Characterization of Receptor Interaction and Transcriptional Repression by the Corepressor SMRT
Hui Li,
Christopher Leo,
Daniel J. Schroen and
J. Don Chen
Department of Pharmacology and Molecular Toxicology University
of Massachusetts Medical School Worcester, Massachusetts
01655-0126
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ABSTRACT
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SMRT (silencing mediator of retinoic acid and
thyroid hormone receptor) and N-CoR (nuclear receptor corepressor) are
two related transcriptional corepressors that contain separable domains
capable of interacting with unliganded nuclear receptors and repressing
basal transcription. To decipher the mechanisms of receptor interaction
and transcriptional repression by SMRT/N-CoR, we have characterized
protein-protein interacting surfaces between SMRT and nuclear receptors
and defined transcriptional repression domains of both SMRT and N-CoR.
Deletional analysis reveals two individual nuclear receptor domains
necessary for stable association with SMRT and a C-terminal helix
essential for corepressor dissociation. Coordinately, two SMRT domains
are found to interact independently with the receptors. Functional
analysis reveals that SMRT contains two distinct repression domains,
and the corresponding regions in N-CoR also repress basal
transcription. Both repression domains in SMRT and N-CoR interact
weakly with mSin3A, which in turn associates with a histone deacetylase
HDAC1 in a mammalian two-hybrid assay. Far-Western analysis
demonstrates a direct protein-protein interaction between two N-CoR
repression domains with mSin3A. Finally we demonstrate that
overexpression of full-length SMRT further represses basal
transcription from natural promoters. Together, these results support a
role of SMRT/N-CoR in corepression through the utilization of multiple
mechanisms for receptor interactions and transcriptional repression.
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INTRODUCTION
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Transcriptional regulation by steroid/thyroid hormones and
retinoids is a critical component in controlling many aspects of animal
development, reproduction, and metabolism (1, 2, 3, 4). The functions of
these hormones are mediated by intracellular receptors, which comprise
a large superfamily of ligand-dependent transcription factors (1). It
has been established that both retinoic acid receptors (RARs) and
thyroid hormone receptors (TRs) function via formation of heterodimeric
complexes with retinoid X receptors (RXRs) (5, 6). Once bound to a DNA
response element, the heterodimer responds to ligand through the
C-terminal ligand-binding domain (LBD), which is known to mediate not
only hormone binding but also receptor dimerization, transcriptional
activation, and repression (7, 8).
Both TR and RAR can function as transcriptional repressors in the
absence of ligands and potent activators upon binding of ligands (7).
DNA-binding assays and functional analysis have demonstrated that the
repressor activities of unliganded receptors depend on DNA response
elements, as well as on the intact LBD of the receptors (7, 9, 10).
In vivo, the TR/RXR heterodimer binds to DNA in the context
of chromatin, and nucleosome assembly enhances the transcriptional
silencing effect (11). Importantly, the oncogenic activity of v-erbA, a
mutated form of TR, is directly linked to transcriptional repression
(12, 13). In addition, deletion of the activation domain of RAR
converts it into a potent transcriptional repressor, and this mutation
was shown to cause defects in cellular differentiation and development
(14, 15, 16). Therefore, transcriptional repression by unliganded nuclear
receptors appears to play an important role in regulating cell growth
and differentiation.
Hormone binding is thought to induce conformational changes that lead
to ligand-dependent transformation of the receptors from repressors to
activators (1). The C terminus of TR, about 20 amino acids, constitutes
the 12th amphipathic helix (helix 12) of the LBD (17, 18, 19), which
functions as a ligand-dependent activation core domain known as the
AF2-AD,
C, or
4 domain (8, 20, 21, 22). Comparison of the LBD
structures of the unliganded (19) and liganded receptors (17, 18)
reveals a striking difference in the relative position of the helix
12/AF2-AD domain. This positional shift is thought to play an important
role in receptor activation, allowing the liganded receptors to
displace corepressors (8, 23, 24, 25) and to interact with coactivators
(see reviews in Refs. 2628).
SMRT (silencing mediator of retinoic acid and thyroid hormone receptor)
and N-CoR (nuclear receptor corepressor) are two related
transcriptional corepressors (24, 25) that are distinct from other
proteins (29). They were shown to utilize the C-terminal domain for
interaction with unliganded receptors (30, 31, 32, 33), and the N-terminal
domain for transcriptional repression (25, 30). In this study, we
investigate mechanisms of protein-protein interactions between SMRT and
nuclear receptors and analyze the modes of repression mediated by
SMRT/N-CoR. To do this, we define the interacting surfaces between SMRT
and nuclear receptors in binding and functional assays. Next, we
compare transcriptional repression mediated by SMRT and N-CoR using
transient transfection assays in mammalian cells. Evidence is presented
that SMRT and N-CoR interact with additional corepressors, and that
histone deacetylation plays a role in SMRT/N-CoR- mediated
repression.
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RESULTS
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Two Receptor Domains Are Essential for Interaction with SMRT
Deletion mutants in the carboxyl and amino termini of TR and RAR
were used to analyze the contribution of different regions in the
receptors for protein-protein interaction with SMRT. Figure 1A
shows the domain structure of TR and
the relative position of individual helices in the LBD as determined by
x-ray crystallography (17, 18). The sequence at the C terminus region
around helices 11 and 12 is also shown for both TR and RAR.
[35S]Methionine-labeled TR or RAR deletion mutants were
hybridized to glutathione S-transferase (GST)-SMRT and
GST-RXR in far-Western analyses in the absence of hormone
(Fig. 1B
). The relative strengths of these interactions are summarized
in Fig. 1C
.

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Figure 1. Two Receptor Domains Interact with SMRT
A, Domain structure of human TRß and the sequences of the C-terminal
helix 11 and 12 (AF2-AD) region of TR and RAR. The relative positions
of individual helices determined by x-ray crystallography (18) are also
indicated. B, Protein-protein interactions between receptors and SMRT
or RXR in far-Western analyses. The full-length TR and RAR and their
deletion derivatives were translated in vitro and
labeled by [35S]methionine. All these deletion mutants
expressed similar amounts of proteins as analyzed by SDS-PAGE and
autoradiography (not shown). The position of GST-C.SMRT (SMRT) and
GST-RXR (RXR) fusion proteins are as indicated (arrows).
Please note that GST-RXR appeared as a doublet in our extract. C,
Summary of relative levels of interactions between receptor mutants and
SMRT or RXR. The relative levels of interactions were scored from
background level (-) to strong (+++). nd, Not done. D, Human RARß
and RAR interact with SMRT in a ligand-reversible manner in
far-Western blots. -, vehicle only; RA, 1 µM of
all-trans-retinoic acid.
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Full-length TR (1456) associates well with both SMRT and RXR, and the
interaction with SMRT can be drastically reduced upon hormone
treatment. A residual weak interaction was observed in the presence of
ligand, consistent with previous observations (24, 30).
Carboxyl-terminal truncation at residue 441, which deletes helix 12,
results in a mutant that interacts normally with RXR but that exhibits
enhanced interaction with SMRT. Further truncation at residue 423,
which removes part of helix 11, reduces the interaction with SMRT
back to a level similar to that of wild type TR. In contrast, this
deletion markedly reduces interaction with RXR. Further deletions that
remove additional helices (helices 8, 9, and 10) result in barely
detectable interaction with SMRT and no interaction with RXR. These
results suggest that helix 12 inhibits SMRT association while helix 11
might promote the association.
Amino-terminal truncation of TR at residue 173, which removes the
DNA-binding domain (DBD), does not affect the interaction with either
SMRT or RXR. Further N-terminal deletion to residue 260, which removes
the first and second helices of the TR LBD, markedly impairs SMRT
association. No interaction with RXR by this mutant was detectable.
Similarly, C-terminal deletion of helix 12 from RAR (1403) also
increases interaction with SMRT as compared with that of wild type RAR
(1462). Further deletion to residue 395, which removes part of helix
11, diminishes the enhanced interaction to a level comparable with that
of full-length RAR, and ligand has little effect on the interaction.
Together, these results identify two distinct interacting domains at
the N-terminal hinge and C-terminal helix 11 regions of the receptor
LBD that might act synergistically to promote interaction with SMRT. We
find that the other two RAR isoforms, ß and
, also interact with
SMRT in a ligand-reversible manner, although the interactions observed
are weaker compared with that with RAR
(Fig. 1D
). The interactions
of both RARß and RAR
with RXR were not affected by ligand
treatment.
Interaction of Helix 12/AF2-AD Deletion Mutants with SMRT in
Yeast
To further understand the role of helix 12/AF2-AD in interaction
with SMRT, we analyzed interactions between AF2-AD deletion mutants of
RAR and RXR with C-terminal receptor-interacting domain of SMRT in a
yeast two-hybrid system (Fig. 2
). The RAR
LBD alone is sufficient to interact with SMRT in a ligand-reversible
manner (Fig. 2A
, column 3), but the resulting activity is much weaker
compared with that of full-length RAR (column 9). Similar to the
far-Western results, SMRT and full-length RAR retain some interaction,
even after treatment of the yeast cells with a saturating amount of
ligand. It is unclear whether this obervation reflects an association
between liganded receptors and SMRT or the existence of a small percent
of unliganded receptors after ligand treatment. Deletion of the AF2-AD
domain results in a RAR mutant that stimulates gene expression in
response to hormone treatment in yeast (columns 4 and 10), as opposed
to the dominant negative activity of this mutant observed in mammalian
cells (14). The ligand-dependent activation of RAR403 is more obvious
in the context of full-length receptor (column 10). A similar effect
has been shown in v-erbA, which normally acts as a constitutive
repressor in mammalian cells, but as a ligand-dependent activator in
yeast (34). Cotransformation of the RAR403 mutants with a Gal4
activation domain-SMRT fusion (Gal4 AD-SMRT) strongly induces
ß-galactosidase expression, even in the absence of hormone
(columns 5 and 11). Furthermore, in contrast to the hormone-dependent
dissociation seen with full-length RAR, hormone treatment does not
interrupt these interactions. Similarly, the Gal4 DBD-SMRT fusion
interacts strongly with the Gal4 AD-RAR403 mutants in a
ligand-insensitive manner (columns 6 and 12). These results are
consistent with the enhanced interaction observed in vitro
and indicate that the AF2-AD domain may act as a negative regulatory
element, controlling hormone-sensitive interaction between SMRT and
nuclear receptors.

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Figure 2. Two-hybrid Interactions between SMRT and Helix
12/AF2-AD Deletion Mutants of Nuclear Receptors
A, Interaction between RAR403 and C- terminal domain of SMRT in yeast
two-hybrid system. The indicated Gal4 AD and Gal4 DBD fusion constructs
were cotransformed into yeast Y190 cells, and the resulting
ß-galactosidase activities were determined from three independent
colonies. The ß-galactosidase activities were determined in the
absence (open bars) or presence (closed
bars) of 1 µM of all-trans-RA. l,
Ligand binding domain; f, full length; 403, RAR403 mutant with
C-terminal truncation at residue 403. B, Interaction of SMRT with
RXR443 and VDR in the absence of hormone (open bars) or
presence (closed bars) of 1 µM
9-cis-RA (for RXR) or 100 nM 1,25
dihydroxyvitamin D3 (for VDR). 443, RXR443 mutant with
C-terminal truncation at residue 443.
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The effect of AF2-AD deletion in RXR on association with SMRT was also
analyzed in the two-hybrid system (Fig. 2B
). Ligand treatment weakly
activates the Gal4 DBD-RXR LBD fusion (column 1), while
cotransformation with Gal4 AD-SMRT enhances reporter gene expression
(column 2), suggesting that SMRT can interact with RXR in either
absence or presence of ligand. Truncation at residue 443 enhances the
association between RXR and SMRT, and treatment with ligand does not
alter this interaction (columns 4 and 5). These results suggest that
SMRT can interact with RXR and that the AF2-AD domain of RXR also acts
negatively in SMRT association. Furthermore, we observed a significant
interaction between vitamin D3 receptor (VDR) and SMRT in
the absence of hormone, and treatment with ligand reduces the
interaction (column 8). This result is consistent with the recent
finding that VDR also contains intrinsic transcriptional repression
activity (35), suggesting that SMRT might mediate transcriptional
repression by VDR.
Two SMRT Domains Mediate Differential Interactions with Nuclear
Receptors
The finding that two regions of TR are essential for
protein-protein interaction with SMRT suggests that SMRT might also
contain duplicated receptor-interacting domains. Several deletion
mutants of SMRT were used to test this possibility in a far-Western
blot, and the results are summarized in Fig. 3A
. The GST fusions of these SMRT mutants
were overexpressed, and the purified proteins (Fig. 3B
, lanes 1 and 2)
or crude extracts (lanes 3, 4, and 5) were analyzed for interaction
with 35S-labeled RAR and TR. SMRT(9811495
) interacts
equally well with both RAR and TR in the absence of ligands. RAR, but
not TR, also interacts with degradation products of SMRT(9811495
).
Similarly, several fast migrating products of SMRT(10861291) also
interact well with RAR, but not with TR (lane 4). These results
indicate that RAR and TR may interact differently with SMRT. Consistent
with this speculation, we find that SMRT(9821291) (lane 2) as well as
SMRT(10861291) interact more strongly with RAR than with TR. In
contrast, the C-terminal fragment (12601495
) interacts better with
TR than wth RAR (lanes 5). All these interactions were found to be
sensitive to hormone treatment (Fig. 3B
and data not shown). Together,
these results identify two independent receptor interacting domains
(RID-1 and RID-2) of SMRT that appear to display different affinities
to TR and RAR.

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Figure 3. Two SMRT Domains Interact with the Receptors
A, Summary of SMRT deletion mutants used in this experiment and their
relative levels of interaction with nuclear receptors in far-Western
analyses shown in panel B. The amino acids encoded by the SMRT mutants
are shown in parentheses. Bound-RAR and TR were detected
by autoradiography, and the relative levels of interaction were scored
from background level (-) to strong (+++). The column numbers in each
panel correspond to constructs shown in panel A. Partially purified GST
fusion proteins were used in lanes 1 and 2 and total cell extracts were
used in lanes 3, 4, and 5. RID, Receptor interacting domain. +
T3, Plus 1 µM T3; Q,
glutamine-rich domain; H, putative helical region; , an internal
deletion between amino acids 1330 and 1375 resulting from alternative
splicing.
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Two SMRT Repression Domains
In addition to the C-terminal receptor interacting domains,
SMRT/N-CoR proteins also contain strong transcriptional repression
activity at their N-terminal regions. To define the minimal region
needed for repression by SMRT, serial SMRT deletion mutants were
generated, and their repression activities were analyzed using
transient transfection (Fig. 4A
).
Consistent with previous observations, full-length as well as N-SMRT
(amino acids 1981) repress basal transcription strongly and in a
dose-dependent fashion (rows 2 and 3), while C-SMRT (amino acids
982-1495
) exhibits minimal repression (row 4) compared with Gal4 DBD
alone (row 1). Further deletion from the C terminus of N-SMRT reveals
that amino acids 743 to 981 are not necessary for repression (row 5),
while deletion to residue 475 reduces the repression effect about
2-fold (row 6). These results suggest that amino acids 475 to 981 may
contribute in part to SMRT repression. Further C-terminal deletion to
residue 337 drastically interferes with repression (row 7), indicating
that the N-terminal boundary of this SMRT repression domain-1 (SRD-1)
is located between amino acids 337 and 475. Truncation from the N
terminus reveals that amino acids 1134 are dispensable for repression
by SRD-1 (row 8), while further deletion to residue 337 abolishes
repression (row 9), indicating that the C-terminal boundary of the
SRD-1 is within amino acids 134337. When the SMRT fragment between
amino acids 475 and 981 was tested for repression, we found that this
fragment also strongly repressed basal transcription (row 10). Together
with the observation that amino acids 743981 are not important for
repression, these results may define amino acids 475743 as a
second, independent SMRT repression domain (SRD-2).

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Figure 4. Multiple Transcriptional Repression Domains
A, Deletion mapping of the repression domains of SMRT. The
transcriptional repression activities were analyzed by transient
transfection in CV-1 cells. The relative levels of repression were
determined from an average of three independent transfections
using 0.1 µg (open bars), 0.2 µg
(hatched bars), or 0.5 µg (closed bars)
of plasmid DNAs. The starting and ending amino acids in each deletion
construct are shown beneath each domain. SRDs, SMRT repression domains.
B, Deletion mapping of the N-CoR repression domains (NRDs). The N-CoR
domains are aligned with those of SMRT in panel A. The relative levels
of repression were determined using 0.5 µg plasmid DNA and comparing
the result to the Gal4 DBD alone. Two new transcriptional repression
domains in N-CoR were found in addition to NRD-1 and NRD-2, which were
identified previously (25). C, SDS-PAGE analysis of in
vitro translated products of SMRT/N-CoR deletion constructs
used in panels A and B. Two microliters of the in vitro
translated products were analyzed in a 12.5% acrylamide gel, which was
exposed overnight. Note that most of these constructs appear to produce
doublet bands, perhaps due to secondary structure of the DNA used in
the translation reaction. D, Western blot analysis of the
repression-defective mutants of SMRT after transient transfection into
293 cells by using anti-Gal4 DBD monoclonal antibody (0.02 µg/ml) and
detected by ECL kit. The gel on the left was resolved in
a 12.5% acrylamide gel while the gel on the right was
resolved in a 10% gel.
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Sequence comparison between SMRT and N-CoR reveals
that they share about 45% identity within both SRD-1 and SRD-2,
suggesting potential functional conservation. Therefore, we tested
whether the two SRD corresponding regions of N-CoR also contain
repression activities. Consistent with a previous observation (25),
amino acids 1312 and 752-1016 of N-CoR exhibit strong repression
activities (Fig. 4B
, rows 2 and 3), and the two N-CoR domains
corresponding to SRD-1 and SRD-2 also yield 10- to 30-fold repression
(rows 4 and 5), similar to the repression effects observed by SRD-1 and
SRD-2. These two additional N-CoR repression domains are termed N-CoR
repression domain 3 and 4 (NRD-3 and NRD-4), and the two N-terminal
repression domains are called NRD-1 and NRD-2. Together, these results
indicate that both SMRT and N-CoR contain multiple, independent
transcriptional repression domains.
To confirm that lack of repression in some of these SMRT/N-CoR
deletion mutants is not due to lack of appropriate protein expression,
we analyzed the expression of these constructs by both in
vitro translation and Western blot analysis after transient
transfection. We find that all constructs used in this experiment
express approximately equal amounts of Gal4 DBD fusion proteins
in vitro (Fig. 4C
) and that the repression-defective mutants
express well in vivo (Fig. 4D
). These results indicate that
lack of repression by certain SMRT/N-CoR deletion mutants are not due
to lack of protein expression.
Multiple Mechanisms of Transcriptional Repression by SMRT/N-CoR
The mechanism of transcriptional activation by nuclear receptors
has been shown to require recruitment of coactivators, including
histone acetyltransferases such as CBP/p300 (36, 37, 38, 39). The opposite of
histone acetylation, histone deacetylation, has recently been
implicated in transcriptional repression by unliganded receptors and
the associated corepressors. Several reports have described a
corepressor complex containing a Mad-dependent corepressor mSin3A, a
histone deacetylase HDAC1 or mRPD3, and the nuclear receptor
corepressor SMRT/N-CoR (40, 41, 42, 43, 44, 45, 46, 47, 48). These results suggest that histone
deacetylation may be a mechanism of transcriptional repression by
unliganded receptors.
To confirm the interaction between mSin3A and the defined
repression domains of SMRT and N-CoR, we tested the interactions
between mSin3A and the individual repression domains of SMRT/N-CoR in a
mammalian two-hybrid system. Coexpression of a VP16 AD-mSin3A fusion
with all Gal4 DBD-SMRT/N-CoR repression domain fusions results in weak
reduction of the repression activities (Fig. 5A
). Coexpression of VP16 AD-mSin3A with
a Gal4 DBD-HDAC1 fusion also results in partial release of repression
mediated by Gal4 DBD-HDAC1 fusion. However, no activation above the
background level was observed even though a VP16 activation domain was
present. Since the weak interaction between SMRT/N-CoR repression
domain with mSin3A in the two-hybrid system may reflect a dominant
effect of repression over activation, we tested the interaction between
mSin3A and individual SMRT/N-CoR repression domains in vitro
by far-Western analysis. Full-length mSin3A was translated and labeled
in vitro and used as a probe for GST fusions of various SRD
and NRD domains. We find that mSin3A interacts specifically and
consistently with NRD-1 and NRD-4 in this assay (Fig. 5B
). In one
experiment, we also detected interaction between SRD-2 and mSin3A (data
not shown). No interaction is observed between SRD-1, NRD-2, and
NRD-3. Therefore, these results suggest that different SMRT and N-CoR
repression domains may repress transcription in a mSin3A-dependent or
-independent manner.

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Figure 5. Multiple Mechanisms of Transcriptional Repression
by SMRT and N-CoR
A, Two-hybrid interactions of mSin3A with SMRT and N-CoR repression
domains and HDAC1. The indicated Gal4 DBD fusion of SMRT and N-CoR
repression domains and HDAC1 were transiently transfected into CV-1
cells together with either Gal4 AD alone or Gal4 AD-mSin3A fusion as
indicated. The relative levels of repression are expressed as the means
of three independent experiments relative to the Gal4 DBD alone. B,
In vitro protein-protein interactions between mSin3A and
SMRT/N-CoR repression domains. The GST fusions of various SRD and NRD
domains were expressed in Escherichia coli and partially
purified. The GST fusion proteins were analyzed by SDS-PAGE
(right bottom panel) and examined for their abilities to
interact with 35S-labeled mSin3A in a far-Western blot
(left upper panel). mSin3A appears to interact
preferentially with intact GST-NRD-1 and GST-NRD-4 domains.
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SMRT Represses Basal Transcription from Natural Promoters
The hypothesis that SMRT/N-CoR proteins are transcriptional
corepressors that facilitate repression by unliganded receptors is
supported by protein-protein interactions and transient transfections
using the Gal4 fusion system. To provide further evidence that SMRT
may be physiologically relevant in transcriptional regulation, we
tested the effect of SMRT overexpression on transcriptional activity of
receptor-responsive promoters. Overexpression of full-length SMRT (Fig. 6
, lane 2), but not that of C-SMRT
lacking the repression domains (lane 3), repressed basal expression
from a mouse RARß2 promoter approximately 2-fold in comparison to the
empty vector (lane 1). The same result is evident with two minimal
response elements in the context of a thymidine kinase promoter in the
absence of hormone (Fig. 5A
). As expected, hormone treatment enhanced
transcription from these promoters, while overexpression of full-length
SMRT reduced slightly this ligand-dependent activation. C-SMRT enhances
the ligand-dependent activation from these promoters (Fig. 5B
). These
results suggest that SMRT may, at least under certain circumstances,
facilitate transcriptional repression of natural promoters.

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Figure 6. SMRT Represses Basal Transcription from RAR- and
TR-Responsive Promoters
The mRARß2 promoter, two copies of the ßRARE (ßRARE-tk-luc), and
the TRE (TRE-tk-luc) response elements were linked to a luciferase
reporter and transiently transfected into CV-1 cells together with
empty vector alone (lanes 1), full-length SMRT expression vector (lanes
2), or C-SMRT expression vector (lanes 3). The relative level of
repression in the absence of hormone is shown in panel A, while the
relative level of activation in the presence of 1 µM
all-trans retinoic acid (atRA) is shown in panel B.
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DISCUSSION
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Transcriptional repression has been recognized as a
critical component of TR and RAR function and is thought to be mediated
by association of unliganded receptors with silencing mediators
(corepressors) such as SMRT and N-CoR. To understand the function of
these putative corepressors, we have characterized their respective
receptor interaction and transcriptional repression properties. Two
distinct receptor-interacting domains of SMRT are identified that may
interact directly with two corresponding regions in the receptor. We
find that SMRT utilizes at least two distinct domains (SRD-1 and SRD-2)
for transcriptional repression, consistent with a recent report (42).
The two SRD-corresponding regions in N-CoR also repress basal
transcription, indicating that N-CoR contains four independent
repression domains. These results demonstrate the existence of multiple
and possibly redundant receptor interaction and transcriptional
repression domains in SMRT and N-CoR. One might expect that this
multiplicity will ensure a reliable targeting of the corepressors and
appropriate repression of target genes before activation.
The hinge region of TR was originally shown to interact directly with
the RID-2 region of N-CoR (25). Our results indicate that TR requires
an additional C-terminal region for efficient association with SMRT.
Nested deletion analyses suggest that helix 11 of the TR LBD plays an
important role in stabilizing SMRT association, presumably by
cooperating with the N-terminal helix 12 region. The interaction of
SMRT with either the N terminus or C terminus of the LBD alone is very
weak but detectable, suggesting that these two potential interacting
surfaces may act synergistically to promote protein-protein
interactions and to ensure appropriate recruitment of the corepressors.
Similarly, two independent regions in the receptor have been shown to
act synergistically for interaction with N-CoR (32, 49, 50). It has
recently been shown that a receptor dimer is required for interaction
with SMRT/N-CoR and that SMRT/N-CoR may contribute to receptor-specific
transcriptional repression (51). Furthermore, an antagonist of the
transcriptional activation by RXR homodimer was shown to promote
association with the corepressor SMRT (52). Together, these studies
suggest that SMRT and N-CoR may utilize similar but distinct mechanisms
for interaction with nuclear receptors.
We cannot exclude the possibility that the tight association with SMRT
by the AF2-AD deletion mutants may weaken hormone binding to the
receptor, but the ability of RAR403 to respond to ligand treatment in
yeast cells indicates that this mutation does not eliminate the
receptors hormone-binding capability, consistent with previous
observations (14, 53). Therefore, the inability of hormone to
dissociate corepressors is likely due to the lack of certain
conformational changes that would normally take place in the presence
of the AF2-AD. It is possible that the assumed shift of AF2-AD upon
hormone binding is a prerequisite for additional structural changes
that are important for corepressor dissociation. Alternatively, the
shift of helix 12 may mask or compete with certain interacting surfaces
required for binding corepressors. The fact that the AF2-AD deletion
creates a mutant that binds tighter to the corepressors favors this
model. We suspect that helix 11 could constitute such an interacting
surface, since disruption of this helix eliminates the enhanced
interaction resulting from deletion of AF2-AD. Our results suggest that
AF2-AD may act to balance the association between nuclear receptors and
the corepressors, by preventing overassociation of unliganded receptors
with corepressors, thereby facilitating ligand-dependent dissociation
of corepressors.
Nested deletion analysis reveals two distinct subdomains in SMRT that
are capable of independent interaction with nuclear receptors. These
two receptor- interacting domains, RID-1 and RID-2, interact
differently with TR and RAR. The N-terminal RID-1 region interacts more
strongly with RAR, and it contains a glutamine-rich domain, while the
C-terminal RID-2 region interacts better with TR and contains a
putative helical domain analogous to that identified previously in
N-CoR (25). The different receptor-interacting properties of these two
domains suggest that SMRT may utilize distinct mechanisms for
interaction with different receptors. The RID-2 region in N-CoR has
been shown to interact directly with the hinge region of TR (25), and
therefore it is reasonable to predict that the N-terminal RID-1 region
might interact with the C-terminal region of the LBD.
Functional analysis of the transcriptional repression activities of
SMRT and N-CoR reveals two independent domains that are capable of
repressing basal transcription. Together, there appear to be four
independent repression domains in N-CoR and two in SMRT. These
repression domains could act independently, and some repress basal
transcription as efficiently as the full-length protein, suggesting
that these domains might act redundantly and possibly through different
mechanisms. Sequence comparison of these repression domains gives
little clue as to possible mechanisms of repression. However, within
SRD-1 and the corresponding NRD-3, four potential repeated motifs
sharing a consensus sequence of GSITQGTPA have been identified (32). In
addition, two other potential repeats with a consensus sequence of
KGHVIYEG are noted. These motifs are well conserved between SMRT and
N-CoR, suggesting that they might contribute to repression.
Recently, several papers reported that mSin3A and the histone
deacetylase HDAC1 form a ternary complex with SMRT and N-CoR (42, 46).
These results indicate that SMRT and N-CoR, while interacting with
unliganded receptors, can also interact with additional corepressors
such as mSin3A and mSin3B (54), as well as the histone deacetylases
HDAC1 (55) and mRPD3 (56). The recruitment of histone deacetylase to
target promoters by unliganded receptors through SMRT, N-CoR, and mSin3
suggests that deacetylation of histones or other factors may play a
role in transcriptional repression, perhaps by establishing an
unfavorable chromatin structure for transcriptional activation (41).
Our results suggest weak two-hybrid interactions between SMRT/N-CoR and
mSin3A, or between mSin3A and HDAC1, even though a VP16 activation
domain was present. Alternatively, these results may suggest that the
repression activity of the corepressor complex is dominant over that of
the VP16 activation domain. An in vitro protein-protein
interaction assay detects association of mSin3A with NRD-1 and NRD-4,
but not with other repression domains. Although our results are
consistent with recent reports, our data also suggest the possibility
of other repression mechanisms.
 |
MATERIALS AND METHODS
|
---|
Plasmids
The GST fusions of C-SMRT (GST-SMRT) and RXR (GST-RXR) were
described previously (24, 30). Serial C-terminal and N-terminal
deletion mutants of human TRß and human RAR
were generated by
appropriate restriction enzyme digestion and/or PCR amplification from
the parental expression construct pCMX-hTRß and pCMX-hRAR
(57).
The GST-SMRT deletion constructs were generated by enzyme digestion at
indicated residues from the parental construct GST-SMRT. The Gal4 DBD
fusions of individual repression domains of SMRT and N-CoR were
generated by PCR amplification and were subsequently transferred to
pGEX vector for expression of GST fusion proteins. The VP16 AD-mSin3A
construct was created by subcloning the ScaI (at residue 56)
to BglII fragment of mSin3A (58) into the pCMX-VP16 vector.
Detailed information regarding these plasmids is available upon
request.
Far-Western Analysis
GST fusion proteins were separated by denaturing protein gels
(SDS-PAGE) and electroblotted onto nitrocellulose filters in transfer
buffer (25 mM Tris-HCl, pH 8.3; 192 mM glycine;
0.01% SDS). After denaturation in 6 M guanidine
hydrochloride (GnHCl), the proteins were renatured by stepwise dilution
of GnHCl to 0.187 M in HB buffer (25 mM HEPES,
pH 7.7; 25 mM NaCl; 5 mM MgCl2; 1
mM dithiothreitol). The filters were then saturated in
blocking buffer (5% nonfat milk, then 1% milk in HB buffer plus
0.05% NP40) at 4 C overnight or at 37 C for 1 h. In
vitro translated 35S-labeled proteins were diluted
into hybridization buffer (20 mM HEPES, pH 7.7; 75
mM KCl; 0.1 mM EDTA; 2.5 mM
MgCl2; 0.05% NP40; 1% milk; 1 mM
dithiothreitol), and the filters were allowed to hybridize overnight at
4 C. After three washes (5 min each) with the hybridization buffer, the
bound proteins were detected by autoradiography.
Yeast Two-Hybrid Assay
The yeast two-hybrid assay was carried out in the Y190 yeast
strain (59). The Gal4 DBD fusion constructs were generated in either
the pAS or pGBT vector (CLONTECH, Palo Alto, CA), and the Gal4 AD
fusion constructs were in the pGAD or pACT vector (CLONTECH). The
ß-galactosidase activities were determined with the
O-nitrophenyl ß-D-galactopyranoside (Sigma,
St. Louis, MO) liquid assay as previously described (30).
Cell Culture and Transient Transfection
African green monkey kidney CV-1 cells were grown in DMEM
supplemented with 10% resin-charcoal stripped FBS, 50 U/ml penicillin
G, and 50 µg/ml streptomycin sulfate at 37 C in 5% CO2.
One day before transfection, cells were plated in a 24-well culture
dish at a density of 50,000 cells per well. Transfection was performed
by standard calcium phosphate precipitation (57). All transfection
experiments were performed in triplicate and were replicated at least
once. Twelve hours after transfection, cells were washed with PBS and
refed fresh medium containing indicated amounts of ligands. After
30 h, cells were harvested for ß-galactosidase and luciferase
assay as described previously (30). The relative luciferase activities
are arbitrary light units normalized to the ß-galactosidase
activities.
In Vitro Translation and Western Blot
In vitro transcription/translation reactions were
carried out in rabbit reticulocyte lysates using the TNT T7 Quick
coupled transcription/translation system (Promega, Madison, WI).
[35S]Methionine (Amersham, Arlington Heights, IL) was
added during the translation reactions, which were performed at 30 C
for 90 min. The translated reactions were analyzed by SDS-PAGE,
followed by autoradiography. For Western blot analysis, transfected
cells were lysed in SDS-sample buffer, and the extracts were separated
by SDS-PAGE. The gels were transferred onto nitrocellulose membranes,
blocked with nonfat milk, and hybridized with anti-Gal4 DBD monoclonal
antibody according to manufacturers recommendation (Santa Cruz
Biotechnology, Santa Cruz, CA). The filters were washed and incubated
with horseradish peroxidase-conjugated anti-mouse IgG secondary
antibody and developed by enhanced chemiluminescent reaction
(Amersham).
 |
ACKNOWLEDGMENTS
|
---|
The authors thank Drs. Neal C. Brown, Kristin Carlson, Carol
Mulder, Douglas R. Waud, and George Wright for critical reading and
insightful suggestions of this manuscript. We are grateful to Drs.
Horlein and M. G. Rosenfeld for providing the N-CoR plasmid. We
are especially grateful to Christian A. Hassig and Dr. Stuart L.
Schreiber for providing the Gal4 DBD-HDAC1 plasmid, and Drs. Ronald A.
Depinho and Margaret S. Halleck for providing the mSin3A construct.
Part of the data presented was initiated by J.D.C. in Dr. Evans
laboratory.
 |
FOOTNOTES
|
---|
Address requests for reprints to: J. Don Chen, Department of Pharmacology and Molecular Toxicology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, Massachusetts 01655-0126.
This work was supported by an American Society of Hematology Junior
Faculty Scholar Award and the USAMRMC Breast Cancer Research Program
Idea Award BC961877 (to J.D.C.) and an Arthritis Foundation
postdoctoral fellowship (to D.J.S.).
Received for publication May 20, 1997.
Revision received August 29, 1997.
Accepted for publication September 5, 1997.
 |
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