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
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ABSTRACT
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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.
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INTRODUCTION
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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.
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RESULTS
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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. 1A
, 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. 1B
).
Furthermore, an ER LBD with mutations (ML543/544A Fig. 1B
) 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. 1C
). 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
DBD Fig. 1D
). 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 DBD) containing both
AF-1 and AF-2.
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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. 2
). When purified bacterially expressed
ER-LBD complexed to estradiol was added, the TR-T3 complex
no longer bound GRIP1, SRC1a, or RIP140 (Fig. 2
, 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. 2
, 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.
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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. 3
, squares). The
T3 response has a half-maximum in the nanomolar range and
levels out at around 10100 nM, consistent with previous
studies. When SRC1a levels are elevated with increasing amounts of an
SRC1a expression vector (Fig. 3A
, 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. 3B
).
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.
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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. 3C
). 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. 1
, top left)
100 ng of TR expression vector were in the range of the linear response
and were far from autoinhibitory. Similarly (Fig. 1
, top
right) 1 µg of expression vector for the coactivator SRC-1
appeared optimal, and was robust but not saturating for GRIP1 (Fig. 1
, 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. 4A
, 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.
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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. 5
, 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. 5
) 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. 4 . Inhibition by ER-LBD was somewhat reduced in this
experiment.
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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. 6A
). Notably, ER AF-2 did not decrease
the residual TR action (compare T3 with
T3+E2 with RIP140, Fig. 6A
, left
panel, and Fig. 6B
). As observed in Fig. 6B
, 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. 6B
). 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. 4 . 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.
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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. 6
, 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. 7
, 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. 4 .
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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. 7
, 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. 8
, 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
DBD, Fig. 8
)
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 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. 4 .
|
|
 |
DISCUSSION
|
---|
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. 9
. 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.

View larger version (14K):
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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
|
---|
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
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, 0.51.0 µg of TR
expression vector, 2 µg of squelcher vector (either ER-LBD or
ER
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-Met297Ser566, 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
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 110
mg/ml (32320 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
DAMD1794-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. 
Received for publication June 15, 1998.
Revision received January 15, 1999.
Accepted for publication February 9, 1999.
 |
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