Titration by Estrogen Receptor Activation Function-2 of Targets That Are Downstream from Coactivators

Gabriela N. Lopez, Paul Webb, Jeanette H. Shinsako, John D. Baxter1, Geoffrey L. Greene and Peter J. Kushner1

Metabolic Research Unit (G.N.L., P.W., J.H.S., J.D.B., P.J.K.) University of California San Francisco, California 94143
Ben May Institute (G.L.G) University of Chicago Chicago, Illinois 60637


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cross-interference (squelching) among nuclear receptors has been proposed to reflect the titration of coactivators that bind the receptors in a hormone-dependent manner. We have tested whether the coactivators are the only target titrated during squelching of one receptor by another, or whether proteins needed for coactivator function are titrated as well. That the coactivators are indeed one target of squelching is apparent. The isolated ligand-binding domain of the estrogen receptor (ER-LBD) squelches transcriptional activation by the thyroid hormone receptor (TR) only when the LBD is bound to ligands that promote coactivator interactions and only when regions of the LBD that promote coactivator interactions are undisturbed. Furthermore, the ER-LBD and the TR compete in vitro for the related p160 coactivators, SRC1a and GRIP1 (glucocorticoid receptor interacting protein 1), or the putative corepressor, RIP140. Finally TR action becomes more potent when coactivator levels are raised. Nonetheless, supplying excess SRC1a or GRIP1 does not abolish squelching by the ER. In fact, squelching becomes even more severe when coactivators are abundant. Supplying combinations of coactivators from the p160 class and the CREB-binding protein (CBP)/p300 class makes squelching most severe. Elevated RIP140 inhibits TR action, but also protects the residual TR action from squelching by the ER-LBD. We conclude that ER-LBD squelches TR both by titrating p160-CBP coactivators and additionally by cooperating with the coactivators to titrate a second factor. The second factor would be needed by the TR for coactivator-mediated transcriptional stimulation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nuclear hormone receptors including the estrogen receptor (ER) and thyroid hormone receptor (TR) are ligand-activated transcription factors that stimulate transcription from hormone response elements in the promoters of target genes (1, 2, 3, 4). Transcriptional activation by the receptors is mediated by two transcriptional activation functions (5, 6, 7). Activation function one (AF-1) is located in the amino-terminal domain of the ER and several other receptors. AF-1 varies greatly in strength from one receptor to another, and in different cellular conditions, but is independent of hormone (8, 9). AF-2 in the C-terminal ligand-binding domain (LBD) is consistently strong in different receptors, but in every case requires the presence of hormone for full activity. For example, AF-2 of the ER is active when the LBD is bound to the agonists estradiol or diethylstilbestrol, but not when the LBD is bound to tamoxifen, raloxifene, ICI 164,384, or other antagonists (6, 7, 10). Similarly the TR AF-2 is active only when bound to T3, TRIAC, or other agonists (3). In addition to the requirement for hormone, AF-2 requires the integrity of conserved residues near the C terminus of the LBD (11). Mutations in this conserved region, which is rich in hydrophobic and charged residues, decrease or abolish AF-2 in estrogen, thyroid, and other receptors (11, 12, 13).

The discovery of the phenomenon of cross-interference or "squelching" between different receptors provided a clue as to how AF-2 function is mediated by hormone. The ER-estrogen complex interferes with the action of the progesterone receptor bound to a progesterone response element even though the ER does not bind to the response element (14). This interference was attributed to the titration by the hormone-bound ER of a common and limiting target factor(s) that is required by the promoter-bound PR. Further analysis demonstrated that the ER is capable of squelching in the absence of an AF-1 domain, that squelching by receptor AF-2 domains requires an agonist hormone and the integrity of the activation helix (12), and that squelching is a widespread phenomenon observed between the ER and almost all members of the steroid/thyroid receptor family (12, 15).

These observations suggested that hormone-bound ER LBD was binding to a limiting factor that was required by all nuclear receptors to mediate AF-2 function. Recently proteins have been identified that bind to the ER and other nuclear receptor LBDs only in the presence of hormone and only when the conserved helix of AF-2 is intact (16, 17). These proteins are thus candidates for mediating AF-2 function (coactivators). The strongest contenders for this coactivator function are three related proteins: SRC1a (N-Co1) (18, 19, 20), GRIP1 (TIF2, N-CoA2) (21, 22, 23), and p/CIP (AIB1, TRAM-1, ACTR, RAC3) (24, 25, 26, 27, 28), which we here refer to as "p160s." p160s bind to intact receptor LBDs, and mutations in the LBD that interfere with p160 binding interfere with AF-2 in exact proportion (29, 30). Overexpression of each of these p160s potentiates AF-2 of nuclear receptors, and antibodies to these proteins when microinjected block AF-2 function. Thus, p160s act as coactivators for AF-2 function. Other proteins, such as RIP140, also bind receptors in an AF-2-dependent fashion, but appear to be inhibitors of AF-2 function (31, 32, 33). They may thus compete with p160s in modulation of receptor action (33).

The p160s are bound in vivo with members of the CREB-binding protein (CBP)/p300 family (19, 34, 35, 36), originally identified as coactivators for CREB, and mediators of E1A effects, but which also appear to play an important role in nuclear receptor AF-2 function. Overexpression of these CBP family proteins synergizes with p160s to potentiate AF-2 (19, 24, 34, 37), and mutations in a particular p160, ACTR, that block its ability to bind CBP block its ability to potentiate AF-2 function (27). In addition, microinjected antibodies to CBP or p300 abolish AF-2 activity (19, 24). Thus, it appears that AF-2 function of nuclear receptors is mediated by a complex of p160 and CBP coactivators (for review see Ref. 38).

How these proteins function as coactivators is currently speculative, but attention centers on their interactions with the basal transcriptional machinery and on their associated histone acetyltransferase (HAT) activity. Both CBP and SRC1a bind to TATA-binding protein (TBP) (20, 39), and the TBP-binding site of CBP serves as an independent activation domain when brought to DNA (40). Both p160s and CBP/p300 appear to interact with TFIIB in vitro (20, 41), although no function for these interactions has been demonstrated as yet. Both CBPs and p160s have acetyltransferase activity in vitro that is active on free histone substrates (27, 42, 43, 44). Furthermore, both p160s and CBP bind to a potent HAT, p/CAF, which, like p160s but unlike CBP, is able to acetylate core nuclesomes in vitro (45). Recent studies with mutants of CBP and p/CAF that are deficient in HAT activity suggest that the integrity of the HAT enzymatic activity of p/CAF is needed for transcriptional activation by the retinoic acid receptor (46). Thus, the associated HAT of these coactivators may enable them to remodel chromatin and allow access to the transcriptional machinery. Indeed, ER and CBP have dramatic effects on transcription in vitro only on chromatin templates and not on naked DNA (47). In addition to targeting histones, the acetyltransferase activity of the coactivators may target the acetylation of basal transcription factors (48).

The proteins of the coactivator complex, p160s, CBP/p300, and p/CAF, are presumed to be the limiting factors for AF-2 function that are titrated when one receptor squelches another. Indeed, studies with SRC1a indicate that p160s are limiting for AF-2 function (18). In addition, p160s overexpression restores the ability of PR to activate transcription in the presence of interfering ER (18). Similarly overexpression of TIF-2 restores ER action when the ER is overexpressed and thus squelching itself (22). Despite these results, it is unclear whether squelching itself is reduced by overexpression of p160 coactivators. It is possible that the p160s are limiting, that elevating them potentiates AF-2 even under squelching conditions, and yet elevating p160s does not relieve squelching because other factors then become limiting.

We have reexamined squelching of the TR by the ER AF-2 to determine whether elevated coactivators relieve squelching. We find they do not, and that squelching becomes more severe when coactivators are elevated. We suggest that the ER-LBD titrates the p160s, but the ER-coactivator complex subsequently titrates a second factor needed by the TR to efficiently activate transcription. We therefore surmise that this second factor may be one of the functional targets for the coactivators.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The ER AF-2 Squelches the Ability of TR to Stimulate Reporter Gene Expression
To study squelching that was mediated solely by AF-2 titration of target proteins and not by competition for DNA sites, we investigated whether the ER-LBD in isolation would be capable of squelching transcriptional activation by the human TRß. We monitored TRß activity on a chloramphenicol acetyltransferase (CAT) reporter gene with a thyroid hormone response element (TREpal) inserted into the core promoter of mouse mammary tumor virus (MMTV). As shown in Fig. 1AGo, thyroid hormone (T3) activated the reporter gene about 10-fold and estradiol (E2) inhibited that activation in a dose-dependent manner. Estrogen did not inhibit TR action in the absence of the ER LBD, indicating a role for the LBD in this phenomenon (Fig. 1BGo). Furthermore, an ER LBD with mutations (ML543/544A Fig. 1BGo) in the activation helix did not inhibit TR action. Even at very high concentrations of expression vector, the mutant ER LBD gave only 10% inhibition (data not shown). The ER-LBD inhibited TR only when bound to the agonist ligands estradiol and diethylstilbestrol (DES) and not when bound to the antagonists tamoxifen (Tam) and ICI 164,384 (ICI) (Fig. 1CGo). Thus, the AF-2 function of the LBD mediates the inhibition. The ER AF-2 is relatively weak at squelching TR action, and squelching rarely exceeds 50%. In contrast, the combined ER AF-1 and AF-2 can squelch TR action almost 90% (ER{Delta}DBD Fig. 1DGo). As expected, the combined ER AF-1 and AF-2 can partly squelch TR action when liganded to tamoxifen, which blocks AF-2, but allows AF-1. These observations indicate the ER AF-2 either by itself or in concert with AF-1 squelches transcriptional activation by TR.



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Figure 1. The Isolated ER-LBD Squelches the Ability of TR to Stimulate Transcription

Cells were transfected with 5 µg MMTV-TREpal-CAT reporter plasmid and 100 ng of hTRß expression vector and treated with thyroid hormone (T3) as indicated. A, Dose-dependent inhibition by estradiol. Cells additionally transfected with 2 µg of an expression vector for ER-LBD were exposed to the indicated concentration of estradiol in the presence of saturating T3. CAT activity driven by the T3-responsive promoter was measured as an indication of gene expression. B, Inhibition requires the integrity of the AF-2 activation helix. Cells transfected by vectors for no protein, the ER-LBD, or an ER-LBD M543A/L544A were tested for their ability to elicit a thyroid hormone response from the reporter gene. Hormones as indicated. C, Inhibition requires an estrogen agonist. Cells transfected with a control vector or a vector for the ER-LBD were exposed to saturating T3 and ER agonists (estradiol and DES), or antagonists (tamoxifen, ICI 164,384) as indicated. D, Inhibition of TR action by an ER derivative (ER{Delta}DBD) containing both AF-1 and AF-2.

 
The ER-LBD Competes with the TR for Binding to Coactivators in Vitro
The above studies demonstrate squelching of the TR by the ER LBD in the presence of agonists. According to the standard model of AF-2 action, such squelching is presumed to reflect the ability of the ER LBD to bind coactivators that are present in limiting amounts and thereby occlude access to the coactivators by the TR. We therefore tested whether the presence of the ER-LBD can block access of the TR to coactivators in vitro. We used a standard "pull down" assay in which a glutathione-S-transferase (GST) fusion to human TRß was fixed to glutathione-Sepharose beads, reacted with labeled target protein and potential competitor, washed, and examined by SDS gels and autoradiography. Consistent with previous studies, GST-TR efficiently bound the coactivators SRC1a and GRIP1, and the putative corepressor RIP140 and did so only in the presence of T3 (compare lane 5 with lane 4, Fig. 2Go). When purified bacterially expressed ER-LBD complexed to estradiol was added, the TR-T3 complex no longer bound GRIP1, SRC1a, or RIP140 (Fig. 2Go, lane 6). The ability of the ER-LBD to compete away binding of the TR to the three target proteins was dependent on the presence of estradiol. ER-LBD complexed to the antagonists tamoxifen or ICI 164,384 was unable to compete with the TR-T3 complex for the target molecules (Fig. 2Go, lanes 7 and 8). The antagonist complexed LBD was, however, able to block homodimerization of ER LBD in a similar pull down assay (data not shown). We conclude that the ER-LBD complexed to agonist, but not to antagonists, efficiently competes with the TR for binding to the coactivators GRIP1 and SRC1a and to the putative corepressor RIP140.



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Figure 2. The ER-LBD-Agonist Complex Competes with the TR for Binding to Coactivators

The figure shows an autoradiograph of the results of a GST-pull down assay in which in vitro translated and labeled SRC1a, GRIP1, or RIP140 were reacted with GST or GST-TR bound to agarose beads in the presence or absence of thyroid hormone as indicated. Binding that occurs in the presence of thyroid hormone (lane 5) was then competed with unlabeled ER-LBD bound to estradiol (lane 6, note competition), ER-LBD bound to tamoxifen (lane 7), or ER-LBD bound to ICI 164,384.

 
Coactivator Concentration Is Limiting for TR Stimulation of Transcription, Even at Low Levels of Hormone-Bound TR
The hypothesis that squelching of TR action by the ER results from titration of coactivators of the p160 class presumes that these coactivators are in some way limiting for TR action. To confirm this, we examined how elevated levels of the coactivators GRIP1 and SRC1a affected the ability of the TR to activate transcription from a TRE-regulated reporter gene. In HeLa cells in the absence of transfected coactivator, the MMTV-TREpal reporter gene is increasingly induced with increasing concentrations of T3 (Fig. 3Go, squares). The T3 response has a half-maximum in the nanomolar range and levels out at around 10–100 nM, consistent with previous studies. When SRC1a levels are elevated with increasing amounts of an SRC1a expression vector (Fig. 3AGo, diamonds, circles, and triangles) there is a dramatic and nearly proportional increase in the ability of T3 to activate transcription. Similar responses are seen with an expression vector for GRIP1 (Fig. 3BGo). Elevated SRC1a or GRIP1 levels also dramatically increased TR-mediated transcription at low levels of T3. This is especially notable with elevated GRIP1 levels, which allow potent activation of transcription in the presence of 0.1 nM T3. Thus, elevating p160 levels potentiates TR action even under conditions where there are low levels of hormone-bound TR. Reporter gene activation by the ER shows a similar dependence on coactivator levels (data not shown). These observations confirm that the levels of SRC1a, GRIP1, and other functionally related coactivators are a limiting determinant for TR and ER action. This is consistent with the notion that receptor contact with coactivator is a critical determinant of receptor action.



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Figure 3. Coactivator Concentration Determines TR Transcriptional Activation at Both High and Low Hormone Levels

HeLa cells were transfected with the MMTV-TRE reporter plasmid and 0.5 µg of TR expression vector and exposed to the indicated concentration of thyroid hormone. A, An expression vector for SRC1a was additionally transfected in the indicated amount. B, An expression vector for GRIP1 was transfected in the indicated amount. C, An expression vector for RIP140 was transfected in the indicated amount. Note expanded scale in panel C.

 
RIP140, which was originally a candidate for a coactivator (31, 49), has more recently been proposed as an inhibitor of AF-2 action (32, 33). We therefore reexamined the effect of exogenous RIP140 expression on TR action. Unlike elevated SRC1a and GRIP1, elevated RIP140 inhibited the ability of the TR to stimulate transcription from the thyroid hormone response element (TRE) reporter gene. The inhibition is dose dependent and at higher levels of RIP140 approaches 90% (Fig. 3CGo). Nonetheless, even with the higher levels of RIP140 (up to 3 µg, data not shown) the inhibition did not increase above 90% (see below). Thus, it is clear that exogenous RIP140 is inhibitory. The plateau in inhibition with increasing RIP140 may suggest that RIP140 is nonetheless able to act as a weak coactivator, one that can compete with more efficient endogenous coactivators when present in elevated concentration (see also Refs. 50, 51).

Elevating SRC1a or GRIP1 Does Not Relieve Squelching Mediated by the ER AF-2
The above studies verify that p160s are limiting for the ability of the TR to stimulate transcription, that ER AF-2 function can squelch the ability of TR to stimulate transcription, and that ER AF-2 can compete with TR for coactivators. These studies thus raise the question whether overexpression of SRC1a or GRIP1 can overcome squelching by the ER AF-2. To probe this question, we studied whether elevating the levels of coactivators affected the ability of ER LBD to interfere with the transcriptional activation by the TR.

To examine these interactions, we first determined levels of the activator, TR, and the coactivators, SRC-1 and GRIP1, that gave robust responses without auto-interference due to overly high expression levels. As shown in the inset (Fig. 1Go, top left) 100 ng of TR expression vector were in the range of the linear response and were far from autoinhibitory. Similarly (Fig. 1Go, top right) 1 µg of expression vector for the coactivator SRC-1 appeared optimal, and was robust but not saturating for GRIP1 (Fig. 1Go, top right). In our initial explorations we employed 0.5 µg of expression vector for ER-LBD, since that amount gave readily measured inhibition, albeit not as consistently as at higher levels. We also varied ER-LBD levels as described below.

In the absence of exogenous p160s, the ER AF-2 reduced activation by TR 49% [Fig. 4AGo, compare activation with T3 and with T3 + E2; the data are also calculated as percent inhibition noted above the downward arrow. Inhibition is calculated as 100% minus (activation with T3 plus E2/activation with T3)]. When SRC1a levels were increased by transfection, activation by TR increased both in the absence and presence of squelching ER AF-2. Increasing SRC1a levels, however, did not decrease inhibition by ER-LBD and estrogen. In fact, fractional inhibition by the ER AF-2 increased from 49% to 66%. Thus, squelching by the ER-LBD became, if anything, more severe in the presence of elevated SRC1a. When increasing amounts of GRIP1 were supplied, a similar pattern was seen. Increasing GRIP1 potentiated TR action either in the absence or presence of squelching ER. Fractional inhibition in the face of squelching ER plus estrogen again increased slightly (from 53% to 72%) with increasing amounts of GRIP1.



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Figure 4. Elevated Levels of SRC1a or GRIP1 Do Not Relieve Squelching Mediated by the ER-LBD Measured as Fractional Inhibition

The activity of the MMTV-TREpal reporter gene in HeLa cells transfected with 0.1 µg of an expression vector for hTRß and exposed to the indicated hormone combinations is shown. Upper inset shows that the chosen level of TR expression vector is in the linear range before saturation or autoinhibition by TR is manifested. The cells were additionally transfected, as indicated, with 0.5 µg of an expression vector for the ER-LBD. A, Ability of elevated SRC1a to relieve squelching. Increasing amounts of an expression vector for SRC1a, as indicated, were transfected along with the expression vector for ER-LBD. CAT activity of the reporter gene is shown as a measure of gene expression. Inhibition by estrogen is shown by the downward arrows and is calculated as: 1-(CAT activity in the presence of estradiol and T3)/(CAT activity in the presence of T3) and is converted to 100% as complete inhibition. B, A similar experiment with an expression vector for GRIP1. In both panels the dashed line indicates expression level in the presence of T3 and absence of estrogen without added coactivator.

 
The observation that elevated p160s did not relieve squelching is counter to expectations. It is possible that the failure of elevated coactivators to completely relieve squelching by ER-LBD reflects an overabundance of the ER-LBD far beyond that needed to induce squelching. To examine this issue, we repeated the study and varied the amount of ER-LBD from none to levels that gave minimal but measurable inhibition. We asked whether GRIP1 supplied at a single, optimal dose could reverse this minimal squelching. As shown in Fig. 5Go, the presence of elevated GRIP1 greatly potentiated TR action. Estrogen inhibited TR action as ER-LBD levels increased. Yet again the effect of estrogen was more pronounced in the presence of elevated GRIP1 than in its absence. This is especially striking at lower levels of ER-LBD (30 ng, 100 ng, Fig. 5Go) at which inhibition is detectable when GRIP1 is elevated, but is undetectable when GRIP1 is not elevated. These studies indicate that supplying p160 coactivators SRC1a or GRIP1 potentiates TR action in HeLa cells, but does not relieve squelching mediated by estrogen and the ER-LBD.



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Figure 5. Elevated GRIP1 Supplied at Optimal Levels Does Not Relieve Squelching Mediated by Minimally Effective Amounts of ER-LBD

The activity of the MMTV-TREpal reporter gene in HeLa cells transfected with 0.1 µg of an expression vector for hTRß and exposed to the indicated hormone combinations is shown. The cells were additionally transfected with 1 µg of GRIP1 expression vector and the indicated amount of expression vector for ER-LBD. Upper inset shows that 0.5 to 1 µg of GRIP1 expression vector is optimal for stimulation of TR-mediated response in the absence of ER. Downward arrows indicate percent inhibition defined as in Fig. 4Go. Inhibition by ER-LBD was somewhat reduced in this experiment.

 
Elevated RIP140 Inhibits TR Action, but Blocks Squelching by the ER AF-2. GRIP1 Reverses These Effects
As noted above, while elevated p160s potentiate TR action, elevated RIP140 inhibits TR action. We therefore tested the effects of increased RIP140 on squelching by ER AF-2. In this experiment ER AF-2 inhibited TR by 50% in the absence of exogenous RIP140. As previously seen, elevating RIP140 severely decreased TR action (Fig. 6AGo). Notably, ER AF-2 did not decrease the residual TR action (compare T3 with T3+E2 with RIP140, Fig. 6AGo, left panel, and Fig. 6BGo). As observed in Fig. 6BGo, increasing ER-LBD while holding RIP140 levels constant leads to estrogen-dependent stimulation of TR action, presumably as ER titrates some of the RIP140 away from TR (Fig. 6BGo). We conclude that in the presence of elevated RIP140, the ER-LBD bound to estrogen no longer inhibits T3-activated MMTV-TRE CAT.



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Figure 6. Elevated RIP140 Reduces Activation by TR and Relieves Squelching by the ER-LBD, and These Actions Are Reversed by Elevated GRIP1

Expression of MMTV-TRE CAT reporter gene in Hela cells cotransfected with 0.1 µg TR and exposed to hormone as indicated. A, Effect of RIP140 on squelching of TR by ER-LBD. Cells were transfected with 0.5 µg of ER-LBD expression vector without or with 1 µg of RIP140 expression vector, or 1 µg of RIP140 and 1 µg of GRIP1 expression vector as indicated. Downward arrows indicate percent inhibition calculated as in Fig. 4Go. B, ER-LBD relieves inhibition of TR action by elevated RIP140. RIP140 expression vector (3 µg) and increasing amounts of ER-LBD expression vector were transfected into cells.

 
The above studies indicate that elevated RIP140 prevents squelching by ER AF-2, whereas elevated p160 makes squelching more severe. These observations suggest that for squelching to occur, the ER must interact with p160 and functionally related coactivators and not with RIP140. To test this, we examined whether elevated GRIP1 could restore squelching when RIP140 levels were elevated. As shown in Fig. 6Go, right panel, elevating GRIP1 both allowed TR to activate transcription in the presence of RIP140 and restored the ability of the ER-LBD to squelch TR action (54% inhibition). These observations suggest that for squelching to occur, the ER-LBD may need to contact GRIP1 or similar coactivators, rather than RIP140 and similar proteins. Below we suggest as an explanation for these phenomena that the ER AF-2 domain complexed with p160s, but not with RIP140, titrates a factor needed for promoter-bound TR to stimulate transcription (see Discussion).

Other Components of the Coactivator Complex and Basal Transcription Factors That Are Known to Contact Them Do Not Relieve Squelching by the ER-LBD
The above studies with expression vectors for p160s suggest that the ER-LBD is able to mediate squelching of TR action when p160s are highly abundant. Thus the question is raised whether some other component of the coactivator complex, such as CBP/p300 or p/CAF, is a limiting factor that is titrated by the ER-LBD. The titration of this second component would mediate the squelching that persists with elevated p160s. We therefore conducted a survey to determine whether among the known targets of p160s there were any that relieved squelching by the ER.

As noted previously, the known targets of p160s include CBP/p300 and p/CAF, which form a coactivator complex with p160s, and components of the basal transcriptional machinery, TBP and TFIIB. As shown in Fig. 7Go, upper panel, CBP cooperated with SRC1a (and with GRIP1, data not shown) to potentiate TR action. Squelching persisted with elevated CBP, and, if anything, became more pronounced, going from 55% to 71%. Similar observations were made with p300 and with p/CAF (data not shown). Thus CBP/p300 do not relieve ER-LBD-mediated inhibition with elevated p160.



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Figure 7. Elevated CBP, TFIIB, or TBP Fail to Prevent Squelching Mediated by ER-LBD in Cooperation with SRC-1

The activity of the MMTV-TREpal reporter gene in HeLa cells transfected with 0.1 µg of an expression vector for hTRß, 0.5 µg of ER-LBD, and, where indicated, 1 µg of expression vector for SRC-1, CBP, TFIIB, or TBP. Hormones were added as indicated, and downward arrows indicate percent inhibition as defined in Fig. 4Go.

 
TFIIB and TBP are both critical components of the basal transcriptional apparatus that have been implicated as targets for transcriptional regulation by transcription factors including the nuclear receptors. As shown in Fig. 7Go, lower panel, overexpression of TBP and TFIIB had little effect on the ability of the ER-LBD to inhibit transcription. Control experiments demonstrated that these factors nevertheless potentiated ER-LBD action in HeLa cells (data not shown and Ref. 52). Thus, these observations indicate that neither the identified components of the coactivator complex, nor the two basal transcription factors that have currently been implicated as candidate targets for coactivator action, are able when overexpressed to relieve inhibition of TR action by ER-LBD.

To examine whether the persistence of squelching in the presence of elevated p160s was limited to the ER, we also tested squelching by the glucocorticoid (GR) and progesterone (PR) receptors, along with squelching mediated by the ER AF-1 and AF-2 working together. As shown in Fig. 8Go, PR efficiently squelched TR action (59%) and did so even more efficiently when GRIP1 was elevated (95%). An ER containing both AF-1 and AF-2 (ER{Delta}DBD, Fig. 8Go) squelched as well (62%) as PR and again squelched more efficiently when GRIP1 was elevated (84%). GR did not squelch TR with quite the same efficiency (16%), but again squelching became more pronounced with elevated GRIP1 (56%). Similar observations were made with elevated SRC-1 (data not shown). We conclude that squelching by PR, ER AF-1/AF-2, and GR, like squelching by ER AF-2, persists and becomes more pronounced when p160 levels are raised.



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Figure 8. Elevating GRIP1 Increases Squelching of TR Mediated by PR, GR, and ER AF-1/AF-2

The activity of the MMTV-TREpal reporter gene in HeLa cells transfected with 0.1 µg of an expression vector for hTRß and, where indicated, 0.5 µg of expression vector for PR, GR, or ER lacking the DNA-binding domain (ER{Delta}DBD) in the absence or presence of 1 µg of GRIP1 expression vector as indicated. Either T3 or T3 plus the appropriate hormone (RU5020 for PR, dexamethasone for GR, estradiol for ER) were added as indicated. Downward arrows indicate percent inhibition as defined in Fig. 4Go.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have examined whether squelching by the hormone-dependent AF-2 function of the ER of the transcription activation abilities of TR is due solely to the titration of the p160 coactivators. That titration of coactivators is part of squelching appears evident from these and previous studies. The p160 coactivators such as SRC1a and GRIP1 are clearly limiting for TR action, even with low levels of hormone-occupied TR. Further, the AF-2 function of ER working in isolation can squelch the TR. Thus squelching of TR by the ER LBD requires agonist and is blocked by mutations that affect AF-2 function. Squelching may occur, in part, when ER-LBD binding to the p160s thereby prevents binding of the TR to the same coactivators. The argument for such surface occlusion as a mechanism of squelching is strengthened by the observation that in vitro the complex of the ER-LBD and estradiol can block T3-dependent interactions of the TR with the coactivators SRC1a and GRIP1 and with the corepressor RIP140. The complex of the ER-LBD and an antagonist is unable to squelch and also unable to compete with TR for coactivator binding. These observations confirm and extend previous studies such as those of Onate and co-workers (18) who showed that p160s were clearly limiting for PR action in HeLa cells. Competition for limiting coactivators related to SRC1a and GRIP1 must in part underlie squelching (for review see Ref. 53).

However, the coactivators do not appear to be the sole target of squelching. When the coactivators SRC1a and GRIP1 are supplied in abundance, there is indeed a dramatic increase in the ability of TR to stimulate transcription in the presence of competing ER-LBD-E2 complex. However, there is also a corresponding and more dramatic increase in the ability of TR to stimulate transcription in the absence of estradiol or in the presence of an ER-LBD-tamoxifen complex. The net result is that squelching by the ER-LBD and estradiol measured as the percentage decrease of TR-mediated activation is not relieved by increasing the amounts of the coactivators SRC1a or GRIP1. Instead, squelching becomes more severe. This potentiation of squelching by elevated coactivators also occurs with very low amounts of ER-LBD. Indeed squelching can be seen with elevated p160s even when there is insufficient ER-LBD to confer squelching with endogenous p160s. Thus, despite the potentiation of TR action by coactivators, there is a component of squelching that appears to require the presence of the coactivators.

This notion, that squelching requires receptor-p160 contact, is underlined by our observations of RIP140. This putative repressor of transcription, binds to nuclear receptors through an LxxLL motif that is similar to the nuclear receptor boxes through which GRIP1 and SRC1a recognize ER and TR (54, 55, 56). Thus, it can be expected that RIP140 and p160s compete with each other for binding to ER and TR. Indeed, such competition for TR binding has recently been demonstrated in vitro betrween TIF2, the human analog of GRIP1, and RIP140 (57). RIP140 decreases the ability of TR to stimulate transcription, and this can be overcome with elevated GRIP1. Thus, inhibition by RIP140 is most likely due to the replacement of p160 coactivators on the TR with RIP140. In this situation, with RIP140 bound to TR, TR action is weak. Nonetheless, the weak TR action when bound to RIP140 is resistant to further inhibition by ER-LBD or is even stimulated by ER-LBD. We surmise that the failure of ER-LBD to squelch the residual TR action reflects that ER is also bound to RIP140 and not to GRIP1. Indeed, elevating GRIP1 allows ER-LBD to once more squelch in the presence of RIP140. These observations with RIP140 again argue that contact between ER-LBD and p160 coactivators may be needed for squelching.

It thus appears there is a component of squelching of TR by ER LBD that is dependent on the presence of p160 coactivators and their contact with ER. We propose that the ER-LBD and the coactivators cooperate in some fashion to squelch a hypothetical second factor. This second factor would play a role in transcriptional activation by TR and its recruited coactivators. This model is illustrated in Fig. 9Go. It should be noted that it is essential to the model that the postulated second factor, depicted as factor X in the cartoon, only associates with the p160 when the p160 is itself complexed with a nuclear receptor (here ER-LBD). This might occur if ER and the coactivator both provided surfaces for factor X to bind or if contact with ER induced a change in coactivator that allowed factor X to bind.



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Figure 9. A Cartoon Model of How Estrogen-Bound ER LBD Complexed with a p160 Coactivator Titrates a Factor Needed for T3-Enhanced Transcription

As shown on the left, activation of transcription by the TR at a TRE is presumed to require a recruited p160 coactivator (here GRIP1) and factor X. Factor X binds to the TR-associated GRIP1 or to another member of the TR- associated GRIP1 complex. An important stipulation is that factor X does not bind to free GRIP1, only to GRIP1 that is in contact with a nuclear receptor. Together, GRIP1 and factor X mediate TR action. As shown on the right, the ER- LBD bound to estrogen (E2) complexes with GRIP1 and promotes binding of factor X to the ER-GRIP1 complex. This titrates factor X away from TR bound at the promoter (dashed arrow), thereby diminishing TR-mediated transcription. In the absence of estrogen the ER LBD is not complexed with GRIP1, factor X is not bound by free GRIP1, and TR-mediated transcription is undisturbed.

 
We further postulate that the ER-LBD complex with RIP140 would not bind factor X. Elevation of RIP140 would thus be predicted to have complex effects. For one, TR at the promoter would recruit mostly RIP140 rather than the p160 coactivator complex. This would result in dramatically decreased transcription by TR. The addition of estrogen would lead to the formation of a complex between ER-LBD and RIP140. Since these complexes would not titrate factor X, transcription by TR, mediated either by RIP140 or by the minority p160 component, would be undiminished. Indeed, since estrogen-ER-LBD might titrate some of the RIP140, an increase in transcription might occur. Slight relief of RIP140 repression by estrogen is, indeed, observed. When GRIP1 levels are coelevated with RIP140, activation by TR is greatly increased, but again squelching by estrogen-ER-LBD is restored. Thus, contact between ER and p160s seems essential for squelching.

Although the postulate of a factor X that interacts with the p160-ER complex may seem unduly complex, it appears difficult to reconcile the present observations with simpler models. For example, it may be tempting to postulate that ER preferentially binds GRIP1 over RIP140 and that the remaining RIP140 is left to interact with TR, leading to the observed repression by estrogen. This alternative model explains the basic observation of squelching. The model also explains the observation that when RIP140 is elevated, ER no longer squelches. This would be due to the complete occupation of TR by RIP140 with no further decrease of GRIP1-TR complexes by titration of GRIP1. The model, however, is difficult to reconcile with the central observation of these studies. Elevating GRIP1 does not relieve squelching; it makes squelching more severe. On the alternative model, elevating GRIP1 should eventually eliminate squelching.

Our observations agree in general, but differ slightly in detail, with those of Onate et al. (18) who found that squelching of PR by ER became somewhat less severe with overexpressed SRC-1. It is possible that these differences can be traced to the different promoters and nuclear receptors used in the two studies. It may also reflect differences in the relative abundance of factor X and p160s in the two experimental systems. Despite the difference in detail, it is noteworthy that in the studies of Onate et al. (18) substantial (85%) squelching by ER persisted in the presence of overexpressed SRC-1. The identity of the coactivator-associated factor X is unknown. We tested the known target proteins of the p160s, which include the associated coactivators CBP/p300 and p/CAF (data not shown), and the two basal transcription factors TFIIB and TBP. None of these factors relieved squelching, and we are forced to conclude that the hypothetical downstream factor is not one of these.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Construction of Recombinant Vectors
An ER-LBD expression vector (pSG5 ER LBD) was constructed by inserting a HindIII/BamHI fragment from HE19G (58) spanning Glycine 400 into the pSG5- based expression vector for the Valine 400 LBD, HE14 (59). A vector for an AF-2 mutant version of the ER LBD (ML543/544AA) was made by site-directed mutagenesis of the ER-LBD expression vector. An expression vector for Glycine 400 ER{Delta}DBD was constructed by inserting a HindIII/BamHI fragment from HE19G into HE11 (59). An expression vector for RIP140 (pEFRIP140) was obtained from Malcolm Parker and has been described (31). Expression vectors for SRC1a (44), CBP (19), p300 (36), and TBP (52) have been described. TFIIB expression vector was a kind gift of Yoel Sadovsky (Washington University, St. Louis, MO). An expression vector for GRIP1 was constructed by transferring the GRIP1 DNA insert from the yeast expression vector pGRIP1.FL (21) into pSG5 (56, 60). The reporter MMTV-TRE-CAT has been described (61) and contains the MMTV long terminal repeat from the -1225 to +135 where a TREpal oligonucleotide has been inserted in an HindIII site at position -190/-88 (61). The human TRß expression has been described (62). The expression of TRß is under control of the human metallothionein promoter in the pLEN vector (62). The GST-TR has been described (62). The GST ER-LBD was constructed by digesting the EcoRI fragment coding the ER-LBD from the pSG5 ER-LBD vector and inserted within a vector from the pGEX series (Pharmacia Biotech, Piscataway, NJ). Expression vectors for PR and GR have been described (63).

Cell Culture Transfection and CAT Assays
Cells were grown as previously described in 10% calf serum (chosen for low estrogen levels) and F12/MD and transfected by electroporation as described previously (29). Transfection typically contained: 5 µg MMTV-TRE-CAT reporter vector, 0.5–1.0 µg of TR expression vector, 2 µg of squelcher vector (either ER-LBD or ER{Delta}DBD), and 1 µg of an actin-hCG expression vector used as a control for transfection. After electroporation, the cells were distributed to six-well culture plates and treated with thyroid hormone (T3, 10-7 M), 17ß-estradiol (10-8 M), or vehicle and after 2 days assayed for CAT activity. Transfection efficiency was monitored by measuring medium hCG from the actin-hCG vector.

In Vitro Protein-Protein Interaction Assays
GST pull down and competition experiments were carried out with 35S- labeled proteins obtained by translation in vitro with rabbit reticulocyte lysate (TNT kit from Promega Corp., Madison, WI). SRC1a and GRIP-1 were translated from the T7-based promoters mentioned above and RIP140 was translated from a T3 promoter (31). The fusion proteins GST-TR or GST-ER-LBD were loaded on glutathione Sepharose beads and incubated with 1 or 2 µl of the labeled protein lysate in a total volume of 150 ml 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). The reaction mix also contained hormones: T3 10-8 M or estradiol 10-8 M and, where indicated, highly purified ER-LBD (2 µg) previously bound to E2, tamoxifen, or ICI. After incubation at 90 min at 4 C with gentle rocking, the beads were washed five times with IPAB without BSA. Beads were then dried under vacuum, resuspended in 20 µl protein loading buffer, and analyzed by SDS-PAGE. Signals were amplified by fluorography for 35S extracts (Amplified, Amersham, Arlington Heights, IL), and gels were exposed at -70 C.

Purification of ER-LBD
Human ER-LBD was purified by affinity chromatography on estradiol-Sepharose (64, 65) essentially as described (66), except that the pET-23d-ERG expression vector was modified to contain the sequence Met-Asp-Pro-Met297–Ser566, in which part of the hinge (D) and carboxy-terminal (F) regions of ER were deleted (a kind gift from Paul Sigler). ER-LBD was overexpressed in BL21 (DE3 {lambda} lysogen) cells, and clarified extracts obtained from sonicated cells were applied to a 10-ml column of estradiol-Sepharose. The column was washed extensively, and bound ER-LBD was eluted either with 3 x 10-5 M estradiol, 4-hydroxytamoxifen, or ICI 182,780 (67) in 30 ml of a buffer that contained 25 mM Tris, 200 mM NaCl, 1 mM EDTA, 4 mM dithiothreitol, 5 M urea, and 10 mM lysine, pH 7.4. Protein concentrations were determined by the method of Bearden (68) with BSA as the protein standard. ER-LBD purity was assessed on silver-stained SDS-polyacrylamide gels and by Western blot analysis with the H222 monoclonal rat antibody (69, 70). The major E-Seph eluate was concentrated by centrifugation through an Amicon Centriprep-10 filter (Amicon, Inc., Beverly, MA) to an ER-LBD concentration of 1–10 mg/ml (32–320 µM; Mr = 31 kDa). Aliquots were snap frozen and stored at -75 C.


    ACKNOWLEDGMENTS
 
We thank Rosalie Uht and Weijun Feng for discussion and Ron Evans (Salk Institute, La Jolla, CA) for MMTV-TRE-CAT plasmid. G.L.G. gratefully acknowledges the technical assistance of Chris Hospelhorn and Lin Cheng.


    FOOTNOTES
 
Address requests for reprints to: Peter Kushner, Metabolic Research Unit, Box 0540, University of California, San Francisco, California 94143-0540. e-Mail: kushner{at}itsa.ucsf.edu

This work was supported by NIH Grant DK-51083 (to P.J.K.) and Grant DAMD17–94-J-44228 from the US Army Medical Research Command (to J.L.G.).

1 Drs. Baxter and Kushner have propietary interests in and serve as consultants to Karo Bio AB, which has commercial interests in this area of research. Back

Received for publication June 15, 1998. Revision received January 15, 1999. Accepted for publication February 9, 1999.


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