The Partial Agonist Activity of Antagonist-Occupied Steroid Receptors Is Controlled by a Novel Hinge Domain-Binding Coactivator L7/SPA and the Corepressors N-CoR or SMRT
Twila A. Jackson,
Jennifer K. Richer,
David L. Bain,
Glenn S. Takimoto,
Lin Tung and
Kathryn B. Horwitz
Departments of Medicine and Pathology and the
Molecular, Biology Program, University of Colorado Health
Sciences Center, Denver, Colorado 80262
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ABSTRACT
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Steroid receptor antagonists, such as the
antiestrogen tamoxifen or the antiprogestin RU486, can have
inappropriate agonist-like effects in tissues and tumors. To explain
this paradox we postulated that coactivators are inadvertently brought
to the promoters of DNA-bound, antagonist-occupied receptors. The human
(h) progesterone receptor (PR) hinge-hormone binding domain (H-HBD) was
used as bait in a two-hybrid screen of a HeLa cDNA library, in which
the yeast cells were treated with RU486. We have isolated and
characterized two interesting steroid receptor-interacting proteins
that regulate transcription in opposite directions. The first is
L7/SPA, a previously described 27-kDa protein containing a basic region
leucine zipper domain, having no known nuclear function. When
coexpressed with tamoxifen-occupied estrogen receptors (hER) or
RU486-occupied hPR or glucocorticoid receptors (hGR), L7/SPA increases
the partial agonist activity of the antagonists by 3- to 10-fold, but
it has no effect on agonist-mediated transcription. The interaction of
L7/SPA with hPR maps to the hinge region, and indeed, the hPR hinge
region squelches L7/SPA-dependent induction of antagonist-mediated
transcription. Interestingly, pure antagonists that lack partial
agonist effects, such as the antiestrogen ICI164,384 or the
antiprogestin ZK98299, cannot be up-regulated by L7/SPA. We also
isolated, cloned, and sequenced the human homolog (hN-CoR) of the
270-kDa mouse (m) thyroid/retinoic acid receptor corepressor. Binding
of hN-CoR maps to the hPR-HBD. mN-CoR, and a related human corepressor,
SMRT, suppress RU486 or tamoxifen-mediated partial agonist activity by
more than 90%. This suppression is completely squelched by
overexpression of the hPR H-HBD. Additionally, both corepressors
reverse the antagonist-dependent transcriptional up-regulation produced
by L7/SPA. Our data suggest that the direction of transcription by
antagonist-occupied steroid receptors can be controlled by the ratio of
coactivators to corepressors recruited to the transcription complex by
promoter-bound receptors. In normal tissues and in hormone-resistant
breast cancers in which the agonist activity of mixed antagonists
predominates, steroid receptors may be preferentially bound by
coactivators. This suggests a strategy by which such partial agonist
activity can be eliminated and by which candidate receptor ligands can
be screened for this activity.
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INTRODUCTION
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Steroid hormone antagonists, such as the antiestrogen tamoxifen or
the antiprogestin RU486, are synthetic pharmaceutical agents that have
been found empirically to suppress the activity of natural steroidal
agonists such as estradiol, progesterone, or glucocorticoids (1, 2, 3).
The ability of antagonists to suppress the transcriptional effects of
agonists has important clinical value (2, 4). However, in some tissues
and tumors, instead of being inhibitory, steroid antagonists can have
inappropriate, agonist-like effects (4, 5, 6, 7, 8). The precise mechanisms by
which antagonists inhibit transcription under some conditions, but
stimulate it in others, are unknown (7).
Steroid hormones and their synthetic analogs bind to steroid receptors,
which are members of a ligand-regulated family of nuclear transcription
factors that includes, in addition to estrogen (ER) and progesterone
(PR) receptors, the receptors for androgens, glucocorticoids (GR), and
mineralocorticoids (9, 10, 11). These receptors belong to a distinct
subgroup of the nuclear receptor superfamily, another subgroup of which
includes the receptors for retinoic acids, vitamin D, and thyroid
hormone (10). A key functional difference between steroid receptors and
retinoic acid/thyroid hormone receptors is that the latter are
constitutive transcriptional repressors, which bind to their cognate
DNA-binding sites in the absence of ligand (12, 13, 14). In contrast,
unliganded steroid receptors have little or no intrinsic DNA-binding
ability or biological activity (12, 15). Instead, they require a ligand
either agonist or antagonist to facilitate receptor-DNA
interactions. The mechanisms by which unliganded retinoic acid/thyroid
hormone receptors repress transcription were unknown until several
recent studies described a new category of modulatory nuclear proteins
having corepressor activity, which interact with the DNA-bound
receptors and actively silence transcription (14, 16, 17, 18, 19, 20, 21, 22). Addition of
ligand destabilizes corepressor binding to these receptors and
activates transcription. These corepressors have been found to interact
specifically only with unliganded members of the retinoic acid/thyroid
hormone receptor subfamily, and they reportedly fail to interact with
either unliganded or agonist-liganded members of the steroid receptor
family (17, 19). No relationship has been known to exist between the
mechanisms by which unliganded retinoic acid/thyroid hormone receptors
repress transcription and the mechanisms by which antagonist-occupied
steroid receptors inhibit the actions of agonists.
Little is known about the mechanisms by which steroid antagonists
inappropriately activate transcription, although several models have
been proposed. Partial agonist activity is often promoter- and cell
type-specific (23, 24), and recently, a number of studies have shown
that cross-talk between antagonist-occupied steroid receptors and cell
surface-signaling pathways, such as activation by cAMP (4, 6, 25, 26),
enhances these partial agonist effects, suggesting that unique receptor
phosphorylation states mediate this activity.
We speculated that an alternative mechanism operates, namely that
unique coactivator proteins are brought to the transcription complex by
antagonist-occupied steroid receptors. To address this possibility, we
used a LexA-human (h) PR hinge (H)-hormone binding domain (HBD) fusion
protein as bait in a yeast two-hybrid screen of a HeLa cell cDNA
library (27, 28). The assay incorporated a novel strategy in which the
yeast cells were treated with the antiprogestin RU486. Using this
screen, we have isolated two interesting proteins that regulate
antagonist-occupied steroid receptors in opposite directions.
The first of these, L7 (29, 30, 31, 32, 33, 34, 35, 36), is a 27-kDa cytoplasmic and nuclear
protein believed to function in translational regulation (33) but of
unknown nuclear function, which contains a canonical RNA- and
DNA-binding leucine zipper bZIP dimerization domain (31). We find that
human L7 or SPA (switch protein for antagonists) is a coactivator that
strongly enhances transcription of RU486-occupied hPR or hGR, and
tamoxifen-occupied hER, but has no effect on agonist-dependent
transcription by these receptors. The second isolate is the human
homolog (hN-CoR) of the mouse (m) retinoic acid/thyroid hormone
receptor corepressor, N-CoR (17). Both mN-CoR and the related human
corepressor SMRT (silencing mediator for retinoid and thyroid hormone
receptors) (19), suppress the agonist-like transcriptional activity of
RU486-occupied hPR and tamoxifen-occupied hER. Transcriptional
repression mediated by N-CoR is reversed by L7/SPA.
We propose that the inhibitory pharmacological effects of
antagonist-occupied steroid receptors involves the adventitious
recruitment of a transcriptional corepressor that has no normal
physiological function in steroid hormone action, whereas the partial
agonist-stimulatory effects of antagonists involves recruitment of
novel coactivators. Thus, the ratio of corepressors to coactivators
that are bound to the transcription complex through the
antagonist-occupied receptors determines whether the outcome is
inhibitory or stimulatory. This property of antagonist-occupied
receptors has therapeutic implications and suggests methods by which
candidate receptor ligands can be screened for their partial
vs. pure antagonist pharmacology.
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RESULTS
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Yeast Two-Hybrid Screening Strategy
To isolate proteins that interact with antagonist-occupied steroid
receptors, we first asked whether agonists and antagonists have the
appropriate transcriptional responses in yeast cells (Fig. 1A
). L40 cells were transformed with a fragment of hPR
(37, 38) consisting of the hinge region (H) and hormone binding domain
(HBD) (amino acids 638933) fused to LexA (pLexA:H-HBD). The cells
were then treated with 1 µM progestin R5020, or 10
µM type II antiprogestins RU486 or ZK112993, or the type
I antiprogestin ZK98299 as shown. R5020 strongly activates
ß-galactosidase transcription from the LexA operator, whereas the
antagonists alone have no effect. However, RU486 and ZK112993
completely abolish R5020-dependent transcription, while ZK98299, which
has a lower binding affinity for hPR, was 85% inhibitory. We conclude
that appropriate antiprogestin-regulated transcriptional inhibition can
be elicited in yeast cells.

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Figure 1. L7/SPA, a Novel Protein That Interacts with
Antagonist-Occupied Steroid Receptors
A, Antiprogestins can inhibit transcription by R5020 in yeast. The
yeast two-hybrid strain L40 carrying a LexA promoter-LacZ reporter was
cotransformed with an expression vector encoding the hPR hinge and
hormone binding domain (hge, HBD) fused to LexA. Yeast cells were
treated 48 h at 30 C with 1 µM of the agonist R5020,
10 µM of the three antiprogestins shown, either alone or
in combination. Yeast colonies were lifted on a nitrocellulose filter
and lysed, and a ß-galactosidase assay was performed. B, The L7/SPA
interaction maps to the hPR hinge region. The yeast two-hybrid strain
L40 was cotransformed with the hPR or hER expression vectors encoding
the fusion proteins shown in the figure and the vector encoding the
Gal4 activation domain:L7/SPA fusion protein recovered from the
library. Transformed yeast cells were grown for 2 days at 30 C on media
containing 10 µM of the indicated antagonists. Colonies
were lifted on a nitrocellulose filter and lysed, and a
ß-galactosidase assay was performed.
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To identify proteins that mediate the agonist and/or antagonist
activity of antiprogestins, pLexA:H-HBD was then used as bait to screen
a HeLa cell cDNA library in yeast cells exposed to 10 µM
RU486. Approximately 10 million recombinants were screened, of which 28
clones, identified as unique by restriction mapping, were classified as
positive in preliminary assays.
The Coactivator, L7/SPA
One clone, TJ48, interacted with the hPR H-HBD but not with a
lamin bait, and had no intrinsic transcriptional activity in the GAL4
AD library vector (data not shown). TJ48 was sequenced and found to be
identical to nucleotide (nt) 54 to 744 of the L7/SPA (35) cDNA, which
encodes a 27-kDa protein originally defined as a potent autoantigen
associated with the large ribosomal subunit (32, 34).The N terminus of
L7/SPA contains a basic region leucine zipper (bZIP) domain (39, 40),
through which it forms stable homodimers that bind to RNA and
double-stranded DNA (31, 35). The protein is detectable in the
cytoplasm and nuclei but not nucleoli of human cell lines (34), and the
transcript is expressed in a variety of adult mouse tissues and in
human T47D and HeLa cell lines (data not shown). It has no known
nuclear function.
Full-length L7/SPA cDNA was isolated by RT-PCR from HeLa cell RNA and
cloned into the pGEX 4T1 glutathione-S-transferase (GST) plasmid and
into the pSG5 mammalian expression vector. The interaction between
L7/SPA and the hPR H-HBD was confirmed by GST pull-down (data not
shown) of in vitro translated L7/SPA. To further map the
L7/SPA-hPR interaction, hPR H-HBD, H, or HBD/LexA bait fusion proteins
were expressed in yeast cells together with the original GAL4 AD-L7/SPA
library fusion protein, which lacks 18 N-terminal amino acids. The
cells were treated or not with RU486, and ß-galactosidase activity
driven by the LexA promoter was measured (Fig. 1B
). As shown,
transcriptional activity in the presence of H-HBD is dependent on
treatment of the cells with RU486. ß-Galactosidase activity is
entirely absent, however, in the presence of HBD alone, regardless of
hormone treatment, and is constitutively active in the presence of the
hinge domain. This suggests that L7/SPA binds to H, but that it is
ordinarily blocked by the HBD, and that this inhibition can be relieved
by RU486 occupancy of the HBD. Similarly, L7/SPA binding to the H-HBD
of hER is dependent on occupancy by the antiestrogen tamoxifen, whereas
the pure antiestrogen, ICI l64,384 (3), does not promote interactions
between L7/SPA and hER.
To test the effect, if any, of L7/SPA on steroid receptor-mediated
transcription in mammalian cells, a
PRE2-TATAtk-CAT reporter was transfected into
HeLa cells, and dexamethasone (Dex) or RU486-regulated transcription
from the endogenous GR was measured (Fig. 2
) in the
absence or presence of exogenous full-length L7/SPA. Dexamethasone
strongly induces transcription (lane 2) which is unaltered by
overexpression of L7/SPA (lane 3). RU486 behaves as a partial agonist
under these conditions (compare lanes 1 and 4). Surprisingly, the
agonist activity of RU486 is enhanced 10-fold by overexpression of
L7/SPA (lane 5), and this extensive up-regulation can be completely
squelched by the hPR hinge domain (lane 6). Similar results are
observed in HeLa cells transiently overexpressing recombinant hGR
(lanes 712). Thus, L7/SPA appears to have the astonishing ability to
strongly enhance the partial agonist activity of a steroid antagonist,
without altering agonist-dependent transcription.

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Figure 2. L7/SPA Enhances the Partial Agonist Activity of
RU486- but Not Dexamethasone-Mediated hGR Transcriptional Activity and
This Activity Is Squelched by the hPR Hinge Region
HeLa cells were cotransfected with 2 µg
PRE2-TATAtk-CAT reporter, with (lanes 712) or
without (lanes 16) 10 ng hGR expression vector, and 5 µg
full-length L7/SPA, plus or minus 5 µg hPR hinge expression vector as
indicated in the figure. Twenty-four hours after transfection, the
medium was changed, and the cells were either untreated or treated with
100 nM RU486 or 10 nM dexamethasone for another
24 h. Cell lysates were normalized to ß-galactosidase activity,
and CAT assays were performed by TLC.
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RU486 is a type II antiglucocorticoid/antiprogestin that promotes
receptor-DNA interactions (41, 42) and has partial agonist activity.
Type I antagonists, such as the antiprogestin ZK98299, lack this
activity (42, 43). The effect of L7/SPA on ZK98299-regulated
transcription was tested using a construct consisting of a truncated
hPR (DBD-H-HBD), lacking the N terminus. As shown in Fig. 3A
, the hPR C terminus (or DBD-H-HBD) is strongly
activated by R5020 (lanes 3 and 4) which is not modified by L7/SPA
(lane 4). On the other hand, the partial agonist activity of RU486
(lane 5) on the PR C terminus is strongly enhanced by L7/SPA (compare
lanes 5 and 6). In contrast, ZK98299 lacks partial agonist activity
(lane 7) and is unaffected by L7/SPA (lane 8). Full-length hPR
B-receptors have the same properties (not shown) as do hPR specificity
mutants (43) in which the PRE-binding specificity of the PR DBD has
been switched to an estrogen response element by mutation of three key
amino acids (44, 45) in the first zinc finger (data not shown). This
was done to rule out any effects of endogenous GR, on recombinant
hPR-regulated transcription.

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Figure 3. L7/SPA Stimulates the Transcriptional Activity of
the RU486-Occupied but Not the ZK98299- or R5020-Occupied hPR
A, HeLa cells were cotransfected with 2 µg
PRE2-TATAtk-CAT reporter, 10 ng hPR C-terminus
consisting of the DNA binding domain linked to the hinge region and
HBD, and 5 µg full-length L7/SPA expression vectors as indicated in
the figure (+). Twenty-four hours after transfection, the medium was
changed, and the cells were either untreated or treated with 100
nM RU486 or ZK98299 or 10 nM R5020. Cell
lysates were normalized to ß-galactosidase activity, and CAT assays
were performed by TLC. B, HeLa cells were transfected as above with
0.1, 1.0, and 10 ng full-length hPR B-receptors and treated with 10
nM R5020.
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The data in Fig. 3A
show that L7/SPA influences antagonist- but not
agonist-mediated transcription. To analyze its effects on the agonist
in more detail, submaximal concentrations of hPR B-receptors were
tested (Fig. 3B
). HeLa cells were transfected with the reporter, with
0.1, 1.0, and 10.0 ng of the B-receptor expression vector in the
presence or absence of the L7/SPA expression vector, and the cells were
treated with saturating concentrations of R5020. At 0.1 and 1.0 ng DNA,
B receptor expression and its ability to transactivate
PRE2-TATAtk-CAT are submaximal, yet L7/SPA is
unable to enhance transcription. These data suggest that the effects of
L7/SPA are indeed antagonist-specific.
Depending on their structure, some antiestrogens (tamoxifen, for
example) possess partial agonist activity, whereas other antiestrogens
(such as ICI 164,384) do not (3). To determine whether L7/SPA modifies
the effects of antiestrogens, HeLa cells were transfected with wild
type hER and the ERE2-TATAtk-CAT reporter in
the presence or absence of the L7/SPA expression vector and were either
left untreated or treated with 17ß-estradiol (Fig. 4A
)
or the antiestrogens shown (Fig. 4B
). Estradiol-dependent hER-mediated
transcription is not influenced by L7/SPA even under submaximal hER
expression levels (Fig. 4A
). In contrast, the partial agonist activity
of tamoxifen is further enhanced by L7/SPA overexpression (Fig. 4B
).
This increase can be squelched (46) by expression of the hPR hinge
region. The extent of L7/SPA squelching by the hPR hinge can not be
gauged without extensive titration studies, but we find that hPR hinge
overexpression can reduce the partial agonist effect of tamoxifen even
in the absence of L7/SPA (Fig. 4B
), suggesting that endogenous cellular
coactivators can also bind the hinge domain. On the other hand, like
ZK98299, the antiestrogen ICI 164,384, which lacks partial agonist
activity, is unaffected by L7/SPA. Thus, the activity of steroid
antagonists that have partial agonist activity can be further enhanced
by expression of L7/SPA, while pure antagonists and agonists are
unaffected by this unusual coactivator. This explains the failure of
ICI 164,384 to promote hER interaction with L7/SPA in the yeast
two-hybrid screen (Fig. 1B
).

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Figure 4. L7/SPA Enhances the Partial Agonist Effects of
Tamoxifen but Has No Effect on Estradiol or the Pure Antagonist ICI
164,384
A, HeLa cells were cotransfected with 2 µg
ERE2-TATAtk-CAT reporter and 0.1 or 10 ng hER
with or without 5 µg L7/SPA as indicated in the figure. Twenty-four
hours after transfection, the medium was changed, and the cells were
either untreated or treated with 10 nM 17ß-estradiol for
another 24 h. Cell lysates were normalized to ß-galactosidase
activity, and CAT assays were performed by TLC and quantitated by
PhosphorImager (Molecular Dynamics, Sunnyvale, CA). B, HeLa cells were
transfected as above, in the presence or absence of 5 µg hPR hinge
(H) expression vector as indicated, and the cells were either untreated
or treated with 100 nM tamoxifen or ICI 164,384. In panel
A, estradiol-mediated CAT activity was set at 100% and in panel B,
tamoxifen-mediated CAT activity was set at 100%. The agonist activity
of tamoxifen is 50% that of estradiol.
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The Corepressors N-CoR and SMRT
A second cDNA clone, TJ53, was isolated by yeast two-hybrid
screening using RU486-occupied hPR H-HBD as bait (Fig. 5
). Sequence analysis showed that this 3300-nt human
cDNA (hN-CoR ID) was homologous to RIP13 (21), which is the interaction
domain (ID) and surrounding C-terminal sequences of the 270-kDa mouse
nuclear receptor corepressor (mN-CoR). Mouse N-CoR mediates
ligand-independent repression by the thyroid hormone/retinoic acid
receptor subfamily (17). Treatment with thyroid hormone or retinoic
acid dissociates mN-CoR from the cognate receptors. It is noteworthy
that the evidence presented in these studies indicates that mN-CoR does
not interact with steroid receptors.

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Figure 5. A Comparison of the Mouse and Human N-CoR
The mN-CoR, as reported by Hörlein et al. (17) is
shown at the top including two repressor domains (RD) at the N terminus
and an interaction domain (ID) at the C terminus. The HeLa cell library
clone isolated by yeast two-hybrid screen, TJ53, is referred to herein
as hN-CoR ID. Also shown is the full-length hN-CoR1, together with its
percent amino acid identity to mN-CoR. Two other clones contain
deletions in RD1: hN-CoR2, an independent isolate from the HeLa cDNA
bacterial library, lacks amino acids 83206; hN-CoR3, an expressed tag
sequence (Genbank accession number N33258), lacks amino acids 83147.
The amino acid sequence and protein structure downstream of amino acid
458 for hN-CoR2 and amino acid 600 for hN-CoR3 are unknown. Numbers
refer to amino acids.
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Using the hN-CoR ID cDNA recovered from the HeLa cell library cloned
into the yeast vector, a HeLa cell cDNA cell library cloned into a
bacterial vector was screened and, together with RT-PCR of HeLa and
T47D cell RNA, the entire human coding sequence was obtained,
sequenced, and compared with the murine protein-coding sequence (Fig. 5
). In addition to a 7359-nt open reading frame that predicts a
2453-amino acid protein (hN-CoR1), two apparent N-terminal splice
variants that result in loss of amino acids 83206 (hN-CoR2) and amino
acids 83147 (hN-CoR3) in the N-terminal repressor domain (17) have
been detected for the human corepressor. The amino acid identity
between mN-CoR and hN-CoR1 is high (98.91%), with the greatest
divergence observed in the second repressor domain (RD2) in which the
identity falls to 80.4%. Because of this identity, we have used mN-CoR
in most studies described below.
We find that the hN-CoR ID interacts with and modulates the activity of
RU486-occupied hPR H-HBD and tamoxifen-occupied hER H-HBD. As shown in
Fig. 6
, the LexA/hPR fusion bait proteins were
coexpressed in yeast cells together with the GAL4-AD/hN-CoR ID in the
absence or presence of three antiprogestins. ß-Galactosidase activity
was dependent on the presence of RU486 when either the H-HBD or the HBD
constructs were present in the cells, but it was uninducible with the H
construct, suggesting that the hN-CoR ID interacts with the hPR HBD but
not the hinge domain. This interaction is promoted by RU486 and by
another type II antiprogestin, ZK112993, but not by the type I
antiprogestin ZK98299. The latter is a pure antagonist that appears to
inhibit hPR interactions with DNA (42, 47). Similarly, the interaction
between the hER H-HBD and hN-CoR ID is very strong with tamoxifen
occupancy, but minimal with the pure antiestrogen, ICI 164,384.

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Figure 6. The hN-CoR ID Interacts with the HBD of Steroid
Receptors Occupied by Antagonists That Have Partial Agonist Activity,
but Not to Pure Antagonists
The yeast two-hybrid strain L40 was cotransformed with the hPR or hER
expression vectors encoding the fusion proteins shown in the figure and
the vector encoding the Gal4 activation domain:hN-CoR ID fusion protein
recovered from the library. Transformed yeast cells were grown for 2
days at 30 C on media containing no hormone addition or 100
µM of the indicated antagonists. Colonies were lifted on
a nitrocellulose filter and lysed and a ß-galactosidase assay was
performed.
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To characterize the function of the full-length corepressor on steroid
receptors, we used mN-CoR (17) and the related corepressor SMRT, a
168-kDa protein described by Chen and Evans (19), having 48% identity
to RIP13 (21), the C terminus of N-CoR. Similar to mN-CoR, the
inhibitory properties of SMRT are restricted to the unliganded thyroid
hormone/retinoic acid receptors. Figure 7
shows that
either mN-CoR or SMRT suppresses the partial agonist activity of
steroid antagonists. COS cells were transfected with the full-length
hPR or hER expression vectors, expression vectors for mN-CoR or SMRT,
the appropriate chloramphenicol acetyl transferase (CAT) reporter, and
treated with RU486 or tamoxifen. As shown, the partial agonist activity
of both steroid antagonists was more than 90% suppressed by either
corepressor. This repression by mN-CoR or SMRT of the partial agonist
effect of tamoxifen can be entirely squelched by coexpression of the
hPR H-HBD (Fig. 8
). Figure 8
also shows that the hPR
H-HBD alone partially suppresses the agonist activity of tamoxifen,
indicating that, in the absence of overexpressed N-CoR, other factors,
possibly coactivators, may bind to the H-HBD.

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Figure 7. Mouse N-CoR and Human SMRT Repress the Partial
Agonist Activity of RU486-Occupied hPRB and Tamoxifen-Occupied hER
COS cells were cotransfected with 2 µg
PRE2-TATAtk-CAT or
ERE2-TATAtk-CAT reporters, 10 ng hPRB or hER,
and 5 µg mN-CoR or hSMRT expression vectors as indicated in the
figure. Twenty-four hours after transfection, the medium was changed,
and cells were either untreated or treated with 100 nM
RU486 or tamoxifen. Cell lysates were normalized to ß-galactosidase
activity, and CAT assays were performed by TLC and quantitated by
phosphorimaging setting the partial agonist activity of RU486 or
tamoxifen (open bars) at 100%.
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Figure 8. Mouse N-CoR and hSMRT Suppress the Partial Agonist
Activity of Tamoxifen-Occupied hER and the Effect of These Corepressors
Is Squelched by the hPR H-HBD
COS cells were cotransfected with 2 µg
ERE2-TATAtk-CAT reporter, 10 ng HEGO with or
without 5 µg mN-CoR or hSMRT, and 5 µg hPR H-HBD expression vectors
as indicated in the figure. Twenty-four hours after transfection, the
medium was changed, and the cells were treated with 100 nM
tamoxifen for 24 h. Cell lysates were normalized to
ß-galactosidase activity, CAT assays were performed by TLC, and
duplicate experimental points were quantitated by phosphorimaging and
averaged. The partial agonist activity of tamoxifen-occupied hER was
set at 100% (open bar). In this study, tamoxifen had
47% the agonist activity of estradiol (not shown).
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Both N-CoR and SMRT are large proteins whose functional domains have
not yet been well characterized. We find, for example, that the hN-CoR
ID contains a transcriptional activation function that localizes to the
C-terminal 240 amino acids (data not shown). Thus, these proteins may
have functions other than the repressor ones described previously (17, 19). We also find (data not shown) that mN-CoR subtly suppresses, while
SMRT subtly increases, the level of basal transcription from the
promoters used in the present studies. This difference may be reflected
in the data shown in Fig. 9
, which show subtle
effects of the corepressors on steroid receptor agonist-dependent
transcription. In this study COS cells were transfected with expression
vectors for hER, hPR A-receptors, and hPR B-receptors lacking 42
C-terminal amino acids (B
42). This truncation converts RU486 into an
agonist (48). Cells were treated with the appropriate agonists (or
RU486 in the case of B
42) in the presence or absence of SMRT or
mN-CoR. We find that under these conditions of agonist-dependent
transcription, SMRT consistently slightly up-regulates transcription
(which rises to 3-fold in the case of B
42 and RU486), whereas mN-CoR
consistently slightly decreases transcription. These subtle effects of
the corepressors with agonists are in sharp contrast to the major
inhibitory effects of both corepressors with antagonists (see Figs. 7
and 8
). We ascribe these agonist-dependent effects of the corepressors
to the influence that they appear to have on basal transcription levels
of the promoters used in the present studies, but other explanations
are possible. In contrast, L7/SPA has no effect on basal transcription
levels and modulates only antagonist-dependent transcription.

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Figure 9. The Effects of mN-CoR and hSMRT on Agonist-Mediated
Transcription
COS cells were cotransfected with 2 µg
PRE2-TATAtk-CAT or
ERE2-TATAtk-CAT reporters, 10 ng hER, hPRA, or
hPRB 42 expression vectors, along with 5 µg hSMRT (A) or mN-CoR (B)
expression vectors as indicated in the figure. Twenty-four hours after
transfection, the medium was changed, and cells were either untreated
or treated with 10 nM of the hormones shown. Cell lysates
were normalized to ß-galactosidase activity, and CAT assays were
performed by TLC and quantitated by phosphorimaging.
Bars represent the average of duplicate experimental
points. Transcription in the absence of corepressors was set at
100%.
|
|
The L7/SPA Coactivator Plus the Corepressors?
Because coactivators and corepressors appear to "coexist" in
cells, it seems reasonable to propose that the ultimate direction of
transcription under control of antagonist-occupied steroid receptors
depends on the relative cellular levels of these two, offsetting
classes of coregulatory proteins. The preliminary study in Fig. 10
suggests that this is indeed the case. Here, HeLa
cells were transfected with an hGR expression vector alone, or together
with L7/SPA or SMRT (either alone or in combination), and the cells
were treated with RU486. As shown, L7/SPA alone enhances
RU486-dependent transcription from the
PRE2-TATAtk-CAT reporter, SMRT alone strongly
inhibits it, but when present together, one coregulator neutralizes the
effects of the other.

View larger version (42K):
[in this window]
[in a new window]
|
Figure 10. The Partial Agonist Activity of RU486-Occupied hGR
Is Controlled by the Ratio of L7/SPA to SMRT Recruited to the
Transcription Complex
COS cells were cotransfected with 2 µg
PRE2-TATAtk-CAT reporter, with 10 ng hGR, and
with 5 µg L7/SPA or hSMRT expression vectors alone or in combination
as indicated in the figure. Twenty-four hours after transfection, the
medium was changed, and the cells were treated with 100 nM
RU486 for 24 h. Cell lysates were normalized to ß-galactosidase
activity, CAT assays were performed by TLC, and duplicate experimental
points were quantitated by phosphorimaging and averaged. The partial
agonist activity of RU486-occupied hGR was set at 100% (open
bar).
|
|
 |
DISCUSSION
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Antagonist-Mediated Transcriptional Inhibition
Steroid receptor antagonists are pharmacological agents that have
been synthesized and selected for clinical use on the basis of
empirical tests that show that they inhibit the actions of the cognate
natural hormonal agonists. Although there is speculation about the
existence of natural antagonists, none have yet been described. The
mechanisms by which synthetic antagonists inhibit transcription of
steroid hormone-regulated genes have been intensively studied.
Antagonists compete with agonists for binding to the HBD of the
receptors and then prevent or modify receptor dimerization or DNA
binding, or facilitate receptor-DNA interactions but form a
transcriptionally nonproductive complex (1, 24, 41, 42). These
scenarios confer a passive role on the inhibitory effects of
antagonists, in which they simply compete for agonist binding, and
therefore block agonist actions.
However, the data presented herein suggest that steroid antagonists can
also actively repress transcription. We show that antagonists are able
to do so, by recruiting to DNA-bound steroid receptors one or more
endogenous corepressors whose normal cellular function is to mediate
gene repression by unrelated transcriptional repressors, such as the
unliganded retinoic acid/thyroid hormone receptors. We speculate that
these corepressors have no normal function with respect to agonist
action, but that they are adventitiously brought to promoter-bound
steroid receptors, when they are occupied by synthetic antagonists.
McDonnell et al. (8) and others (48, 49, 50) have proposed that
agonists and antagonists stabilize different conformational states of
steroid receptors. If so, it is possible that a subset of synthetic
antagonists freeze DNA-bound steroid receptors in a unique
conformational state that enhances the binding affinity of corepressors
for the HBD of the receptors. Furthermore, it is possible that on
promoters in which steroid receptors repress transcription (16),
recruitment of corepressors comes into play.
We have taken advantage of the partial agonist property of RU486 and
tamoxifen to demonstrate the recruitment of corepressors to steroid
receptors and the resultant transcriptional inhibition. On the other
hand, steroids such as ZK98299 (Fig. 1
) and ICI 164,384 are also
competent antagonists, yet our data suggest that they do not promote
receptor-corepressor interactions. It is possible that two, quite
different, mechanisms are involved in antagonist-dependent
transcriptional inhibition: type I inhibitors may function passively by
sequestering the receptors away from the transcription complex, whereas
type II inhibitors, which foster receptor binding to DNA, function
actively by recruiting corepressors that block transcription. However,
no definitive mechanism can be advanced until the controversy
surrounding the DNA binding properties, or lack thereof, of
antagonist-occupied receptors is resolved.
Recruitment of corepressors explains another physiological puzzle,
i.e. the ability of some steroid antagonists to suppress
gene transcription even in the absence of a hormonal agonist (51). As
described above, current assumptions hold that antagonist-occupied
receptors suppress agonist-regulated transcription by competitive
inhibition. Our model predicts that a gene that contains a steroid
hormone response element, but is up-regulated by any signal, including
a nonsteroidal one, can be suppressed by recruitment of a corepressor
to antagonist-occupied complexes bound to the hormone response element
of that gene, leading to inhibition in the absence of an agonist.
Partial Agonists and the Coactivator, L7/SPA
When steroid antagonists are used therapeutically, two problems
commonly arise. The first is that the drug may have the desired effect
in one tissue, but the opposite effect in another. Tamoxifen is a case
in point. It is appropriately antiestrogenic in the breast but acts
like an estrogen in the uterus, where it induces endometrial cancers
(52, 53, 54, 55, 56). The mechanisms underlying these undesirable tissue-specific
agonist effects are unclear. The second problem arises in
tamoxifen-responsive breast cancers, which not only acquire resistance
to tamoxifen treatment after a period of time, but in which tamoxifen
actively switches to an agonist (7). We have speculated that the
mechanisms involved in tissue-specific agonist effects of antagonists,
and in the acquired resistance of tumors to tamoxifen treatment, are
similar and that both are mediated by coactivators recruited to the
transcription complex by the antagonists. Note that tamoxifen-resistant
tumors often respond to second-line treatment with a pure antiestrogen
or other hormone therapies (57, 58, 59), underscoring our contention that
pure antagonists operate through mechanisms that differ from those of
antagonists with partial agonist activity.
We have now isolated a protein, L7/SPA (29, 30, 31, 32, 33, 34, 35, 36), that distinguishes
between these two classes of steroid antagonists. In the cytoplasm,
L7/SPA associates with the large ribosomal subunit (30), where it
inhibits cell-free translation (33). Like other ribosomal proteins, it
is a potent autoantigen (32, 34). However, L7/SPA is also an
extranucleolar nuclear protein of unknown function (34). Recently an
-helical leucine zipper domain (bZIP) was mapped to the N-terminal
1549 amino acids of the 248-amino acid protein, through which it
homodimerizes and binds to DNA and RNA (31, 35).
We isolated L7/SPA by its ability to bind the H-HBD of hPR,
mapped that binding to the hPR hinge region, and showed that L7/SPA
strongly enhances transcription by antagonist-occupied hGR, hER, and
hPR, but interestingly, that it has no effect on agonist-mediated
transcription. L7/SPA therefore exhibits the novel property of being an
antagonist-specific transcriptional coactivator whose binding maps to
the hinge region. This is the first description, to our knowledge, of
an activation function in this region, although an inhibitory function
has previously been described (60). Moreover, YL8A (36), the
Saccharomyces cerevisiae homolog of mammalian L7/SPA, lacks
the canonical N-terminal bZIP domain, but the remainder of the molecule
shares 56% amino acid identity and 81% conservation with the human
protein. As we show in Fig. 1A
, RU486 has no partial agonist activity
in yeast, suggesting perhaps that the bZIP domain of L7/SPA is
important for its coactivator activity, and studies to address this
hypothesis are in progress.
There are multiple examples, particularly with tamoxifen, demonstrating
agonist activity of antagonists. Several groups have shown that
tamoxifen agonism is especially strong on unusual EREs, including the
raloxifene response element (61), AP-1 sites (62), and cooperating weak
EREs (63). It is interesting to speculate that L7/SPA might be a very
potent coactivator at such elements.
Steroid Antagonists and the Combined Effects of Coregulators
The present studies show that antagonist-occupied steroid
receptors are targets for the actions of both corepressors and
coactivators. It seems logical to suppose that the sum of the combined
effects of these coregulatory proteins, determined by their relative
cellular concentrations and binding affinities for the receptors, will
control the direction of transcription by a particular ligand. This
model predicts that the inhibitory or stimulatory efficacy of an
antagonist will vary among tissues and tumors depending on the levels
and availability of the endogenous coregulators, and suggests that by
modulating those levels, it may be possible to control the direction of
transcription by the antagonist. Moreover, if the ability to bind an
antagonist-specific coactivator is the mark of an antagonist having
partial agonist activity, this property should be useful for the
pharmacological screening of candidate ligands.
 |
MATERIALS AND METHODS
|
---|
Plasmid Construction
The hPR H-HBD and hinge region (H or hge) alone, including the
complete endogenous nuclear localization signal (NLS), were amplified
by PCR and cloned in-frame into the 5' EcoRI and 3'
BamHI sites of the pBTM116 (64) bait plasmid (a gift of Stan
Hollenberg, Oregon Health Sciences University, was constructed by
Stanley Fields, University of Washington, Seattle, WA and Paul Bartel).
The resulting vectors, pLEXA:H-HBD and pLEXA:hge were used in yeast
two-hybrid experiments. A third yeast two-hybrid bait vector
(pLEXA:HBD) encoding only the HBD of hPR was also constructed by PCR
amplification of the HBD as described above and insertion of this
fragment into a modified pBTM116 containing the hPR NLS inserted
in-frame into the PstI site. A vector encoding the GST
fusion protein GST-H-HBD was generated by PCR amplification of the
hinge and hormone binding domains of hPR including the entire NLS using
primers containing 5' EcoRI and 3' BamHI sites
which were cloned in frame into pGEX 4T1 (Pharmacia, Piscataway, NJ)
cut with EcoRI and BamHI. The pCMX:mN-CoR
construct was a gift from Andreas Hörlein and M. G. Rosenfeld,
University of California, San Diego. Full-length L7/SPA was PCR
amplified from reverse transcribed HeLa cell cDNA and cloned into the
5' EcoRI and 3' BamHI sites of the mammalian
expression vector pSG5 to create pSG5:L7/SPA. The construct,
pLEXA:hN-CoR ID, was made by PCR amplification from the yeast HeLa cell
library hN-CoR ID clone of the regions indicated in Fig. 5
and cloned
into the 5' EcoRI and 3' BamHI sites of pBTM116.
The hPR1
42 mutant was constructed by PCR amplification of a
HindIII-BglII fragment in the hPR HBD located
between amino acids 810 and 891, which was inserted into
HindIII and BglII-cut hPR1. Wild type hPR and hER
expression vectors were obtained from Pierre Chambon (Strasbourg,
France), and the hGR expression vector was from John Cidlowski (NIEHS,
Research Triangle Park, NC); pCMX-SMRT was a gift from Ron Evans (The
Salk Institute, La Jolla, CA). The reporters,
PRE2-TATAtk-CAT used for hPR and hGR and
ERE2-TATAtk-CAT used for hER, were previously
described (65).
Yeast Two-Hybrid System
The plasmid pLEXA:H-HBD was transformed into the yeast
two-hybrid reporter strain L40 (64) (MATa his3
200 trp1901 leu23,
112 ade2 LYS2::(lexAop)4-HIS3
URA3::(lexAop)8-lacZ GAL4 gal80), a gift from S.
Hollenberg, yielding a strain called L40-LEXA:H-HBD. This strain was
transfected with a HeLa cell cDNA fusion library cloned into the GAL4
activation domain (AD) vector pGADGH (Clontech, Palo Alto, CA) and
plated on appropriate selective media containing 10 µM of
the antiprogestin RU486 (Roussel-Uclaf, Romainville, France). Ten
million primary transformants were screened for two-hybrid interactions
and were detected by growth on histidine drop-out plates and confirmed
by ß-galactosidase assay. The large-scale library transformation
protocol was supplied by Stan Hollenberg and is a modification of
published methods (66, 67). Modifications include an overnight growth
in liquid media before the histidine selection is applied and the
addition of 10 µM RU486 to all growth steps in the
transformation protocol.
Yeast ß-Galactosidase Assay
Colonies were lifted from original library transformation plates
with nitrocellulose filters. Filters were immersed in liquid nitrogen
for 15 sec to lyse cells and then placed in petri dishes containing
Whatman filters soaked in Buffer ZX (60 mM
Na2HPO4, 40 mM
Na2HPO4, 10 mM KCl, 1
mM MgSO4, 0.4 mg/ml X-gal, pH 7.0). Reactions
were carried out at 30 C for 8 h.
False-Positive Tests
pLEXA:lamin (64) (a gift of Paul Bartel and Stan Fields) was
used to test for nonspecific interactions. The positive GAL4 AD library
clones were tested for autonomous activation of reporter genes by
ß-galactosidase assay and by growth on histidine drop-out media in
L40. The GST fusion protein GST-H-HBD was expressed and purified
according to published methods (68, 69). The hN-CoR ID and L7/SPA
proteins were synthesized and labeled in vitro (70, 71).
Labeled hNCo-R ID and L7/SPA were incubated with purified GST-H-HBD and
glutathione Sepharose 4B matrix, pelleted, and extracted, and protein
binding was assessed by SDS-PAGE and autoradiography.
Shuttling Positive GAL AD Clones into E. coli
A single positive yeast colony was swirled into ice-cold
electro-competent HB101 E. coli in a 2-cm electroporation
cuvette (Bio-Rad Labs, Hercules, CA). Conditions for electroporation
were a pulse at 1500 V, 100 W, and 25 milliFarads (mF) followed by a
second pulse 30 sec later at 2500 V, 200 W, and 25 mF in a Gene Pulser
(Bio-Rad). Bacteria were plated on M9 media lacking leucine.
Cloning of hN-CoR
To obtain full-length hN-CoR, a human HeLa cell 5' Stretch Plus
cDNA Library (Clonetech, Palo Alto, CA) was screened using two probes.
The first probe was the original hN-CoR ID isolated by the two-yeast
hybrid screen. The second probe was an N-terminal 1540-bp fragment
obtained by RT-PCR from HeLa cell total RNA Template, a sense primer
based on the mN-CoR sequence (Genbank MMMU35312) beginning at nt 117
(the translation start site), and an antisense primer designed from a
human expressed tagged sequence cDNA (Genbank accession N33258, Genome
Systems, St. Louis, MO) corresponding to nt 1540 of the mN-CoR. From
this screen two N-terminal clones of approximately 2 kb were obtained
that contained 350 bp of 5'-untranslated region, and one 1.8-kb
C-terminal clone was obtained that started at nt 6890 in the
corresponding mN-CoR sequences and contained approximately 1330 bp of
3'-UT. In addition, by RT-PCR of both HeLa and T47D cell total RNA, a
5564-bp fragment was obtained using the Expand Long Template PCR system
(Boehringer Mannheim, Indianapolis, IN), and MuLV reverse transcriptase
(Perkin Elmer, Branchburg, NJ), using a sense primer beginning at the
translation initiation codon of hN-CoR and an antisense primer
initiating within the hN-CoR ID obtained from the yeast two-hybrid
clone, and corresponding to nt 5564 of mN-CoR. All of the above
fragments were sequenced either manually using Sequenase Version 2.0
(Amersham, Cleveland, OH) or with an ABI 377 sequencer (University of
Colorado Health Science Center, Cancer Center DNA Sequencing and
Analysis Core). The sequence of hN-CoR was assembled using AssemblyLIGN
sequence assembly software (Eastman Kodak Company, Rochester, NY), and
compared with mN-CoR using MacVector (Kodak Scientific Imaging, New
Haven, CT).
Cell Culture and Transfections
HeLa or COS cells were plated in 100-mm dishes in MEM
supplemented with twice charcoal-stripped FCS. Cells were cotransfected
by calcium phosphate precipitation (6) with 2 µg reporter plasmid,
expression vector (amounts indicated in figure legends), and 3 µg
ß-galactosidase expression vector pHC110 (Pharmacia-LKB
Biotechnology) to normalize for transfection efficiency and carrier DNA
for a total of 15 µg/plate. Twenty-four hours after transfection, the
cell medium was changed and ligands were added. The following ligand
concentrations were used throughout: 100 nM synthetic
antagonists RU486 (Roussel Uclaf), ZK98299, ZK112993 (Schering Corp.,
Berlin, Germany), tamoxifen or ICI164,384 (ICI Pharmaceuticals,
Mecclesfield, England) and 10 nM concentrations of the
agonists R5020, 17ß-estradiol, or dexamethasone. Cells were treated
with ligand for 24 h and then harvested. Cell lysates were
normalized to ß-galactosidase activity, then assayed for
chloramphenicol acetyl transferase (CAT) activity by TLC and quantified
by phosphorimaging and autoradiography.
 |
ACKNOWLEDGMENTS
|
---|
We thank S. Hollenberg, R. Evans, M. Rosenfeld, J. Cidlowski, and P.
Chambon for reagents; J. Jaehning and R. Sclafani and members of their
laboratories for useful discussions and yeast reagents; D. Graham and
C. Clarke for the mouse tissue RNA blot; and Roger Powell for expert
technical assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Kathryn Horwitz, Department of Medicine, Division of Endocrinology, University of Colorado Health Center, 4200 East Ninth Avenue, Medicine, Box B151, Denver, Colorado 80262.
Supported by NIH Grants CA-2686G and DK-48238, by Grant DAMD
1794-J-4391 from the U.S. Army, and by a graduate student stipend
from the Lucille P. Markey Charitable Trust (to T.A.J.).
Dedicated to "Grandpa" Bert OMalley on the happy
occasion of his 60th birthday and to the memory of his first Fellow, William L. McGuire,
my teacher and mentor K.B.H.
Effects of hN-CoR ID and L7/SPA with steroid antagonists were reported
at the Steroid/Thyroid/Retinoic Acid Gene Family Keystone Meeting, Lake
Tahoe, CA, March 1723, 1996.
Received for publication February 5, 1997.
Accepted for publication March 13, 1997.
 |
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