Constitutive Activation of Transcription and Binding of Coactivator by Estrogen-Related Receptors 1 and 2
Wen Xie,
Heng Hong,
Na N. Yang1,
Richard J. Lin,
Cynthia M. Simon,
Michael R. Stallcup and
Ronald M. Evans
Howard Hughes Medical Institute (W.X., R.M.E.) Gene Expression
Laboratory (W.X., N.N.Y., R.J.L., C.M.S., R.M.E.) The Salk
Institute for Biological Studies La Jolla, California 92037
Department of Pathology (H.H., M.R.S.) University of Southern
California Los Angeles, California 90033
 |
ABSTRACT
|
---|
In this report, we demonstrate that, in contrast
to most previously characterized nuclear receptors, hERR1 and hERR2
(human estrogen receptor-related protein 1 and -2) are constitutive
activators of the classic estrogen response element (ERE) as well as
the palindromic thyroid hormone response element
(TREpal) but not the glucocorticoid response
element (GRE). This intrinsically activated state of hERR1 and hERR2
resides in the ligand-binding domains of the two genes and is
transferable to a heterologous receptor. In addition, we show that
members of the p160 family of nuclear receptor coactivators, ACTR
(activator of thyroid and retinoic acid receptors), GRIP1
(glucocorticoid receptor interacting protein 1), and SRC-1 (steroid
receptor coactivator 1), potentiate the transcriptional activity by
hERR1 and hERR2 in mammalian cells, and that both orphan receptors bind
the coactivators in a ligand-independent manner. Together, these
results suggest that hERR1 and hERR2 activate gene transcription
through a mechanism different from most of the previously characterized
steroid hormone receptors.
 |
INTRODUCTION
|
---|
Our understanding of the action of steroid and thyroid hormones,
vitamin D, and retinoid acid has undergone a rapid development as a
consequence of the molecular characterization of their cognate
receptors and characterization of the specific recognition of the
target DNA element (for review, see Refs. 1, 2, 3). Nearly all members of
the nuclear receptor (NR) superfamily share similar structural
features, most notably, a DNA-binding domain (DBD) composed of two zinc
fingers and an associated carboxyl-terminal stretch of approximately
250 amino acids, which comprise the ligand binding domain (LBD, for
review, see Refs. 1, 2, 3). Embedded within the LBD is a hormone-dependent
transcription activation domain (AF2, or E domain), and a transcription
repression domain that functions only in the absence of hormone. In
some NR family members, such as the two estrogen receptors (ER
and
ß) and steroidogenic factor 1 (SF-1), an additional activation
function (AF-1, or A/B domain) resides in the NH2-terminal
region of the NRs (4, 5, 6, 7). It is possible to exchange the domains
between receptors to create chimeras with predictable altered DNA
and/or ligand binding properties (8).
An important conceptual advance for NRs was the isolation and
characterization of the orphan receptors whose cognate ligands are
either unknown or unnecessary. Most orphan receptors exhibit domain
structure similar to that of classic NRs (3, 9, 10, 11). At present, orphan
receptors are by far the largest subclass of the family, and ligands
and synthetic drugs have been identified for some of these (for review,
see Refs. 3, 9, 12).
Recent advances have identified several classes of NR cofactors
that appear to play key roles in transcriptional activation (for
review, see Refs. 13, 14). One well characterized family of
coactivators for NRs are the three related p160 proteins: steroid
receptor coactivator 1 (SRC-1) (15, 16), glucocorticoid receptor
interacting protein 1 (GRIP1)/transcriptional intermediary factor 2
(TIF2) (17, 18, 19), and activator of thyroid and retinoic acid receptors
(ACTR) (20) [also known as p/CIP (p300/CBP interacting protein) (21),
RAC3 (receptor-associated coactivator 3) (22), AIB1 (amplified in
breast cancer 1) (23), and TRAM-1 (thyroid hormone receptor activator
molecule-1)(24)]. Coactivators are targeted to the LBD via specific
receptor interaction domains or RIDs (for review, see Refs. 13, 14).
Both structural and mutational analysis of multiple RIDs revealed a
core LXXLL motif (where L is leucine and X can be any amino acid) that
mediates the direct association. The LXXLL motif-containing
-helices
located in the central region of the p160 coactivators function as
protein-protein interaction modules and contact a hydrophobic groove on
the surface of an agonist bound LBD (19, 21, 26, 27, 28). Therefore, the
LBD serves as a molecular switch that recruits activator proteins in
the presence of ligand and releases them in the absence of ligand.
While classical NRs require binding of ligand before they can activate
transcription (16, 18, 19), some receptors display constitutive
activity and bind coactivator in the absence of any apparently added
inducer. For example, orphan receptor CARß apparently functions as a
transcriptional activator and associates with coactivator SRC-1 in the
absence of ligand, while the binding of its ligand androstane
metabolites results in the dissociation of bound coactivators, thus
providing a negative regulatory mechanism for the ligand (29).
Even though human estrogen receptor-related protein 1 and 2 (hERR1 and
-2) were the first orphan receptors identified, their properties and
mechanisms of transcriptional activation remain to be defined. We now
describe the characterization of ligand-independent gene activation by
each of these receptors and suggest that members of the p160
family of NR coactivators potentiate this activity.
 |
RESULTS
|
---|
Ligand-Independent Activation of Estrogen Response Element (ERE)-
and Palindromic Thyroid Hormone Response Element
(TREpal)-Controlled Reporter Genes by hERR1 and
hERR2
To analyze the transactivation of hERR1 and hERR2,
expression vectors pRShERR1 and pRShERR2 were cotransfected into CV-1
cells with a series of hormone-responsive reporter plasmids based on
the thymidine kinase (tk) promoter driving luciferase or
chloramphenicol acetyltransferase (CAT) gene expression. As shown in
Fig. 1A
, both hERR1 and hERR2
unexpectedly activate transcription through both the ERE and the
palindromic thyroid hormone response element (TREpal) (Fig. 1A
) reporters in a hormone- and serum-independent fashion. Under the
same serum-free conditions, other NRs including the ER and TR (Fig. 1A
)
and GR (Fig. 1B
), activate gene transcription only in the presence of
their cognate ligands. To demonstrate that this constitutive activity
is promoter independent, another series of reporters based on the
glucocorticoid-inducible mammary tumor virus (MTV)-CAT (10) was
used in the cotransfection experiments. To generate
MTV-ERE-CAT and
MTV-TREpal-CAT (30), the glucocorticoid response element
(GRE) in the MTV sequence was replaced by the ERE or
TREpal, respectively. As shown in Fig. 1B
, when this series
of reporters were cotransfected in HeLa cells together with the hERR2
expression plasmid, gene transcription is again activated independently
of exogenous hormones or serum. In addition, similar activation curves
were seen with the ERE-containing Xenopus vitellogenin A2
gene promoter (vitA2 -331/-87CAT) (31) when cotransfected with hERR1
and hERR2 expression vector (data not shown). Induction is specific to
ERE and TRE reporter genes as the two hERRs fail to stimulate MTV, a
reporter carrying the glucocorticoid response element under the same
experimental conditions (Fig. 1B
). This hormone-independent
transactivation activity of hERR2 depends upon the amount of the
expression vector transfected into the recipient cells; the activity
increased with the amount of hERR1 or hERR2 expression vector
transfected. Shown in Fig. 2
is a
saturation curve revealing a dose-dependent transactivation
profile.

View larger version (43K):
[in this window]
[in a new window]
|
Figure 1. Ligand-Independent Activation of ERE- and
TREpal-Controlled Reporter Genes by hERR1 and hERR2
A, Two micrograms of expression plasmids encoding ER, TR, hERR1, and
hERR2 were cotransfected into CV-1 cells together with 5 µg of either
a thyroid-hormone-responsive (tk-TRE-Luc) or an estrogen-responsive
(tk-ERE-Luc) reporter plasmid. Luciferase activity was measured and
graphed. Where indicated transfected cells were grown with
10-7 M T3 or 10-8
M E2. C, Control expression plasmid. The amount
of expression plasmid in each reaction was compensated to a total of 10
µg by addition of pRSerbA-1 control plasmid. The transfections were
done under serum-free condition; cells were split into serum-free DMEM
1 day before transfection. B, HeLa cells were cotransfected with 2 µg
of the expression plasmid and 5 µg of the reporter plasmid by calcium
phosphate precipitation method as indicated. MTV, MTV, ERE, and
TREpal represent the reporter plasmids MTV-CAT, MTV-CAT,
MTV-ERE-CAT, and MTV-TREpal-CAT (10 ), respectively.
Cells were treated with ethanol (-), 10-7 M
dexamethasone (Dex), or 10-8 M estradiol
(E2) or 10-7 M T3
after transfection. CAT activity was normalized by ß-galactosidase
activity.
|
|

View larger version (41K):
[in this window]
[in a new window]
|
Figure 2. Dose-Dependent Activation of hERR2
A, Increasing amounts (0.00110.0 µg) of pRShERR2 plasmid were
transfected into HeLa cells (lanes 28) as indicated under each
reaction. Five micrograms of MTV-TREpal-CAT reporter
plasmid were cotransfected. Two micrograms of pRShTR (68) were
transfected in lanes 9 and 10. T3 (10-7
M) was added to reaction 10 after calcium phosphate
transfection while ethanol was added to reaction 19. B, Quantitation
of the CAT activity. Percentage of conversion of CAT activity was
calculated and graphed. Activity was measured relative to the activity
of 10 µg pRShERR2 (maximum conversion was arbitrarily defined as
100%). Relative activity of each conversion was then plotted against
the amount of plasmid in the transfection.
|
|
The Intrinsically Activated State of hERR1 and hERR2 Resides in
Their LBDs
To further confirm their constitutively active phenotype and
to localize the regions that transmit this property, a series of domain
swap mutants were generated. Site-directed mutagenesis was used to
introduce common NotI and XhoI restriction sites
into the receptor cDNA sequences to generate hGRNX,
hERR1NX, and hERR2NX, as illustrated in Fig. 3A
. The DBDs of hERR1 and hERR2 were then
swapped with that of hGR to generate chimeric receptor GE1G and GE2G
(Fig. 3A
). When the resulting chimeric receptor GE2G was transfected
into HeLa cells, it functioned as a glucocorticoid-dependent activator
of reporter vectors harboring ERE and TRE sequences (Fig. 3B
). This
indicates that the DBD of hERR2 is important for response element
recognition but does not embody the dominant constitutive activity of
the parental receptor.

View larger version (37K):
[in this window]
[in a new window]
|
Figure 3. Construction and Activity of GR/ERR Chimera
A, Unique NotI and XhoI sites were
introduced into the sequences of hGR, hERR1, and hERR2 flanking the
DBDs to create mutants hGRNX, hERR1NX, and
hERR2NX. Numbers above the boxes correspond
to the amino acid positions. Chimeric receptors were created by
exchanging domains through the common XhoI and
NotI sites. Hybrids are named by three letters referring
to the origins of each domain. All the expression plasmids used in
transfections performed in this paper were constructed in the pRS
vector. Note the diagrams are not necessarily proportional to
the actual domain sizes. B, GE2G activates gene transcription through
ERE and TRE DNA sequences. HeLa cells were transfected with 2 µg
control plasmid (lanes 1, 2, 5, 6, 9, and 10) or 2 µg of pRSGE2G
(lanes 3, 4, 7, 8, 11, and 12) and 5 µg of the reporter plasmids,
MTV-CAT (lanes 14), MTV-TREpal-CAT reporter (lanes
58), and MTV-ERE-CAT reporter (lanes 912). Dexamethasone (Dex)
was administered after calcium phosphate transfection at
10-7 M as indicated. C, GGE1 and GGE2 activity
is hormone independent. HeLa cells were cotransfected with 2 µg of
the expression plasmids pRShGRNX (lanes 3 and 4), pRSGGE1
(lanes 5 and 6), and pRSGGE2 (lanes 7 and 8) and 5 µg of the reporter
plasmid MTV-CAT by the calcium phosphate coprecipitation method. Cells
were then treated with minus (-) or 10-7 M
dexamethasone (Dex) as indicated.
|
|
In the reciprocal experiments, the putative LBDs of the hERR1 and hERR2
were substituted with that from the GR to generate GGE1 and GGE2 (Fig. 3A
). The resulting hybrids should recognize the GRE of the MTV-LTR.
When cotransfected with MTV-CAT reporter genes into HeLa cells, GGE1
and GGE2 activated gene transcription through MTV-LTR in the absence of
any exogenous inducers (Fig. 3C
, lanes 5 and 7) in contrast to the
parental hGRNX molecule, which activated MTV only in a
hormone-dependent fashion (Fig. 3C
, lane 3 and 4). Together, these data
indicate that the LBDs of the hERR1 and hERR2 are in an intrinsically
activated state and that this property is transferable to a
heterologous receptor.
The Intact LBDs of hERR1 and hERR2 Are Essential for
Transcriptional Activation
In the case of the GR and ER, the LBDs function as
a contiguous unit such that disruption of this region by deletion or
insertions leads to loss of hormone binding. Such mutants are expected
to become transcriptionally inactive. In contrast, complete truncation
of the hormone- binding domain can lead to constitutively active
mutants as previously described for both GR and ER (4). Accordingly, a
series of mutations were generated in the LBD of hERR1 to examine their
potential effects on transactivation (Fig. 4
). These include a C-terminal truncation
mutant 2681, an internal deletion mutant,
268373, and a linker
insertion mutant I373 (Fig. 4
). Mutant 2681 has the entire LBD deleted
after amino acid position 268 with an addition of six extra amino acids
from the inserted linker. Mutant
268373 has an in-frame deletion
from amino acid 268 to 373, and mutant I373 contains a linker encoding
five amino acids, PHRWG, inserted in-frame after amino acid 373 without
disrupting any other parts of the molecule. When each of the mutants
was tested, all were transcriptionally silent (Fig. 4
). Thus, an intact
LBD is apparently required for the constitutive activation phenotype.
Furthermore, the failure of these mutants in transactivation indicates
that, unlike ER, the putative AF-1 domain in the N terminus of hERR1
and 2 may not contribute to their transcription activity.

View larger version (22K):
[in this window]
[in a new window]
|
Figure 4. Structure and Activity of Mutant hERR1 Gene
Products
HERR1 gene and mutants are shown. Construction of the mutants are
described in Materials and Methods.
Numbers indicate the amino acid positions. CAT activity
was assayed using HeLa cell extracts obtained by cotransfection with
the indicated mutant hERR1 genes and MTV-TREpal-CAT
reporter plasmid. The transfection procedures were as described in Fig. 1B . C, Control; AC, activation.
|
|
ACTR, GRIP1, and SRC-1 Are Transcriptional Coactivators of hERR1
and hERR2
NR interacting proteins, including members of the
p160 family of coactivators, play key roles in hormone-induced
transcription. ACTR, GRIP1, and SRC-1 function as transcriptional
coactivators for a wide group of the NRs, both in yeast and in
mammalian cells (16, 18, 20, 23, 32, 33). We performed transfection
assays to examine whether the p160 family members would also serve as
transcriptional coactivators for hERR1 and hERR2. Indeed, in
transiently transfected CV-1 cells, the activation of the
ERE-controlled reporter gene (
MTV-ERE-Luc) by hERR1 (Fig. 5A
) and hERR2 (Fig. 5B
) was enhanced
significantly by cotransfection of ACTR, GRIP1, or SRC-1, while these
coactivators did not activate the reporter gene in the absence of hERRs
(Fig. 5A
). Among the examined coactivators, GRIP1 exhibited relatively
higher potency as compared with ACTR and SRC-1. In the case of GRIP1,
while the wild-type proteins potentiate the activation of transcription
by hERR1 (Fig. 5D
) or by hERR2 (Fig. 5E
), a mutant GRIP1 containing
mutations in NR boxes II and III failed to function as a coactivator
(Fig. 5
, D and E). In the NR box mutant, the second and third LXXLL
motifs are changed to LXXAA; these mutations are known to disrupt
binding to the hydrophobic pocket created by the carboxyl-terminal AF-2
domain (Ref. 32 and data not shown). In the coactivator transfection
assays, treatment with 17ß-estradiol (E2) had no effect
on either hERR1 or -2 activation (data not shown).

View larger version (24K):
[in this window]
[in a new window]
|
Figure 5. ACTR, GRIP1, and SRC-1 Are Transcriptional
Coactivators for hERR1 and hERR2
A and B, CV-1 cells were transiently transfected with MTV-ERE-LUC
reporter gene (0.5 µg), pCMX-hERR1 (panel A) or pCMX-hERR2 (panel B)
(0.5 µg), together with 0.5 µg of expression vectors of indicated
coactivators, ACTR, GRIP1, and SRC-1a. Total DNA used in each
transfection was adjusted to 2 µg by adding the appropriate amount of
empty expression vectors. Cells were grown in charcoal/dextran-treated
serum with no added hormone after transfection (34 ). Luciferase assays
were performed 48 h after transfection. C, Diagram of GRIP1
structure. The vertical filled boxes represent NR boxes
(NRB) I, II, and III, each composed of a LXXLL motif. The CBP/p300
interaction domain of GRIP1 is also indicated. D and E, CV-1 cells were
transiently transfected with MTV-ERE-LUC reporter gene (0.5 µg),
pCMX-hERR1 (panel D), or pCMX-hERR2 (panel E) (0.5 µg), together with
0.5 µg of wild-type GRIP1 or a mutant GRIP1 bearing mutations in the
LXXLL motifs of NR Boxes II and III.
|
|
Thus, both hERR1 and hERR2, like classical nuclear hormone receptors,
can utilize all three p160 coactivator family members, ACTR, GRIP1, and
SRC-1, as transcriptional coactivators.
Ligand-Independent Binding of Coactivators by hERR1 and hERR2
We used a glutathione-S-transferase GST pull-down assay
to determine whether hERR1 and -2 can directly interact with p160
proteins in vitro. As shown in Fig. 6A
, while GST does not bind the
full-length hERR1 or hERR2 synthesized in vitro, the
bead-bound GST-ACTR-RID621821 (20) is able to bind the
hERRs efficiently. The binding between ACTR and hERRs can also be
demonstrated when the LBD of the hERRs, instead of the full-length
proteins, was used (data not shown). In the same GST pull-down assays,
GST-GRIP15631121 also bound full-length hERR2 efficiently
(Fig. 6B
). No hormone was added either in the synthesis of the proteins
or in the binding reactions, further supporting the notion that
activation of transcription by hERRs is ligand independent.

View larger version (42K):
[in this window]
[in a new window]
|
Figure 6. Ligand-Independent Binding of Coactivators by hERR1
and hERR2
A and B, The full-length hERR1 or hERR2 was synthesized in
vitro with [35S]methionine and then incubated
with Sepharose bead-bound GST, GST-ACTR-RID621821 (panel
A), or GST-GRIP15631121 (panel B), in the absence of any
added hormone. After washing, the bead-bound hERR proteins were eluted
and analyzed by SDS-PAGE and autoradiography. For reference a sample
equivalent to 10% of the labeled protein in the binding assay is shown
along with the total amount of labeled protein bound to the beads. C,
The LBD of hERR1 (VP-L-hERR1) or hERR2 (VP-L-hERR2) interacts with
ACTR-RID621821 in mammalian two-hybrid assays (20 ). CV-1
cells were cotransfected with expression plasmids for
tk-(MH100)4(UAS)-Luc and ACTR-RID621821,
together with expression vector of VP control, or VP-L-hERR1 and
VP-L-hERR2. No exogenous ligand was added in this assay.
|
|
The interaction between ACTR and hERRs was further demonstrated in a
mammalian cell two-hybrid assay. Using tk-(MH100)4(UAS)-Luc
(29) as reporter and GAL-ACTR-RID621821 as bait, VP16
activation domain (VP) itself results only in a low level of basal
activation. In contrast, the fusion of VP16 with the LBD of hERR1 or -2
(VP-L-hERR1 and VP-L-hERR2) exhibited substantial activation in the
absence of exogenously added ligand (Fig. 6C
).
 |
DISCUSSION
|
---|
Orphan NRs are continuing to increase in number and diversity (3, 9, 12). The large number of currently known orphan NRs, guided by the
well characterized paradigm of the classical nuclear hormone receptors,
provides an invaluable resource for the elucidation of new aspects of
developmental and regulatory biology.
HERR1 and -2 were originally isolated by low-stringency hybridization
probed with the DBD of human ER and were the first two orphan receptors
identified. Indeed, ERR is also an expanding family of proteins; we
have recently cloned the closely related ERR3 gene as a GRIP1
interacting protein (34). While ERRs display significant homology to
the ER (hERR1 has 91% amino acid identity with hERR2, 68% with hER in
DBD), none bind either natural or synthetic estrogens in
vitro (35). In addition, initial ligand screen has ruled out some
other traditional hormones as ERR ligands (N. N. Yang and R.
M. Evans, data not shown). Interestingly, individual ERRs appear to
have unique expression patterns: ERR1 expresses widely in later stages
of mouse embryos, although its expression in the heart, skeletal
muscles, and nervous system is relatively abundant (36). The expression
of ERR2 is restricted to the trophoblast progenitor cells between
embryonic days 6.5 and 7.5. Restricted low levels of ERR2 expression
were also detected in adult mouse tissues (35). The highest expression
of ERR3 occurs around days 1115 post coitum, a period of very active
organogenesis (34). The restricted patterns of their expression suggest
ERRs have unique potential significance in developmental and
physiological processes. Indeed, ERR1 has been shown to be a
transcriptional regulator of the human lactoferrin (37), the human
medium-chain acyl coenzyme A dehydrogenase (38), thyroid hormone
receptor
(TR
) (39), and the osteopontin (40) genes. Homologous
recombination studies revealed that ERR2 has an important role in early
embryogenesis where ERR2 null mice die at 10.5 days post coitum due to
placental abnormalities (41).
Although many members of the steroid receptor superfamily are
ligand-dependent transcription factors, the results reported here
support the notion that some members may manifest constitutive activity
in absence of the addition of a specific ligand. Transfection studies
indicate that hERR1 and hERR2 have similar DNA binding properties and
are effective activators of estrogen and thyroid hormone target genes.
The activation of ERE-controlled gene expression by hERR2 is consistent
with the previous observation that ERR2 protein is able to bind ERE
in vitro as revealed by gel mobility shift assay (42).
Whether either hERR1 or -2 manifests any estrogenic or thyroimimetic
type properties in vivo is not yet known. Even though the
LBDs of hERR1 and hERR2, like classic hormone receptors, harbor
transcriptional activation functions, added ligands are apparently not
needed to manifest this property. This appears to be a consequence of
the ability of the p160 cofactors to be able to directly bind the LBDs
in a ligand-independent fashion.
Based upon these studies, we can consider at least four possible, but
not exclusive, models to explain the transcription properties of the
hERRs. First, the hERRs may not be exceptions to the rule that all
receptors are hormone dependent in activation. Rather, the activating
ligands may be produced endogenously by the recipient cells leading to
an apparent constitutive property. In this case, the ligand could
either be secreted and reabsorbed by the cells in an autocrine fashion
or could represent an internal molecule that is not released but is
able to interact with the intracellular receptor directly. Such
intracellular regulatory systems might have value in modulating
metabolic pathways as well as providing direct feedback regulation for
intracellular homeostatic systems. Furthermore, even though hERRs
exhibit transcriptional activity in cell-based assays in the absence of
any exogenously added ligand, this does not exclude the potential
existence of an endogenous ligand that modulates this function. For
example, the orphan receptor LXR
manifests some constitutive
activity (high basal) on certain target sequences that can be
substantially potentiated by the addition of ligands such as
24(S),25-epoxycholesterol and 24(S)-hydroxycholesterol (43). In
addition, two other orphan receptors, SF-1 and hepatic nuclear factor 4
(HNF-4), which also display constitutive activity, have recently been
proposed to be further activated by oxysterols and fatty acyl-CoA
thioesters, respectively (44, 45). However, direct binding of the
ligands to the receptor has yet to be confirmed. A second model would
suggest that the receptors are ligand-dependent repressors such that in
the absence of the appropriate inducing molecule, target genes are
constitutively activated. Indeed, we have recently shown that the
constitutive activity of the CAR-ß results from a ligand-independent
recruitment of transcriptional coactivators, while androstane
metabolites bind to and deactivate CAR-ß by promoting coactivator
release from the LBD (29). It remains possible that a mechanism of
potential ligand-mediated receptor deactivation also applies to hERRs.
A third model is that hERRs may be activated by agents other than
steroid hormones. Indeed, a number of steroid receptors including
ER
, ERß, and SF-1 have been shown to be activated by nonsteroid
agents such as growth factors, protein kinase A, and dopamine
(for review, see Ref. 46), and the AF-1 domain of these NRs appears to
be the target. It has been shown recently that the mitogen-activated
protein kinase-mediated phosphorylation of the AF-1 domain of
ERß and SF-1 promotes ligand-independent recruitment of NR
coactivators (7, 47). Indeed, most if not all, of the members of the NR
family are phosphoproteins (for review, see Ref. 46). However, two
pieces of evidence seem to go against the possibility that the
phosphorylation mechanism also applies to hERRs: 1) Unlike ERs, the
constitutive activation property of hERRs can be destroyed by
truncations, deletions, and insertional mutations in the LBD,
indicating that the putative AF-1 domain may not contribute the
transcription activity of the hERRs; 2) The transactivation activity of
hERRs can be observed in serum-free conditions. The fourth
possibility is that some receptors do not bind classic ligands and,
rather, represent a subgroup of the gene family that have evolved as
hormone-independent transcription factors. Interestingly, classical NRs
undergo conformational changes after hormone binding, and the resulting
conformation allows them to interact with transcriptional coactivators
via the LXXLL motif (26, 28, 32, 48). Mutation of this motif blocks ERR
activation. The coactivator binding with hERR1 and -2 would appears to
reflect a similar conformation to that of an activated receptor. How
this is achieved in the absence of ligand is unknown. Therefore, it
will be of interest to investigate the structural features of hERRs
that enable their constitutive interaction with coactivators. In either
case, these orphan receptors provide a unique opportunity to further
investigate the diverse mechanisms by which the NRs may contribute to
endocrine function.
The fact that hERRs can activate synthetic ERE-controlled genes raises
several interesting questions. Is the ERE a natural response element of
hERRs? And if so, what is the role of ERRs, relative to ER, in gene
regulation. Alternatively, do hERRs have other perfect or imperfect
response elements? For the role of hERRs in ERE-controlled gene
expression, one possible regulatory mechanism could involve the
formation of ER-ERR heterodimers, which may have a different level of
activity from ER-ER homodimers. Indeed, cotransfection of hERR1
increased estrogen-dependent activation mediated by the ERE in the
lactoferrin promoter, suggesting that heterodimerization between ERRs
and ER could play a role in the attenuation or potentiation of
estrogen-dependent transcription (37). On the other hand, ERR1 has been
shown to bind a monomeric response element (38, 39, 49, 50), and the
technique of selected and amplified binding (SAAB) (51) predicts that
ERR1 can bind a response element containing a single consensus
half-site, 5'-TNAAGGTCA-3' (ERRE). Indeed, most of the previously
characterized ERR1 response elements in the promoters of its responsive
genes are this single core element (38, 39, 49, 50). We observed that
all three ERR family members can activate gene transcription when
tk-ERRE-Luc was used as a reporter (W. Xie and R. M. Evans, data
not shown). Interestingly, in some promoters, such as those of the
oxytocin, PRL, and lactoferrin genes, a putative ERRE overlaps a known
ERE (for review, see Ref. 49). Therefore, it is conceivable that ERRs
may play a role as transcriptional modulators whose contribution to
promoter activity could be determined both by the context of the ERE or
ERRE within a complex hormone response element and by potential
interactions between ERRs and other nuclear hormone receptors.
 |
MATERIALS AND METHODS
|
---|
Plasmids
To generate pRShERR1 construct, hERR1 coding sequence (35) was
constructed into a BamHI-digested and Klenow filled-in
vector fragment derived from a double BamHI linker-insertion
mutant pRShER
1601825 (V. Giguere, unpublished data). The construct
with the correct orientation was named pRShERR11. To generate
pRShERR1, a SacII-ATtIII fragment of pRShERR11
was inserted into a BamHI and ATtIII-digested
pRShGR214 (10) vector. To generate pRShERR2, the coding sequence of the
hERR2 (35) was prepared as a KpnI-BamHI fragment
and ligated into the same pair of enzymes digested vector of
pRSER1825 (30). In the assays of coactivators, the coding sequences of
hERR1 or hERR2 were released from pRShERR1 or pRShERR2 as an
Asp718-BamHI fragment, or an
EcoRI-BamHI fragment, respectively, and cloned
into the same enzyme-digested pCMX-PL2 expression vector (20) to
generate pCMX-hERR1 and pCMX-hERR2 constructs. To generate the NX
mutants, NotI and XhoI sites were introduced into
the cDNA sequence of hERR1 and hERR2 by site-directed mutagenesis as
described previously (8).
For the construction of hERR1 mutants, the C-terminal truncated
hERR12681 was generated by digesting pRShERR1 with SmaI
and Eco47III enzymes, which recognize two unique sites in the
ligand-binding region of hERR1. Ligation of the larger fragment
resulted in a plasmid coding for hERR1 that stops at amino acid
position 268. Six extra amino acids were added upon the sequence before
it runs into a stop codon. The in-frame internal deletion mutant
hERR1-
268373 was created by inserting an oligo linker coding for
ClaI recognition site (CATCGATG) into the SmaI
and Eco47III enzymes double-digested pRShERR1 fragment. The linker
was ligated with the larger fragment. The linker restores the reading
frame; therefore,
268373 encodes an hERR1 protein with the
sequence between amino acid 268 and 374 in the LBD replaced by a
sequence of four amino acids, Pro-Ile-Glu-Gly. HERR1-I373 was generated
by inserting an oligo linker with ClaI recognition site
(CCCATCGATGGG) into the Eco47III site in the LBD. The resulting hERR1
mutant protein, therefore, has four extra amino acid residues,
Pro-His-Arg-Trp, inserted between amino acid 372 and 373.
MMTV-LUC, MTV-ERE-LUC, and MTV-TREpal-LUC (30), tk-ERE-Luc
and tk-TREpal-Luc (52), MTV-CAT,
MTV-CAT,
MTV-ERE-CAT, and
MTV-TREpal-CAT (10), and expression
vectors pSG5-GRIP1 (32), pCMX-ACTR, pCMX-SRC-1a (20),
tk-(MH100)4(UAS)-Luc (29), and pRShTR
(8) were described
previously.
Bacterial expression vector for GST-GRIP1 fusion protein,
pGEX.2TK.GRIP15631121, was made by inserting a
PCR-amplified GRIP1 fragment into BamHI/EcoRI
sites of pGEX.2TK (Pharmacia). pGEX-ACTR-RID 621821 was
described before (20).
Cell Culture and Transient Transfection
CV-1 cells and HeLa cells were maintained in phenol red-free
DMEM supplemented with 10% FBS (Gemini Bio-Products, Inc.). The serum
condition after transfection is presented in the figure legends.
Calcium phosphate precipitation-based (
Figs. 14


) or liposome-based
(Figs. 5
and 6
) transient transfections were performed as described
previously (8, 17, 20). Total DNA used in each transfection was
adjusted to the same amount by adding the appropriate amount of empty
expression vectors. Luciferase assays (30, 34) or CAT assay (10) on
cell extracts were performed 48 h after transfection. Data shown
represent the mean and SD for three or four transfected
cultures.
In vitro transcription and translation of proteins and GST
pull-down assays were performed as described previously (20, 53). The
cDNAs of hERR1 and 2 were subcloned into pCMX vector (20) to generate
vectors for in vitro transcription and translation.
Mammalian two-hybrid assays were performed as described previously
(20). pCMX-VP16-L-hERR1 and pCMX-VP16-L-hERR2 were constructed by
cloning LBDs from hERRs into the pCMX-VP16 vector.
 |
ACKNOWLEDGMENTS
|
---|
We thank Hongwu Chen and Hung-Ying Kao for SRC-1 expression
vector; Henry Juguilon for help in tissue culture; and Elaine Stevens
and Lita Ong for administrative assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Ronald M. Evans, Howard Hughes Medical Institute, Gene Expression Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037.
This work was supported by grants from NIH. M.R.S is supported by
NIH Grant DK-43093. W.X. is supported by the California Breast Cancer
Research Program (5FB-0117). R.M.E. is an Investigator of the Howard
Hughes Medical Institute at the Salk Institute for Biological Studies
and March of Dimes Chair in Molecular and Developmental Biology.
1 Present address: Endocrine Research, Lilly Research Laboratories,
Eli Lilly & Co., Indianapolis, Indiana 46285. 
Received for publication July 9, 1999.
Revision received August 12, 1999.
Accepted for publication August 19, 1999.
 |
REFERENCES
|
---|
-
Evans RM 1988 The steroid and thyroid hormone receptor
superfamily. Science 240:889895[Medline]
-
Mangelsdorf DJ, Evans RM 1995 The RXR heterodimers and orphan
receptors. Cell 83:841850[Medline]
-
Enmark E, Gustafsson JA 1996 Orphan nuclear receptorsthe
first eight years. Mol Endocrinol 10:12931307[Medline]
-
Kumar V, Green S, Stack G, Berry M, Jin JR, Chambon P 1987 Functional domains of the human estrogen receptor. Cell 51:941951[Medline]
-
Berry M, Metzger D, Chambon P 1990 Role of the two activating
domains of the oestrogen receptor in the cell-type and promoter-context
dependent agonistic activity of the anti-oestrogen 4-hydroxytamoxifen.
EMBO J 9:28112818[Abstract]
-
Crawford PA, Polish JA, Ganpule G, Sadovsky Y 1997 The
activation function-2 hexamer of steroidogenic factor-1 is required,
but not sufficient for potentiation by SRC-1. Mol Endocrinol 11:16261635[Abstract/Free Full Text]
-
Hammer GD, Krylova I, Zhang Y, Darimont BD, Simpson K, Weigel
NL, Ingraham HA 1999 Phosphorylation of the nuclear receptor SF-1
modulates cofactor recruitment: integration of hormone signaling in
reproduction and stress. Mol Cell 3:521526[Medline]
-
Thompson CC, Evans RM 1989 Trans-activation by thyroid
hormone receptors: functional parallels with steroid hormone receptors.
Proc Natl Acad Sci USA 86:34943498[Abstract]
-
Blumberg B, Evans RM 1998 Orphan nuclear receptorsnew
ligands and new possibilities. Genes Dev 12:31493155[Free Full Text]
-
Hollenberg SM, Evans RM 1988 Multiple and cooperative
trans-activation domains of the human glucocorticoid receptor. Cell 55:899906[Medline]
-
Durand B, Saunders M, Gaudon C, Roy B, Losson R, Chambon P 1994 Activation function 2 (AF-2) of retinoic acid receptor and 9-cis
retinoic acid receptor: presence of a conserved autonomous constitutive
activating domain and influence of the nature of the response element
on AF-2 activity. EMBO J 13:53705382[Abstract]
-
OMalley BW, Conneely OM 1992 Orphan receptors: in search of
a unifying hypothesis for activation. Mol Endocrinol 6:13591361[Medline]
-
Torchia J, Glass C, Rosenfeld MG 1998 Co-activators and
co-repressors in the integration of transcriptional responses. Curr
Opin Cell Biol 10:373383[CrossRef][Medline]
-
Perlmann T, Evans RM 1997 Nuclear receptors in Sicily: all in
the famiglia. Cell 90:391397[Medline]
-
Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Lin
S-C, Heyman RA, Rose DW, Glass CK, Rosenfeld MG 1996 A CBP integrator
complex mediates transcriptional activation and AP-1 inhibition by
nuclear receptors. Cell 85:403414[Medline]
-
Oñate SA, Tsai SY, Tsai M-J, OMalley BW 1995 Sequence
and characterization of a coactivator for the steroid hormone receptor
superfamily. Science 270:13541357[Abstract]
-
Hong H, Kohli K, Trivedi A, Johnson DL, Stallcup MR 1996 GRIP1, a novel mouse protein that serves as a transcriptional
coactivator in yeast for the hormone binding domains of steroid
receptors. Proc Natl Acad Sci USA 93:49484952[Abstract/Free Full Text]
-
Hong H, Kohli K, Garabedian MJ, Stallcup MR 1997 GRIP1, a
transcriptional coactivator or the AF-2 transactivation domain of
steroid, thyroid, retinoid, and vitamin D receptors. Mol Cell Biol 17:27352744[Abstract]
-
Voegel JJ, Heine MJS, Zechel C, Chambon P, Gronemeyer H 1996 TIF2, a 160 kDa transcriptional mediator for the ligand-dependent
activation function AF-2 of nuclear receptors. EMBO J 15:36673675[Abstract]
-
Chen H, Lin RJ, Schiltz RL, Chakravarti D, Nash A, Nagy L,
Privalsky ML, Nakatani Y, Evans RM 1997 Nuclear receptor coactivator
ACTR is a novel histone acetyltransferase and forms a multimeric
activation complex with P/CAF and CBP/p300. Cell 90:569580[Medline]
-
Torchia J, Rose DW, Inostroza J, Kamei Y, Westin S, Glass CK,
Rosenfeld MG 1997 The transcriptional co-activator p/CIP binds CBP and
mediates nuclear-receptor function. Nature 387:677684[CrossRef][Medline]
-
Li H, Gomes PJ, Chen JD 1997 RAC3, a steroid/nuclear
receptor-associated coactivator that is related to SRC-1 and TIF2. Proc
Natl Acad Sci USA 94:84798484[Abstract/Free Full Text]
-
Anzick SL, Kononen J, Walker RL, Azorsa DO, Tanner MM, Guan
X-Y, Sauter G, Kallioniemi O-P, Trent JM, Meltzer PS 1997 AIB1, a
steroid receptor coactivator amplified in breast and ovarian cancer.
Science 277:965968[Abstract/Free Full Text]
-
Takeshita A, Cardona GR, Koibuchi N, Suen C-S, Chin WW 1997 TRAM-1, A novel 160-kDa thyroid hormone receptor activator molecule,
exhibits distinct properties from steroid receptor coactivator-1.
J Biol Chem 272:2762927634[Abstract/Free Full Text]
-
Heery DM, Kalkhoven E, Hoare S, Parker MG 1997 A signature
motif in transcriptional co-activators mediates binding to nuclear
receptors. Nature 387:733736[CrossRef][Medline]
-
Darimont BD, Wagner RL, Apriletti JW, Stallcup MR, Kushner PJ,
Baxter JD, Fletterick RJ, Yamamoto KR 1998 Structure and specificity of
nuclear receptor-coactivator interactions. Genes Dev 12:33433356[Abstract/Free Full Text]
-
Westin S, Kurokawa R, Nolte RT, Wisely GB, McInerney EM, Rose
DW, Milburn MV, Rosenfeld MG, Glass CK 1998 Interactions controlling
the assembly of nuclear-receptor heterodimers and co-activators. Nature 395:199202[CrossRef][Medline]
-
Nolte RT, Wisely GB, Westin S, Cobb JE, Lambert MH, Kurokawa
R, Rosenfeld MG, Willson TM, Glass CK, Milburn MV 1998 Ligand binding
and co-activator assembly of the peroxisome proliferator-activated
receptor-
. Nature 395:137143[CrossRef][Medline]
-
Forman BM, Tzameli I, Choi HS, Chen J, Simha D, Seol W, Evans
RM, Moore DD 1998 Androstane metabolites bind to and deactivate the
nuclear receptor CAR-ß. Nature 395:612615[CrossRef][Medline]
-
Umesono K, Evans RM 1989 Determinants of target gene
specificity for steroid/thyroid hormone receptors. Cell 57:11391146[Medline]
-
Klein-Hitpass L, Schorpp M, Wagner U, Ryffel GU 1986 An
estrogen-responsive element derived from the 5' flanking region of the
Xenopus vitellogenin A2 gene functions in transfected human
cells. Cell 46:10531061[Medline]
-
Ding XF, Anderson CM, Ma H, Hong H, Uht RM, Kushner PJ,
Stallcup MR 1998 Nuclear receptor-binding sites of coactivators
glucocorticoid receptor interacting protein 1 (GRIP1) and steroid
receptor coactivator 1 (SRC-1): multiple motifs with different binding
specificities. Mol Endocrinol 12:302313[Abstract/Free Full Text]
-
Voegel JJ, Heine MJS, Tini M, Vivat V, Chambon P, Gronemeyer H 1998 The coactivator TIF2 contains three nuclear receptor-binding
motifs and mediates transactivation through CBP binding-dependent and
-independent pathways. EMBO J 17:507519[Abstract/Free Full Text]
-
Hong H, Yang L, Stallcup MR 1999 Hormone-independent
transcriptional activation and coactivator binding by novel orphan
nuclear receptor ERR3. J Biol Chem 274:2261822626[Abstract/Free Full Text]
-
Giguere V, Yang N, Segui P, Evans RM 1988 Identification of a
new class of steroid hormone receptors. Nature 331:9194[CrossRef][Medline]
-
Bonnelye E, Vanacker JM, Spruyt N, Alric S, Fournier B,
Desbiens X, Laudet V 1997 Expression of the estrogen-related receptor 1
(ERR-1) orphan receptor during mouse development. Mech Dev 65:7185[CrossRef][Medline]
-
Yang N, Shigeta H, Shi H, Teng CT 1996 Estrogen-related
receptor, hERR1, modulates estrogen receptor-mediated response of human
lactoferrin gene promoter. J Biol Chem 271:57955804[Abstract/Free Full Text]
-
Sladek R, Bader JA, Giguere V 1997 The orphan nuclear receptor
estrogen-related receptor
is a transcriptional regulator of the
human medium-chain acyl coenzyme A dehydrogenase gene. Mol Cell Biol 17:54005409[Abstract]
-
Vanacker JM, Bonnelye E, Delmarre C, Laudet V 1998 Activation
of the thyroid hormone receptor alpha gene promoter by the orphan
nuclear receptor ERR a. Oncogene 17:24292435[CrossRef][Medline]
-
Vanacker JM, Delmarre C, Guo X, Laudet V 1998 Activation of
the osteopontin promoter by the orphan nuclear receptor estrogen
receptor related alpha. Cell Growth Differ 9:10071014[Abstract]
-
Luo J, Sladek R, Bader JA, Matthyssen A, Rossant J, Giguere V 1997 Placental abnormalities in mouse embryos lacking the orphan
nuclear receptor ERR-ß. Nature 388:778782[CrossRef][Medline]
-
Petterson K, Svensson K, Mattsson R, Carlsson B, Ohlsson R,
Berkenstam A 1996 Expression of a novel member of estrogen response
element-binding nuclear receptor is restricted to the early stages of
chorion formation during mouse embryogenesis. Mech Dev 54:211223[CrossRef][Medline]
-
Lehmann JM, Kliewer SA, Moore LB, Smith-Oliver TA, Oliver BB,
Su JL, Sundseth SS, Winegar DA, Blanchard DE, Spencer TA, Willson TM 1997 Activation of the nuclear receptor LXR by oxysterols defines a new
hormone response pathway. J Biol Chem 272:31373140[Abstract/Free Full Text]
-
Lala DS, Syka PM, Lazarchik SB, Mangelsdorf DJ, Parker KL,
Heyman RA 1997 Activation of the orphan nuclear receptor steroidogenic
factor 1 by oxysterols. Proc Natl Acad Sci USA 94:48954900[Abstract/Free Full Text]
-
Hertz R, Magenheim J, Berman I, Bar-Tana J 1998 Fatty acyl-CoA
thioesters are ligands of hepatic nuclear factor-4
. Nature 392:512516[CrossRef][Medline]
-
Weigel NL, Zhang Y 1998 Ligand-independent activation of
steroid hormone receptors. J Mol Med 76:469479[CrossRef][Medline]
-
Tremblay A, Tremblay GB, Labrie F, Giguere V 1999 Ligand-independent recruitment of SRC-1 to estrogen receptor ß
through phosphorylation of activation function AF-1. Mol Cell 3:513519[Medline]
-
Brzozowski AM, Pike AC, Dauter Z, Hubbard RE, Bonn T,
Engstrom O, Ohman L, Greene GL, Gustafsson JA, Carlquist M 1997 Molecular basis of agonism and antagonism in the oestrogen receptor.
Nature 389:753758[CrossRef][Medline]
-
Johnston SD, Liu X, Zuo F, Eisenbraun TL, Wiley SR, Kraus
RJ, Mertz JE 1997 Estrogen-related receptor
1 functionally binds as
a monomer to extended half-site sequences including ones contained
within estrogen-response elements. Mol Endocrinol 11:342352[Abstract/Free Full Text]
-
Bonnelye E, Vanacker JM, Dittmar T, Begue A, Desbiens X,
Denhardt DT, Aubin JE, Laudet V, Fournier B 1997 The ERR-1 orphan
receptor is a transcriptional activator expressed during bone
development. Mol Endocrinol 11:905916[Abstract/Free Full Text]
-
Blackwell TK, Kretzner L, Blackwood EM, Eisenman RN, Weintraub
H 1990 Sequence- specific DNA binding by the c-Myc protein. Science 250:11491151[Medline]
-
Umesono K, Giguere V, Glass CK, Rosenfeld MG, Evans RM 1988 Retinoic acid and thyroid hormone induce gene expression through a
common responsive element. Nature 336:262265[CrossRef][Medline]
-
Hong H, Darimont BD, Ma H, Yang L, Yamamoto KR, Stallcup MR 1999 An additional region of coactivator GRIP1 required for interaction
with the hormone-binding domains of a subset of nuclear receptors.
J Biol Chem 274:34963502[Abstract/Free Full Text]