RIP 140 Enhances Nuclear Receptor-Dependent Transcription in Vivo in Yeast

A. Joyeux, V. Cavaillès, P. Balaguer and J. C. Nicolas

INSERM U439 (A.J., P.B., J.C.N.) and INSERM U148 (V.C.), 34090 Montpellier, France


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RIP140 has previoulsy been cloned as a factor that interacts with the estrogen receptor (ER) in vitro. We demonstrate in this study that RIP140 is a cofactor for nuclear receptor in yeast. RIP140 enhances the ER transcriptional activity by increasing 1.5- to 4-fold the induction factor of the reporter gene response at saturating hormone concentrations, this effect being magnified at suboptimal doses of estradiol. Moreover, RIP140 decreases the ED50 of the dose-response curve. These effects are recovered with an N-terminal truncated ER, but impaired by point mutations that abolish AF2-AD activity. We did not observe any modulation of the partial agonist 4-hydroxytamoxifen activity in the presence of RIP140. Thus, RIP140 modulates transcriptional activity of ER through the AF2-AD domain and in a agonist-dependent fashion. RIP140 is also a strong coactivator for the retinoid pathway, as its expression enhances 10-fold the transactivation of a chimeric retinoic acid-{alpha} receptor at saturant hormone concentration and left shifted 5-fold the ED50 of the dose-response curve. We have investigated whether RIP140 could be involved in cross-talk between estrogenic and retinoid pathways.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Steroid/thyroid/retinoid nuclear receptors share a common modular structure that can be divided in five to six (from A to E or F) domains. The most conserved region is the central domain C that allows DNA recognition on specific hormone-responsive sequences in the promoter of target genes. Receptors bind to DNA in a homo- or heterodimeric form due to dimerization interfaces recovered in the C and E regions. The E region encompasses the ligand-binding domain and contains a ligand-dependent transcriptional activation function (AF2). An autonomous activation function (AF1) is recovered in the N-terminal part (domain A/B) of the protein (1, 2, 3).

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 {alpha}-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-{alpha} (RAR{alpha}) agonist-dependent transactivation and could participate in cross-talk between nuclear receptors.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RIP 140 Enhances Estrogen Receptor Transcriptional Activity in Yeast
In vitro interaction assays suggest that RIP140 could play a role in the estrogen receptor-induced transcription (23). We decided to test this hypothesis in yeast, in an estrogen-inducible system. We first used the PL3{alpha} strain in which an estrogen-responsive reporter gene (3EREs-URA3) has been stably integrated in one copy at a single locus in the genome (37).

By far-Western blotting, in our PL3{alpha} 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{alpha} 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{alpha} 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 (15–20 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. 1AGo, 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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Figure 1. RIP140 Enhances ER Transcriptional Activity in Yeast

PL3{alpha} strain was transformed with ER in pRS315 and Yep10 (solid circles) or RIP140 in Yep10 (open circles). Estradiol-dependent OMPdecase induction was measured by growth assay as described in Materials and Methods (A) or enzymatic activity (B).

 
To further investigate this apparent enhancement of estrogen receptor transcription activity by RIP140 in the PL3{alpha} strain, we performed orotidine-5'-monophosphate decarboxylase (OMPdecase) enzymatic assay, which gave a direct evaluation of the URA3 reporter gene transcriptional activation. In this model, the reporter gene is present in single copy, and the amount of receptor is critical to obtain maximal transactivation (37). Thus we checked the effect of RIP 140 on the activity of ER when expressed from a low or high copy number vector. With a low ER level, we were able to observe a left shift of the dose-response curve, from 10 nM to 5 nM, and an increase in the activity of the reporter gene in the presence of RIP140 (Fig. 1BGo). At a saturant hormone concentration (1 µM), RIP140 expression led to a 1.5-fold enhancement of the OMPdecase activity. This effect was higher at a suboptimal estradiol concentration (1 nM) where it reached 3.6-fold. When ER was expressed from a high copy number vector, the shift of the dose response was less important, but the enhancement of reporter activity reached more than 2-fold in the presence of RIP140 at 1 µM estradiol (data not shown). In the two sets of experiments, the basal activity of the estrogen-response element (ERE)-containing reporter remained unchanged by RIP140 expression.

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. 2AGo). 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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Figure 2. RIP140 Enhancement of ER Activity Is Specifically Agonist Dependent

A, YHP250 strain was transformed with the pRL{Delta}21 vector carrying the LacZ gene driven by the URA3 promoter and three EREs, ER in pRS315 and Yep10 (solid circles), or RIP140 in Yep10 (open circles). The dose-response curve of estradiol-dependent ß-galactosidase induction was established as described in Materials and Methods. B, The reporter activity of the same strains (without RIP140, hatched bars; with RIP140, open bars) was assayed in the presence of saturating concentrations of agonist (1 µM E2) or antagonist (10 µM OH-Tam) as indicated. C, Vehicle-treated cells.

 
We also tested whether RIP140 expression could modulate the ER-mediated transactivation in the presence of antiestrogen such as 4-hydroxytamoxifen (OH-Tam). Agonist activity has been recovered with OH-Tam in mammalian and yeast cells, in a cellular and promoter-dependent manner, probably due to a functional AF1 in these contexts (12, 38, 42). In our transactivation system (Fig. 2BGo), this agonist activity is weak but significantly different from basal activation. RIP140 expression has no effect on this partial agonist activity. This reinforces the previous observations 1) that RIP140 binds ER preferentially in the presence of an agonist and 2) that RIP140 has no effect on AF1-dependent transactivation (23).

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 121–599, in which most of the N-terminal domain was deleted (Fig. 3AGo). As described in this promoter context, the ß-galactosidase activity was lower than in the presence of wild type ER (compare Figs. 3AGo and 2AGo) 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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Figure 3. RIP140 Effects on ER Transactivation Requires a Functional AF2 Domain

A, YHP250 was transformed with the estrogen-responsive LacZ reporter gene (as in Fig. 2Go), the N-terminal-truncated mutant of mER (DBD-AF2 wt) in pYER6 and Yep10 (solid circles), or RIP140 in Yep10 (open circles). Estradiol-dependent ß-galactosidase induction was assayed as in Fig. 1Go. B, YHP250 was transformed with the estrogen-responsive LacZ reporter gene and the indicated vectors (DBD-AF2wt in pYER6, DBD-AF2mut in pYER15, RIP140 in Yep10, or the corresponding empty vector). ß-Galactosidase activity was assayed in the presence of ethanol or 1 µM E2.

 
We then introduced in the AF2 core domain of the truncated mutant the two point mutations M547A/L548A, which abolished 1) the estrogen-dependent transactivation in mammalian cells (4) and 2) the interaction between ER and RIP140 in an in vitro assay (22). Tested in our yeast transactivation assay, this mutant had no transcriptional activity (data not shown), and RIP140 cotransformation led to no further increase in transactivation (Fig. 3BGo), reinforcing the idea that RIP140 requires a functional AF2 core domain to increase the transcription of the reporter gene.

RIP140 Is Also a Coactivator for Retinoic Acid Receptor-{alpha}
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{alpha}-mediated transcription, using a yeast heterologous retinoid-inducible transcription system. The chimeric RAR{alpha}-ERCas receptor was cotransformed with the ERE-LacZ reporter gene in the YHP250 strain, in the presence or absence of RIP140. RAR{alpha}-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 4AGo 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{alpha}-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{alpha} system, in growth or enzymatic assays (data not shown).



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Figure 4. RIP140 Is a Cofactor for the Retinoid Pathway in Yeast

A, YHP250 strain was transformed with the estrogen-responsive LacZ gene as in Fig. 1Go, the chimeric retinoid receptor RAR{alpha}-ERCas in Yep90 and Yep10 (solid circles), or RIP140 in Yep10 (open circles). ß-Galactosidase activity was assayed in the presence of growing concentration of all-trans-retinoic acid. B, The same strains as in panel A) (solid bars in the absence of RIP140 and open bars in its presence) were assayed for reporter activity in the presence of ethanol (C), all-trans-retinoic acid (RA), 1 µM, or Ro 41–5253, 1 µM.

 
We examined the effect of RIP140 on RAR{alpha}-ERCas-mediated transactivation in the presence of an antagonist (Fig. 4BGo). The synthetic retinoid analog Ro 41–5253 has been described as a specific RAR{alpha} pure antagonist in mammalian cells (43). Tested in our yeast system, in the absence of RIP140, this compound had a partial agonist activity: at 1 µM, the ß-galactosidase activity reached 30% of all-trans-RA-induced activity at the same concentration. Similar observations have previously been made for other antagonists tested in yeast (36, 38, 44). The expression of RIP140 enhanced the basal level of transcription, but the partial agonist effect of Ro 41–5253 at 1 µM then represented less than 5% of the maximum agonist activity. This suggests that the interaction between RIP140 and the retinoid receptor could not be reinforced in the presence of this antagonist.

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. 5Go, GST-RIP140 interacted specifically with ER and RAR{alpha}, this interaction being greatly reinforced in the presence of hormone agonist.



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Figure 5. In Vitro Interaction of RIP140 with Nuclear Receptors

In vitro 35S-labeled receptors were incubated with Sepharose gluthatione beads preloaded with the fusion protein GST-RIP140. The interaction assay was performed in the absence of hormone (NH, no hormone) or in the presence of estradiol (E2) or all-trans-retinoic acid (RA). As control, incubation with GST alone and 1/10 of the receptor input used in the assay are shown.

 
These data suggest that RIP140 is at least specifically involved in ER and retinoid receptor signaling, in yeast, by specifically increasing the transactivation induced by agonist ligands. As a control for the specificity of RIP140 action, we have determined that RIP140 does not alter the transcriptional activity of Gal4 activator in yeast (data not shown).

Cross-Talk between Estrogenic and Retinoid Pathways in Yeast
Because RIP140 is a common factor to estrogenic and retinoid pathways, we investigated whether RAR{alpha}, 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{alpha} in multicopy, and RIP140 in multicopy or the corresponding empty vectors. As shown in Table 1Go, the basal activity of all the strains in the presence of ethanol is identical. In the strains expressing RAR{alpha} 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{alpha} 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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Table 1. Agonist-Activated RAR{alpha} Transactivates an ERE in the Presence of RIP140

 
We tested the cross-talk between RAR{alpha} and ER on an estrogenic response in the presence of RIP140 in the PL3{alpha} strain. Figure 6Go shows that retinoic acid potentiated significantly the estrogenic response, in the presence of RAR{alpha} and RIP140. Retinoic acid (1 µM) increased the transcription activity (by 150%) and left shifted the dose-response curve by a factor of 2. Interestingly, these effects were not produced with the RAR{alpha} antagonist (Fig. 6BGo) or in the absence of RIP140 (Fig. 6AGo).



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Figure 6. Agonist-Activated RAR{alpha} Potentiates an Estrogenic Response in the Presence of RIP140

The PL3{alpha} strain was transformed with ER in pRS315, RAR{alpha} in Yep90, and Yep10 (A) or RIP140 in Yep10 (B). The estrogenic reporter induction was assayed in the presence of ethanol (open circles), of 1 µM of Ro 41–5253 (solid circles and dotted line), or of 1 µM of all-trans-retinoic acid (black squares).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RIP140 Is a Cofactor for a Subclass of Nuclear Receptors in Yeast
In the present paper, we have studied in vivo the effect of RIP140 on nuclear receptor-mediated transcription. We show that RIP140 actually enhances ER-mediated transcription, by increasing the induction factor and decreasing the ED50 of the dose-response curve. Interestingly, the maximum enhancement of ER activity by RIP140 was obtained at a limiting concentration of estradiol (1 nM). At a saturant level of hormones (1 µM), the effects were less pronounced. Similar observations have been made previously for TIF1 on retinoid responses (24).

RIP140 effects were recovered in two different cellular contexts. Compared with the estrogen-responsive reconstituted system in the PL3{alpha} 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-{alpha}. Because these results are in agreement with in vitro interaction assays, it would appear that RIP140 is a specific coactivator of ER and RAR{alpha} 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{alpha}-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{alpha}-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{alpha} (Ro 41–5253) 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{alpha}, 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{alpha} antagonist did not have such an effect. The binding of RAR{alpha} on the ERE could be stabilized by RIP140, by bridging the RAR{alpha} 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{alpha} 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{alpha}, 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Strains and Media
The Saccharomyces cerevisiae strains used were PL3{alpha} (Mat {alpha} ura3-{Delta}1 his3-{Delta}200 leu2-{Delta}1 trp1:: 3ERE-URA3) and YHP250 (Mat a ura3-52 lys2-801 ade2-101 trp1-{Delta}1 his3-{Delta}200 leu2-{Delta}1) kindly provided dy Dr R. Losson (U184 INSERM, IGBMC Illkirch, France). In PL3{alpha}, the reporter gene (URA3) coding for the OMPdecase was placed under the control of three EREs from Xenopus laevis vitellogenin and inserted at the trp1 locus in the RL15 strain as previously described (37).

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), hRAR{alpha}1 and hRAR{alpha}1-ERCas in Yep90 (32), the multicopy pRL{Delta}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{alpha} 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{alpha}) in pSG5 were transcribed, translated, and 35S-labeled in rabbit reticulocyte lysate (Promega, Madison, WI) following the manufacturer’s 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.


    ACKNOWLEDGMENTS
 
We thank Dr. Losson for providing yeast strains and plasmids. We are grateful to Drs. Wakeling and Klaus for the gift of ligands.


    FOOTNOTES
 
Address requests for reprints to: J. C. Nicolas, Pathologie Moleculaire, Institut National de la Sante et de la Recherche, INSERM U439, 70 rue de navacelles, 34090 Montpellier, France.

Received for publication August 2, 1996. Revision received October 28, 1996. Accepted for publication November 1, 1996.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Beato M 1989 Gene regulation by steroid hormones. Cell 56:325–344
  2. Evans RM 1988 The steroid and thyroid hormone receptor superfamily. Science 240:889–895[Medline]
  3. Tsai MJ, O’Malley BW 1994 Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451–486[CrossRef][Medline]
  4. Danielian PS, White R, Lees JA, Parker MG 1992 Identification of a conserved region required for hormone dependent transcriptional activation by steroid hormone receptors. EMBO J 11:1025–1033[Abstract]
  5. 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 13:5370–5382[Abstract]
  6. Bourguet W, Ruff M, Chambon P, Gronemeyer H, Moras D 1995 Crystal structure of the ligand-binding domain of the human nuclear receptor RXR-{alpha}. Nature 375:377–382[CrossRef][Medline]
  7. Wagner RL, Apriletti JW, McGrath ME, West BL, Baxter JD, Fletterick RJ 1995 A structural role for hormone in the thyroid hormone receptor. Nature 378:690–697[CrossRef][Medline]
  8. Wurtz JM, Bourget W, Renaud JP, Vivat V, Chambon P, Moras D, Gronemeyer H 1996 A canonical structure for the ligand-binding domain of nuclear receptors. Nature Struct Biol 3:87–94[Medline]
  9. Renaud JP, Rochel N, Ruff M, Vivat V, Chambon P, Gronemeyer H, Moras D 1995 Crystal structure of the RAR-{gamma} ligand-binding domain bound to all-trans retinoic acid. Nature 378:681–689[CrossRef][Medline]
  10. Bocquel MT, Kumar V, Stricker C, Chambon P, Gronemeyer H 1989 The contribution of the N- and C-terminal regions of steroid receptors to activation of transcription is both receptor and cell specific. Nucleic Acids Res 17:2581–2595[Abstract]
  11. Nagpal S, Saunders M, Kastner P, Durand B, Nakshatri H, Chambon P 1992 Promoter context- and response element-dependent specificity of the transcriptional activation and modulating functions of retinoid acid receptors. Cell 70:1007–1019[Medline]
  12. Tzukerman MT, Esty A, Santiso-Mere D, Danielian P, Parker MG, Stein RB, Pike JW, McDonnell DP 1994 Human estrogen receptor transactivational capacity is determined by both cellular and promoter context and mediated by two functionally distinct intramolecular regions. Mol Endocrinol 8:21–30[Abstract]
  13. McEwan IJ, Amlöf T, Wikström AC, Dahlman-Wright K, Wright APH, Gustafsson JA 1994 The glucocorticoid receptor functions at multiple steps during transcription initiation by RNA polymerase II. J Biol Chem 269:25629–25636[Abstract/Free Full Text]
  14. Schulman IG, Chakravarti D, Juguilon H, Romo A, Evans RM 1995 Interactions between the retinoid X receptor and a conserved region of the TATA-binding protein mediate hormone-dependent transactivation. Proc Natl Acad Sci USA 92:8288–8292[Abstract]
  15. Baniahmad A, Ha I, Reinberg D, Tsai S, Tsai MJ, O’Malley BW 1993 Interaction of human thyroid hormone receptor ß with transcription factor TFIIB may mediate target gene derepression and activation by thyroid hormone. Proc Natl Acad Sci USA 90:8832–8836[Abstract]
  16. Blanco JCG, Wang IM, Tsai SY, Tsai MJ, O’Malley BW, Jurutka PW, Haussler MR, Ozato K 1995 Transcription factor TFIIB and the vitamin D receptor cooperatively activate ligand-dependent transcription. Proc Natl Acad Sci USA 92:1535–1539[Abstract]
  17. Ing NH, Beekman JM, Tsai SY, Tsai MJ, O’Malley BW 1992 Members of the steroid hormone receptor superfamily interact with TFIIB (S300-II). J Biol Chem 267:17617–17623[Abstract/Free Full Text]
  18. Jacq X, Brou C, Lutz Y, Davidson I, Chambon P, Tora L 1994 Human TAFII30 is present in a distinct TFIID complex and is required for transcriptional activation by the estrogen. Cell 79:107–117[Medline]
  19. Lee JW, Ryan F, Swaffield JC, Johnston SA, Moore DD 1995 Interaction of thyroid-hormone receptor with a conserved transcriptional mediator. Nature 374:91–94[CrossRef][Medline]
  20. vom Baur E, Zechel C, Heery D, Heine MJS, Garnier JM, Viviat V, Le Douarin B, Gronemeyer H, Chambon P, Losson R 1996 Differential ligand-dependent interactions between the AF-2 activating domain of nuclear receptors and the putative transcriptional intermediary factors mSUG1 and TIF1. EMBO J 15:110–124[Abstract]
  21. Halachmi S, Marden E, Martin G, MacKay H, Abbondanza C, Brown M 1994 Estrogen receptor-associated proteins: possible mediators of hormone-induced transcription. Science 264:1455–1458[Medline]
  22. Cavaillès V, Dauvois S, Danielian PS, Parker MG 1994 Interaction of proteins with transcriptionally active estrogen receptors. Proc Natl Acad Sci USA 91:10009–10013[Abstract/Free Full Text]
  23. Cavaillès V, Dauvois S, L’Horset F, Lopez G, Hoare S, Kushner PJ, Parker MG 1995 Nuclear factor RIP140 modulates transcriptional activation by the estrogen receptor. EMBO J 14:3741–3751[Abstract]
  24. Le Douarin B, Zechel C, Garnier JM, Lutz Y, Tora L, Pierrat B, Heery D, Gronemeyer H, Chambon P, Losson R 1995 The N-terminal part of TIF1, a putative mediator of the ligand-dependent activation function (AF-2) of nuclear receptors, is fused to B-raf in the oncogenic protein T18. EMBO J 14:2020–2033[Abstract]
  25. 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 93:4948–4952[Abstract/Free Full Text]
  26. Onate SA, Tsai SY, Tsai MJ, O’Malley BW 1995 Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 270:1354–1357[Abstract]
  27. Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Lin SC, Heyman RA, Rose DW, Glass CK, Rosenfeld MG 1996 A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell 85:403–414[Medline]
  28. 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 15:3667–3675[Abstract]
  29. Conaway RC, Conawaay JW 1993 General initiation factors for RNA polymerase II. Annu Rev Biochem 62:161–190[CrossRef][Medline]
  30. Kakidani H, Patshne M 1988 Gal4 activates gene expression in mammalian cells. Cell 52:161–167[Medline]
  31. Schena M, Yamamoto KR 1988 Mammalian glucocorticoid receptor derivatives enhance transcription in yeast. Science 24:965–967
  32. Heery DM, Zacharewski T, Pierrat B, Gronemeyer H, Chambon P, Losson R 1993 Efficient transactivation by retinoic acid receptors in yeast requires retinoid X receptors. Proc Natl Acad Sci USA 90:4281–4285[Abstract]
  33. McDonnell DP, Pike JW, Drutz DJ, Butt TR, O’Malley BW 1989 Reconstitution of the vitamin D-responsive osteocalcin transcription unit in Saccharomyces cerevisiae. Mol Cell Biol 9:3517–3523[Medline]
  34. Metzger D, White JH, Chambon P 1988 The human oestrogen receptor functions in yeast. Nature 334:31–36[CrossRef][Medline]
  35. Hall BL, Smit-McBride Z, Privalsky ML 1993 Reconstitution of retinoid X receptor function and combinatorial regulation of other nuclear hormone receptors in the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci USA 90:6929–6933[Abstract]
  36. Pham TA, Hwung YP, Santiso MD, McDonnell DP, O’Malley BW 1992 Ligand-dependent and -independent function of the transactivation regions of the human estrogen receptor in yeast. Mol Endocrinol 6:1043–1050[Abstract]
  37. Pierrat B, Heery DM, Lemoine Y, Losson R 1992 Functional analysis of the human estrogen receptor using a phenotypic transactivation assay in yeast. Gene 119:237–245[CrossRef][Medline]
  38. Metzger D, Losson R, Bornert JM, Lemoine Y, Chambon P 1992 Promoter specificity of the two transcriptional activation functions of the human oestrogen receptor in yeast. Nucleic Acids Res 20:2813–2817[Abstract]
  39. Allegretto EA, McClurg MR, Lazarchik SB, Clemm DL, Kerner SA, Elgort MG, Boehm MF, White SK, Pike JW, Heyman RA 1993 Transactivation properties of retinoic acid and retinoid X receptors in mammalian cells and yeast. Correlation with hormone binding and effects of metabolism. J Biol Chem 268:26625–26633[Abstract/Free Full Text]
  40. Heery DM, Pierrat B, Gronemeyer H, Chambon P, Losson R 1994 Homo- and heterodimers of the retinoid X receptor (RXR) activate transcription in yeast. Nucleic Acids Res 22:726–731[Abstract]
  41. Jin CH, Pike JW 1996 Human vitamin D receptor-dependent transactivation in Saccharomyces cerevisiae requires retinoid X receptor. Mol Endocrinol 10:196–205[Abstract]
  42. Berry M, Metzger D, Chambon P 1990 Role of the two activating domains of the estrogen receptor in the cell-type and promoter-context dependent agonistic activity of the anti-oestrogen 4-hydroxytamoxifen. EMBO J 9:2811–2818[Abstract]
  43. Apfel C, Bauer F, Crettaz M, Forni L, Kamber M, Kaufmann F, LeMotte P, Pirson W, Klaus M 1992 A retinoic acid receptor {alpha} antagonist selectively counteracts retinoic acid effects. Proc Natl Acad Sci USA 89:7129–7133[Abstract]
  44. Kohno H, Gandini O, Curtis SW, Korach KS 1994 Anti-estrogen activity in the yeast transcription system: estrogen receptor mediated agonist response. Steroids 59:572–578[CrossRef][Medline]
  45. Demirpence E, Balaguer P, Trousse F, Nicolas JC, Pons M, Gagne D 1994 Antiestrogenic effects of all-trans-retinoic acid and 1,25-dihydroxyvitamin D3 in breast cancer cells occur at the estrogen response element level but through different molecular mechanisms. Cancer Res 54:1458–1464[Abstract]
  46. Fontana JA, Burrows Mezu A, Cooper BN, Miranda D 1990 Retinoid modulation of estradiol-stimulated growth and of protein synthesis and secretion in human breast carcinoma cells. Cancer Res 50:1997–2002[Abstract]
  47. Imhof MA, McDonnell DP 1996 Yeast RSP5 and its human homolog hRPF1 potentiate hormone-dependent activation of transcription by human progesterone and glucocorticoid receptors. Mol Cell Biol 16:2594–2605[Abstract]
  48. Lee JW, Moore DD, Heyman RA 1994 A chimeric thyroid hormone receptor constitutively bound to DNA requires retinoid X receptor for hormone-dependent transcriptional activation in yeast. Mol Endocrinol 8:1245–1252[Abstract]
  49. Schulman IG, Juguilon H, Evans RM 1996 Activation and repression by nuclear hormone receptors: hormone modulates an equilibrium between active and repressive states. Mol Cell Biol 16:3807–3813[Abstract]
  50. Hörlein AJ, Näär AM, Heinzel T, Torchia J, Gloss B, Kurokawa R, Ryan A, Kamei Y, Söderström M, Glass CK, Rosenfeld MG 1995 Ligand-independent repression by the thyroid hormone receptor mediated by a nuclear receptor co-repressor. Nature 377:397–404[CrossRef][Medline]
  51. Kurokawa R, Söderström M, Hörlein A, Halachmi S, Brown M, Rosenfeld MG, Glass CK 1995 Polarity-specific activities of retinoic acid receptors determined by a co-repressor. Nature 377:451–454[CrossRef][Medline]
  52. Chen JD, Evans RM 1995 A transcriptional co-repressor that interacts with nuclear hormone receptors. Nature 377:454–457[CrossRef][Medline]
  53. Joyeux A, Balaguer P, Gagne D, Nicolas JC 1996 In vitro and in vivo interactions between nuclear receptors at estrogen response elements. J Steroid Biochem Mol Biol 58:507–515[CrossRef][Medline]
  54. Berger SL, Pina B, Silverman N, Marcus GA, Agapite J, Regier JL, Triezenberg SJ, Guarente L 1992 Genetic isolation of ADA2: a potential transcriptional adaptator required for function of certain acidic activation domains. Cell 70:251–265[Medline]
  55. Baniahmad C, Nawaz Z, Baniahmad A, Gleeson MAG, Tsai MJ, O’Malley BW 1995 Enhancement of human estrogen receptor activity by SPT6: a potential coactivator. Mol Endocrinol 9:34–43[Abstract]
  56. Lee JW, Choi HS, Gyuris J, Brent R, Moore DD 1995 Two classes of proteins dependent of either the presence or absence of thyroid hormone for interaction with the thyroid hormone receptor. Mol Endocrinol 9:243–254[Abstract]
  57. Rubin DM, Coux O, Wefes I, Hengartner C, Young RA, Goldberg AL, Finley D 1996 Identification of the gal4 suppressor Sug1 as a subunit of the yeast 26S proteasome. Nature 379:655–657[CrossRef][Medline]
  58. Fields S, Song O 1989 A novel genetic system to detect protein-protein interactions. Nature 340:245–246[CrossRef][Medline]
  59. O’Malley BW, Schrader WT, Mani S, Smith C, Weigel NL, Conneely OM, Clark JH 1995 An alternative ligand-independent pathway for activation of steroid receptors. In: Wayne Bardin C (ed) Recent Progress in Hormone Research. Academic Press, San Diego, CA, pp 333–347
  60. Rose MD, Winston F, Hieter P 1990 Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  61. Sikorski RS, Hieter P 1989 A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:18–27
  62. Dalton S, Treisman R 1992 Characterization of SAP-1, a protein recruited by serum response factor to the c-fos serum response element. Cell 68:597–612[Medline]