INSERM U439 (A.J., P.B., J.C.N.) and INSERM U148 (V.C.), 34090 Montpellier, France
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ABSTRACT |
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INTRODUCTION |
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While nuclear receptors are hormone-dependent transcription factors,
the mechanism(s) that triggers the transcriptional activation remains
unknown. The AF2-activating domain (AF2 AD) has been shown to be
restricted to a limited sequence of amino acids (4, 5), whose
amphiphatic -helix structure and residue composition are well
conserved among the superfamily (6, 7, 8, 9). AF1 and AF2 activate the
transcription in a promoter- and cellular-dependent way, suggesting the
existence of other factors that play a role in transactivation (5, 10, 11, 12). Furthermore, it has been shown that nuclear receptors enhance
the formation of the preinitiation complex of basal transcription
factors (13). In vitro interaction studies have identified
TATA binding protein (14), the TFIIB factor (15, 16, 17), and some TATA
binding protein-associated factors (18) as receptor target proteins.
The search for receptor partners has led to the identification of a
growing set of new proteins, called coactivators or transcription
intermediary factors, such as TRIP1/SUG1 (19, 20), ERAP140 (21), RIP140
(22, 23), TIF1 (24), and SRC-1 related proteins (25, 26, 27, 28). These are
shown to bind the receptors in a ligand-dependent way, suggesting a
putative role in ligand-induced transcription.
RIP140 was cloned as a factor that associates with estrogen receptor in the presence of estrogen but not in the presence of antiestrogen (22). This interaction requires a fonctional AF2 domain and is impaired by point mutations that inhibit the receptor hormone-dependent transcriptional properties. Two-hybrid experiments in mammalian cells suggest that this interaction occurs in intact cells (23). As the core domain of AF2 appears to be conserved in other nuclear receptors (8), it is conceivable that RIP140 interacts with other members of the superfamily. However, the physiological implications of this protein in activated transcription remain to be clearly established in vivo.
Yeast is an eukaryotic microorganism system in which the major basic mammalian functions would appear to be conserved. Components of the basic RNA polymerase II are highly homologous, and in some cases functionally interchangeable (29). In mammalian cells, yeast activators such as GAL4 protein activate the transcription of genes under the control of their cognate DNA recognition sequences (30). Moreover, members of the steroid/thyroid/retinoid superfamily induce transcription of chimeric genes under the control of their respective hormone response element in yeast (31, 32, 33, 34, 35). These cells are devoid of nuclear receptor proteins, enabling studies to be performed without any cellular background. The estrogen receptor characteristics are maintained in yeast: the two activation functions are able to synergize and have promoter-dependent activity (36, 37, 38). Yeast has been used to analyze the molecular mechanism of action of the retinoid receptor (32, 39, 40) and to emphasize the cofactor role of retinoid X receptor (RXR) in vitamin D (41) and thyroid signaling (35).
In the present study, yeast systems have been used to study the effect
of RIP 140 on nuclear receptor-mediated transcription. We show that
RIP140 actually enhances in vivo estrogen receptor (ER) and
retinoic acid receptor- (RAR
) agonist-dependent transactivation
and could participate in cross-talk between nuclear receptors.
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RESULTS |
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By far-Western blotting, in our PL3 strain, we were unable to detect
any protein in the range of 140 kDa that could interact with a
GST-ER(AF2) probe (data not shown). This suggests that, in these
extracts, a yeast homolog of RIP140 is probably expressed not at all or
only in an amount undetectable in our assay.
We constructed PL3 strain transformants, expressing estrogen
receptor, from a mono- (pRS315) or multicopy vector (Yep90), in the
presence or absence of RIP140 expressed from a multicopy vector
(Yep10). Empty vectors were transformed as control. When RIP140 was
expressed, a protein of the expected size was detected by far-Western
assay (data not shown).
In the PL3 strain transformed with ER, growth induction is strictly
dependent upon the reporter gene (URA3) transcriptional induction,
i.e. upon the estrogen receptor-mediated transcriptional
activation (37). This system is very sensitive, since the level of
reporter activity required for full growth is quite low (
0.4 U),
compared with the maximum activity of the reporter in enzymatic assay
(1520 U). Therefore, the growth assay measures only the variation in
reporter gene induction below the critical threshold of reporter
activity required for full growth.
As shown in Fig. 1A, when ER was expressed from a
monocopy vector, an estradiol concentration of 0.5 nM led
to growth induction whereas at this concentration estradiol had no
effect on the strain not expressing RIP140. A comparable shift was
observed when ER was expressed from a multicopy vector, except that the
estradiol concentrations required for growth were lower, due to the
higher ER expression level (data not shown).
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These results were confirmed in another estrogen-responsive system in
the YHP250 strain. The reporter gene was the LacZ gene linked to the
URA3 promoter and under the control of three EREs. When ER was
expressed from a low copy vector, the half-maximal ß-galactosidase
activity was reached at 4 nM in the absence of RIP140 and 2
nM in its presence (Fig. 2A). Moreover, the
effects of RIP140 were even more pronounced than in the former system,
because RIP140 triggered a 4- and 9-fold enhancement of transcription
at saturant (1 µM) or limiting (1 nM)
estradiol concentrations, respectively.
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The Enhancement of ER Transactivation by RIP140 Requires a
Functional AF2-Activating Domain
In vitro glutathione-S-transferase (GST)
pull-down assays have previously shown that the interaction between
RIP140 and ER is efficient in the presence of the AF2 domain of the
receptor and was inhibited by point mutations that affect
transcriptional capacity of AF2 core domain (23). This suggests that
AF2 function is necessary for RIP140 to mediate its effect. To test
this hypothesis, we studied the RIP140-mediated transcriptional
modulation of a truncated form of mouse ER, MOR 121599, in which most
of the N-terminal domain was deleted (Fig. 3A). As
described in this promoter context, the ß-galactosidase activity was
lower than in the presence of wild type ER (compare Figs. 3A
and 2A
)
due to the absence of AF1 activation function, thus eliminating the
synergism between AF1 and AF2 (37, 38). RIP140 expression increased the
activated transcriptional level 10-fold and the basal level of this
truncated ER mutant 4-fold.
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RIP140 Is Also a Coactivator for Retinoic Acid Receptor-
Recently, other cofactors for nuclear receptors have been
described (20, 21, 24, 26). They have been cloned for their interaction
with a particular receptor, but further analyses have shown that they
are common to several pathways. The above results show that the AF2 AD
core domain is involved in the interaction between ER and RIP140.
Sequence alignments of the nuclear receptor genes revealed that the AF2
core domain is highly conserved in the superfamily (8). We thus tested
whether RIP140 could be a coactivator for another member of the
superfamily.
We tested the hypothesis that RIP140 is involved in RAR-mediated
transcription, using a yeast heterologous retinoid-inducible
transcription system. The chimeric RAR
-ERCas receptor was
cotransformed with the ERE-LacZ reporter gene in the YHP250 strain, in
the presence or absence of RIP140. RAR
-ERCas is a chimeric retinoid
receptor, whose DNA-binding domain was replaced by the ER-binding
domain. This receptor is able to bind an ERE and to induce a
transcriptional activation in the presence of retinoic acid. In the
absence of ligand, this receptor is probably bound to DNA because its
expression can induce a weak basal activity (32).
Figure 4A shows that RIP140 expression enhanced a
10-fold retinoic acid-induced ß-galactosidase activity but also the
ligand-independent reporter activity. Furthermore, the potency of
all-trans-retinoic acid as an agonist was increased as the
half-maximal activity of RAR
-ERCas was reached at about 10
nM in the absence of RIP140, whereas it occurred at 2
nM in its presence. Similar results were found in the
PL3
system, in growth or enzymatic assays (data not shown).
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We used pull-down experiments to test whether the effects of RIP140 on
estrogenic and retinoid pathways could be recovered in an in
vitro interaction assay. A GST-RIP140 fusion protein retained on
glutathione Sepharose beads was incubated with in vitro35S-labeled nuclear receptor, with or without the
presence of cognate hormone. As shown in Fig. 5, GST-RIP140 interacted specifically with ER and RAR
, this interaction
being greatly reinforced in the presence of hormone agonist.
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Cross-Talk between Estrogenic and Retinoid Pathways in Yeast
Because RIP140 is a common factor to estrogenic and retinoid
pathways, we investigated whether RAR, in the presence of RIP140 and
in a ligand-dependent fashion, could modulate an estrogenic response.
Interferences between estrogenic and retinoid transduction pathways
have actually been described in mammalian cells, as retinoid ligands
are able to inhibit estrogenic responses (45, 46).
We constructed several YHP250-derived strains, by transformation of the
ERE-LacZ reporter gene, estrogen receptor in low copy, RAR in
multicopy, and RIP140 in multicopy or the corresponding empty vectors.
As shown in Table 1
, the basal activity of all the
strains in the presence of ethanol is identical. In the strains
expressing RAR
and RIP140, in the presence or in the absence of ER,
retinoic acid induced a 25-fold increase in activity, whereas in the
absence of RIP140, retinoic acid had no effect. This suggests that
RAR
interacts with ERE and that in the presence of retinoic acid,
RIP140 could reinforce this interaction, thus allowing a significant
transactivation to occur.
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DISCUSSION |
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RIP140 effects were recovered in two different cellular contexts.
Compared with the estrogen-responsive reconstituted system in the
PL3 strain, the system developed in the YHP250 strain appeared to be
more sensitive, as ED50 values were lower and the effect of
RIP140 on the induction factor was more pronounced. This could be due
to the higher accessibility of the reporter gene in the YHP250-derived
strains since the LacZ reporter is carried on a multicopy vector.
RIP140 also has a strong positive effect on the transactivation
mediated by retinoid receptor-. Because these results are in
agreement with in vitro interaction assays, it would appear
that RIP140 is a specific coactivator of ER and RAR
in yeast.
RIP140 Effects on Transactivation Require a Functional AF2
Domain
The positive effect of RIP140 on transactivation is recovered with
a truncated mutant of ER lacking the N-terminal region but abolished
when AF2 AD transactivation function is impaired by two-point
mutations. Previous in vitro interaction assays have led to
similar conclusions (23). Taken together, these results show that a
functional AF2 AD region, at least, is necessary for RIP140 to interact
with nuclear receptors and to act as a positive cofactor for
agonist-liganded receptors.
RIP140 led to a higher enhancement of agonist transactivation mediated
by the AF1-truncated ER than the full-length receptor. But, in contrast
to what we observed with the wild type ER receptor, the
cotransformation of the N-terminal-truncated mouse ER receptor or
RAR-ERCas with RIP140 led to an increase in the basal activation of
the reporter gene. A similar effect has been reported in yeast on a
progesterone-response element/glucocorticoid response element with the
yeast RSP5 factor (47). This protein potentiates the progesterone
transactivation without influencing the ligand-independent reporter
activity. However, when RSP5 was expressed with the glucocorticoid
receptor, the basal transcription of the same reporter gene was
dramatically increased.
It has been shown in yeast that the AF1-deleted ER receptor (37), the
chimeric receptor RAR-ERCas (32), and wild type retinoic acid and
thyroid receptors (35, 40, 48) significantly activate the transcription
in the absence of ligand. This supposes that a small proportion of the
receptor population is in an active conformation under unliganded
conditions. Higher basal activity of truncated ER receptors and of
retinoid receptors in yeast could be due to the absence of repression
either by the N-terminal domain of ER or by RAR-specific repressors.
This is in agreement with the recent hypothesis that receptors exist in
equilibrium, shifting between inactive-repressive and active
transcriptional states, in the absence of hormone (49). This unstable
unliganded active status can be detected in yeast by weak constitutive
transcriptional activity, due to the absence of the repression
machinery coupled to nuclear receptors. In yeast, although retinoid and
thyroid receptors are able to induce transactivation in the presence of
hormone, promoter silencing in the absence of ligand has not been
observed (32, 35, 40, 48). This silencing activity has been attributed
in mammalian cells to such specific repressors as nuclear receptor
corepressor (N-CoR) and silencing mediator for retinoid and thyroid
hormone receptors (SMRT), which are associated with receptor in the
absence of hormones but are released by the binding of agonist ligands
(50, 51, 52). This class of repressor is thus thought to be absent in
yeast. The weak constitutive activity observed in yeast has been
correlated to the presence of a functional AF2 domain, believed to be
in a conformational active form (49). It is thus not surprising that
this activity could be enhanced by RIP140 expression.
RIP140 as a Factor That Discriminates between Agonist and
Antagonist Activities
Although basal activity of receptor was increased in the presence
of RIP140, the activity observed in the presence of antagonist of ER
(OH-Tam) and of RAR (Ro 415253) was not enhanced by RIP140. These
data are in agreement with 1) in vitro assays showing that
RIP140 interacts only with nuclear receptors in the presence of agonist
and 2) transactivation assays suggesting that RIP140 has no effect on
ER AF1 function (23). This RIP140 property was clearly demonstrated
with the retinoid antagonist. In the absence of RIP140, the antagonist
activity represented more than 30% of the agonist activity, whereas in
the presence of RIP140 this activity reached only 5% of the maximum
reporter activity. It would thus appear that RIP140 is a cellular
factor implicated in the discrimination between agonist and
antagonists, by specifically enhancing the agonist transactivation.
Cross-Talk between Estrogenic and Retinoid Signaling
Because RIP140 is involved in estrogenic and retinoid pathways, it
could be implicated in the inhibition of estrogenic response by
retinoid as described previously (45, 46). In our yeast experiments,
due to an inappropriate ratio between reporter gene, nuclear receptor,
and RIP140, we did not observe a squelching effect. On the contrary,
activated retinoid receptor enhanced the transcription of a reporter
gene under the control of ERE, in the presence of RIP140.
The enhancement of ER transactivation could reflect a binding
cooperativity of retinoid and ER on the response element since we
demonstrated that RAR, in the presence of RIP140 and retinoic acid,
induced a moderate expression of an ERE-dependent gene reporter, even
in the absence of expressed ER. It is noteworthy that the RAR
antagonist did not have such an effect. The binding of RAR
on the
ERE could be stabilized by RIP140, by bridging the RAR
receptor to
the transcriptional machinery.
In mammalian cells, inhibition of estrogenic response by retinoic acid could be due to both a squelching effect and to binding of inactive receptors on the ERE. We have previously shown that, in mammalian cells, ligand-bound RAR/RXR heterodimer is able to inhibit chimeric receptors containing the DNA- binding domain of ER (45). This implies that competition at the level of ERE occurs between retinoid receptors and ER. We also have demonstrated that RAR or RXR binds to an ERE, but that this binding was not sufficient to inhibit the estrogenic response in yeast (53). The present study suggests that cofactors such as RIP140 could reinforce the interaction of retinoid receptors with the transcriptional apparatus. In mammalian cells, the inhibitory effect could be due to the existence of specific repressors, absent in yeast, that block the transactivation of retinoid receptors bound to certain response elements. This mechanism has been described on a DR1 motif, where the heterodimer RAR/RXR cannot transactivate due to the binding of the N-CoR repressor, even in the presence of retinoid agonists (51).
Nuclear Receptors and Coactivators
It is now clear that the transcription factors require
coactivators to mediate their effects. Such coactivators exist in yeast
(54), and a genetic selection recently successfully isolated RSP5, a
yeast protein that seems involved in progesterone and glucocorticoid
signaling (47). Other yeast factors have been described that enhance
the transactivation by ER as ySPT6 (55) or ySUG1. Mammalian homolog of
ySUG1 (mSUG1/hTRIP1) is known to interact with nuclear receptors (19, 20, 56). Except for mSUG1, direct transcriptional assays have been
performed in yeast; these cofactors enhanced 4-fold (for SPT6) and
7-fold (for RSP5) the transactivation of the agonist-bound receptor.
However, RSP5 is able to significantly increase the partial agonist
activity of the antiprogestin RU486 (47), and mSUG1 interacts with
RAR bound to either retinoid agonists or antagonists (20). These
proteins thus appear to play a role in the general enhancement of
nuclear receptor transcription but do not seem to be specific for
agonist pathways. Because SUG1 and RSP5 are homologous to proteins
involved in the proteasome complex (57) or in the ubiquitination
pathway (47), they could act on the turnover of receptors or associated
proteins.
With the development of the two-hybrid assay (58), yeast has been widely used to clone mammalian factors, using chimeric receptor constructs as a bait.
The mTIF1 factor has been cloned for its interaction with RXR, and
further investigations have shown that this factor is also shared by
estrogenic, retinoic, vitamin D3 hormonal pathways (24).
Tested in a direct transcription assay in yeast, mTIF1 expression led
to a increase of RXR transactivation, more pronounced at suboptimal
ligand concentrations. RIP140 and mTIF1 would therefore appear to have
similar properties, despite being unrelated proteins: they have common
nuclear receptor targets, and they potentiate the transcription of
receptor through the AF2-AD domain, in an agonist-dependent manner with
a greater efficiency at limiting concentration of ligand.
The recent cloning of mammalian proteins of the SRC-1 family (SRC-1, GRIP1, p160, TIF2) (25, 26, 27, 28) reinforces the idea that all classes of nuclear receptors could have a set of cofactors involved in transactivation. SRC-1 and GRIP1 factors have been selected for their involvement in the glucocorticoid and progesterone pathway, whereas TIF2, which is closely homologous to SRC-1, has been cloned for its interaction with estrogen and retinoid receptors. When this property has been tested, all these mammalian coactivators specifically increase the agonist-induced transcription and are without any effect if the receptor is bound to antagonists. The role of this class of protein could be to selectively enhance the transactivation of receptor by discrimination between agonist and antagonist activities.
It thus appeared that transactivation through nuclear receptor requires various complexes of coactivators, either specific for one pathway or common to several receptors. This could be a molecular basis for cross-talk between the different signalings, as has been described for the estrogen-dependent inhibition of PR activity (26). Furthermore, nuclear receptors have been shown to be activated by hormone-independent events such as phosphorylation after growth factor stimulation (59). Factors common to the different transduction pathways could serve as signal integrators in the cell, as recently described for the CBP/p300 family of proteins (27). Future work will be necessary before the global complex mechanism of transcriptional regulation can be elucidated.
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MATERIALS AND METHODS |
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Yeast strains were grown in selective medium (Difco Laboratories, Detroit, MI) and transformed by the lithium acetate protocol (60).
Receptor Expression Vectors
Yep10 (40) and Yep90 (37) are 2 µ-derived multicopy vectors
carrying TRP1 and HIS3, respectively, as selection markers. pRS315 is a
LEU-2-containing centromeric vector (61). All these vectors carry a PGK
promoter and terminator cassette with a unique EcoRI-cloning
site. hER in pRS315 and Yep90 (37), hRAR1 and hRAR
1-ERCas in
Yep90 (32), the multicopy pRL
21-U-3EREs vector (38), carrying the
LacZ gene linked to the URA3 promoter and under the control of three
EREs, were a gift from Dr Losson.
Yeast vectors allowing the expression of mouse ER variants lacking most of the A/B region were generated by inserting a BamHI/EcoRI fragment from pJ13MOR121-599, either wild type (DBD-AF2wt in pYER6) or with the M547A/L548A mutations (DBD-AF2mut in pYER15), into the BamHI/XbaI sites of pSDO8 multicopy vector carrying the LEU2 marker (62).
To express RIP140 in yeast, the RIP cDNA from pBRIP140 (23) was digested by SpeI, blunted and subcloned in the Yep10 vector digested by EcoRI, and blunted.
Growth Assay
PL3 strains were seeded at low density in 96-well multiplates
and grown in selective media lacking uracil but in the presence of
growing concentrations of hormone. After 24 h, growth was
evaluated by reading absorbance at 600 nm on a microplate
spectrophotometer. Data present results of a typical experiment
performed in triplicate and recovered at least on two independent
transformants.
Transactivation Assay
The yeast double or triple transformants were allowed to grow
exponentially in selective media with uracil for four generations in
dim light, in the presence or absence of hormone. Estradiol was
purchased from Sigma Chemical Co. (St. Quentin Fallairer, France),
OH-Tam was provided by Dr Wakeling (Zeneca Laboratory, Macclesfield,
England), retinoid ligands were a gift from Dr Klaus (Hoffman-La-Roche,
Basel, Switzerland).
OMPdecase activity was measured as previously described and expressed as nanomoles of substrate transformed per mg of protein per min (37). ß-Galactosidase activity was assayed in a permeabilized cell assay. Briefly, cells were collected by centrifugation, washed in water, resuspended in 1 ml of lysis buffer (0.1 M Tris, pH 7.5, 0,05% Triton) and frozen in liquid nitrogen. An aliquot (usually 25 µl) of this cell suspension was assayed for ß-galactosidase activity and units of ß-galactosidase activity are defined by the formula 1000 x A420/(txVxA600) where A420 is from the o-nitrophenyl ß-galactosidase/hydrolysis assay, t is the incubation time, V the volume of yeast suspension assayed, and A600 the absorbance at 600 nm of 1 ml of the yeast suspension. All the data represent results from a typical experiment recovered at least for three independent transformants (mean ± SEM).
GST-Pull Down Assay
Recombinant nuclear receptors (ER, RAR) in pSG5 were
transcribed, translated, and 35S-labeled in rabbit
reticulocyte lysate (Promega, Madison, WI) following the
manufacturers instructions. GST-RIP140, which encoded a fusion
between GST and residues 752-1158 of hRIP140, was generated by
inserting a BamHI/BglII fragment from
pBRIP3 (23) into the BamHI site of pGEX-2TK (Pharmacia,
Piscataway, NJ). The fusion protein was expressed in Escherichia
coli and purified as previouly described (22). The fusion protein
loaded onto gluthathione-Sepharose beads was incubated with the
35S-labeled receptors in the presence of ethanol (no
hormone), E2, or all-trans-RA at 1
µM for 1.5 h at 4 C in a total volume of 150 µl
IPAB buffer (150 mM KCl, 0,02 mg/ml BSA, 0.1% Triton,
0.1% NP40, 5 mM MgCl2, 20 mM
HEPES, pH 7.9, protease inhibitors). Beads were washed five times with
IPAB without BSA. The beads were then dried under vacuum, resuspended
in 20 µl loading buffer, and analyzed by SDS-PAGE. Signals were
amplified by fluorography (Amplify, Amersham, Arlington Heights, IL),
and gels were exposed at -70 C.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication August 2, 1996. Revision received October 28, 1996. Accepted for publication November 1, 1996.
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REFERENCES |
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