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Gene Responsible for the Transient Transcription of a Variant Receptor
Université de Rennes I, Interactions Cellulaires et Moléculaires, Equipe Information et Programmation Cellulaires, Centre National de la Recherche Scientifique UMR 6026, Campus de Beaulieu, Rennes Cedex 35042, France
Address all correspondence and requests for reprints to: Dr. M.-L. Thieulant, Equipe Information et Programmation Cellulaires, UMR 6026, Bat 13, Campus de Beaulieu, 35042 Rennes cedex, France. E-mail: Marie-Lise.Thieulant{at}univ-rennes1.fr
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Abstract |
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gene. Analysis of the promoter sequence revealed the
presence of a major transcriptional start site, a canonical TATA
box and putative cis regulatory elements for pituitary
specific expression as well as an E-responsive element. In transient
transfection, the truncated ER product-1 promoter was transcriptionally
the most active in the lactotrope cell lines (MMQ). Analysis of
truncated ER product-1 functionality showed that: 1) the protein
inhibited ER binding to the E-responsive element in
electromobility shift assays, 2) inhibited the E2 binding to ER
in binding assays, 3) the truncated ER product-1/ER complex
antagonized the transcriptional activity elicited by E2, 4) nuclear
localization of green fluorescent protein-ER was altered in Chinese
hamster ovary cell lines stably expressing truncated ER product-1.
Collectively, these data demonstrated that the protein exerts full
dominant negative activity against ER. Moreover, truncated ER
product-1/ER complex also repressed the activity of all promoters
tested to date, suggesting a general inhibitory effect toward
transcription. In conclusion, the data suggest that truncated ER
product-1 could regulate estrogen signaling via a specific promoter
in lactotrope cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß (ER and ß) (1). Like other members of the
superfamily, the ERs have a modular structure consisting of distinct
functional domains (2). The DNA-binding domain enables the
receptor to bind to its cognate target site. The carboxy-terminal
domain contains the ligand-binding domain as well as sequences
necessary for nuclear localization, receptor dimerization, and
transcriptional function (AF-2) (2, 3, 4). A second
activation function, AF-1, is present in the amino-terminal domain of
the receptor (4, 5). Although the full transcriptional
activation is mediated through both AF-1 and AF-2, only activation
through AF-2 requires hormone binding (2, 6). The
transactivation mediated by estrogen-occupied receptor is a complex
process, involving interactions with transcription factors and other
components of the basal transcriptional machinery, and finally
modulating gene transcription (6).
Multiple forms are known to exist for the members of the nuclear
receptor superfamily. Regarding ER, multiple variants have been
described in human breast cancer biopsies, cell lines, and pituitary
tumors (7, 8, 9, 10). Mutations, deletions, or rearrangements of
the ER gene as well as alternative splicing, yielding precise
deletions of either a single exon or two exons, have been found
(7, 8, 9). In addition, human and rat ER genes are
transcribed from different promoters, yielding different mRNA isoforms
(11, 12, 13). Several groups have attempted to establish a
relationship between overexpression of ER variants and tumor
progression. However, their data have been conflicting
(14). These ER variants do not seem to be restricted to
malignant tissues because they have been found in normal tissues
expressing the wild-type (WT) ER (7, 8, 9, 15).
Interestingly, it has been shown that a non-DNA-binding ER isoform,
missing exon 3, suppressed E2-stimulated gene expression in breast
cancer and that its relative loss may be important in carcinogenesis
(16). Alternative translational start sites for ER may
also account for the formation of truncated ER proteins (17, 18). Although the majority of data for ER variant expression
are at the RNA level, accumulating data support the detection of
proteins corresponding to some of the previously identified ER
variant mRNAs, but their function still remains unclear.
The presence of ER mRNA variants or truncated proteins has been
previously described in both male and female rat pituitaries
(19, 20, 21). After E2 injection, Friend et al.
(20) cloned two variants missing exons 1–4 with a unique
31-bp untranslated sequence upstream of exon 5 (named TERP-1 and -2,
for the female and male pituitary, respectively). Our laboratory
previously identified such an ER variant mRNA in a female rat
pituitary gland (21). The TERP-1 mRNA appears to be
restricted to the pituitary gland on proestrous stage. This mRNA is
inducible by E2 or gonadotrophins and localized in lactotrope
populations (20, 21). A protein product of 20–24 kDa was
described (22, 23). Although TERP-1 lacks the A/B-, DNA
binding, and hinge region, it can form heterodimers with WT-ER both
in vitro (24) and in vivo
(25) and inhibits ER binding to its cognate DNA
response element (24). On the other hand, contradictory
results have been published concerning the transcriptional effect of
TERP-1 on WT-ER transactivation (24, 26, 27). TERP-1
physiological function remains unknown.
Because TERP-1 expression is transient and pituitary cell specific, we
hypothesized that it could be generated by the use of an alternative
promoter. As an initial step to understand how TERP-1 expression may be
regulated, we isolated genomic clones encoding a specific promoter. In
this report, we demonstrate that TERP-1 originates from an intronic
promoter into the ER gene. The truncated protein has also been
functionally characterized. Specifically, we reexamine the
transcriptional control exerted by TERP-1 on WT-ER stimulated activity
using various E-responsive element (ERE) reporter gene constructs. We
confirm that TERP-1 interferes with the ability of ER to bind to a
consensus ERE. At transcriptional level, we observed the TERP-1/ER
complexes have an inhibitory effect on corresponding reporter genes. We
demonstrate that the effects of TERP-1 could be due to an
alteration of the E2 binding to ER. Moreover, the TERP-1/ER
complex can apparently block general transcriptional activity,
suggesting that it could work as a negative regulator of nuclear
receptor activity in vivo. Taken together, data herein show
that TERP-1 potentially functions as a dominant negative inhibitor of
ER activity and suggest that TERP-1 could regulate estrogen
signaling via a specific promoter in lactotrope cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cloning TERP-1
A set of primers was designated according to the published
TERP-1 sequence (20) and ER sequence (30).
The sequences were as follows: TERP-1,
5'-CGGGATCCATTTCTTGAGCTTGTTGAACAGC-3'; exon 8,
5'-GGAATTCCGATTCGCAGAACCTTGTGGG-3'. Single-strand cDNA was
reverse transcribed from pituitary total RNA from rats on proestrous
stage. PCR cycling was performed as following : 1) 94 C, 45 sec; 60 C,
45 sec, and 72 C, 1 min (5 cycles); and 2) 94 C, 45 sec; 66 C, 45 sec,
and 72 C, 1 min (35 cycles). The resulting PCR fragments were subcloned
into the EcoRI/BamHI sites of plasmid and carrier
DNA (pcDNA) 3.0 (Invitrogen, Gröningen, The
Netherlands) and were completely sequenced from both
strands.
Cloning of the 5'-flanking region of the TERP-1 exon
We have cloned the 5'-flanking region of the TERP-1 exon with
the PCR-based gene walking method (Genome Walker kit; CLONTECH Laboratories, Inc., Palo Alto, CA). The 5'-flanking region of
the TERP-1 sequence was amplified from the five rat Genome Walker
genomic libraries using Expand High Fidelity Polymerase (Roche Diagnostics, Meylan, France). Two consecutive rounds of
PCR using the adaptor primers AP1 and AP2 and the gene-specific reverse
primers S-TERP-1 (5'-CTGGTCGCTGTTCAACAAGCTC-3', nucleotides at
positions 34–12 of specific TERP-1 sequence) and FL-TERP-1 (5'
CTGGTCGCTGTTCAACAAGCTCAAGAAATGG-3', at positions 34–3 of specific
TERP-1 sequence) were performed. The first amplification was performed
using AP1 and S-TERP-1. The reaction involved 2 sec of denaturation at
94 C, followed by 40 cycles consisting of 2 sec of denaturation at 94 C
and 3.5 min of annealing and extension at 68 C. The PCR products were
diluted to 1/50 and then subjected to the secondary PCR with the nested
primers AP2 and FL-TERP-1 using the same protocol. The PCR products
were subcloned either into the BlueScript vector
(Stratagene, Amsterdamzuidoost, The Netherlands) for
sequencing or pGL3 vector (Promega Corp.,
Charbonnières, France), respectively.
Southern blot analysis
Ten micrograms of rat genomic DNA were digested overnight with
restriction endonucleases, electrophoresed on a 0.8% agarose gel, and
subjected to Southern blot analysis using either P-labeled rat ER
cDNA (exons 4–5) or 726-bp fragment of the 5'-flanking region of the
TERP-1 gene (corresponding to residues +34 -692 of the rat DNA library
1 fragment) as probes. Hybridization was carried out for 16 h at
55 C in 1 mM EDTA, 1% BSA, 7% SDS, 0.5 M
phosphate buffer, pH 7.2. Filters were washed under high stringency
conditions in 0.2x salt sodium citrate, 0.1% SDS for 30 min each at
25 C, 55 C, and 68 C.
Primer extension
A 22-oligonucleotide primer selected to be 57 bp downstream of
the translational start codon (ATG) (-57 to -78 bp) was used for
primer extension. The primer was end-labeled with
32P using T4 polynucleotide kinase (Roche Diagnostics). Total RNA (50 or 100 µg) from the MMQ lactotrope
pituitary cell line or rat liver and spleen was isolated and was
hybridized to the primer for 16 h at 50 C in 80% Formamide, 0.4
M NaCl, 1 mM EDTA, and 40 mM PIPES
pH 6.4. Extension was initiated with reverse transcriptase (Superscript
II, Life Technologies, Inc.), and the reaction was
allowed to proceed for 1 h at 42 C. The primer extension reaction
products were analyzed on a 4% polyacrylamide-7 M urea gel
and their lengths determined by comparison with a sequencing
ladder.
Construction of the GFP-ER expression vector
The rat ER cDNA was generously provided by Dr. M. Muramatsu,
isolated from pUC-ER (30), and mutated in the first ATG
of the ER coding region. The pEGFPC3 vector, which encodes a green
fluorescent protein (GFP) variant (S65T-GFP) was purchased from
CLONTECH Laboratories, Inc. EcoRI and
BamHI restriction sites were added to the 5' and 3' ends of
ER using PCR. In GFP-ER, GFP was fused at the C terminus of the rat
ER.
Construction of stable cell lines expressing TERP-1
CHO-K1 cells were transfected in 25-cm2
flasks using Fugene-6 (Roche Diagnostics) with 3 µg
TERP-1 pcDNA. Twenty-four hours after transfection, the medium was
changed, and G-418 (Roche Diagnostics, Inc.) was added to
a final concentration of 500 µg/ml. After 24 h, the cells were
trypsinized, diluted 1:100 in complete medium plus G418, plated in
25-cm2 flasks, and incubated until cell clones
became visible. The clones were characterized by RT-PCR for TERP-1
expression.
Transient transfection assays
For TERP-1/ER heterodimer transactivation activities, 1 x
105 cells were plated on 6-well plates 24 h
before transfection in phenol red-free media and charcoal-stripped
serum. Precipitates were formed using expression vectors in the amounts
indicated in the figures, 3.5 µg of reporter pcDNA (pcDNA3 or pCMV5)
to a total of 4.5 µg. Following transfection, cells received fresh
medium containing twice charcoal-stripped serum and were incubated with
or without E2 (10 nM). For TERP-1 promoter activity, medium
with complete serum was used. Cells were transfected with Fugene-6
according to manufacturer’s instructions (Roche Diagnostics). Then 0.7 µg of RDL-4-LUC vector and 0.3 µg
ß-galactosidase expression plasmid (pCMV-ßgal, Promega Corp.) as internal control were transfected. For all studies,
cells were harvested 30 h after removal of the precipitates. All
transfections were done in triplicates. Protein concentrations in cell
lysates were determined using the colorimetric protein assay system
(Bio-Rad Laboratories, Inc., Ivry/Seine, France).
Chloramphenicol acetyl transferase assays (31) and
ß-galactosidase assays were done using the method essentially as
described (32). Luciferase activity (31) was
determined with reagents according to the manufacturer’s instructions
(Promega Corp.) using either a luminometer ML 3000
(Dynatech Corp., Guyancourt, France) or a reporter
microplate luminometer system (Promega Corp.). According
to the experiments, luciferase activities were normalized either per
100-µg proteins or relative to ß-galactosidase activity.
Analysis of TERP-1 by RT-PCR
Tissues or cells were collected and total RNA prepared using
Trizol (Life Technologies, Inc.) according to the
manufacturer’s instructions. Total RNA was reverse transcribed (400 U;
Superscript, Life Technologies, Inc.) as previously
described (21). Briefly, amplification conditions were 94
C for 45 sec, annealing for 45 sec, 72 C for 1 min for 30 cycles, and a
final cycle of a 5-min extension step. The PCR products were
electrophoresed on 1.2% agarose gel, visualized by ethidium bromide
staining. The oligonucleotides (Genset, SA, Paris, France)
TERP-1, 5'-CCATTTCTTGAGCTTGTTG-3' and exon 8, 5'-CGTTTCAGGGATTCGCAG-3'
were used for amplification of TERP-1 mRNA. The oligonucleotides, exon
1, CAGCAGCGAGAAGGGAAACA-3', and exon 4, 5'-GGGCGGGGCTATTCTTCTTA-3' were
used for amplification of ER mRNA. The oligonucleotides, RL19–1,
5'-GGACCCCAATGAAACCAACG-3' and RL19–2, 5'-CCTTCCTCTTCCCTATGCCC-3' were
used for amplification of control RL19 mRNA as previously described
(21). In a few experiments, the oligonucleotides, Actin-1,
5'-TGGTGGGTATGGGTCAGA-3', and Actin-2, 5'-CCAGCATAGAGCCACCAA-3' were
also used as controls.
In vitro translation
Recombinant ER and TERP-1 cDNAs were in vitro
transcribed and translated in TNT-T7 coupled rabbit reticulocytes
lysates (Promega Corp.) from the T7 promoter following the
manufacturer’s guidelines. Typically, the reactions were carried out
in 12.5-µl TNT lysate containing 0.5 µg plasmid, 1-µl TNT buffer,
0.5 µl of amino acid mixture minus methionine, 0.5 µl
[35S] methionine (1175 Ci/mmol, ICN), 0.5 µl
T7 RNA polymerase, and completed up to 25 µl with DNasefree
water.
Electrophoretic mobility shift assay
Electrophoretic assays were performed in a 20-µl binding
reaction in a binding buffer (10 mM Tris-HCl pH 7.5, 1
mM EDTA, 0.5 mM dithiothreitol, 5% glycerol,
and 50 mM NaCl). Each binding reaction contained 10 µl
ER and/or TERP-1 cotranslated in vitro as indicated
above, 0.5 µg of poly(dI-dC), and 20,000 cpm of consensus ERE probe
(33) labeled with [
-32P] ATP
(4000 Ci/mmol, ICN) using T4 polynucleotide kinase (Roche Diagnostics). Preincubations containing proteins and/or cold
competitor as indicated were performed at 4 C for 20 min. After the
incubation step, the probe was added, and binding was conducted for 20
min at room temperature. Protein-DNA complexes and free probe were then
separated on a 6% native polyacrylamide gel using 0.5x
Tris-borate-EDTA buffer.
Ligand binding analysis
Ligand binding studies were conducted essentially as previously
described (34) with the following modifications: ER and
TERP-1 proteins were synthesized in vitro either alone or
cotranslated in a 1:5 ratio. Translation reaction mixtures were diluted
10 times with TET (10 mM Tris-HCl, pH 7.4, 1.5
mM EDTA, 10 mM
thioglycerol, containing protease inhibitors, 50 µg/ml leupeptin, 5
µg/ml phenylmethyl sulfonyl fluoride, and 5 µg/ml aprotinin) and
0.1-ml aliquots were incubated at 4 C overnight with [2, 4, 6,
7-3H] 17ß-estradiol (110 Ci/mmol,
Amersham Pharmacia Biotech, Orsay, France) ranging from
0.1 to 50 nM. Nonspecific binding was assessed by
including a 200-fold excess of unlabeled E2. Binding was measured by
the protamine sulfate assay (34).
Western blotting
Pituitary cell extracts (75 µg) were resolved by 13% SDS-PAGE
and then electrotransferred to a nitrocellulose membrane (Hybond C,
Amersham Pharmacia Biotech). Filters were blocked
overnight with PBS containing 2% nonfat milk and 0.1% Tween 20
(Sigma, Inc.), incubated with primary anti-ER
monoclonal antibody F3 (35) for 2 h at room
temperature. Protein bands were visualized by enhanced
chemiluminescence (ECL, Amersham Pharmacia Biotech) using
a horseradish peroxidase-conjugated goat antirabbit secondary
antibody. Filters were washed in PBS-Tween four times for 5 min between
each step.
Data analyses
The values were expressed as mean ± SEM of at
least three independent experiments. A t test was used to
determine statistical significance. Differences between groups were
considered to be significant at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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exon 8 (20, 30). A schematic
representation of TERP-1 structural organization is presented in Fig. 1A
. Figure 1B shows the pattern of PCR
products appearing during the estrous cycle, confirming data previously
observed using Northern blot analysis (21). According to
the high sensitivity of the PCR technique, a very low-level signal was
detected on metestrous day. This signal increased up to a maximal level
on proestrous day, in which two TERP products were clearly identified.
No PCR product was amplified at estrus. One of these amplified products
corresponds to TERP-1; the second product, TERP-C, was cloned and
sequenced. It contains the TERP-1 specific 31 bp and an additional
insertion of 99 bp between exons 5 and 6 of ER. Figure 1C shows the
expression pattern of TERP-1 in two pituitary cell lines: GH4-C1, a
mammosomatotrope tumor cell line (29), and MMQ, a pure
lactotrope tumor cell line (28). Bands corresponding
either to the expected size of ER or TERP-1 transcripts were
detectable in both lactotrope cell lines (Fig. 1C). The two variant
TERPs were detected as the major ER in MMQ cells, confirming the
specific expression in the lactotrope cells (21). No PCR
product was detected for TERP-1 either in gonadotrope cell lines
(T3–1) or in other tissues tested, (uterus and hypothalamus [data
not shown]).
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. Finally, the nucleotide sequence
contains 2001 bp upstream the TERP-1 sequence and 507 bp
downstream.
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) was labeled and hybridized to total
RNA from MMQ lactotrope pituitary cell line and extended with a reverse
transcriptase deprived of RNase H activity. Negative controls were
performed with total RNA isolated from rat liver and spleen. Several
primer-extended products were generated from pituitary but not from
liver, spleen, or yeast tRNA (Fig. 3B). A major extended product mapped
transcription start site at position 99 bp upstream from the ATG
initiation codon. This site is referenced in the sequence as position
+1 (Fig. 3). Three additional minor extended products were found, one
of them in the proximal region.
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). A
computer-assisted search for consensus transcription factor-binding
sites localized a putative palindromic ERE at positions -196/205. For
11 out of 12 bp, this ERE is similar to the EREc (AGGTCAnnnTGACCT)
described in the vitellogenin A2 gene of Xenopus laevis
(33). Four perfect half-EREs were also found in this
sequence. The identification of eight potential Pit-1 sites
(36), a pituitary-specific transcription factor, is of
particular interest. In addition, the promoter region contains three
consensus motifs for pituitary homeobox 1 and 2 factors, Pitx1/2
(37), and four potential binding sites for the specific
pituitary factor neural zinc finger factor-1 (NZF-1) (38).
Other consensus binding sites for some factors known to regulate
eukaryotic promoters were also found. Notably, two potential activator
protein-1 (AP-1) recognition sites were also present.
To investigate the exon TERP-1 position, we carried out a Southern blot
analysis of the rat genomic DNA using two different
32P-labeled probes corresponding either to the
cloned genomic fragment RDL-1 (Fig. 2) or to exons 4–5 of rat ER
cDNA (nucleotides 908-1331). Rat genomic DNA was digested with various
restriction endonucleases, separated on agarose gels, and hybridized
under high stringency conditions with the two probes. As shown in Fig. 4A, it clearly appears that the fragments
of the HindIII and BamHI digests hybridized both
probes. These results indicate that the TERP-1-exon is localized within
intron 4 of ER gene (Fig. 4B).
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T3–1 and MMQ)
and nonpituitary (MCF-7 human mammary tumor cells and CHO-K1 cells)
origins were transiently transfected with RDL-4-LUC. Comparisons were
made with the -LUC promoter (containing the -846/+50 promoter
fragment of human - subunit) as a pituitary-specific control,
and the rainbow trout ER gene promoter inserted into a pGL2 vector
(containing the -40/-248 ER promoter fragment of rainbow trout,
rtER) as an ER control promoter, and the promoterless control
luciferase vectors, pGL3 and pGL2, which served as controls for basal
levels of luciferase expression. Our results indicate that the 2.0-kb
5'-flanking region of exon TERP-1 was transcriptionally active in all
the tested cell lines (Fig. 5).
Transcriptional activity of RDL-4-LUC in MMQ cells (which contain
endogenous ER and Pit-1) increased by 30.2 ± 3.0-fold
relative to the activity of the promoterless vector, pGL3 (n = 6,
P < 0.0001). In contrast, RDL-4-LUC activity in the
T3–1 gonadotrope cell line (which expresses endogenous ER but
not Pit-1) showed only a 5.3 ± 0.5-fold increase (n = 6,
P < 0.0001) above the pGL3 control. When RDL-4-LUC was
transfected into the nonpituitary cell line MCF-7 (ER positive),
luciferase activity was 8.6 ± 0.4-fold (n = 3,
P < 0.0001) higher than pGL3 controls.
Transfection of TERP-1 promoter led to a modest but significant
1.8 ± 0.2-fold (n = 3, P < 0.01) increase
in luciferase activity over the parent vector pGL3-LUC in CHO cells
(ER negative) (Fig. 5). The activity of the TERP-1 promoter was 25%
higher than that of promoter LUC in both MMQ and T3–1 (n =
3). Comparatively, the activity of the well-characterized rtER
promoter was 3.0 ± 0.3-fold (n = 2) in MCF-7 cells and
4-fold in MMQ cells. TERP-1 promoter therefore directed luciferase gene
expression in the MCF-7 and T3–1 cells at a level superior to that
of both the rtER and -LUC. Transient transfection studies thus
indicate that the putative TERP-1 promoter is functional. In addition,
the data show that maximal cell-specific expression was conferred
in lactotrope cells, suggesting that regulatory elements for
cell-specific expression are present within the studied promoter
sequence.
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shows the in
vitro translation protein products from ER and TERP-1 cDNA.
[35S]-methionine-labeled protein products
migrated at the expected sizes of 66 kDa for WT-ER and 24 and 22 kDa
for TERP-1. The same pattern was observed when TERP-1 and ER were
cotranslated in a 1:1 ratio. To identify TERP-1 in vivo,
Western blot analyses were performed using F3 (35), a
monoclonal antibody specifically directed against the F-domain of
ER. Notably, a protein of approximately 24 kDa was detected in
whole-cell extracts of pituitaries from 28-d-old PMSG-primed female
rats, a treatment inducing a proestrous-like (21) (Fig. 6B, lane 1). The 24-kDa protein was not detected in control rats (lane
2) or by ER 715, a polyclonal antibody directed against the D domain of
ER (39) (data not shown). The 32- to 40-kDa bands
observed in these studies were nonspecific because they were also
present in the spleen used as a negative ER control. Consequently,
the data strongly suggest that the in vivo translation
product of TERP-1 mRNA is the 24-kDa protein. Consequently, the
methionine 393 of ER is the apparent translation initiation site of
TERP-1. The 22-kDa protein observed in in vitro-translated
preparations is presumably translated from a second in-frame start
codon corresponding to methionine 401, leading in vitro to
the production of two alternative forms of the TERP-1 (Fig. 6A).
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and TERP-1
interaction, we examined the possibility for TERP-1 to specifically
modulate the ability of WT-ER to bind to its cognate DNA-binding motif,
a consensus ERE, using EMSAs. The ERE used in these experiments
contained a 35-bp palindromic sequence identified in the
Xenopus vitellogenin A2 promoter
(33). ER and TERP-1 proteins were produced by in
vitro translation, incubated with a
32P-labeled double-stranded ERE oligonucleotide
and analyzed in EMSAs. The translational efficiencies of the
proteins were determined to be equivalent by performing parallel
reactions in the presence of (35S) methionine
(data not shown). As expected, the WT-ER translated in lysate was able
to form specific ER-ERE complexes, which were completely displaced with
an excess of unlabeled ERE. Neither an excess of ARE nor an excess of
GRE inhibited WT-ER binding (Fig. 7A). Control experiments showed that
TERP-1 did not bind this element (data not shown).
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). On the other hand, when increasing ratios
of WT-ER were used, the binding activity was maintained constant (data
not shown).
The ability of TERP-1 to antagonize the binding of WT-ER to ERE was
further evaluated in experiments in which different molar ratios
(0.5:1.0, 1:1, 5:1, and 10:1) of TERP-1 and WT-ER were used. At a 1:1
molar ratio of the two plasmids, TERP-1 highly inhibited the formation
of WT-ER-ERE complexes (Fig. 7C, lane 3). These results demonstrate
that TERP-1 prevents WT-ER from binding to its response element under
standard experimental conditions when cotranslated with ER.
TERP-1 inhibits E2 binding to WT-ER in vitro
To ascertain whether TERP-1 was able to interfere with E2 binding
to WT-ER, binding studies were performed on
in vitro-translated TERP-1, ER or cotranslated TERP-1 and
ER. As shown in Fig. 8A, when TERP-1
and WT-ER were cotranslated in a ratio of 5:1, the presence of TERP-1
inhibited by 90% the E2 specific binding to WT-ER. The number of
binding sites was very low for TERP-1 translated alone and represented
only 10% of the capacity of WT-ER binding. Scatchard plots revealed a
dissociation constant (Kd) of 0.18
nM for ER similar to that measured in uterine
cell extracts (Kd = 0.22
nM) whereas TERP-1 showed a
Kd of 0.8 ± 0.27 nM
(n = 3) (Fig. 8B).
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expression vector increased up to 250 ng/well, only a 20% decrease of
the maximal activity was observed. TERP-1 had no constitutive or
E2-stimulated effects on reporter gene transcription when expressed
alone in the CHO cell line (data not shown).
WT-ER plasmid was cotransfected with TERP-1 at different molar ratios
using various ERE reporter genes: (ERE)2-tk-CAT, p(ERE)2-TATA-CAT,
ERE-tk-LUC, and ERE-SV-LUC. Data displayed in Fig. 9A show that TERP-1 inhibits
E2-dependent transcription of all ERE gene reporters tested. When WT-ER
and TERP-1 were coexpressed at a molar ratio 5:1, E2-dependent
transcription from the (ERE)2-TATA-CAT reporter was inhibited by
48 ± 4% (n = 3). Coexpression of a 10-, 15-, or 20-fold
molar excess of TERP-1 over WT-ER did not induce a further decrease. A
similar pattern of response was reproduced with the (ERE)2-tk-CAT
reporter. Inhibition was even more marked on simple ERE-tk-LUC or
ERE-SV-LUC.
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significantly repressed the activity of either tk (Fig. 10A) or CMV promoters (Fig. 10B), resulting in a dose-dependent decrease of promoter activities.
Therefore, the TERP-1/ER complex could have a more general
inhibitory effect toward transcription.
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). Similar results were observed in CHO
cell lines using hAR expression vector, suggesting a repressing
activity toward various nuclear receptors.
Qualitative analysis of ER subcellular localization in stable
cell lines expressing TERP-1
To follow the subcellular localization of the ER in living
cells expressing TERP-1, we tagged the amino terminus of the rat ER
with the S65T variant of GFP (pGFP-ER). The TERP-1 vector was stably
transfected in ER negative CHO cell lines (CHO-TERP-1). In a first
set of experiments, original CHO cells were transfected with either
pGFP or pGFP-ER to verify the localization of GFP alone or GFP-ER in
the absence of TERP-1. As expected, fluorescence in CHO cells
transiently transfected with GFP alone appears uniformly distributed
throughout the cytoplasm and nucleus (Fig. 11A) whereas CHO cells transfected with
pGFP-ER show green fluorescence restricted to the nucleus (Fig. 11B).
Thus, GFP-ER localization is consistent with previously reported data
for the endogenous ER. The same distribution of ER was observed
in CHO cells using immunocytochemistry clearly demonstrating the
validity of our conditions. These data also show that the presence
of the GFP tag has little effect on the localization of WT-ER. In the
second set of experiments, TERP-1 localization was assessed by
immunocytochemistry in CHO-TERP-1 cells. Results (not shown) confirmed
those observed after transient transfection by Resnick et
al. (24) demonstrating that TERP-1 is present in both
the cytoplasm and nucleus of cells. Then CHO-TERP-1 cells were
transiently transfected with pGFP-ER. Surprisingly, part of the
fluorescent GFP-ER receptor was diffusely distributed throughout the
cytoplasm (Fig. 11, D through F). As a control, GFP-COUP-TFI
transiently introduced in the stable line CHO-TERP-1 showed an
exclusive nuclear localization (Fig. 11F). Similar data were observed
with pGFP-GR (data not shown). These results suggest a retentional
activity of TERP-1 toward the ER in the cytoplasm.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene, which controls
the expression of TERP-1 mRNA, and analyzed the promoter region. We
also provide functional evidence demonstrating that TERP-1 can
antagonize ER.
We have isolated three overlapping genomic clones encoding 2.5 kb in
the flanking regions upstream and downstream the sequence TERP-1.
Genomic Southern blot analysis showed that TERP-1 sequence corresponds
to a noncoding leader exon localized within the intron 4 of ER gene.
Therefore, we demonstrate that TERP-1 is alternatively spliced from an
intronic promoter within the ER gene. The existence of a tight
transcriptional control of the expression of the TERP-1 mRNA through
this promoter could explain the fluctuations observed in the pattern of
expression during the estrous cycle (21, 22). The
triggering mechanism for the choice between the "classical"
alternate exons 1 (10, 11, 12) and exon TERP-1 remains
unknown. However, transient and cell-specific expression of the variant
suggests that a distinct activating signal is necessary to drive the
TERP-1 promoter during the estrous cycle.
Analysis of the 5'-flanking region of the gene showed a major
transcription initiation site and revealed the presence of a TATA box
motif residing near the transcription initiation site. The presence of
consensus TATA sequences is unusual among the steroid receptor family.
Consensus binding sites for the POU-homeodomain transcription factor,
Pit-1, may play an important role in the cell-specific expression of
TERP-1. In addition, the promoter region contains three consensus
motifs for bicoid-related homeobox 1 and/or 2 factors (Pitx-1/2), and
four potential binding sites for the pituitary NZF-1, which have been
shown to be involved in pituitary-specific expression and development
(36, 37, 38). The presence of these sites has not been yet
described in the promoter of nuclear receptor and could suggest an
involvement of TERP-1 in differentiation of the lactotrope and
somatotrope lineages. Of note, expression of Pit-1 and of the latter
three factors is maintained in the mature pituitary gland (38, 40). Most E-responsive genes, including the classical promoter
of the ER gene, lack the canonical ERE and contain one or more
imperfect EREs or multiple copies of ERE half-sites. The presence of
consensus palindromic ERE sequence suggests that estrogen may have a
direct effect on the TERP-1 promoter and play a role in the regulation
of TERP-1. Several half-EREs are also present in the TERP-1 promoter
sequence and could be active individually or cooperatively
(41, 42, 43). Moreover, cooperative interactions between the
Pit-1 protein and the ER have been shown to be important in the
regulation of PRL gene transcription (44) and could also
be involved in the regulation of such a promoter.
The 2.0-kb promoter is functional and directs strong basal expression
in pituitary cells. It is transcriptionally more active in lactotrope
than in gonadotrope cell lines. The transcriptional activity of TERP-1
promoter is even slightly higher in MCF-7 cells than in gonadotrope
cell lines. The higher basal activity observed in MMQ, compared with
T3–1, cells could be explained by the absence of any endogenous
Pit-1 in the latter cells. In addition, the promoter activity in
ER-positive T3–1 and MCF-7 cells is probably attributable to the
presence of the EREc. These results once again agree with the
cell-specific distribution of TERP-1 mRNA previously observed in rat
lactotrope cells (21); we also observed a high expression
of TERP-1 mRNA in lactotrope cell lines as described by Schreihofer
et al. (45). However, in contrast to their
findings, we did not observe any expression in gonadotrope -T3 cell
line. Taken together, these results suggest a cell-specific pituitary
regulation of the TERP-1 promoter.
The expression of the truncated protein correlates with our previous data showing TERP-1 mRNA expression in pituitary cells on proestrous day (21). Studies from Friend et al. (22) and Mitchner et al. (23) have also described a 20- to 24-kDa TERP-1 protein in female estrogen-treated pituitaries. In this report, we characterized a 24-kDa protein in a proestrous-like situation. The methionine 393 is the apparent translation initiation site of TERP-1 in vivo. In contrast, Mitchner et al. (23) have suggested that the methionine 426 could be the initiation site of TERP-1 translation. The reasons for this discrepancy may be attributed to different experimental procedures.
Previous experiments have clearly demonstrated heterodimerization
between ER and TERP-1 both in vitro (24) and
in vivo (25). Although TERP-1 is unable to bind
to an ERE by itself, our experiments clearly showed that an equimolar
concentration of TERP-1 effectively inhibits WT-ER binding to EREc,
confirming the recent findings of Resnik et al.
(24). Because contradictory results have been published
concerning transcriptional effect of TERP-1 on WT-ER transactivation
(24, 26, 27), we reexamined the transcriptional control
exerted by TERP-1 on WT-ER stimulated activity of various ERE-reporter
gene constructs. We used reporters containing various promoters to
compare the effects of TERP-1/ER heterodimer on single or multiple
tandem copies of EREs. In this work, we confirm in CHO cells that
TERP-1 is able to repress ER-mediated activation. In addition, the
inhibition varies according to the strength of the promoter and the
presence of single or double EREc.
To obtain a transrepression mediated by TERP-1, molar ratios 5:1
(TERP-1/ER) of the expression plasmids are required. No effect was
obtained at lower ratios (equimolar or submolar ratios). However, the
biphasic action of TERP-1 on transcription obtained by Schreihofer
et al. (26) was not observed under the
conditions employed in the present study, action involving
transcriptional repression only when WT-ER and TERP-1 are coexpressed
at high concentrations of transfected vectors. The reasons for these
discrepancies could be due either to the total amount of expression
plasmids employed or to the two involved cell types (CHO vs.
COS cell lines). On the other hand, Mitchner et al.
(27) were unable to demonstrate any effect of TERP-1
overexpression in GH3 cells on either PRL or a triple-vitellogenin
ERE/luciferase construct. Under the conditions used in the present
study, maximal transcriptional activity required approximately 25 ng
ER expression plasmid. At the efficient ratio 5:1, the TERP-1
plasmid represents only 125 ng. Transfection of 10-fold higher levels
of WT-ER resulted in no significant change in transcriptional activity.
Therefore, it is unlikely that the transcriptional repression was due
to sequestering of cellular factors. In addition, these data were
extended using cell lines expressing normal AR and showed similar
results. All together, the data demonstrate that TERP-1 exerts full
dominant-negative activity. Because such a transcriptional repression
was also observed using ERß (24), TERP-1 therefore might
be a natural antagonist of nuclear receptors. Furthermore, an important
finding of this study is that the TERP-1/ER heterodimer can repress
the basal activity of all promoters tested to date, suggesting that it
could exert a more general inhibitory effect toward transcription.
The exact mechanism responsible for the inhibitory activity of TERP-1
in the presence of ER is still unclear. TERP-1 could suppress
estrogenic action by squelching the WT receptor, preventing either ER
binding to DNA, WT-ER transcriptional activity, or nuclear transport.
Our findings concerning the inhibition of E2 binding to WT-ER by TERP-1
are also of great importance and contribute to the repressive effect
that we observed. ER partition between the cytoplasmic and nuclear
compartments in living cells stably transfected with TERP-1 strengthens
these results. TERP-1 could also interact with the plasma membrane ER,
a receptor for which strong evidence of its existence has now emerged
(46), and able to mediate estrogen-stimulated PRL release
from rat pituitary tumor cells (47). Another possible
hypothesis would be that TERP-1/ER heterodimer may inhibit
transcription by squelching receptor coactivators and/or general
transcription factors. TERP-1 was recently shown to be able to bind
SRC-1, steroid receptor coactivator (24). Therefore,
TERP-1 suppression of ER transcription could occur both by formation
of inactive heterodimers unable to bind the hormone and by competition
for transcription factors.
The results presented in this report provide important insights into
the pituitary regulation of ER gene expression during the
reproductive cycle. Because TERP-1 expression is tightly regulated
throughout the estrous cycle and is controlled through a specific
promoter, we propose that expression of the TERP-1 isoform represents a
novel mechanism for the modulation of lactotrope hormonal
responsiveness through protein-protein interaction. The relative ratio
of the ER and TERP-1 proteins in vivo may be a critical
factor regulating lactotrope cell responsiveness to estrogenic stimuli.
At proestrus, the level of TERP mRNA is 45% of the normal ER mRNA
in the pituitary gland (21). TERP-1 could modulate ER
activity, exerting a protective role against the high level of E2 on
proestrus suppressing E2 binding to WT-ER. On the other hand, our
results do not rule out that an E2-independent pathway could regulate
target genes via the TERP-1/ER heterodimer. Lactotrope cells exhibit
unique features that distinguish them from other anterior pituitary
cells: only lactotrope cells indeed exhibit a change in proliferation
rates through out the estrous cycle with a peak on the estrous morning
(48). Moreover, the progesterone receptor induction, a
typical estrogenic response, is not observed in lactotrope cells
(49, 50). Therefore, TERP-1 could exert either a role in
the control of lactotrope proliferation or a repressing role toward the
progesterone receptor.
In conclusion, the interest of our findings is that TERP-1 is a natural
variant expressed in a cell type-specific manner, transiently during
the proestrous stage and regulated by an intronic promoter within rat
ER gene. The identification and partial characterization of TERP-1
promoter is a first step in the study of the mechanisms involved in the
transcriptional regulation of ER/TERP-1 expression and their role in
the pituitary gland. Further study is required to determine the precise
elements involved in tissue-specific expression as well as the
identification of factors that bind to these response elements.
|
Acknowledgments |
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-LUC promoter (containing the -846/+50 promoter fragment of
human -subunit), Dr. P. Chambon (IGBMC, University L. Pasteur,
Strasbourg, France), and Dr. J. D. Parlow (National Hormone and
Pituitary Program, Harbor-UCLA Medical Centre) for the gifts of
monoclonal antibody F3 and polyclonal antibody ER 715,
respectively. We thank Dr. C. H. Le Dréan (University of
Rennes 1, France) and Dr. C. L. Hew (Research Institute, Toronto,
Canada), for kindly providing us with the p(ERE)2 [(ERE)2-TATA-CAT].
We thank Dr. W. Wahli (IBM, University of Lausanne) for the ERE-tk-LUC,
Dr. O. Jänne (University of Helsinki) for the p(ARE)4-tk-LUC
construct and hAR expression vector, Dr. Lefebvre (INSERM U 459,
University of Lille) for GFP-GR, and Dr. M. Muramatsu (University of
Tokyo, Japan) for the rat ER cDNA. We thank our colleagues Dr. F.
Pakdel for the (ERE)2-tk-CAT and the trER promoter and Dr. G. Salbert
for the plasmid GFP-COUP-TF. We also thank Dr. P. Mellon (University of
California, La Jolla, CA) for supply of T3-1 pituitary cells. This
work was supported by grants from the Association Recherche pour le
Cancer (Agreement No. 9898) and from the Fondation Langlois. |
Footnotes |
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Abbreviations: AF-2, Transcriptional function; AP-1, activator protein-1; ARE, androgen-responsive element; GRE, glucocorticoid-responsive element; GFP, green fluorescent protein; ATG, translational start codon; CHO, Chinese hamster ovary; ERE, E-responsive element; NZF-1, neural zinc finger factor-1; RDL, rat DNA library; TERR-1, truncated ER product-1; pcDNA, plasmid and carrier DNA; TERP, truncated ER product; WT, wild-type; WT-ER, wild-type ER.
Received January 25, 2001.
Accepted for publication May 28, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß
isoforms in human pituitary tumors. J Clin Endocrinol Metab 83:3965–3972 gene are generated by alternative splicing
and promoter usage. Mol Endocrinol 12:1939–1954 
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receptors. FASEB J 14:157–165This article has been cited by other articles:
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), ERE-tk-LUC (
), or
ERE-SV-LUC (
) plasmids. Twenty-five nanograms WT-ER expression
vector and 25–500 ng/dish of TERP-1 expression vector were employed.
The total amount of plasmid was equalized in each well at 4.5 µg
using the empty expression vector. Transfected cells were treated for
48 h with 10 nM E2. LUC or CAT activities were
normalized for protein. The results are expressed vs.
ER
