7. The Sequences 3' to the Rat Osteocalcin VDRE
Generate Two High Mol Wt Protein-DNA Complexes in ROS 17/2.8 (A) and
UMR-106 (B) Nuclear Extracts
-D14D3 was employed as a probe (Table 3 ) in gel
retardation assays. Competitor oligonucleotides (10- and 100-fold molar
excess) were preincubated with nuclear extracts before probe addition,
as outlined in Materials and Methods. The two high mol
wt protein-DNA complexes are indicated by the arrows.
|
|
 |
DISCUSSION
|
---|
1,25-(OH)2D3-mediated
transactivation of the rat osteocalcin gene is initiated by the binding
of the hormone-bound VDR/RXR heterodimer to the VDRE (2, 3, 4). The bases
3' to the VDRE have previously been shown to influence the level of
1,25-(OH)2D3 responsiveness of this gene (5).
The experiments presented herein demonstrate that the bases between
-420 to -414 of the rat osteocalcin gene are essential for the
1,25-(OH)2D3 responsiveness of the rat
osteocalcin gene in the context of the native promoter, although this
region does not independently bind the VDR, nor does it confer
1,25-(OH)2D3 responsiveness independently of
the VDRE (5). Furthermore, because these DNA sequences increase
1,25-(OH)2D3 responsiveness in the context of
both the native rat osteocalcin promoter and the heterologous HSV-tk
promoter, the positions of these bases, with respect to the
transcription preinitiation complex, are not crucial. The
transcriptional activity of this region is not limited to VDRE-mediated
transactivation, as we have shown that it is capable of augmenting
transcription in concert with either an ERE or a CRE.
Accessory factors have been shown to contribute to transcriptional
activation mediated by the estrogen, thyroid hormone, and retinoic acid
receptors (13, 14, 15, 16, 17, 21, 22). Several positive cofactors have been
identified, including TRIP1 (23), TIF1 (24), RIP140, RIP160 (14, 16),
and SRC-1 (17). These proteins appear to mediate their positive effects
on transcriptional activation through an interaction with the
C-terminal transactivation domain of the steroid receptor. Although
these proteins have not been shown to bind DNA, sequences adjacent to
the steroid response element in question may contribute to binding or
transactivation by these accessory proteins.
The mechanism by which the identified sequences in the rat osteocalcin
gene enhance transcriptional responsiveness has not yet been
determined. Gel retardation assays indicate that this region does not
affect the affinity of the VDR/RXR heterodimer for the VDRE in either
porcine intestinal (5) or ROS 17/2.8 nuclear extracts (data not shown).
Gel retardation assays indicate that the sequences between -434 and
-410 (3' to the rat osteocalcin VDRE) generate two protein-DNA
complexes when incubated with ROS 17/2.8 and UMR-106 nuclear extracts.
The identified bases may, therefore, bind a transcription factor that
promotes 1,25-(OH)2D3-mediated transactivation
by facilitating interactions between the receptor-occupied VDRE and the
basal transcription apparatus. The VDR has been shown to interact with
transcription factor IIB via its carboxyl-terminal region (25), which
is also involved in receptor heterodimerization and ligand-induced
transactivation (26). When bound to their respective sites, the VDR/RXR
heterodimer and the factor that interacts with the sequences identified
may recruit unique coactivator proteins to the transcription initiation
complex or, together, efficiently recruit a single protein, resulting
in synergistic transcriptional activation (27). To date, the GGTTTGG
sequence present from -420 to -414 has not been identified as a
transcription factor-binding site. In composite mutations, upstream
sequences have also been identified as functionally relevant,
suggesting that the response element extends beyond the GGTTTGG
motif.
The GGTTTGG sequence that is critical for the
1,25-(OH)2D3 responsiveness of the rat
osteocalcin gene is also present in the human osteocalcin gene (10),
and we have shown that the region 3' to the VDRE in the human
osteocalcin gene enhances ligand-dependent transactivation 2-fold.
Comparison of the rat and human osteocalcin sequences immediately 3' to
their respective VDREs (Table 3
) indicates that of the eight bases
identified by mutagenesis experiments (between -425 and -416 of the
rat sequence) as being critical for
1,25-(OH)2D3 responsiveness in the context of
the native promoter, seven are conserved. In contrast, in the region
immediately 5' to the functionally important sequences, corresponding
to between -434 and -426 of the rat sequence, only four of nine bases
are conserved. Taken together, these data suggest that the boundaries
for the identified element are likely to lie between -429 and -414 of
the rat osteocalcin gene. Analysis of other
1,25-(OH)2D3- (7, 8, 9) and estrogen-responsive
genes (19, 22, 28, 29, 30, 31) failed to reveal a homologous sequence. However,
further definition of the exact base requirements for ligand-activated
transcription of the rat osteocalcin gene may yield new and important
information.
We have identified an element, distinct from the VDRE, that is
required for induction of rat osteocalcin gene transcription by
1,25-(OH)2D3. Our data lend credence to the
hypothesis that transactivation by steroid hormone receptors involves
more than the occupied receptor binding to its response element.
Characterization of accessory proteins and identification of novel
enhancer elements will provide important insights into the mechanism of
transcriptional activation by 1,25-(OH)2D3 and
other steroid hormones.
 |
MATERIALS AND METHODS
|
---|
Synthesis of CAT Fusion Genes
For experiments using a heterologous promoter, oligonucleotides
were inserted into the BamHI site of pUTKAT3 (18) (a gift
from Dr. David Moore, Massachusetts General Hospital, Boston, MA).
Oligonucleotides were synthesized (on an Applied Biosystems model 380A
synthesizer, Foster City, CA) corresponding to the sequences of
interest, with the addition of 5'-bases (GATC) to permit cloning into
the BamHI site of pUTKAT3. Orientation and copy number of
the oligonucleotides were determined by DNA sequencing.
For experiments using the native rat osteocalcin promoter, the plasmid
522-CAT, which contains 522 bp upstream from the transcriptional start
site of the rat osteocalcin gene fused to the CAT gene, was employed
(4). Mutations in regions of interest were introduced by site-directed
mutagenesis using the U.S.E. Mutagenesis kit (Pharmacia, Piscataway,
NJ). Two oligonucleotide primers were employed in this procedure. A
oligonucleotide (50-mer) that eliminated the XbaI site in
the polylinker of pUC18 (CGACTCTAGA to
CGACgaTAGA) was used as a selection primer. The mutation of
interest was introduced into a second primer. The mismatched bases were
located in the central portion of an oligonucleotide of 54 bases. The
position of the mutation was between bases -420 and -416 of the rat
osteocalcin gene (CCTGGGGTTTGGCTCC to
CCTGGttTggGGaTCC). The conversion of C
to A in position 413 created a BamHI site, which was used to
screen for the introduction of the mutation.
1,25-(OH)2D3 responsiveness in cells
transfected with the fusion gene that contained this base change alone
in the context of the native (data not shown) or HSV-tk promoters (Fig. 2
, M6-14D3) was the wild type. The sequences from -522 to
-306 (SacI site) were sequenced to confirm the introduction
of these mutations and to exclude the presence of other undesired
mutations. This region was then substituted for the identical
nonmutated region in the wild-type parent plasmid (OC-CAT) to yield
M3,M4-OC-CAT. A similar procedure was employed to introduce a mutation
in bases between -425 to -421
(CCTGGGGTTTGGCTCC to
aaTttGGTTTGGaTCC) to yield
M1,M2-OC-CAT.
Cell Culture and Transfections
ROS 17/2.8 cells were maintained in Hams F-12 medium with
L-glutamine (Life Technologies, Grand Island, NY)
supplemented with 10% (vol/vol) FBS, penicillin, and streptomycin.
From 24 h before transfection until harvesting, cells were
cultured in medium containing charcoal-stripped FBS. Transfections were
performed using the calcium phosphate method, as previously described
(32). UMR-106 cells were maintained in DMEM (Life Technologies)
supplemented with 10% (vol/vol) FBS, penicillin, and streptomycin.
From 24 h before transfection until harvesting, cells were
maintained in medium containing charcoal-stripped FBS. Transfections
were performed by lipofection (Life Technologies) with 10 µg test
plasmid/well of a six-well plate. As with the ROS 17/2.8 cells, UMR-106
cells were treated with 10-8 M
1,25-(OH)2D3 immediately after transfection and
again the following day. The cells were fed and stimulated 40 h
posttransfection and harvested 24 h later. A similar time course
of hormone treatment was used in the studies in which ROS 17/2.8 cells
were treated with 10-8 M 17ß-estradiol and
10-6 M forskolin. For the transfection assays
employing the ERE-tkCAT fusion genes, ROS 17/2.8 cells were maintained
in phenol red-free Hams F-12 medium with L-glutamine
(Life Technologies). CAT activity was assessed as previously described
(33). All test plasmids were cotransfected with a control plasmid
containing the luciferase gene under the control of the Rous sarcoma
virus (RSV) promoter. Luciferase activity was measured using a standard
protocol (34). The presence of 1,25-(OH)2D3,
17ß-estradiol, or forskolin did not affect the level of luciferase
activity. CAT activity was assessed by densitometric scanning of TLC
plate autoradiograms, and each replicate was normalized for
luminescence units. Relative CAT activity, presented as fold
stimulation, reflects the ratio of corrected CAT activity in the
presence or absence of inducer.
DNA Sequencing
All DNA sequencing was carried out by the dideoxynucleotide
chain termination method after subcloning into M13 vectors (35).
Gel Retardation Assays
Oligonucleotides were labeled by filling in recessed ends with
the large fragment of DNA polymerase I and
[
-32P]deoxy-ATP. The oligonucleotide used as a probe
for these studies was the sequence 3' to the rat osteocalcin VDRE
between bases -434 and -410 (-D14D3). The sequence of
its sense strand and those of competitor oligonucleotides are shown in
Table 3
. Gel retardation assay buffer composition and nuclear extract
preparation were described previously (5). ROS 17/2.8 or UMR-106 cell
nuclear extracts were preincubated with poly(dI-dC) at a concentration
of 0.5 µg/µg extract protein along with 10 or 100 ng of each
unlabeled competitor, as indicated, for 15 min at 22 C. Subsequently, 1
ng probe was added for an additional 15 min. The mixture was brought to
10% (vol/vol) glycerol and electrophoresed on a 4% polyacrylamide
gel.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Marie Demay, M.D., Endocrine Unit Wellman 501, Massachusetts General Hospital, Boston, Massachusetts 02114.
This work was supported by NIH Grant DK-36597.
1 Present address: Instituto de Investigaciones Metabolicas, Buenos
Aires, Argentina. 
2 Present address: University of Vermont Medical School, Burlington,
Vermont. 
Received for publication April 10, 1996.
Revision received October 28, 1996.
Accepted for publication October 30, 1996.
 |
REFERENCES
|
---|
-
Beato M 1989 Gene regulation by steroid hormones. Cell 56:335344[Medline]
-
Yu VC, Delsert C, Anderson B, Holloway JM, Devary OV,
Näär AM, Kim SY, Boutin J-M, Glass CK, Rosenfeld MG 1991 RXRß: a coregulator that enhances binding of retinoic acid, thyroid
hormone, and vitamin D to their cognate response elements. Cell 67:12511266[Medline]
-
Kliewer SA, Umesono K, Heyman RA, Evans RM 1992 Retinoid X
receptor interacts with nuclear receptors in retinoic acid, thyroid
hormone, and vitamin D3 signalling. Nature 355:446449[CrossRef][Medline]
-
Demay MB, Kiernan MS, DeLuca HF, Kronenberg HM 1992 Characterization of 1,25-dihydroxyvitamin D3 receptor
interactions with target sequences in the rat osteocalcin gene. Mol
Endocrinol 6:557562[Abstract]
-
Demay MB, Gerardi JM, DeLuca HF, Kronenberg HM 1990 DNA
sequences in the rat osteocalcin gene that bind the
1,25-dihydroxyvitamin D3 receptor and confer responsiveness
to 1,25-dihydroxyvitamin D3. Proc Natl Acad Sci USA 87:369373[Abstract]
-
Liu M, Freedman LP 1994 Transcriptional synergism between the
vitamin D3 receptor and other nonreceptor transcription
factors. Mol Endocrinol 8:15931604[Abstract]
-
Gill RK, Christakos S 1993 Identification of sequence
elements in mouse calbindin-D28k gene that confer
1,25-dihydroxyvitamin D3- and butyrate-inducible responses.
Proc Natl Acad Sci USA 90:29842988[Abstract]
-
Cao X, Ross FP, Zhang L, MacDonald PN, Chappel J, Teitelbaum
SL 1993 Cloning of the promoter for the avian integrin ß3
subunit gene and its regulation by 1,25-dihydroxyvitamin
D3. J Biol Chem 268:2737127380[Abstract/Free Full Text]
-
Chen K-S, DeLuca HF 1995 Cloning of the human
1-alpha,25-dihydroxyvitamin D3 24-hydroxylase gene promoter
and identification of two vitamin D-responsive elements. Biochim
Biophys Acta Gene Struct Express 1263:19[Medline]
-
Morrison NA, Shine J, Fragonas J-C, Verkest V, McMenemy ML,
Eisman JA 1989 1,25-Dihydroxyvitamin D-responsive element and
glucocorticoid repression in the osteocalcin gene. Science 246:11581161[Medline]
-
Noda M, Vogel RL, Craig AM, Prahl J, DeLuca HF, Denhardt
DT 1990 Identification of a DNA sequence responsible for binding of the
1,25-dihydroxyvitamin D3 receptor and 1,25-dihydroxyvitamin
D3 enhancement of mouse secreted phosphoprotein 1
(Spp-1 or osteopontin) gene expression. Proc Natl Aca Sci
USA 87:99959999[Abstract]
-
Ducy P, Karsenty G 1995 Two distinct osteoblast-specific
cis-acting elements control expression of a mouse
osteocalcin gene. Mol Cell Biol 15:18581869[Abstract]
-
Cavailles V, Dauvois S, Danielian PS, Parker MG 1994 Interaction of proteins with transcriptionally active estrogen
receptors. Proc Natl Acad Sci USA 91:1000910013[Abstract/Free Full Text]
-
Halachmi S, Marden E, Martin G, MacKay H, Abbondanza C, Brown
M 1994 Estrogen receptor-associated proteins-possible mediators of
hormone-induced transcription. Science 264:14551458[Medline]
-
Kurokawa R, Soderstrom M, Horlein A, Halachmi S, Brown M,
Rosenfeld MG, Glass CK 1995 Polarity-specific activities of retinoic
acid receptors determined by a co-repressor. Nature 377:451454[CrossRef][Medline]
-
Cavailles V, Dauvois S, LHorset F, Lopez G, Hoare S, Kushner
PJ, Parker MG 1995 Nuclear factor RIP140 modulates transcriptional
activation by the estrogen receptor. EMBO J 14:37413751[Abstract]
-
Oñate SA, Tsai SY, Tsai M-J, OMalley BW 1995 Sequence and characterization of a coactivator for the
steroid hormone receptor superfamily. Science 270:13541357[Abstract]
-
Prost E, Moore DD 1986 CAT vectors for the analysis of
eukaryotic promoters and enhancers. Gene 45:107111[CrossRef][Medline]
-
Burch JBE 1984 Identification and sequence analysis of the 5'
end of the major chicken vitellogenin gene. Nucleic Acids Res 12:11171135[Abstract]
-
Montminy MR, Sevarino KA, Wagner JA, Mandel G, Goodman RH 1986 Identification of the cyclic-AMP-responsive element within the rat
somatostatin gene. Proc Natl Acad Sci USA 83:66826686[Abstract]
-
Suen C, Chin WW 1995 A potential transcriptional
adaptor(s) may be required in thyroid hormone-stimulated gene
transcription in vitro. Endocrinology 136:27762783[Abstract]
-
Maurer RA, Notides AC 1987 Identification of an
estrogen-responsive element from the 5' flanking region of the rat
prolactin gene. Mol Cell Biol 7:42474254[Medline]
-
Lee JW, Ryan F, Swaffield JC, Johnston SA, Moore DD 1995 Interaction of thyroid hormone receptor with a conserved
transcriptional mediator. Nature 374:9194[CrossRef][Medline]
-
LeDouarin B, Zechel C, Garnier JM, Lutz Y, Tora L, Pierrat B,
Heery D, Gronemeyer H, Chambon P, Losson R 1995 The N-terminal part of
TIF1, a putative mediator of the ligand-dependent activation function
(AF-2) of nuclear receptors, is fused to B-raf in the
oncogenic protein T18. EMBO J 14:20202033[Abstract]
-
MacDonald PN, Sherman DR, Dowd DR, Jefcoat Jr SC, DeLisle RK 1995 The vitamin D receptor interacts with general transcription factor
IIB. J Biol Chem 270:47484752[Abstract/Free Full Text]
-
Whitfield GK, Hsieh J, Nakajima S, MacDonald PN,
Thompson PD, Jurutka PW, Haussler CA, Haussler MR 1995 A highly
conserved region in the hormone-binding domain of the human vitamin D
receptor contains residues vital for heterodimerization with retinoid X
receptor and for transcriptional activation. Mol Endocrinol 9:11661179[Abstract]
-
Guarente L 1995 Transcriptional coactivators in yeast and
beyond. Trends Biochem Sci. 20:517521
-
Walker P, Germond J-E, Brown-Luedi M, Givel F, Wahli W 1984 Sequence homologies in the region preceding the transcription
initiation site of the liver estrogen-responsive vitellogenin and
apo-VLDLII genes. Nucleic Acids Res 12:86118626[Abstract]
-
Berwaer M, Monget P, Peers B, Mathy-Hartert M, Bellefroid E,
Davis JRE, Belayew A, Martial JA 1991 Multihormonal regulation of the
human prolactin gene expression from 5000 bp of its upstream sequence.
Mol Cell Endocrinol 80:5364[CrossRef][Medline]
-
Shupnik MA, Weinmann CM, Notides AC, Chin WW 1989 An upstream
region of the rat leutinizing hormone ß gene binds estrogen receptor
and confers estrogen responsiveness. J Biol Chem 264:8086[Abstract/Free Full Text]
-
Hobson GM, Molloy ER, Benfield PA 1990 Identification of
cis-acting regulatory elements in the promoter region of the
rat brain creatine kinase gene. Mol Cell Biol 10:65336543[Medline]
-
Demay MB, Roth DA, Kronenberg HM 1989 Regions of the rat
osteocalcin gene which mediate the effect of 1,25-dihydroxyvitamin
D3 on gene transcription. J Biol Chem 264:22792282[Abstract/Free Full Text]
-
Demay MB, Kiernan MS, DeLuca HF, Kronenberg HM 1992 Sequences
in the human parathyroid hormone gene that bind the
1,25-dihydroxyvitamin D3 receptor and mediate
transcriptional repression in response to 1,25-dihydroxyvitamin
D3. Proc Natl Acad Sci USA 89:80978101[Abstract]
-
Ausubel FM, Brent R, Kingston RE, et al. 1992 Current
Protocols in Molecular Biology. Greene and Wiley-Interscience, New
York, pp 9.7.129.7.14
-
Sanger F, Nicklen S, Coulson AR 1977 DNA sequencing with
chain-terminating inhibitors. Proc Natl Acad Sci USA 74:54635467[Abstract]