Transcriptional Activities of the Orphan Nuclear Receptor ERR
(Estrogen Receptor-Related Receptor-
)
Jean-Marc Vanacker,
Edith Bonnelye1,
Sandrine Chopin-Delannoy,
Cateline Delmarre,
Vincent Cavaillès and
Vincent Laudet
Centre Nationale de la Recherche Scientifique UMR 49 (J-M.V.,
V.L.) Ecole Normale Supérieure de Lyon 69364 Lyon,
France
Centre Nationale de la Recherche Scientifique UMR
319 (S.C.-D., C.D.) Institut de Biologie de Lille Institut
Pasteur de Lille 59021 Lille, France
INSERM U148
(V.C) 34090 Montpellier, France
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ABSTRACT
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Estrogen receptor-related receptor
(ERR
) is
an orphan nuclear receptor closely related to the estrogen receptor
(ER), whose expression covers various stages of embryonic development
and persists in certain adult tissues. We show that ERR
binds as a
homodimer on a specific target sequence, the SFRE (SF-1 response
element), already known to respond to the orphan nuclear receptor SF-1.
Target sequences that are related to the SFRE and that discriminate
between ERR
and SF-1 were identified. We have also analyzed the
transcriptional properties of the ERR
originating from various
species. All ERR
orthologs act as potent transactivators through the
consensus SFRE. ERR
activity depends on the putative AF2AD domain,
as well as on a serum compound that is withdrawn by charcoal treatment,
suggesting the existence of a critical regulating factor brought
by serum.
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INTRODUCTION
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The superfamily of nuclear receptors includes transcription
factors whose activity depends on the presence of a specific ligand
(reviewed in Refs. 1, 2, 3). These ligands are of various chemical natures
and include, among others, steroid and thyroid hormones and retinoic
and fatty acids. All nuclear receptors share the same overall protein
organization. In the N-terminal domain (A/B domain), a
hormone-independent transactivation function (AF-1 region) has been
identified in certain receptors (1). Immediately downstream, the C
domain is strongly conserved among receptors and is responsible for the
specific DNA-binding activities. The DNA sequences recognized by
nuclear receptors generally consist of a core element (sequence
AGGTCA), either single or present as a direct or inverted repeat.
In the latter cases, the spacing between both core elements contributes
to the specificity of recognition by a given receptor (4).
Although also participating to some extent in the DNA-binding function,
the D domain is mainly considered a hinge region, bridging the C domain
to the most C-terminal ligand-binding domain (E domain). The
ligand-binding domain contains the hormone-binding and
hormone-dependent transactivation (AF-2 region) functions and is
evolutionarily conserved among the receptors (5). On the basis of this
domain organization, a growing number of so-called orphan receptors
(i.e. for which no ligand has yet been identified) have been
characterized (6). It is unclear whether these receptors are regulated
by a ligand still to be identified or whether they are constitutively
active.
The orphan receptor estrogen receptor-related
(ERR
also called
ERR1) was one of the first orphan receptors to be identified (7). It is
expressed in several tissues during embryonic development (8) and in
the adult (7, 9), suggesting a role of prime importance for this
receptor throughout life. ERR
displays high sequence identity with
the estrogen receptor (ER), hence its name (7). Indeed, ER and ERR
share 68% similarity of amino acids in their C domain and 36% in
their E domain. Despite this sequence similarity, ERR
does not bind
17ß-estradiol (E2), the natural ligand of ER. Moreover,
ER and ERR
have been reported to bind to different DNA targets. The
estrogen response element (ERE) consists in an inverted repeat of the
core elements separated by three variable nucleotides, on which ER
binds as a homodimer (10). On the other hand, we and others have shown
that ERR
binds to a single core element extended in its 5'-side by
TCA trinucleotide (11, 12, 13). This sequence was originally found
to mediate SF-1 transcriptional activity, and consequently termed SF-1
response element (SFRE) (14, 15, 16).
The nature of the transcriptional control exerted by ERR
is still a
matter of controversy. Fusion of the C-terminal part of ERR
to the
DNA-binding domain of the progesterone receptor (PR) generates a potent
transactivating protein acting through the PR response element, which
indicates that the E domain of ERR
is transcriptionally functional
(17). Moreover, we have shown that a trimer of the SFRE confers a
positive ERR
response to a minimal promoter in a cell-specific
manner (11). ERR
also contributes to the induction of the
lactoferrin promoter by ER (18), an effect that is likely to require
the physical interaction demonstrated to occur between these two
receptors. On another hand, a negative regulation of the SV40 late
promoter has been reported to occur through elements that are different
from the SFRE (12, 19). Sladek et al. (13) documented a
negative effect of ERR
on the induction of the MCAD gene promoter by
retinoic acid. This effect is mediated through a retinoic acid response
element, consisting of two core elements separated by five nucleotides.
The same report also demonstrated that ERR
is inactive on a single
copy of the SFRE, cloned in front of a minimal promoter.
These contradictory results, together with the potential importance of
ERR
, as judged by its broad expression pattern and its capacity of
interaction with ER, prompted us to analyze in more detail the
transcriptional properties of this receptor. Analysis of the
DNA-binding capacities of ERR
show that it forms a homodimer on the
SFRE site in vitro. In addition, the ability of both ERR
and SF-1 to bind to and to activate transcription through derivatives
of the SFRE was evaluated, ascribing a consensus recognition sequence
to each of these receptors. The positive effect of ERR
on promoter
activity depends on the number of responsive sites, but not on the
reporter gene nor on the minimal promoter used. cDNAs corresponding to
ERR
originating from several species (human, mouse, and zebra fish)
all efficiently enhance transcription in transient transfection assays
via a consensus SFRE. This effect requires the integrity of the AF-2
domain, which may suggest a ligand-mediated regulation. In agreement
with this, cells cultivated in charcoal-stripped medium do not support
ERR
-induced transactivation. In conclusion, this report demonstrates
that ERR
is a potent transcriptional activator.
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RESULTS
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Target Site Requirements of ERR
We questioned in more detail the DNA-binding mode of ERR
using
electrophoretic mobility shift assay (EMSA, Fig. 1A
). Mouse (m) ERR
(lane 2) formed a
single complex with the SFRE probe (sequence shown in Fig. 1B
as
oligonucleotide A). Using the deletion construct
A/BmERR
in the
same assay (lane 3) resulted in the appearance of a faster migrating
complex. As previously reported (11), a superposition of these patterns
was obtained when mixing the independently translated proteins
(lane 5). Surprisingly, cotranslation of the two products generated a
third, intermediate migrating complex (lane 4, arrow),
formed from a heterodimer between mERR
and
A/BmERR
, bound on
the SFRE probe. The intensity of this complex could be modulated by
varying the respective amounts of mERR
and
A/BmERR
plasmid
used in cotranslation (lanes 610), reaching its maximum in a 50:50
ratio. We thus reinterpret the complex formed between the SFRE and
ERR
as containing a homodimer of this receptor. Identical results
were obtained using the oligonucleotides displayed in Fig. 1B
as
probes, suggesting irrelevence of the sequences flanking the SFRE for
homodimer binding. Since ERR
forms a single complex with the SFRE
probe, we conclude that this receptor preferentially binds as a
homodimer on the SFRE.

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Figure 1. Dimerization of ERR
A, EMSA. mERR (wt, lane 2) or A/BmERR ( A/B, lane 3) were
synthesized in vitro and allowed to bind on
32P-labeled consensus SFRE probe (first sequence displayed
on panel B) in EMSA. Proteins were tested together either after
cotransfection (cotr., lane 4) or as a mixture of separately translated
products (indep. tr., lane 5). Proteins originating from cotranslation
in a mERR to A/BmERR plasmid ratio of 0:100, 25:75, 50:50,
75:25, or 100:0 (lanes 6 to 10) were used in EMSA. Lane 1, Unprogrammed
reticulocyte used as a control. The arrow indicates a
heterodimer between wt- and A/BmERR . This figure represents one
of three independent experiments. B, Sequence of the oligonucleotides
supporting ERR homodimer binding. Sequence of the probes that have
been tested is displayed. Core SFRE appears in capital
letters.
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Contradictory results have been obtained on the transcriptional effects
of ERR
using different systems. For instance, the lack of
transcriptional activity of ERR
reported by Sladek et al.
(13) was documented using a single copy of the SFRE. In contrast, the
reporter plasmid that we used in our published experiments (8, 11)
contains three copies of the consensus SFRE sequence. To investigate
this discrepancy, we analyzed the importance of the number of
responsive sites in the reporter plasmid. To this end,
SFRE-encompassing oligonucleotides were inserted as mono-, di-, or
trimer, upstream of the thymidine kinase (tk) minimal
promoter driving the chloramphenicol acetyltransferase (CAT) reporter
gene (plasmid pBLCAT4; Ref 20 and Fig. 2A
). These constructs were cotransfected
in ROS 17/2.8 (rat osteosarcoma) cells together with increasing amounts
of mERR
-encoding plasmid (Fig. 2B
). In agreement with Sladek
et al. (13), we did not find any effect of ERR
on a
single copy of the SFRE. Indeed, three copies of this DNA fragment were
required to yield a full transactivation by ERR
. Inserting two
copies of the SFRE generated only a moderate ERR
-response, if any.
We previously performed (11) transactivation experiments with a
SV40-derived promoter driving the Luciferase gene, whereas the present
ones were obtained with a tk promoter driving the CAT gene.
We thus conclude that the transcriptional effect of ERR
is
independent of the reporter and of the minimal promoter used. However,
we constantly observed a lower effect of ERR
on pSFREx3CAT than on
pSFREx3Luc. Essentially the same results were obtained using HeLa cells
(data not shown).

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Figure 2. Influence of the Number of Responsive Sites on
ERR Transactivation
A, Structure of the constructs used in this study. Consensus SFRE
sequences (represented by an arrow) were concatemerized
and inserted in front of the minimal tk promoter driving the CAT
reporter gene (plasmid pBLCAT4). All constructs were verified by
sequencing. B, Cotransfection experiment. ROS 17/2.8 cells were
cotransfected with 0.1 µg of the indicated reporter plasmid together
with the indicated molar excess (activator over reporter) of
mERR -encoding plasmid. CAT activities were determined 48 h
after transfection. The results are expressed as fold activation over
the reporter activity without transactivator and represent the average
of three independent experiments. Errors bars are indicated.
White bars, Basal reporter activity without
transactivator. Gray and black bars, 5- and 10 fold
(respectively) molar excess of receptor over reporter.
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SF-1 and ERR
were reported to bind to the same DNA sequence (SFRE;
Refs. 14, 15, 16). We determined their respective binding specificity by
competition EMSAs. To this end, we designed oligonucleotides, each
bearing a single difference in the SFRE, relative to the consensus
sequence (see Table 1
). Systematic
changes of the initial three and last two nucleotides of the SFRE were
generated. In vitro translated SF-1 or ERR
proteins were
incubated with a consensus SFRE radioactive probe in the presence of
unlabeled mutant oligonucleotides. Table 1
summarizes the competition
properties of the oligonucleotides, as determined with this method. For
both receptors, bases 1, 8, and 9 of the SFRE appeared irrelevant. In
contrast, changing bases 2 and 3 of the SFRE not only affected the
binding of SF-1 and ERR
, but also revealed subtle affinity
differences between the two receptors. For example,
TAAAGGTCA displayed ERR
- but not SF-1 binding, whereas
TGAAGGTCA behaved in the reverse manner. This allowed us to
define the indicated consensus binding sequences for SF-1 and ERR
.
We then tried to correlate the binding capacities of the two receptors
with their transcriptional activation capacities, taking the two
above-mentioned sequences as models. To this end, oligonucleotides were
inserted as trimers in front of the minimal SV40 promoter contained in
plasmid pGL2-prm. The resulting plasmids (pTAAx3Luc and pTGAx3Luc,
respectively) were transiently introduced in HeLa cells together with
varying amounts of pSG5-SF-1 or pSG5-ERR
. As presented in Fig. 3
, ERR
efficiently enhanced the
reporter activity driven by plasmid pTAAx3Luc, but not by plasmid
pTGAx3Luc. SF-1 displayed an opposite behavior. As a positive control,
plasmid pSFREx3Luc (which contains a trimer of the consensus SFRE
in the same plasmid context) was transactivated by both receptors.
These results are in agreement with the EMSA experiments summarized
above and suggest that, although generally binding to the SFRE, SF-1
and ERR
can discriminate between derivatives of this sequence and
thus exert convergent but also different transcriptional
activities.

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Figure 3. Effect of ERR and SF-1 on SFRE Derivatives
A, Structure of the constructions used in this study. Sequences of the
SFRE derivatives are displayed. Bold characters indicate
the divergence with respect to the consensus SFRE. B, Cotransfection
experiment. HeLa cells were cotransfected with 0.1 µg of the
indicated reporter plasmid together with the indicated molar excess
(activator over reporter) of mERR -encoding or SF-1-encoding plasmid,
as stated. Luc activites were determined 48 h after transfection.
The results are expressed as fold activation over the reporter activity
without transactivator and represent the average of three independent
experiments. Errors bars are indicated. White bars,
Basal reporter activity without transactivator. Gray and black
bars, 5- and 10-fold (respectively) molar excess of receptor
over reporter.
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Receptor Domain Requirements of ERR
Since the discrepancies concerning the transcriptional activities
of ERR
might be due to differences in the species from which the
ERR
cDNA originates, we isolated cDNAs corresponding to ERR
in
human and zebra fish (Danio rerio), hERR
and zfERR
,
respectively, and analyzed their activity. The transcriptional effects
of these constructs were compared with those (previously described in
Ref. 11) of the mERR
cDNA. Increasing amounts of these constructs
were cotransfected in ROS 17/2.8 cells together with pSFREx3Luc
reporter plasmid. The three ERR
efficiently enhance the expression
of the reporter gene in a dose-dependent manner, indicating that the
transactivation properties of ERR
are evolutionarily conserved, at
least from fish to human (Fig. 4
).
Comparable results were obtained when HeLa cells were transfected (data
not shown).

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Figure 4. Transcriptional Activities of ERR Originating
from Various Species
ROS 17/2.8 cells were cotransfected with 0.1 µg of pSFREx3Luc plasmid
together with the indicated molar excess (activator over reporter) of
ERR -encoding plasmid. Luciferase activities were determined 48
h after transfection. The results are expressed as fold activation over
the reporter activity without transactivator and represent the average
of three independent experiments. Errors bars are indicated.
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Nuclear receptors are reported to contain two transactivation domains
(1). In particular, this is true for the estrogen receptor (ER), which
is closely related to ERR
. The AF-1 function is located in the A/B
domain, is hormone independent but promoter and cell type dependent.
The AF-2 region is located in the extreme part of the E domain and is
hormone dependent in classical receptors. Since these regions are
present in ERR
, we questioned their importance in transactivation
driven by this receptor. The mERR
cDNA was deleted of its A/B
domain, and the zfERR
was truncated in its AF-2 part (resulting in
A/BmERR
and
AF-2zfERR
, respectively; see Fig. 5A
). We first determined that both
recombinants were not impaired in their DNA-binding ability (Ref. 11
and data not shown). Increasing amounts of these constructs were
then independently cotransfected together with the pSFREx3Luc
plasmid in ROS 17/2.8 cells. As seen in Fig. 5B
, the A/B domain
(encompassing the putative AF-1 function) was not required for
transactivation in our experimental conditions. In contrast, deleting
the extreme 3'-part of the E domain, and thus the putative AF-2 region,
abolished the transcriptional activity of zfERR
. Deleting the AF-2
region of nuclear receptors generally results in creating a dominant
negative molecule. We thus tested the ability of
AF-2zfERR
to
repress the activation induced by wild-type ERR
. pSFREx3Luc plasmid
was cotransfected with increasing amounts of zfERR
, in the presence
or absence of plasmid
AF-2zfERR
. As shown in Fig. 5C
, the latter
construct totally inhibited the activation driven by wtERR
,
demonstrating its dominant negative feature. As a control,
AF-2zfERR
was unable to repress the activation exerted by
retinoid X receptor (RXR)
on DR1-containing constructs (right
panel in Fig. 5C
), indicating that the dominant negative effect is
specific.
Since the AF-2 region is also required for the hormone-binding
properties of nuclear receptors, this suggests that ERR
might be
ligand regulated. To address this hypothesis, we tested the serum
dependence of the transactivation driven by ERR
. ER
, whose
transcriptional activity on the ERE is known to rely on the presence of
estrogen in the culture medium, was used as a control. ROS 17/2.8 cells
were cultured for 2 weeks in phenol red-free medium supplemented with
charcoal-treated (hormone-free) serum or, alternatively, in native
medium and serum. These conditions are referred to as "stripped" or
"normal medium," respectively, on Fig. 6
. pSFREx3Luc or pEREx2Luc plasmid was
then transiently introduced into the cells together with ERR
- or
ER
-expressing plasmid, respectively (Fig. 6A
). After transfection,
cells were further cultivated for 48 h in the same medium as
before transfection. Both ER
and ERR
were unable to exert any
transactivation on their cognate response element when cells were
cultivated in charcoal-treated serum. As expected, transfection
performed in normal medium conditions resulted in transactivation by
ER
or ERR
. The antiestrogen ICI 164,384 inhibited the
transactivation ability of ER
in normal medium, confirming that
these culture conditions stimulate this receptor. On the contrary, ICI
164,384 has no effect on ERR
-driven transactivation. It is
noteworthy that ER
achieved the same level of transcriptional
enhancement as when the cells were maintained in stripped serum
conditions supplemented with pure E2 (data not shown),
suggesting that the amount of estrogen present in the culture medium
was fully sufficient. Our data indicate that charcoal treatment
withdrew a factor that is essential to the activity of ERR
. This
factor could be a direct ligand (or precursor of a ligand) for ERR
or, alternatively, could intervene in any other way in ERR
-induced
transactivation (e.g. through phosphorylation). We supposed
that if a ligand for ERR
is present in the serum (as is
E2 for ER
), the transcriptional activity of ERR
would
be restored upon the shift from stripped to normal serum conditions.
Transient transfections (pSFREx3Luc minus or plus ERR
) were thus
performed on cells initially cultivated for 2 weeks in stripped serum
conditions and shifted to normal serum at various times relative to
transfection (Fig. 6B
). ER
(cotransfected together with pEREx2Luc)
was again used as a control. Changing the medium immediately after
transfection (D0) resulted in a moderate enhancement of reporter
activity induced by ER
or ERR
. The transactivation potential of
these receptors increased when normal medium was added 24 h (D-1)
or 48 h (D-2) before transfection. Interestingly, the potential
enhancement of ERR
as a transactivator paralleled that of ER
,
suggesting that the same phenomenon (i.e. accumulation of a
ligand in the cells) occurred in both cases.

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Figure 6. Serum Requirements for ERR -Induced
Transactivation
ROS 17/2.8 cells were cotransfected with 0.1 µg pSFREx3Luc or
pEREx2Luc plasmid together with a 10-fold molar excess of
mERR -encoding plasmid or hER -encoding plasmid (respectively), as
indicated. Luciferase activities were determined and are expressed as
fold activation over the reporter activity without transactivator.
Results represent the average of three independent experiments. Errors
bars are indicated. Stripped medium, Phenol red-free DMEM supplemented
with charcoal-treated serum. Normal medium, DMEM supplemented with
native serum. White bars, Basal reporter activity
without transactivator. Black bars, 10-fold molar excess
of receptor over reporter. A, Effect of serum depletion. Cells were
cultivated for 2 weeks in untreated serum-containing medium or stripped
serum-containing medium, as indicated. Same medium was added after
transfection with 10-8 M ICI 164,384 where
indicated. B, Kinetics of serum effect. Stripped culture medium was
shifted to normal medium at the indicated time relative to transfection
(D0, immediately after transfection; D-1, 24 h before
transfection; D-2, 48 h before transfection).
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DISCUSSION
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ERR
Binds as a Homodimer in Vitro
Earlier reports have described the SFRE sequence as a preferred
binding target for ERR
(11, 12, 13). Based on experiments in which
ERR
and
A/BERR
proteins were mixed after translation, we
concluded that this receptor binds as a monomer on the SFRE (11). Here
we have used another experimental protocol, namely cotranslation of a
wild-type and a mutant form of ERR
. This generated three
complexes with the SFRE probe, the intermediately migrating one
representing an association of both protein products. The two other
complexes are formed of bound homodimers of either wild-type or
deletion-mutant ERR
. It is noteworthy that these three complexes
were also observed upon cotranslation of mouse and human ERR
, but
not upon mixing of their separated translation (data not shown). The
same situation has also been observed for Xenopus ER, where
a heterodimer between two isoforms can be observed only upon
cotranslation of the mRNAs (21). Since we repeatedly observed a single
complex between ERR
and the SFRE probe, our data suggest that this
receptor can only bind in vitro as a homodimer on this probe
and not as a monomer as previously claimed.
Evidence for monomer binding also came from deoxyribonuclease (DNase)
footprinting experiments on the SV40 major late promoter (12). These
data demonstrated that ERR
contacts a single extented half-site on
the promoter. Taken together with the present data obtained with an
SFRE probe (also encompassing a single extended half-site), this
suggests that only one member of the ERR
homodimer binds to DNA.
The other member of the dimer would develop protein-protein
contact but no DNA-protein contact. In this hypothesis, the sequences
flanking the SFRE core are predicted to be irrelevant for dimer
binding. This is indeed the case since all the probes shown in Fig. 1B
supported ERR
dimer binding. In particular, the shorter size of
oligonucleotide D excludes the existence of a cryptic second half-site
that could be necessary for homodimerization. The SFRE, although
monovalent, is thus the only critical determinant in the homodimer
binding of ERR
. Such a situation could be reminiscent of the
NGF1-B-RXR heterodimer bound on the monovalent AAAGGTCA sequence, in
which RXR does not contact DNA (22). However this observation has not
been confirmed (23), and a divalent NGFI-B response element that is a
lot more potent has been found in the POMC gene promoter (24). Dimer
binding of ERR
on a monovalent site could thus represent a new,
unique feature among nuclear receptors.
The fact that homodimerization can only be observed upon cotranslation
suggests that the ERR
-dimer is a very stable complex as has been
pointed out for dimers of the Xenopus ER (21).
Alternatively, it could be that physical association between two ERR
peptides can only be initiated during protein synthesis. Once
translation is finished, the conformation adopted by the
completed protein could prevent the association between the two
species. As the proteins were translated in an in vitro
system that is devoid of DNA (other than the plasmid carrying the
ERR
constructs), we suggest that dimerization does not require DNA.
However, we must emphasize that the in vivo relevance and
significance of this peculiar dimeric mode of binding are presently
unknown and will require more investigation.
ERR
Is an Activator of Transcription
We here describe the positive transcriptional activity of the
orphan nuclear receptor ERR
on the SFRE site. This activity is
apparently independent of the A/B domain of the receptor. However, it
should be noted that the AF-1 region of nuclear receptors (located in
the A/B domain) functions in a promoter- and cell-dependent manner (1).
The
A/B mutant that we used may thus behave in a different manner
under other experimental conditions. On the contrary, ERR
transcriptional activity is dependent on the putative AF-2AD region.
This is consistent with published data demonstrating that the putative
transactivation domain (domain E) of ERR
is fully competent since
fusion of the E region of this receptor to the DNA-binding domain of
the PR generates a chimeric transcriptional activator acting on PR
response elements (17).
We have previously reported that ERR
enhances the expression
of the luciferase reporter gene under the control of the SV40 minimal
promoter supplemented with three copies of the SFRE (11). Our present
data show that the transactivation driven by ERR
is independent of
the minimal promoter and of the reporter gene used. However, it should
be noted that the level of transactivation achieved is always lower
with the tkCAT system than with SVLuc. Interestingly, three copies of
the SFRE sequence must be cloned in front of the minimal promoter to
ensure maximum efficiency. In particular, a single copy of the SFRE is
unable to confer any response to ERR
. Our results are consistent
with those reported by Sladek et al. (13) who could not
demonstrate any ERR
-driven transactivation, using a single copy of
the SFRE sequence. We have shown that the promoter driving the
expression of the thyroid hormone receptor-
is transactivated by
ERR
(9). Interestingly enough, this effect depends on the single
SFRE site present on this promoter, pointing to the importance of the
promoter context that surrounds the cis- acting sequence. On
the contrary, two imperfect SFRE sites are required for maximal
activation of the osteopontin promoter by ERR
(25).
The situation seems to be different when considering the effect of
ERR
on responsive sites other than the SFRE. For instance, it has
also been reported that this receptor represses the activation of the
MCAD gene promoter by the retinoic acid receptor (13). A DR5 sequence
present on the promoter is necessary and sufficient to mediate this
activity. Similarly, ERR
has a negative effect on the SV40 late
promoter, apparently through an ERE present in the genome of this virus
(12, 19). On the other hand, activation of the lactoferrin promoter by
ERR
requires both the SFRE and the imperfect ERE present on this
promoter (18). Our data show that ERR
can act as a potent
transcription activator in given conditions. Together with results
published by others, we conclude, however, that this receptor is a
pleiotropic modulator of transcription. This type of phenomenon has
already been emphasized for other nuclear receptors and is therefore
not surprising. For instance, members of the FTZ-F1/SF-1 subfamily
mediate various transcriptional effects, ranging from inhibition to low
and high levels of activation (16, 26, 27). A number of potential
target genes of ERR
have been suggested (as reviewed above), and
this receptor has been suspected to act as a modulator of bone (8, 25)
and fat metabolism (13), where it is highly expressed. However, in the
absence of knockout and/or transgenic studies, the precise in
vivo functions of ERR
remain largely unknown.
Is ERR
a True Orphan Receptor?
ERR
was (together with ERRß) the first receptor described as
an orphan and is, up to now, still viewed as such (7). Whether true
orphan receptors indeed exist, or if proteins that are considered as
such simply await the identification of their natural ligand, is a
question that remains open. ERR
makes no exception to this rule. The
fact that the putative AF-2AD domain of ERR
is required for
transactivation could suggest that this activity is ligand dependent.
Indeed, determination of the crystal structure of classical nuclear
receptors has shown that the AF-2AD domain blocks the ligand into the
hydrophobic pocket, thereby stabilizing the receptor in an "active"
conformation (28). However, the AF-2AD domain is also necessary to
establish contacts with coactivators (29, 30, 31, 32, 33). The lack of
transactivation in the absence of the putative AF-2AD domain in ERR
might be due to an inability to interact with a coactivator and thus is
not a decisive argument in favor of the existence of a ligand.
Nevertheless, the experiments presented here suggest that a regulator
of ERR
transcriptional activity is indeed present in the serum and
is withdrawn by charcoal treatment. The identity of this factor is
still unknown although some hormones can be excluded. Indeed, the lack
of activity of ERR
under stripped serum conditions could not be
challenged by addition of thyroid hormone, retinoic acids,
E2, raloxifene (a synthetic antiestrogen), vitamin D,
dexamethasone, or pregnenolone (data not shown). How the compound
present in the serum acts is also an unresolved question. Our
experiments suggest only that ERR
has an activator, and not
necessarily a ligand in the sense of E2 for ER for example.
The effect can also be indirect and, for example, could potentiate a
coactivator (or any factor that contributes to ERR
-driven
transactivation) rather than ERR
itself. It must be admitted that
the effect is ERR
specific and dispensable for ER
. The latter is
indeed affected by the same charcoal treatment, but E2
addition is sufficient to reverse the inhibition of its transcriptional
activity. The lack of activity of ERR
and ER
in stripped serum
conditions can be overcome by addition of normal medium. Interestingly,
the kinetics with which this restoration occurs is the same for ERR
and ER
. Since the activator of ER
in the medium is a direct
ligand, our data strongly suggest that ERR
is also a
ligand-regulated receptor and not an orphan one.
SF-1 and ERR
Both SF1 and ERR
have been shown to bind to the same
general element, namely, the SFRE. Nevertheless, our experiments reveal
subtle differences in the preferential binding of these receptors on
derivatives of the SFRE. This allows the definition of a consensus
binding sequence for each of these factors. The reason for these
differences is at present unclear. It has been shown that the T/A box
of nuclear receptors, a region located immediately downstream of the C
domain, directs the specific recognition of the 5'-extension of the
core sequence (15). Comparison of the T/A boxes of SF-1 and ERR
has
revealed strong similarities, consistent with their common interaction
with the consensus SFRE (15). However, the sequence divergences of
these two factors in their T/A box are likely to be responsible for the
preferential binding to SFRE derivatives.
At least in two cases (the TGA and TAA variants of SFRE), the
difference between SF-1 and ERR
observed in the EMSA is reflected in
their transcriptional behavior. This suggests that naturally occurring
SFRE-like sequences might respond to one or the other receptor as a
function of its binding affinity to the target site. As an example,
seven SFRE-like sequences can be found on the osteopontin promoter,
none of which represent a perfect consensus (34). Two of these
sequences are bound by ERR
but none are bound by SF-1 (25).
Consistently, we have found that the osteopontin promoter is
transactivated by ERR
but not by SF-1. Conversely, the analysis of
SF-1-responsive gene promoters (and particularily those implicated in
steroid hydroxylase cascade; Refs. 1, 35 and references therein)
could indicate whether these genes are likely to be transactivated by
ERR
.
Several parameters thus contribute to the specificity of response to
SF-1 or ERR
. In addition to the differences in the pattern of
expression of these two receptors, which have already been noted (8, 36), the relative binding affinity for an SFRE derivative can thus
contribute to the specificity of response to SF-1 or ERR
. Given the
cell specificity of action displayed by both receptors and the
pleiotropic effects that can be observed according to various
parameters, we conclude that, although initially described as binding
to the same consensus sequence, SF-1 and ERR
can mediate a vast
array of transcriptional responses.
 |
MATERIALS AND METHODS
|
---|
Plasmid Constructions
The zebra fish homolog of ERR
was isolated by cDNA
library screening and will be described elsewhere (E. Bonnelye, V.
Laudet, and B. Thisse, manuscript in preparation). Deletion of the
putative AF2AD region was performed using the PCR technique. cDNAs were
subsequently subcloned in the EcoRI site of plasmid pSG5.
Plasmid
A/BmERR
has been described previously (11). Partial human
ERR
cDNA was isolated from a library of K562 cells using the mERR
cDNA as a probe. An in-frame ATG was added by PCR. PCR was performed
using Gold Taq polymerase (Perkin Elmer Corp.,
Norwalk, CT). PCR fragments were sequenced to confirm the lack of
undesired mutations introduced during the amplification process.
SFRE derivatives containing 30-bp long oligonucleotides flanked by
BglII and BamHI restriction sites were ligated in
the presence of both restriction enzymes to ensure a correct
orientation. Concatemers were purified on a native polyacrylamide gel.
Products were inserted in the BglII site of plasmid pGL2 PRM
(Promega Corp., Madison, WI) or in the BamHI
site of plasmid pBLCAT4 (20). All constructions were verified by
sequencing. In all cases, SFREs are separated by 21 nucleotides that
are identical from one construct to another.
EMSAs
ERR
or SF-1 proteins were translated in vitro
using TNT kit (Promega Corp.). Consensus SFRE probe (A
sequence in Fig. 1B
) was end labeled with
-32P ATP.
Reticulocyte lysates were incubated with 40 103 cpm of
probe as described previously (37). Unlabeled oligonucleotides were
added as competitor where stated. Reactions were run on a native
polyacrylamide gel.
In the dimerization experiment, mERR
and
A/BmERR
plasmid were
cotranslated in a 1:1 (cotr. lane in Fig. 1
) or on a 0:100, 25:75,
50:50, 75:25, or 100:0 ratio as indicated. Equal quantities of
individually translated mERR
and
A/BmERR
were used in the
"indep. tr." lane in Fig. 1
.
Cells and Transfections
Cell lines (HeLa and rat osteosarcoma ROS17/2.8) were maintained
in DMEM with 10% FCS. For serum test, cells were maintained for 2
weeks in phenol red-free medium supplemented with charcoal-treated
serum. Transient transfection was performed using ExGen (Euromedex,
Soufflersheim, France), in which 0.1 µg of the reporter
plasmid was introduced together with the indicated molar excess of
receptor-producing plasmid. When necessary, pSG5 plasmid was added as a
carrier up to 1 µg per transfection. Cells were washed after 4 h
and fresh medium was added. Cells were then lysed 48 h after
transfection and processed for reporter quantitation using standard
methods.
 |
ACKNOWLEDGMENTS
|
---|
We thank Vincent Giguère for the generous gift of mERR
,
Philippe Berta for SF-1, and Frank Delaunay for ER
. We are indebted
to Olivier Gandrillon, Marc Robinson, and Hector Escriva-Garcia for
critical reading of the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Jean-Marc Vanacker, Centre National de la Recherche Scientifique, UMR 49, Ecole Normale Supérieure de Lyon, 46 allée dItalie, F-69364 Lyon Cedex 07, France. E-mail:
Jean-Marc.Vanacker{at}ens-lyon.fr
This work was supported by Centre Nationale de la Recherche
Scientifique, Ministère de lEnseignement Supérieur et de
la Recherche and Association pour la Recherche Contre le
Cancer.
1 Present address: Department of Anatomy and Cell Biology, University
of Toronto, M5S 1A8 Canada. 
Received for publication November 3, 1998.
Revision received February 1, 1999.
Accepted for publication February 22, 1999.
 |
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