The Specificity of Interactions between Nuclear Hormone Receptors and Corepressors Is Mediated by Distinct Amino Acid Sequences within the Interacting Domains
Ronald N. Cohen,
Sabrina Brzostek,
Brian Kim,
Michael Chorev,
Fredric E. Wondisford and
Anthony N. Hollenberg
Section of Endocrinology (R.N.C., F.E.W.) Department of
Medicine University of Chicago Chicago, Illinois 60637
Thyroid Unit (S.B., B.K., A.N.H.) Division of Endocrinology
and Division of Bone and Mineral Metabolism (M.C.) Beth Israel
Deaconess Medical Center Boston, Massachusetts 02215
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ABSTRACT
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The thyroid hormone receptor (TR) and retinoic
acid receptor (RAR) isoforms interact with the nuclear corepressors
[NCoR (nuclear corepressor protein) and SMRT (silencing mediator for
retinoid and thyroid hormone receptors)] in the absence of ligand to
silence transcription. NCoR and SMRT contain C-terminal nuclear hormone
receptor (NHR) interacting domains that each contain variations of the
consensus sequence I/L-x-x-I/V-I (CoRNR box). We have previously
demonstrated that TRß1 preferentially interacts with NCoR, whereas
RAR
prefers SMRT. Here, we demonstrate that this is due, in part, to
the presence of a novel NCoR interacting domain, termed N3, upstream of
the previously described domains. An analogous domain is not present in
SMRT. This domain is specific for TR and interacts poorly with RAR. Our
data suggest that the presence of two corepressor interacting domains
are necessary for full interactions with nuclear receptors in cells.
Interestingly, mutation of N3 alone specifically decreases binding of
NCoR to TR in cells but does not decrease NCoR-RAR interactions. In
addition, while the exact CoRNR box sequence of a SMRT interacting
domain is critical for recruitment of SMRT by RAR, the CoRNR box
sequences themselves do not explain the strong interaction of the N2
domain with TRß1. Additional regions distal to the CoRNR box sequence
are needed for optimal binding. Thus, through sequence differences in
known interacting domains and the presence of a newly identified
interacting domain, NCoR is able to preferentially bind TRß1. These
preferences are likely to be important in corepressor action in
vivo.
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INTRODUCTION
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The thyroid hormone receptor (TR) and retinoic acid receptor (RAR)
are members of the nuclear hormone receptor (NHR) superfamily (1). In
the absence of their respective ligands, TR and RAR repress gene
transcription by binding to nuclear corepressor proteins (2, 3, 4, 5, 6, 7, 8). These
proteins, such as NCoR (nuclear corepressor protein) and SMRT
(silencing mediator for retinoid and thyroid hormone receptors) mediate
ligand-independent repression by binding a complex with histone
deacetylase activity (9, 10, 11). NCoR and SMRT share a similar structure
(Fig. 1
), containing C-terminal nuclear
receptor-interacting domains (IDs) and at least three N-terminal
repressing domains. The IDs share limited homology with each other,
suggesting that there may be specificity in terms of NHR recruitment of
corepressors.

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Figure 1. Nuclear Corepressor Interacting Domains
Amino acid sequences of mNCoR and hSMRT are indicated. The NCoR IDs are
outlined and numbered, and corresponding regions of SMRT
are identified. The CoRNR box sequences of each interacting domain are
indicated and numbered.
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In fact, data from a number of groups suggest that such specificity
does exist. Previously we have demonstrated that TRß1 prefers to
interact with NCoR, and RAR
prefers to interact with SMRT, on DNA
response elements (12). These preferences are mediated through the
proximal IDs of NCoR and SMRT, N2 and S2, respectively (see Fig. 1
).
Additionally, Zamir et al. (13) have shown that RevErb, an
orphan nuclear receptor, interacts with NCoR, but does not interact
with SMRT. Wong and Privalsky (14) have demonstrated that the various
RAR isoforms recruit SMRT with different affinities, such that RARß
is deficient in interactions. In fact, knock-out of NCoR is embryonic
lethal, possibly due to a key role of NCoR in TR-mediated erythrocyte
development; therefore, SMRT cannot compensate for a lack of NCoR
in vivo (15).
Recent work has established that I/L-x-x-I/V-I motifs (or CoRNR boxes)
and adjacent helical structure within the nuclear corepressor IDs are
critical for mediating interactions with NHRs (16, 17, 18). While these
domains exhibit similarity to LXDs (L-x-x-L-L motifs) found in
coactivator IDs (19), their structure results in a conformation that
favors release on ligand binding. In fact, changing the LXD of a
coactivator to a CoRNR box results in a alteration in ligand
dependence, such that the coactivator is bound in the absence of ligand
(16).
Data from a number of groups suggest that a single corepressor molecule
binds a NHR dimer complex (homodimer or heterodimer) (12, 17, 18).
These data suggest that each interacting domain may contact a single
NHR. However, the specific roles of the different IDs in mediating this
process have not been well defined. To identify the functions of the
distinct interacting domains, we first searched for additional
I/L-x-x-I/V-I motifs in NCoR and SMRT. In fact, we found that NCoR
contains a novel ID, proximal to its other two known IDs. This region
(termed N3) binds TR well. This domain corresponds to the recently
described interacting domain described by Webb et al. (20).
N3 is not present in SMRT, suggesting a potential mechanism for the
preferential interaction of TRß1 complexes with NCoR over SMRT.
Whether the distinct sequences within the CoRNR box domains mediate the
specificity of interactions with NHRs is unknown. In fact, each of the
NCoR or SMRT IDs contains a unique CoRNR box (Fig. 1
), suggesting the
possibility that differences in corepressor binding by NHRs may depend
on the distinct CoRNR box sequences within the IDs. For example, the
distal CoRNR box in NCoR and SMRT appears to be important for retinoid
X receptor (RXR) binding, whereas RXR binds the proximal CoRNR box
sequences poorly (16). This is likely to be important in the binding of
NCoR and SMRT to NHR heterodimers. It has previously been shown that
regions of coactivators adjacent to LXDs dictate specificity in terms
of binding to NHRs (21, 22, 23). We therefore studied the regions within
the corepressor IDs to determine what portions contribute to the
specific preferences of TRß1 for NCoR, and of RAR
for SMRT. We
focused on N2 and S2, as these domains appear to be important in
mediating preferences, and share some degree of homology (12). Our data
suggest that specificity of corepressor recruitment by TR and RAR may
depend on distinct mechanisms.
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RESULTS
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NCoR Contains a Novel Interacting Domain, Not Present in SMRT
Analysis of the NCoR amino acid sequence suggests the presence of
an additional CoRNR box motif I-D-V-I-I at amino acids 19491953 of
murine (m)NCoR. Interestingly, although this sequence contains an
I/L-x-x-I/V-I sequence (16, 18), it does not have a full extended
helical motif as described by Perissi et al. (17),
L-x-x-I/H-I-x-x-x-I/L. Therefore, to identify whether a region
containing this sequence could function as an independent interacting
domain in vitro, we analyzed interactions of NCoR amino
acids 19392021 (termed N3) with TRß1 in electrophoretic mobility
shift assay (EMSA) (Fig. 2A
).
Glutathione-S-transferase (GST)-N3 was designed to be
similar in size to GST-N2. As shown in lanes 26, GST-N3 binds
strongly to TRß1 on a DR+4 element. In isolation, GST-N3 and GST-N2
bind TRß1 with high affinities (compare lanes 26 with 812),
although N2 binding is stronger (compare particularly lanes 2 and 3
with 8 and 9). Both N3 and N2 are dissociated from TRß1 in the
presence of its ligand, T3 (data not shown). To
determine whether this interaction is specifically dependent on the
I-D-V-I-I CoRNR box sequence, N3mut was made. This protein contains
NCoR amino acids 1,9392,021, but with a mutated CoRNR box sequence,
A-D-V-I-I. In fact, N3mut does not bind TRß1 on a DR+4 element (Fig. 2B
, lanes 4 and 5). Therefore, the CoRNR box sequence of N3 is
vital for its interactions with the TR.

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Figure 2. NCoR Contains a Third Interacting Domain
A, Gel mobility shift assay with 4 µl in vitro
translated (IVT) TRß1; 0.02 µg (lanes 2, 8), 0.1 µg (lanes 3, 9),
0.5 µg (lanes 4, 10), 1 µg (lanes 5, 11), or 1.5 µg (lanes 6, 12)
of indicated GST-CoR construct; and DR+4 radiolabeled probe. B, Gel
mobility shift assay with 4 µl IVT TRß1; 0.02 µg (lanes 2, 4) or
0.1 µg (lanes 3, 5) of indicated GST-CoR construct; and DR+4
radiolabeled probe. C, CV-1 cells were cotransfected with 1.7 µg UAS
reporter; 80 ng of the indicated Gal 4 construct; and 80 ng of VP16-
TRß1 (or empty vector-VP16). Cells were also cotransfected with 20 ng
of a CMV-ß-galactosidase vector to control for transfection
efficiency. The experiment was performed in triplicate, and repeated
three times. Data are expressed as fold luciferase activity, in the
presence vs. absence of cotransfected VP16-TRß1
(mean ± SE). D, Western blot of Gal4 constructs. CV-1
cells were transfected with 10 µg of indicated Gal4 constructs.
Protein nuclear extracts were analyzed by SDS-PAGE, transferred to
nitrocellulose, and blotted with an anti-Gal4 antibody. Panel A, NCoR
(1,9392,142); panel B, NCoR (1,9592,142); panel C, NCoR
(20632142).
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To determine whether N3 is important for interactions of NCoR with
TRß1 in cells, we employed a mammalian two-hybrid assay (Fig. 2C
).
Portions of NCoR with and without N3 were placed downstream of the Gal4
DNA-binding domain and tested for the ability to interact with
VP16-TRß1. In particular, Gal4- NCoR 1,5792,454, which contains N3,
N2, and N1, interacts more than 20-fold with TRß1. Gal4-NCoR
1,9412,142, which contains N3 and N2 (but not N1), also
interacts strongly with TRß1. In contrast, deletion of just 20
further amino acids (Gal4-NCoR 1,9612,142), which contains N2
but not N3, interacts minimally with TRß1, as does a smaller
N2-containing region (Gal4-NCoR 2,0632,142). Interestingly, this
region (containing only N2) binds TRß1 strongly in EMSA (12). These
data suggest that a single NCoR ID does not optimally bind TRß1 in
cells. Furthermore, N3, in the context of N2, allows for strong
interaction with TRß1. A Western blot of the Gal4 constructs (Fig. 2D
), probed with an antibody to the Gal4 DNA-binding domain,
shows that the Gal4 chimeric proteins are produced in CV-1 cells and
that the decreased interactions with NCoR 1,9412,142 and NCoR
2,0632,142 are not due to decreased protein production.
The Presence of Two CoRNR Box Motifs Are Necessary for Full
Structural and Functional Interactions of NCoR with the TR
To more fully define the role of individual IDs in the context of
a larger portion of NCoR, a variety of CoRNR box mutations were
introduced into NCoR amino acids 1,5792,454. This fragment of NCoR
contains the IDs of NCoR, but deletes the major repressing domains. It
reverses basal repression when transfected into cells, by competing
with endogenous NCoR for TR binding. It has therefore been termed NCoR
inhibitor, or NCoRI (24). Mutations were made in each CoRNR box of
NCoRI, either alone or in combination. These mutations each substitute
an alanine for the initial amino acid of the I/L-x-x-I/V-I motif, such
that N3m contains NCoR amino acids 1,5792,454, but with an A-D-V-I-I
in place of I-D-V-I-I. Similar mutations were made to create N2m and
N1m. Combinations of these mutations were made to create N32 m, N31
m, and N21 m. Finally, the N32-1 m construct has similar mutations
in all three CoRNR box motifs.
To determine whether these mutations decreased interactions with
TRß1, GST interaction assays were performed. In these studies,
35S-labeled NCoRI constructs were tested for
their ability to interact with GST-TRß1. As shown in Fig. 3A
, wild-type NCoR AA 1,5792,454
interacted strongly with GST-TRß1. This interaction was decreased
(although not entirely eliminated) in the presence of
T3 (lane 1). Both N3m (lanes 2) and N2m (lanes 3)
have significantly decreased interactions with TRß1. In contrast, N1m
maintains strong interactions with TRß1, consistent with the
decreased ability of N1 in isolation to interact with TRß1 in EMSA
(12). Mutation of any two CoRNR box sequences (but in particularly
N32 m and N31 m) results in a dramatic decrease in TRß1 binding,
suggesting that the one remaining ID is not sufficient for strong
interactions with the TR. Although an isolated, single interacting
domain is capable of binding the TR in EMSA (see Fig. 2A
), the
experiments in Fig. 3A
were done in the context of a larger portion of
NCoR, as well as a smaller amount of protein. Thus, the presence of two
IDs is necessary for full TR-NCoR interactions. Not surprisingly,
mutation of all three CoRNR box motifs results in compete loss of
binding.

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Figure 3. Two Intact CoRNR Box Sequences Are Necessary for
Interactions with TRß1
A, GST interaction assay of NCoRI mutants. Four microliters of
in vitro translated 35S-methionine-labeled
NCoRI constructs were incubated with GST-TRß1, washed extensively,
and analyzed by SDS-PAGE. B, Gel mobility shift was carried out using 4
µl in vitro translated TRß1; 4 µl in
vitro translated mutant or wild-type NCoRI construct; and DR+4
radiolabeled probe. Shown below is an SDS-PAGE of the NCoRI constructs.
Constructs were in vitro translated with 35S
methionine; 2 µl of the reaction mixture was analyzed by SDS-PAGE. C,
CV-1 cells were cotransfected with 1.7 µg DR+4-luciferase reporter,
80 ng of TRß1, and 160 ng of pKCR2-NCoRI construct (or empty vector
pKCR2). Data were performed in triplicate and repeated twice.
Transfection efficiency was controlled using a ß-galactosidase
expression vector. Data are expressed as relative luciferase activity
(mean ± SE).
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To examine these interactions in the context of an
underlying thyroid hormone response element, EMSAs were
employed, using a 32P-radiolabeled DR+4 probe,
and in vitro translated TRß1 and mutant or wild-type NCoRI
constructs (Fig. 3B
, upper panel). As shown in lane 2,
wild-type NCoRI (NCoR amino acids 1,5792,454) binds strongly to the
TRß1 homodimer. In contrast, N2m and N1m bind weakly (lanes 23),
and N3m not at all (lane 1), suggesting that N3 plays a critical role
in binding the TR. In addition, mutation of any two CoRNR box motifs
results in loss of all binding in this assay (lanes 46), again
suggesting that two intact IDs are necessary for strong binding to
TRß1 in the context of NCoR. All NCoRI constructs were in
vitro translated similarly (Fig. 3B
, lower panel),
suggesting that differences seen in the EMSA reflect actual differences
in binding.
We used the ability of NCoRI to reverse basal repression to provide an
assay of the function of the distinct NCoR IDs (24). The ability of the
mutated NCoRI variants to reverse repression on a DR+4 element was
tested in transient transfections in CV-1 cells. As shown in Fig. 3C
, transfection of TRß1 results in approximately 4-fold
ligand-independent repression (from
40,000 light units to 10,000).
Cotransfection of pKCR2-NCoRI decreases this repression by more than
half, i.e. luciferase activity was decreased, but was still
greater than half of basal activity. In contrast, cotransfection of
either pKCR2-N3mut or pKCR2-N2mut results in an intermediate level of
repression, whereas the level of repression with pKCR2-N1mut is no less
than that of pKCR2-NCoRI itself. Mutation of two IDs results in further
loss of NCoRI function, if N3 is one of the mutated domains, and
mutation of all three IDs yields a construct with minimal, if any,
function. These data again suggest that two IDs are necessary for full
interactions of NCoR with TRß1 in cells, although they do suggest the
possibility of residual function in the presence of a single N3 ID,
perhaps through an alternative mechanism. In sum, these data indicate
that N3 is important for interactions with the TR in cells; the
combination of N3 and N2 allows for full interactions.
N3 Contributes to NCoR Specificity of Binding to Nuclear Hormone
Receptors
To determine whether N3 binds RAR as well as TR, an EMSA paradigm
was again used. In this EMSA, performed on a DR+5 element, RAR
is
present in all lanes; RXR
additionally is present in even-numbered
lanes. The presence of RAR-RXR heterodimers is noted in the
even-numbered lanes. In fact, in contrast to its interactions with
TRß1, N3 does not bind RAR
well in EMSA (Fig. 4A
, lanes 910). In contrast, S2 binds
well to the RAR-RXR heterodimer (Fig. 4A
, lane 14). Since a region
homologous to N3 is not present in SMRT, these data suggest that N3
plays a role in determining specificity of interactions with TR and
RAR.
A mammalian two-hybrid assay was used to determine whether N3 dictated
such specificity in cells. In this experiment, Gal4-NCoR increased
interactions 140-fold with VP16-TRß1, whereas interactions were only
increased 40-fold for VP16-RAR
(Fig. 4B
, upper panel),
consistent with previous data (12). As shown in Fig. 4B
(middle), Gal4-NCoRI interacts strongly with TRß1-VP16. As
expected, this interaction is dramatically decreased by mutation of the
N3 CoRNR box (Gal4-N3mut).
The baseline interaction of Gal4-NCoRI and RAR-VP16 is significantly
less than with TRß1-VP16 (Fig. 4B
, upper panel; and Ref.
12). In contrast to TR, however, Gal4-N3mut interacts with RAR-VP16 as
strongly as does wild-type NCoRI. However, Gal4-N2mut was markedly
deficient in interactions with RAR-VP16. Therefore, N3 dictates the
preference of TR for NCoR. NCoR binds RAR more weakly, and N2, not N3,
is required.
Specificity of Corepressor Recruitment by TR and RAR Depends on
Distinct Mechanisms
To examine the roles of the sequences within the individual IDs in
modulating interactions, we focused on the N2 and S2 domains. These
particular IDs were chosen because they contain some degree of sequence
homology; however, their differences suggest that there may be
sequence-specific mechanisms governing the specificity of corepressor
recruitment. We have previously shown that TRß1 binds N2 more
strongly than S2, whereas RAR
prefers S2; these interactions help
explain the preferences of TRß1 for NCoR and RAR
for SMRT (12). N3
was not used in the following studies because there is no analogous
domain in SMRT. We therefore next studied what portions of N2 are
important for TR binding. As shown in Fig. 5
, various deletion constructs of N2 were
made as GST fusion proteins. These constructs were used in EMSA on a
DR+4 element, to determine their interactions with TRß1. When the C
terminus of N2 is gradually deleted, interactions with TRß1 are
reduced dramatically (compare lanes 25). In particular, there is a
significant decrease in binding when amino acids 2,106 to 2,119 are
deleted (compare lanes 3 and 4). However, some binding is still clearly
observed (lane 3). In contrast, deletion of even the proximal 11 amino
acid residues of N2 abolished binding to TRß1 (lane 7), suggesting
that this region is necessary for binding. To determine whether the
proximal portion of N2 was sufficient for binding as well, we made
constructs containing only the proximal portion of N2. In fact,
constructs containing the proximal 27 amino acids of N2 bound to TR on
the DR+4 element (data not shown). These data suggest that the proximal
region of N2 is both necessary and sufficient for TR binding,
consistent with the results of other groups in that it contains a CoRNR
box motif (16, 17, 18). However, the marked decrease in binding of the
C-terminal deletion constructs (e.g. Fig. 5
, lanes 3 and 4)
suggests that an additional (more distal) portion of N2 also
contributes to TR binding.

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Figure 5. Proximal and Distal Portions of N2 Play Distinct
Roles in Binding TR
Gel mobility shift assay was carried out using 4 µl in
vitro translated (IVT) TRß1; 100 ng of the indicated GST-CoR
construct; and DR+4 radiolabeled probe. A schematic illustration of the
GST-CoR constructs is also shown; hatched marking
indicates position of ICQII motif. The numbering shown
is based on the mNCoR sequence. hNCoR numbering is as follows: mNCoR
2,0632,142 correspond to hNCoR 2,0452,128; mNCoR 2,0632,105
correspond to hNCoR 2,0452,091; mNCoR 2,0632,119 correspond to
hNCoR 2,0452,105; mNCoR 2,0632,132 correspond to hNCoR
2,0452,118; mNCoR 2,0742,142 correspond to hNCoR 2,0562,128;
mNCoR 2,0842,142 correspond to hNCoR 2,0662,128; mNCoR 2,0932,142
correspond to hNCoR 2,1792,128; mNCoR 2,1072,142 correspond to
hNCoR 2,1932,128.
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As discussed previously, the most proximal portion of N2 contains the
I-C-Q-I-I CoRNR box sequence. It is known that mutations of this region
abolish binding to TR (16, 17, 18). However, it is not clear if the
differences in the distinct ID CoRNR box sequences contribute to the
specificity of corepressor recruitment. In fact, an examination of the
corepressor IDs shows that each contains a unique CoRNR box (Fig. 1A
).
For example, the N2 CoRNR box sequence is I-C-Q-I-I-, whereas the S2
CoRNR box sequence is I-S-E-V-I.
To define the importance of the N2 and S2 CoRNR box sequences in
mediating the specificity of interactions with NHRs, we made GST fusion
proteins composed of constructs that swap these domains. Thus, N2*
contains the S2 CoRNR box in the context of the full N2, and S2*
contains the N2 CoRNR box in the context of the full S2 (Fig. 6A
). As demonstrated in Fig. 6B
, on a
DR+5 element, RAR
binds S2 much more strongly than N2, both in the
context of the supershift and the decrease in the remaining RAR/RXR
heterodimer (compare lanes 6 and 14). Changing the S2 CoRNR box to that
of N2 (S2*) markedly decreases these interactions (lanes 14 and 18). In
contrast, changing the N2 CoRNR box to that of S2 (N2*) increases
interactions with RAR (lanes 6 and 10). Therefore, the S2 CoRNR box
plays a pivotal role in mediating the preference of RAR
for S2 (over
N2).

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Figure 6. The S2 CoRNR Box Mediates Specificity with RAR
A, Amino acid sequences of the proximal 27 amino acids of N2 and S2 are
indicated, along with sequences of the corresponding portions of mutant
constructs. B, Gel mobility shift assay carried out with 4 µl IVT
RAR ; 2 µl IVT RXR in even numbered lanes (or
unprogrammed reticulocyte lysate in odd numbered lanes);
0.02 µg (lanes 3, 4, 7, 8, 11, 12, 15, 16) or 1 µg (lanes 5, 6, 9,
10, 13, 14, 17, 18) GST-CoR construct; and DR+5 radiolabeled probe. C,
Gel mobility shift carried out with 4 µl IVT TRß1; 2 µl IVT RXR
in even numbered lanes (or unprogrammed reticulocyte
lysate in odd numbered lanes); 0.02 µg indicated
GST-CoR construct; and DR+4 radiolabeled probe. D, SDS-PAGE of GST-CoR
constructs. After analysis by SDS-PAGE, protein quantification was
performed by Bradford assay. Panel A, N2*; panel B, S2; panel C, S2*;
panel D, N2. Equivalent amounts of protein were used in each EMSA, as
indicated.
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In contrast, TRß1 binds N2 much more strongly than S2 on a DR+4
element (Fig. 6C
, lanes 3 and 7). Mutating N2 to be N2* does not,
however, decrease interactions with TR (lanes 3 and 5). Moreover,
changing S2 to S2* decreases, not increases, binding to TR (lanes 7 and
9). Therefore, even though the N2 CoRNR box is vital in the binding of
TR to NCoR, the specific N2 CoRNR box sequence (I-C-Q-I-I) does not
mediate the specific preference of TR for N2 (over S2). In fact, the TR
appears to bind the S2 CoRNR box sequence (I-S-E-V-I) as well as the N2
CoRNR box. Therefore, the region of N2 important for mediating this
specificity must reside elsewhere in N2. Analysis of GST constructs by
SDS-PAGE showed that all the constructs were produced appropriately
(Fig. 6D
); after SDS-PAGE, a Bradford assay was performed, so that
equivalent amounts of GST construct could be used in each
experiment.
As the motif just proximal to the N2 and S2 CoRNR boxes have also been
shown to be important in binding these IDs to NHRs (17), we next wanted
to determine whether these regions explained the phenomenon of
corepressor specificity. We therefore mutated N2 so that it would
contain the proximal domain and CoRNR box of S2 (m1); and S2 so it
would contain the proximal domain and CoRNR box of N2 (m2). As shown in
Fig. 7A
, however, these changes did not
in themselves alter the specificity of TR for N2; in particular, TR
binds m1 much stronger than m2 (see lanes 45). These data are in
agreement with the hypothesis that regions of N2 C-terminal to the
CoRNR box are also important in binding TR.

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Figure 7. A Distal Domain in N2 Is Required for Specificity
A, Gel mobility shift assay carried out with 4 µl IVT TRß1, 20 ng
indicated GST-CoR construct, and DR+4 radiolabeled probe. B, Gel
mobility shift assay carried out with 4 µl IVT TRß1, 20 ng (lanes
2, 3, 4, 5) or 1 µg (lanes 6, 7, 8, 9) indicated GST-CoR construct,
and DR+4 radiolabeled probe.
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To further define the region of N2 important for mediating specificity,
additional chimeric proteins were made, containing regions common to N2
or S2. m3 contains the proximal 23 amino acids of N2, followed by the
remainder of S2. m4 contains the proximal 23 amino acids of S2,
followed by the distal portion of N2 (Fig. 6A
). The first 23 amino
acids of N2 and S2 contain not only the CoRNR box sequences, but also
both regions immediately proximal and distal to this motif. As shown in
Fig. 7B
, N2 again binds TRß1 more strongly than S2 on a DR+4 response
element (compare lanes 2 and 3, or 6 and 7). When N2 is changed to m3
(so that it contains distal S2), significant TR binding is lost
(compare lanes 6 and 8). In contrast, when S2 is changed to m4 (so that
it contains the N2 distal region), binding is regained (compare lane 7
with lane 9). Thus, a region in the distal domain of N2 is important in
mediating the preference of TRß1 for N2. However, both m3 and m4 bind
TR more weakly than does N2, suggesting that both portions of N2 are
important in specifying strong TR binding (compare lanes 4 and 5 with
lane 2; or lanes 8 and 9 with lane 6).
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DISCUSSION
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The TRs and RARs repress gene transcription in the absence of
ligand. This action is mediated by the nuclear corepressor proteins,
NCoR and SMRT. NCoR and SMRT bind TR and RAR via their C-terminal
interacting domains. Ligand binding causes a conformation change in the
receptor, such that helix 12 rotates. This repositioning of the NHR
releases bound corepressor (8) and forms a hydrophobic
coactivator-binding surface (25). The L-x-x-L-L motifs present in
coactivators mediate binding to this pocket formed in the NHR
ligand-binding domain (LBD). Amino acids adjacent to this motif are
important in mediating the specificity of interactions with distinct
NHRs (21, 22, 23). In contrast, the amino acid sequences within the
corepressor IDs that dictate specificity in terms of interactions with
NHRs are less well defined.
Corepressor IDs are characterized by CoRNR box motifs: regions
containing the sequence I/L-x-x-I/V-I (16, 17, 18). It appears that the
portion of the NHR that contacts the corepressors is similar to the
portion that makes contact with coactivators, but that the position of
helix 12 is important to distinguish which class of cofactor is able to
bind. Thus TR and RAR bind corepressors in the absence of ligand, but
upon ligand binding, corepressors are released and coactivators are
recruited. In contrast, the estrogen receptor binds corepressors only
in the presence of antagonists such as 4-OH tamoxifen (26, 27, 28). This
antagonist binds the ligand-binding domain in such a way as to rotate
helix 12 to stabilize corepressor binding. Finally, RevErb binds NCoR
and has no known ligand. RevErb lacks a helix 12, thereby stabilizing
NCoR binding; moreover, side chains fill the ligand-binding cavity, and
it may therefore bind NCoR constitutively (29).
Specificity in cofactor recruitment is likely to play an important role
in NHR action. For example, distinct roles for coactivators are
becoming better understood through recent gene knockout experiments.
Knockout of steroid receptor coactivator 1 (SRC-1) causes a variety of
defects, including impaired decidual stimulation (a
progesterone-mediated response), impaired prostate growth in response
to androgen, and decreased mammary gland ductal branching (30). In
addition, these animals appear to have a degree of thyroid hormone
resistance (31). Although the lack of SRC-1 (NCoA-1) may be partially
replaced by an up-regulation in glucocorticoid receptor-interacting
protein 1 (GRIP-1) (TIF2/SRC-2), it is clear that not all SRC-1 action
is replaced (30). Interestingly, knock out of p/CIP
(SRC-3/TRAM-1/AIB1/ACTR) results in a more severe phenotype, including
growth retardation and delayed puberty (32). Thus, although
coactivators appear to have redundant roles in vitro, they
play more specific roles in vivo. In terms of the
corepressors, deletion of NCoR is embryonic lethal, suggesting that
NCoR and SMRT play distinct roles in development (15). In turn,
specificity in terms of corepressor recruitment by the distinct NHRs
may play an important role in mediating these distinct functions.
Whereas the region adjacent to the L-x-x-L-L motifs in coactivators
appear to play an important role in mediating specificity in terms of
interactions with NHRs, the domains important for specificity in terms
of corepressor recruitment are less well defined. Hu and Lazar (16)
have shown that RXR preferentially binds to N1 over N2 (16). We have
shown that TRß1 prefers to bind NCoR, and RAR
prefers to bind
SMRT, and that this preference is mediated by the proximal IDs (12). In
this report, we have examined the regions of N2 and S2 that mediate
preferential interactions. We have found that the I-S-E-V-I CoRNR box
motif in S2 is vital in dictating the preference of RAR
for S2. In
contrast, the I-C-Q-I-I CoRNR box motif in N2, while important for
binding, does not in itself dictate the preference of TRß1 for N2.
Regions downstream of the CoRNR box play a pivotal role in this
respect. Moreover, deletion of this C-terminal region of N2 impairs
binding to TRß1. Therefore, the TR requires additional sequences in
the corepressor IDs, distal to the CoRNR box, for optimal binding.
Our data also show that NCoR contains a third interacting domain,
N-terminal to the other two domains. This domain, which we term N3,
binds TRß1 strongly; it has also recently been described by another
group (20). Our data confirm the importance of N3 as an NCoR
interaction domain and also extends previous findings to establish a
role for N3 in mediating the specificity of corepressor interactions.
Our data suggest that N3 contributes to the preferential recruitment of
NCoR by TR.
In isolation, N3 binds TR almost as well as N2, and more strongly than
N1. In addition, mutation of the N3 CoRNR box impairs binding of NCoR
to TR to a greater degree than does mutation of N2 or N1. Therefore, N3
is likely to be an important domain for interactions of NCoR with TR.
Interestingly, a region analogous to N3 is not present in SMRT.
Therefore, the presence of N3 provides an important mechanism to
explain the TRß1 preference for NCoR over SMRT.
Not only does TR bind NCoR over SMRT, but the reverse is also true:
NCoR binds TR more strongly than it binds RAR. This is, in part, due to
the presence of the N3 domain. Although N3 binds TRß1 well, it binds
RAR
poorly. Mutating the N3 domain dramatically decreases
interactions of NCoRI with TR in cells but has no effect on the binding
of NCoR to RAR. Therefore, the N3 domain also plays an important role
in mediating the preferences of corepressors for the distinct NHRs.
Although N2 binds TRß1 well in EMSA, the isolated domain interacts
weakly with TRß1 in cells, as measured by the mammalian two-hybrid
system. However, constructs containing N3 and N2 interact strongly with
TRß1 in mammalian cells. These data suggest that two IDs are
necessary for full interactions with TRß1 in vivo. Prior
studies showed that deletion of N2 or N1 (in the presence of N3)
results in preserved functional activity of a dominant inhibitory form
of NCoR (NCoRI) in cells (33). Our current study extends this finding
and shows that mutation of any two ID CoRNR boxes blocks NCoR binding
to TR in EMSA, and significantly impairs NCoRI function in transient
transfections, particularly if N3 is one of the domains mutated. This
is consistent with the N3-N2 combination being sufficient for
functional interactions with the TR homodimer complex in
vivo. In fact, data from our group and others suggest that the NHR
complexes (homodimers and heterodimers) each bind a single NCoR with a
stoichiometry of one receptor dimer to one NCoR corepressor (12, 17, 18), with each interacting domain contacting a member of the dimer
pair.
If two NCoR IDs are necessary for binding, why are there three such
domains in NCoR? Our data suggest that the N3-N2 ID combination may be
the optimal pair to bind the TR-TR homodimer. It has been suggested
that RXR has a preference for the distal corepressor IDs (16).
Therefore, the binding of NCoR to TR-RXR heterodimers or TR-TR
homodimers may rely on different sets of IDs (Fig. 8
). In contrast, the preference of SMRT
for receptor heterodimers may depend, in part, on the lack of a region
homologous to N3 in SMRT.

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|
Figure 8. Binding of NCoR to Homodimers and Heterodimers Is
Distinct
Schematic model of the interactions between NCoR and TR-TR homodimers
and TR-RXR heterodimers. The presence of N3 allows for strong binding
of NCoR to the TR homodimer, whereas N1 may play a role specifically in
binding the TR-RXR heterodimer.
|
|
Thus these data support a model in which the specificity of
interactions between corepressors and NHRs depends on sequences within
the corepressor interacting domains, both within the CoRNR box motifs
(for RAR) and outside that motif. Moreover, these data suggest the
presence of a novel interacting domain (N3) that plays a key role in
binding TR and mediating the preference of TR homodimers for NCoR. The
differences between the NCoR and SMRT IDs may explain corepressor
specificity, and thus the distinct physiological roles that
corepressors have in vivo (15). A more detailed
understanding of the mechanisms by which the corepressor IDs mediate
this specificity will allow an opportunity to modulate these
effects.
 |
MATERIALS AND METHODS
|
---|
Plasmids
All GST fusion proteins were cloned into the vector PGEX-4T1 as
EcoRI fragments or EcoRI-XhoI
fragments. PCR was used to amplify the indicated amino acid sequences
from either human (h)NCoR or hSMRT, which were then placed downstream
of the GST moiety. A schematic illustration of these constructs is
shown in Fig. 6A
. GST-N2 includes amino acids (aa) from hNCoR
corresponding to mNCoR aa 2,0632,142 (34). GST-S2 includes hSMRT aa
2,1192,266. GST-N2* includes the same amino acid residues as GST-N2,
but with aa 2,0732,077 mutated to be ISEVI instead of ICQII.
Similarly, GST-S2* contains the same amino acids as GST-S2, but mutated
such that it contains ICQII instead of ISEVI (aa 2,0292,033). m1 is
similar to N2*, but additionally with Q in position 2071; similarly, m2
is equivalent to S2* but with D in position 2117. m3 contains the
proximal 23 aa of N2 and the distal 125 aa of S2; similarly, m4
contains the proximal 23 aa of S2 and the distal portion of N2.
PKCR2-NCoRI consists of a portion of hNCoR corresponding to amino acids
15792454 of mNCoR (23). Mutations of pKCR2-NCoRI were made using the
QuikChange Site Directed Mutagenesis Kit (Stratagene, La
Jolla, CA); the codon coding for the initial isoleucine of each CoRNR
box was mutated to code for alanine, either separately or in
combination.
Gal4-NCoR was made by cloning aa 1,5792,454 of NCoR downstream of the
sequence encoding the GAL4 DNA-binding domain in the SV40-driven
expression vector pECE. All other Gal4-N2 and Gal4-N3 constructs were
made by PCR and placed downstream of the GAl4-DNA binding domain as
either ECoRI or EcoRI-PstI fragments. Construct
integrity was confirmed by restriction endonuclease digestion and
dideoxy sequencing.
GST Fusion Proteins
GST fusion proteins were expressed in DH5
or BL21
Escherichia coli expressing thioredoxin by induction with
0.1 mM
isopropylthio-ß-D-galactosidase, as described
previously (12). Proteins were isolated by lysis with lysozyme and
purified on Sepharose beads. For experiments using EMSA, the bound
proteins were eluted using a glutathione buffer. Verification of
protein synthesis was obtained on SDS-PAGE. The amount of protein
generated was quantified by Bradford assay, such that equivalent
amounts of protein could be used in each EMSA.
EMSA
EMSAs were carried out as previously described (12, 35) with
either a 32P-radiolabeled DR+4 or DR+5 probe.
GST-corepressor proteins (GST-CoRs) were purified on Sepharose beads
and eluted using a glutathione buffer. Nuclear receptors were in
vitro translated in reticulocyte lysate (Promega Corp., Madison WI) using T7 polymerase. For each EMSA, 4 µl of
in vitro translated TR or RAR were used. For experiments
with RXR, 2 µl were used, or an equivalent amount of unprogrammed
reticulocyte lysate as a control. When NCoRI mutant constructs were
used, 4 µl of in vitro translated protein were added. When
GST-CoR constructs were used, the amount of GST protein used is
indicated in each figure. Quantification was determined by Bradford
assay. Incubations were carried out for 20 min, and complexes were
resolved on a 5% nondenaturing gel, followed by
autoradiography.
Cell Culture and Transfection
All transient transfections were performed in CV-1 cells, which
were maintained as previously described (33). Mammalian two-hybrid
transient transfections were performed in six-well plates using the
calcium phosphate technique, with each well receiving 1.7 µg of
upstream activating sequence-thymidine kinase luciferase reporter; 80
ng of Gal4-corepressor construct; 80 ng of VP16-TRß1 or VP16-RAR (or
empty vector VP16 as a control); and 20 ng of a cytomegalovirus (CMV)
ß-galactosidase construct. Fifteen to 18 h after transfection,
cells were washed in PBS and refed with 10% steroid hormone-depleted
FBS, as previously described. Forty to 44 h after transfection,
cells were lysed and assayed for luciferase and ß-galactosidase
activity. ß-Galactosidase was used to control for transfection
efficiency. Experiments were performed in triplicate. Data are
expressed as fold stimulation ± SEM.
For Western blot experiments of the Gal4 constructs, 10 µg of the
indicated construct were transiently transfected into CV-1 cells.
Twenty-four hours after transfections, cells were washed in PBS and
changed to fresh media; 24 h later, proteins from nuclear extracts
were isolated, run on SDS-PAGE, transferred to nitrocellulose, blotted
using a Gal4 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), and visualized by ECL+
(Amersham Pharmacia Biotech, Arlington Heights, IL).
For transient transfections analyzing the ability of NCoRI or NCoRI
mutants to reverse basal repression, 1.7 µg DR+4-pA3Luc, 80 ng of
pKCR2-TRß1, 160 ng of pKCR2-NCoRI construct (or empty vector pKCR2),
and 20 ng of a CMV ß-galactosidase construct were transfected. After
transfection, the procedure was the same as outlined above for the
mammalian two-hybrid assay. Data are expressed as relative luciferase
activity, after correcting for ß-galactosidase activity, ±
SEM.
GST Protein Interaction Assay
GST-TR was expressed in BL21 E. coli expressing
thioredoxin by induction with 0.1 mM
isopropylthio-ß-D-galactosidase. The proteins
were isolated with lysis by lysozyme and purified on sepharose beads.
Verification of protein synthesis was obtained on SDS-PAGE. GST-TR was
incubated with 4 µl 35S methionine-labeled
in vitro translated NCoRI construct. The concentration of
T3 used was 10-6
M. After extensive washing, the bound proteins
were eluted by boiling in loading buffer and run on SDS-PAGE.
 |
ACKNOWLEDGMENTS
|
---|
We would like to thank R. Evans, C. Glass, and N. Moghal for
plasmids, and A. Takeshita for thioredoxin-expressing BL21 E.
coli.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Anthony N. Hollenberg, Thyroid Unit, Department of Medicine, Beth Israel-Deaconess Medical Center, 330 Brookline Avenue Research North 325, Boston, Massachusetts 02215. E-mail: THollenb{at}caregroup.harvard.edu
This work was supported by NIH Grants to R. Cohen (DK-02581), F.
Wondisford (DK-49126 and DK-53036), and A. Hollenberg (DK-56123).
Received for publication February 7, 2001.
Revision received March 29, 2001.
Accepted for publication April 2, 2001.
 |
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