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Departments of Pharmacology and Psychiatry and Behavioral Sciences, University of Washington, Seattle, Washington 98195
Address all correspondence and requests for reprints to: Tracy L. Bale, Box 357280, Department of Pharmacology, University of Washington, Seattle, Washington 98195. E-mail: tbale{at}u.washington.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine how estrogen might influence transcription of the OR gene, we isolated and sequenced 4.5 kilobases (kb) of upstream sequence of a rat genomic clone. In doing so, we discovered a discrepancy in nucleic acid content and length between our clone and that which was previously reported (22). This novel promoter sequence was then analyzed for regulatory elements and used in transfection assays to determine OR gene regulation by estrogen.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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phage GT10 rat genomic testes library was screened using
oligonucleotides based on human OR sequence. Positive clones were
digested with EcoRI and analyzed by Southern blotting.
Membranes were probed with the same 32P-labeled
oligonucleotides used for screening. Positive fragments were then
isolated, purified, and subcloned into pGEM7 (Promega). Sequencing was
performed according to the Sequenase (U.S. Biochemical Corp.,
Cleveland, OH) protocol, using primers corresponding to flanking vector
sequences and internal rat DNA sequences.
Verification of novel sequence in rat genome
As the genomic clone examined contained an additional
sequence internal to that reported by Rozen et al. (22),
oligonucleotides were synthesized by PCR to verify the presence of our
novel sequence in rat genomic DNA. Genomic DNA isolated from brain
tissue in two rat strains (Long-Evans and Sprague-Dawley) was used for
amplification. The sense oligonucleotide was located within the novel
sequence (nucleotide 3396–3416, 5'-ACATACGTGGTGCTGCAGCC-3'), whereas
the antisense oligonucleotide was located within a previously
reported sequence (nucleotide 4304–4324,
5'-AAAGTGGTTCATCGCAAGCCT-3'). The control reaction contained
oligonucleotides synthesized from the coding region sequence (sense,
oligonucleotides 4530–4550; antisense, oligonucleotides 5900–5920).
The PCR reaction was carried out under standard conditions for
Perkin-Elmer thermocycling: 1.5 mM MgCl2,
1 x PCR buffer, 0.5 µM oligonucleotide DNA, 1 µg
template DNA, and 0.25 mM nucleotides in a 100-µl
reaction for 35 cycles at temperatures of 95 C for 2.5 min, 57 C for
2.0 min, and 72 C for 1.5 min. PCR products were run on a 1% agarose
gel for size determination. Further PCR amplification included using
sense and antisense primers based on a previously reported sequence
that was amplified across the internal region of the novel
sequence.
Reporter gene constructs
Three reporter constructs were designed for use in
transfection experiments (see Fig. 3
). Portions of the OR promoter were
subcloned into the luciferase plasmid pGL2B (Promega, Madison, WI). As
shown in Fig. 3, clone ORfp contained the full-length promoter, 4.5 kb
upstream of the ATG translation start site. DNA clone ORp+
was a truncated form of the promoter, with an internal deletion of 3480
bp, bringing the upstream palindromic ERE into closer proximity to the
transcription start site, previously reported by Rozen et
al. (22). Clone ORp- was the same clone as
ORp+, except the upstream region containing the ERE was
removed by restriction enzyme digestion (NdeI).
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Transfection experiments
Transfection was performed by introduction of 3 µg
plasmid DNA/well into six-well plates containing MCF7 cells at 80%
confluency using the lipid transfection agent, DOTAP (Boehringer
Mannheim, Indianapolis, IN). Cells were treated with steroid hormone
for 8 h and harvested after 15-min incubation in lysis buffer
(Promega), unless otherwise specified. Cell extracts were used in
luciferase assays according to the Promega luciferase assay protocol.
The native pGL2 plasmid was used as a negative control for these
experiments, such that each well’s luciferase activity was subtracted
by the negative control’s luciferase activity for each experiment. For
experiments involving the ERE as an enhancer in the pGL3 plasmid, the
negative control was the native pGL3 plasmid. Results were normalized
by cotransfection of ß-galactosidase DNA, such that for each well the
resulting luciferase light units were divided by the ß-galactosidase
activity for that well.
OR binding
Binding was performed on whole cell extracts from treated MCF7
cells. Cells were distributed into the desired number of flasks, with
equal numbers of cells per flask. Flasks were randomly assigned to
treatment groups and were treated at 90–95% confluence for 24 h.
Cells were harvested (2.5–3 x 106 cells/flask) by
trypsinization, pelleted by centrifugation, resuspended in PBS for
washing, repelleted by centrifugation, and resuspended in a final
volume of 3 ml PBS. Cells were frozen at -80 C until assay was
performed.
Binding was performed using a cell harvester apparatus. GF-C membranes
(Whatman, Clifton, NJ) were used for attachment of cells on harvester.
Membranes were soaked in PBSBT (0.1% BSA, 0.02% tyrosine, and 0.1%
MgCl2 in PBS) plus 6 mg/ml BSA and 1 µm OT at room
temperature for 30 min. Binding reactions consisted of triplicate
samples for specific and nonspecific conditions for each cell
treatment. Each reaction contained approximately 2.5 x
105 cells, 0.03 nM
[125I]ornithine vasotocin (OTA) (New England Nuclear,
Boston, MA), and PBSBT buffer to a volume of 500 µl. Nonspecific
samples also contained 1 µM unlabeled OT. Reactions were
incubated for 15 min at 37 C before loading onto the harvester. Before
loading cells, membranes were first rinsed with 250 ml of 0.2%
polyethyleneimine. After cells were loaded onto membranes, incubation
tubes and membranes were rinsed four times with 3 ml 50 mM
Tris, pH 7.5. Membranes were then counted on a 
-counter for
quantitation of bound [125I]OTA. Averages of triplicates
for nonspecific binding values were subtracted from averages of
specific binding values for each treatment to determine specific
binding.
Western blot analysis
MCF7 cells were treated with vehicle (ethanol) or 10
nM estradiol (E2) for 24 h, then harvested
by trypsinization and resuspended in 10 mM Tris-HCl (pH
7.4), 100 mM NaCl, and 1 mM dithiothreitol
containing aprotinin (10 µg/ml), leupeptin (10 µg/ml), and
pepstatin (1 µM; homogenization buffer). Cells were
homogenized and centrifuged for 5 min at 700 x g, and
the supernatant was removed and centrifuged at 100,000 x
g for 30 min. Membrane pellets were resuspended in 1 ml
homogenization buffer, and the protein concentration was determined by
Bradford analysis. Membrane proteins (10 µg/lane) were separated by
SDS-PAGE on a 10% Laemmli minigel and electrotransferred to
nitrocellulose in a semidry transfer apparatus (Bio-Rad Laboratories,
Richmond, CA) for 2 h at 15 V (constant voltage) with 25
mM Tris (pH 8.3), 192 mM glycine, 20%
methanol, and 0.004% SDS as the transfer buffer. Unbound sites were
blocked overnight at 4 C in 10 mM Tris and 0.15
M NaCl (pH 7.4; TBS) containing 5% (wt/vol) skim milk
powder. Blots were washed three times for 10 min each time with TBS
containing 0.05% Tween-20 (TBST) and incubated for 1 h with a
1:10,000 dilution of anti-OR antibody (25) in TBST. Blots were then
washed three times for 10 min each time with TBST and incubated with
horseradish-linked goat antirabbit antibody (1:20,000) in TBST for
1 h. Blots were again washed three times with TBST as described
above, and immunoreactive bands were visualized using the Amersham ECL
immunoblotting detection system (Amersham Corp., Arlington Heights,
IL).
Mobility gel shift
The oligonucleotides synthesized were: the ERE of the
vitellogenin promoter as a positive control
(5'-GATCCAGGTCACTGTGACCTG-3'), the ERE for the OR
(5'-CCTTGGTCCAGATGACCCAGC-3'), and a scrambled form of the OR ERE as a
negative control (5'-CACTCGACATCAGCGTGTCGC-3'). Cytosolic extracts of
parturient uterine tissue were prepared as reported previously (26).
Aliquots of extracts were incubated with 250 ng poly(dI-dC) in 20 µl
buffer [10 mM Tris-HCl (pH 7.5), 1 mM
dithiothreitol, 100 mM KCl, 10% glycerol, and 3 µg/µl
BSA] plus 32P 5'-end-labeled oligonucleotides (20,000 cpm)
for 15 min at room temperature (27). Complexes were resolved by
electrophoresis at 4 C on a 5% nondenaturing acrylamide gel
equilibrated in 0.5 x TBE (5 mM Tris and 0.5
mM EDTA). For supershift assays, 1 µl of antiestrogen
receptor (anti-ER) antibody (SRA1000, Stressgen, Vancouver, British,
Columbia) was preincubated with cell extract overnight at 37 C before
adding labeled oligonucleotides.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
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, A and B). DNA from two rat strains were used to
verify that the discrepancy in sequence data was not due to a
difference in OR genes between rat strains. A control PCR reaction was
used to amplify coding region product of known length (Fig. 2C).
Further PCR reactions were performed to determine whether both this
novel gene and the originally reported gene were present in the genome,
suggesting the presence of two rat OR genes. None of these reactions
yielded PCR products consistent with the previously reported sequence.
The length of novel sequence is 1.26 kb and lies between the first two
large tg repeats found in the 5'-flanking region of this genomic clone
(Fig. 1
).
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). This distal sequence contains several
potential transcription factor-binding sites, including several
activating protein-1 (AP-1), AP-2, AP-3, and AP-4 sites and a
half-serum response element site. Included in the novel internal
sequence was a cAMP response element (CRE). A palindromic ERE,
differing by 1 base from the consensus, was found 4 kb 5' of the
translation start site. No typical CCAAT box was found in the novel
sequence; however, a TATA-like motif was localized to a region
approximately 100 bp from the transcription start site identified
previously by Rozen et al. (22) using rapid amplification of
3'-cDNA ends cloning. Other putative elements of interest in the novel
sequence include several half-glucocorticoid response elements and
retinoic acid response elements.
Effects of estrogen on reporter gene constructs
Several different reporter gene constructs were created for use in
transfection experiments to assess the effects of E2 on OR
promoter activity (Fig. 3). Our initial experiments used
the full-length construct (ORfp), and the two truncated constructs
(ORp+ and ORp-), which enabled us to examine
the potential activity of the palindromic ERE located 4 kb upstream.
Basal transcription was consistently enhanced in the full-length
construct. In a typical experiment, when corrected for transfection
efficiency, basal luciferase activity for the full-length construct in
MCF7 cells was 2000 vs. 250 light units for the luciferase
plasmid alone. Little induction in response to E2 treatment
was evident in the full-length (ORfp) construct. In contrast, exposure
to 10 nM E2 promoted a small, but consistent,
1.5-fold induction over vehicle levels of the ORp+
construct in MCF7 cells. This response was absent in the
ORp- construct in which the upstream ERE-containing
segment had been removed (Fig. 4). Transfection assays
using increasing concentrations of E2 (from 10–200
nM) showed no further effect on transcription (data not
shown). As these truncated constructs were determined to not contain
the newly identified TATA-like motif, we tested the induction
capability of the ERE by subcloning it as an enhancer into a basal
simian virus 40 reporter construct (pGL3).
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). Mutation of only
2 bases in this ERE completely eliminated its ability to act as an
enhancer after E2 treatment.
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). Levels
of specific binding in vehicle-treated cells were low (37.6 cpm/tube),
indicating a low basal state of expression of the OR in these
cells.
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). This band has a mol wt similar to that previously
shown for the OR (28, 29).
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). The OR ERE (OR-ERE)
showed a somewhat lower binding affinity for the ER than the
vitellogenen ERE, but was much greater than the scrambled sequence
negative control. The anti-ER antibody abolished protein binding to
both the vit-ERE and the OR-ERE.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transfection experiments in MCF7 cells using the OR 5'-flanking DNA, including the newly identified segments, demonstrated that this promoter exhibits both basal and inducible transcriptional activity. E2 exposure produced a variable response in MCF7 cells, enhancing transcription of the truncated ERE-containing construct (ORp+) 1.5 times that of vehicle, whereas induction was absent in the same construct lacking the ERE (ORp-). No induction of transcription by E2 was noted when the full-length construct (ORfp) was tested, suggesting that proximity of the distal ERE to the basal transcriptional machinery may be essential for E2 inducibility.
Although the in vitro reporter gene assays failed to show the expected large induction of transcription by E2, the receptor binding studies demonstrated that E2 can increase the number of endogenous OR-binding sites in MCF7 cells. Measurement of specific binding in vehicle- and E2-treated cells using [125I]OTA revealed a dramatic increase (17-fold) in OR binding. These results were supported by an increase in OR protein with E2 treatment of MCF7 cells, as shown by Western blot analysis. It has previously been shown that MCF7 cells endogenously express ORs, but only at levels that require extremely sensitive methods for detection (30, 31, 32, 33). The increase in OTA-binding sites and protein induced by E2 in these cells supports in vivo data suggesting that transcription of this gene can be regulated by E2. These data also argue that the small estrogenic induction of transcription of the OR reporter constructs we have seen cannot be attributed to the use of a heterologous cell line that does not express ORs or the factors necessary to induce transcription of the OR gene.
Mobility gel shift assays showed that the OR ERE does bind ER, and this shifted complex can be eliminated by an anti-ER antibody. This protein-binding activity was specific, as no binding was seen for a scrambled oligonucleotide composed of the same length and gc content. Because the truncated reporter constructs used did not contain the newly identified TATA-like motif, we assessed the enhancer activity of the upstream palindromic ERE in a basal promoter. Interestingly, in the context of a basal promoter, the OR ERE-induced transcription in an E2 concentration-dependent manner, whereas a mutant form of this ERE was nonresponsive to E2. These results suggest that if this ERE is involved in E2-induced gene transcription, it may be necessary that it be brought into closer proximity to the basal transcriptional apparatus, perhaps through DNA folding.
Several other genes known to be strongly regulated by E2in vivo exhibit only weak E2 inducibility
when exposed to E2 in vitro (27, 34). These
results suggest several hypotheses. First, the effect of E2in vivo may be indirect and may be mediated by
activation of protein kinase C and/or protein kinase A pathways.
Several studies have been reported that support this possibility
(35, 36, 37, 38). Alternatively, the effects of E2 in
vivo may not be mimicked in vitro due to a requirement
for a specific DNA configuration not achievable in our plasmid
constructs. We have noted evidence of secondary structure formation
caused by the three large tg repeat segments in this promoter. It is
conceivable that these tg repeats serve important roles in promoter
function and may allow response elements that are more distal to the
transcription start site to be brought into closer proximity. Evidence
for Z-DNA structure folding associated with promoter activation has
been suggested for other genes, including the rat cholesterol
7
-hydroxylase gene (39). Steroid hormones are known to influence the
formation of secondary structure required for promoter activity. For
example, the chromatin structure of the mouse mammary tumor virus is
dependent not only on the presence of certain steroid hormones, but
also on the cell type and receptor status of the recipient cell into
which it is transfected (40, 41, 42). Based on these reports, it is
possible that the OR gene may rely on an induced secondary structure
formed by the three tg repeats to exhibit E2 induction.
Lastly, it is reasonable to suggest the existence of a second OR gene,
which may be responsive to E2. The existence of two OR
genes might also provide an explanation for discrepancies that have
been reported between OR mRNA and protein localization in the brain. OR
binding studies have shown that OR binding is eliminated in the VMH
when female or male rats are gonadectomized, whereas binding is not
affected in the central nucleus of the amygdala (12, 43). It is
possible then that the gene we have isolated and studied here is that
which is responsible for this amygdala expression and, therefore, does
not require E2 for its induction.
In summary, we have sequenced and analyzed the rat OR promoter, including a novel 1.26-kb segment that had been deleted from the original published rat OR sequence, in addition to 2 kb of promoter sequence 5' of that previously reported (22). Sequence analyses revealed a palindromic ERE in addition to numerous potential response elements within this sequence. Reporter constructs of this promoter demonstrated only a small transcriptional response to E2. However, reporter constructs containing the OR ERE as an enhancer in the context of a basal promoter demonstrated an E2-concentration dependent induction. The palindromic ERE identified in the distal portion of the OR promoter showed ER-binding activity in gel shift assays. Receptor binding and Western blot assays showed that E2 treatment of MCF7 cells increased endogenous OR levels. Further studies are required to fully characterize modes of transcriptional regulation of this gene and their potential roles in OR expression in the brain and periphery.
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Acknowledgments |
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Footnotes |
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Received October 7, 1997.
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-hydroxylase gene.
Biochim Biophys Acta 1132:337–339[Medline]
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