The Nuclear Corepressors Recognize Distinct Nuclear Receptor Complexes
Ronald N. Cohen,
Andrew Putney,
Fredric E. Wondisford and
Anthony N. Hollenberg
Thyroid Unit Department of Medicine Beth Israel Deaconess
Medical Center and Harvard Medical School Boston, Massachusetts
02215
 |
ABSTRACT
|
---|
The thyroid hormone receptor (TR) and retinoic
acid receptor (RAR) isoforms have the capacity to silence gene
expression in the absence of their ligands on target response elements.
This active repression is mediated by the ability of the corepressors,
nuclear receptor corepressor (NCoR) and silencing mediator of retinoid
and thyroid hormone receptors (SMRT), to recruit a complex
containing histone deacetylase activity. Interestingly, NCoR and SMRT
share significant differences in the their two nuclear
receptor-interacting domains (IDs), suggesting that they may recruit
receptors with different affinities. In addition, the role of the
receptor complex bound to a response element has not been fully
evaluated in its ability to recruit separate corepressors. We
demonstrate in this report that the proximal ID in NCoR and SMRT, which
share only 23% homology, allows preferential recognition of nuclear
receptors, such that TR prefers to recruit NCoR, and RAR prefers to
recruit SMRT, to DNA response elements. However, mutations in the TR
found in the syndromes of resistance to thyroid hormone can
change the corepressor recruited by changing the complex (homodimer or
heterodimer) formed on the TRE. These results demonstrate that the
corepressor complex recruited can be both nuclear receptor- and
receptor complex-specific.
 |
INTRODUCTION
|
---|
The thyroid hormone (TR) and retinoic acid receptor (RAR) isoforms
are members of the nuclear receptor superfamily (1). Unlike the
majority of the members of this family, the TR and RAR possess
ligand-independent activity that leads to the silencing of positively
regulated target genes. This silencing activity has been shown to be
due to the recruitment of at least two nuclear corepressor proteins,
nuclear receptor corepressor (NCoR) and silencing mediator of retinoid
and thyroid hormone receptors (SMRT) (2, 3, 4, 5, 6, 7), which, in turn,
recruit a multiprotein complex with histone deacetylase activity that
appears to modify chromatin to prevent transcription (8, 9, 10). In the
presence of their cognate ligands, the TR and RAR isoforms release the
nuclear corepressors and recruit members of the coactivator family,
which include the p160 family members [steroid receptor coactivator-1
(SRC-1), TIF II, and ACTR], CREB-binding protein (CBP) and
p300, pCAF (11, 12, 13, 14, 15, 16, 17, 18) (reviewed in Ref. 19), and other coactivators such
as p120 (20). Unlike the nuclear corepressors, the coactivator complex
possesses histone acetyl transferase activity, which allows for
transcriptional activation.
NCoR and SMRT are modular proteins (see Fig. 1
) that contain at least three repressing
domains in their N termini, and two domains that mediate interactions
with the TR and RAR isoforms in their C termini (21, 22, 23, 24). In addition
to mediating interactions with mSin3, Sun-CoR (25), and other members
of the corepressor complex, the central domains of NCoR and SMRT also
appear to mediate interactions with the AML-ETO product, which may
prevent normal differentiation and lead to acute myelogenous leukemia
in patients with this chromosomal translocation (26). In addition, the
extreme amino-terminal domain of NCoR appears to be important in the
regulation of the mature protein through its interaction with mSIAH2,
which allows for proteolytic degradation of NCoR (27).

View larger version (15K):
[in this window]
[in a new window]
|
Figure 1. The Nuclear Corepressor Family
Amino acid sequences of NCoR and SMRTe are indicated. The NCoR
interacting domains are outlined and numbered (22 ).
Corresponding portions of the homologous regions of SMRTe are also
identified.
|
|
Both NCoR and SMRT contain two C-terminal interacting domains that
mediate interactions with both the TR isoforms and the RAR isoforms.
While similar in structure, the more proximal of the interacting
domains (ID 2) shares only 23% amino acid homology, while the more
distal interacting domains (ID 1) are 53% homologous. Consistent with
these differences in structure are results from several different
groups, including our own, which suggest that NCoR and SMRT interact
differently with nuclear receptors and that specificity may exist in
the recruitment of nuclear corepressors by nuclear complexes. For
example, it has been demonstrated by Zamir et al. (22) that
the orphan receptor RevErb can interact only with NCoR on its DNA
response element and is unable to recruit SMRT. We have demonstrated
that the TRß1 isoform preferentially recruits NCoR rather than SMRT
to a DR + 4 response element (24). In addition, using
glutathione-S-transferase (GST) pull-down assays and the mammalian
two-hybrid system, Wong and Privalsky (28) have shown that separate RAR
isoforms can recruit SMRT with different affinities. Taken together,
these data indicate that the polypeptides present in the interacting
domains of NCoR and SMRT allow for specific interactions with nuclear
receptors that may allow for separate biological actions in
vivo.
The interactions between the corepressors and the TR isoforms are also
influenced by complex formation. We and others have demonstrated that
the TRß1 isoform recruits NCoR preferentially as a homodimer on DNA
and that the addition of retinoid X receptor (RXR) causes a diminution
in corepressor binding. Indeed, NCoR appears to stabilize the homodimer
complex in solution where it normally does not form (24). In contrast,
work using the mammalian two-hybrid system and the TR
1
ligand-binding domain (LBD) has demonstrated that RXR can enhance
interactions with nuclear corepressors (29). However, this study
investigated TR-corepressor interactions in the absence of an
underlying thyroid hormone response element, and did not utilize
full-length TRs. To address these issues, we studied TRß1 and RAR
and examined their ability to recruit either NCoR or SMRT to their
cognate response elements. In addition, we examined the ability of RXR
to influence corepressor recruitment in the context of specific nuclear
receptors. By using a TR mutant that is defective in its ability to
homodimerize, we demonstrate that the TR complex present on a native
response element determines the nature of the corepressor recruited.
Our data demonstrate that the polypeptides that represent the
corepressor interacting domains appear to recognize both specific
nuclear receptors and the complexes that they form.
 |
RESULTS
|
---|
The Proximal Corepressor Interacting Domains Allow for Specific
Interactions with TRs and RARs
To delineate the specificity of NCoR and SMRT for the nuclear
hormone receptors, TR and RAR, we constructed plasmids that express
either both receptor-interacting domains as GST fusions, or the
individual interacting domains as GST fusion proteins (Fig. 2
). We have shown previously using
in vitro translated (IVT) proteins that the TRß1 isoform
prefers NCoR over SMRT (24). Similar data are seen in electrophoretic
mobility shift assay (EMSA) using the GST fusion proteins representing
both interacting domains of NCoR and SMRT on a DR+4 element (Fig. 3A
; compare, for example, lanes 3 and 5,
or 7 and 9). Both NCoR and SMRT bind TRß1 on this thyroid hormone
response element (TRE), but NCoR is bound significantly more avidly
than SMRT. This preference is seen over a wide range of amount of GST
protein used, although it is seen most dramatically with lower amounts
of protein (20100 ng). The shifts associated with corepressor binding
result in a decreased amount of remaining TRß1 homodimer (compare
lane 1 with lanes 3, 7, and 11). The addition of RXR causes a loss of
corepressor binding (lanes 4, 6, 8, 10, 12, and 14). There is no
further supershift in the presence of RXR (see especially lanes 4 and
8), consistent with specific binding to the TR homodimer. These data
suggest that the TRß1 homodimer binds corepressors more avidly than
does the TRß1-RXR heterodimer.

View larger version (24K):
[in this window]
[in a new window]
|
Figure 2. NCoR and SMRT-Interacting Domain Constructs
A, Schematic illustration of GST-corepressor (GST-CoR) constructs.
The amino acid sequences of the interacting domains are indicated. The
locations of the GST-CoR constructs are indicated by
bars, and their specific amino acid sequences are
identified in Materials and Methods. In particular, the
amino acid sequences of the individual interacting domain constructs
are as follows: GST-N1 (aa 22392300); GST-N2 (aa 20632142); GST-S1
(aa 22672507); GST-S2 (aa 20982266). B, SDS-PAGE of GST-CoR
constructs. After analysis by SDS-PAGE, protein quantification was
performed by Bradford assay, so that equivalent amounts of protein
constructs could be used in each EMSA. 1, ladder; 2, GST alone; 3,
GST-N1; 4, GST-N2; 5, GST-S1; 6, GST-S2; 7, GST-N2S1; 8,
GST-S2N1; 9, GST-NCoR; 10, GST-SMRT.
|
|

View larger version (50K):
[in this window]
[in a new window]
|
Figure 3. TRß and RAR Show Distinct Preferences for the
Nuclear Corepressors
A, Gel mobility shift assays were carried out using 4 µl IVT
TRß1; 2 µl IVT RXR; 20 ng, 100 ng, or 1 µg GST-CoR complex (as
noted); and DR+4 radiolabeled probe. B, Gel mobility shift assays
were carried out using 4 µl IVT RAR ; 2 µl IVT RXR; 20 ng, 1
µg, or 2 µg GST-CoR complex (as noted); and DR+5 radiolabeled
probe. When no RXR was used, an equal amount of unprogrammed (UP)
reticulocyte lysate was added in its place. CoR shift indicates
mobility shift(s) caused by the binding of indicated corepressor
interacting domain constructs.
|
|
In contrast, on a DR+5 element (Figure 3B
), which is a retinoic acid
response element, RAR
binds corepressor solely when heterodimerized
to RXR (compare lanes 9 and 10, and 13 and 14). Furthermore, the
RAR
/RXR heterodimer binds SMRT, but does not interact well with NCoR
(compare, for example, lanes 78 with 910). The shifts associated
with SMRT binding result in a decreased amount of remaining RAR
/RXR
heterodimer (compare lane 2 with lanes 10 and 14). In contrast to
TRß, when 20 ng (lanes 36) or 100 ng (data not shown) of GST
protein are used, no specific binding is detected, and it is only at
higher amounts (lanes 714) that the preferential binding of the
RAR/RXR heterodimer to SMRT is identified. However, when identical
amounts of protein are used (e.g. 1 µg), the RAR-RXR
heterodimer prefers to interact with SMRT, while the TRß1 homodimer
prefers to interact with NCoR.
To delineate which of the interacting domains (IDs) mediates this
specificity, we next performed EMSA with TRß1 or RAR
using
bacterially expressed GST proteins containing individual interacting
domains (Fig. 4
, A and B). As Fig. 3
had
demonstrated that differences in corepressor binding to TRß1 are most
apparent with 20 ng of GST fusion protein (lanes 36), this amount of
protein was used in Fig. 4A
; similar data were obtained when 1
µg of GST fusion proteins was used (data not shown). In contrast,
since binding to RAR could only be clearly detected using higher
amounts of GST fusion proteins, 1 µg of GST protein was used in the
EMSA in Fig. 4B
.
As is demonstrated in Fig. 4A
, the proximal ID 2 allows for specificity
in that N2 strongly binds the TRß1 homodimer (lane 5) while S2 does
not bind the TRß1 isoform well (lane 7) on the DR+4 element. In
contrast, on the retinoic acid response element (RARE) (Fig. 4B
), RAR interacts well with S2 (lanes 78) and does not bind N2
well (lanes 56). These equivalent domains share only 23% homology at
the amino acid level, suggesting that specificity is encoded for within
the ID 2 polypeptide. With the amount of GST protein used in each of
these experiments, neither ID 1 from NCoR or SMRT was able to bind to
either receptor complex on DNA response elements. However, binding of
N1 to the TR homodimer could be seen when greater amounts of protein (1
µg) were used. In addition, N1 binding was enhanced by sequences 3'
to its defined boundary (N1', see Fig. 2
), but never approached the
strength of N2 binding. Interestingly, the individual interacting
domains (for example N2 and S2) bound optimally to TR and RAR in the
absence of RXR (compare Fig. 4A
, lanes 5 and 6, and Fig. 4B
, lanes 7
and 8). Although the RAR homodimer is not believed to exist in
vivo, these data suggest that under certain circumstances it might
be stabilized by interacting proteins. Importantly, however, in the
context of both interacting domains together, RAR binds corepressors
solely as a heterodimer with RXR (Fig. 3B
, lanes 10 and 14).
To confirm that indeed the proximal ID 2 was responsible for
receptor specificity, we next swapped the distal ID 1 region among the
NCoR and SMRT constructs, creating chimeric interacting domains
consisting of N2S1 and S2N1 (Fig. 4
, C and D). As expected, the TRß1
isoform homodimer preferred the N2S1 chimera (Fig. 4C
, lane 3), while
the RAR/RXR heterodimer preferred to interact with the S2N1 chimera
(Fig. 4D
, lane 6). Note that since the chimeric constructs bound TR and
RAR more weakly than did wild-type constructs, 2 µg of chimeric
constructs were used in Fig. 4D
. However, these data again suggest that
the proximal interacting domains mediate the preferential interactions
of TR and RAR for NCoR and SMRT, respectively, and that that the distal
IDs do not exert restrictive properties in this context. Moreover,
while the individual S2 domain bound RAR well in the absence of RXR
(see above), the addition of either S1 or N1 resulted in enhanced
binding to the RAR-RXR heterodimer (see Fig. 3B
, lane 10; and
Fig. 4D
, lane 6). These data suggest that portions of the NCoR and SMRT
interacting domains aid in the recognition of receptor complexes
(homodimer or heterodimer).
To define the effects of ligand on the interactions between nuclear
receptors and the individual corepressor interacting domains, we
performed an EMSA with TRß1 in the presence or absence of
T3. As shown in Fig. 5A
, TRß1 prefers to bind NCoR over SMRT
(compare lanes 5 and 9). The addition of T3
results in decreased homodimer formation (lanes 1 and 3). In addition,
both NCoR and SMRT are released in the presence of
T3 (compare lanes 5 and 7, and lanes 9 and 11).
As shown in Fig. 5B
, TRß1 binds strongly to N2 (lane 1), and this
interaction is disrupted by T3 (lane 3). Finally,
TRß1 binds weakly to S2, and only in the absence of
T3 (lanes 5 and 7).

View larger version (40K):
[in this window]
[in a new window]
|
Figure 5. The Binding of CoR-Interacting Domains to TR Is
Blocked by Ligand
A, Gel mobility shift assay was carried out using 4 µl IVT TRß1; 2
µl RXR (or an equivalent amount of unprogrammed reticulocyte lysate);
20 ng of indicated GST-CoR construct; DR+4 radiolabeled probe; and
T3, where indicated. The concentration of T3
used was 100 nM. B, Gel mobility shift assay carried out as
in panel A, using 20 ng of indicated GST-CoR construct.
|
|
Given the ability of TRß1 to interact well with NCoR in EMSA,
we next asked whether the TRß1 homodimer recruits a single NCoR, or
whether more than one NCoR binds the homodimer (i.e. perhaps
each partner independently binds a separate NCoR molecule). To do this,
we used the EMSA assay with two different sized proteins, each
representing both interacting domains of NCoR as shown in Fig. 6A
. GST-NCoR includes NCoR amino acids
20632300 downstream of GST. IVT-NCoR consists of NCoR amino acids
15792454. If the TRß1 homodimer bound two NCoR molecules, we would
expect a mobility shift between the two individual bands produced
(consisting of a TRß1 homodimer binding one GST-NCoR and one
IVT-NCoR). As the TRß1 homodimer forms separate complexes with each
NCoR protein, it appears more likely that the
TRß1 homodimer binds a single NCoR molecule. We next performed a
similar experiment using two different sized N2 constructs: GST-N2
(which contains amino acids 20632142) and IVT-N2 (which contains
amino acids 15792211). As shown in Fig. 6B
, again no intermediate
band is seen, suggesting that each homodimer complex is able to bind a
single interacting domain. This is specific to the TR homodimer, as
Figs. 3B
and 4D
above suggest that the presence of a second interacting
domain does influence receptor complex recruitment.

View larger version (40K):
[in this window]
[in a new window]
|
Figure 6. The TRß1 Homodimer Binds a Single NCoR
A, Gel mobility shift assay was carried out using 4 µl TRß1; 4 µl
IVT human NCoR construct (IVT NCoR) or unprogrammed reticulocyte lysate
(UP); 20 ng GST-NCoR; and a DR+4 radiolabeled probe. The IVT hNCoR
construct (IVT NCoR) spans NCoR aa 15792454 and has been previously
described (24 ). B, Gel mobility shift assay using 4 µl IVT TRß1; 4
µl IVT human N2 construct (IVT N2) or unprogrammed reticulocyte
lysate (UP); 20 ng GST-N2; and a DR+4 radiolabeled probe. The IVT N2
spans aa 15792211 and has been previously described (24 ).
|
|
Mutant TRs Found in Syndromes of Resistance to Thyroid Hormone
Suggest that the TR Complex Also Determines the Corepressor
Recruited
The syndromes of resistance to thyroid hormone
(RTH) have been linked to mutations in the TRß gene and are generally
inherited in an autosomal dominant fashion (30). Affected patients
have elevated free thyroid hormone levels, with a nonsuppressed
TSH, and exhibit variable tissue resistance to the actions of thyroid
hormone. To determine whether specificity in the context of corepressor
recruitment is complex-specific, as well as nuclear hormone
receptor-specific, we took advantage of mutations in TRß found in
kindreds with RTH that affect receptor complex formation on thyroid
hormone response elements. We first employed a natural mutation of the
ninth heptad of the TR, R429Q. This mutation has been shown to be
defective in its ability to homodimerize but to still retain its
ability to silence gene expression on positive TREs (31, 32). Patients
with this mutation clinically have impaired central
T3 action, but preserved peripheral
T3 action, resulting in selective pituitary
resistance to thyroid hormone, or PRTH. We analyzed the ability of
R429Q TRß1 to recruit either NCoR or SMRT using EMSA. As is shown in
Fig. 7A
, the R429Q mutation in context of
the TRß1 isoform is unable to homodimerize well and does not recruit
NCoR either as a homodimer or a heterodimer (lanes 56). However, the
R429Q-RXR heterodimer is able to recruit the SMRT interacting domains
to the DR+4 binding site (lane 8). Of note, the R429Q mutant does not
bind either NCoR or SMRT well at low amounts (i.e. 20 ng;
lanes 14) and required higher amounts of GST corepressor construct
than did wild-type TRß1 to exhibit strong corepressor binding in
EMSA. However, these data are consistent with the hypothesis that the
structure of the complex influences specific corepressor
recruitment.

View larger version (35K):
[in this window]
[in a new window]
|
Figure 7. Certain TR Mutants Exhibit Altered Interactions
with Corepressors
A, Gel mobility shift assay was carried out using 4 µl IVT R429Q
TRß1; 2 µl RXR (or unprogrammed reticulocyte lysate); and 20 ng or
1 µg of indicated GST-CoR construct (as indicated); and a DR+4
radiolabeled probe. B, Gel mobility shift assay was carried out
using 4 µl IVT 337T TRß1, 2 µl RXR (or unprogrammed
reticulocyte lysate); 20 ng or 1 µg of indicated GST-CoR construct
(as indicated); and a DR+4 radiolabeled probe.
|
|
We next examined another mutant TR found in certain kindreds
with RTH,
337T. This mutant TR does not bind
T3, and patients with this mutation have severe
generalized resistance to thyroid hormone (GRTH) (30). In contrast to
R429Q TRß1, the
337T TRß1 mutant homodimerizes well on thyroid
hormone response elements. As shown in Fig. 7B
,
337T TRß1 binds
NCoR more strongly than SMRT (compare lanes 1 and 3). As with wild-type
TRß1, this preference is seen most dramatically at lower amounts of
GST fusion proteins used (compare lanes 14 with 58), although it is
also seen at higher amounts (compare lanes 5 and 7). Thus,
337T
TRß1 is an example of a TR mutant that exhibits enhanced homodimer
formation and preserved corepressor specificity.
SMRT and NCoR Recruit Distinct Nuclear Receptor Complexes in
Cells
To complement the EMSA assay and determine whether SMRT and NCoR
would prefer to interact with homo- or heterodimers, we employed a
two-hybrid assay in mammalian cells. In this system, we fused the
interacting domains of NCoR and SMRT to the Gal4-DNA-binding domain and
fused full-length nuclear receptors downstream of the VP-16 activation
domain. Full-length nuclear hormone receptors were used instead of the
corresponding LBDs, as we and others have demonstrated that the amino
termini influence complex formation (33, 34). In Fig. 8A
, the basal activity of Gal4-NCoR or
Gal4-SMRT is set at 1; the interactions between the Gal4-corepressor
constructs and the nuclear receptor-VP16 constructs is then expressed
as relative luciferase activity. As shown in Figure 8B
, the experiments
were next done in the presence of cotransfected RXR-VP16 to examine the
effect of heterodimerization on corepressor interactions. These data
are presented as fold expression in the presence vs. absence
of cotransfected RXR-VP16. A ratio greater than 1 implies greater
luciferase activity in the presence of RXR.

View larger version (35K):
[in this window]
[in a new window]
|
Figure 8. Corepressors Recognize Receptor Complexes
A, CV-1 cells were cotransfected with 1.7 µg of UAS reporter; 80
ng of the indicated Gal4 construct; and 80 ng of indicated TR-VP16 or
RAR-VP16 construct. Data are expressed as the relative luciferase
activity compared with basal levels of Gal4-NCoR (upper
panel) or Gal4-SMRT (lower panel) alone
(mean ± SE). B, CV-1 cells were transfected as in
panel A, but additionally in the presence of 80 ng RXR-VP16 (or
EV-VP16). Data are expressed as the fold enhancement of activation of
luciferase activity in the presence vs. absence of
cotransfected RXR-VP16 (mean ± SE). C, CV-1
cells were transfected as in panel B, but instead in the presence of
320 ng RXR-pKCR2 (or EV-pKCR2). Data are expressed as the fold
enhancement of activation of luciferase activity in the presence
vs. absence of cotransfected RXR-pKCR2 (mean ±
SE). D, CV-1 cells were transfected with 1.7 µg of a UAS
reporter; 80 ng Gal4-NCoR or Gal4-SMRT; 80 ng of the LBDs of wild-type
TRß or R429Q TRß placed downstream of VP16; and 80 ng of RXR-VP16
or EV-VP16. In the upper panel, data are expressed as
the relative luciferase activity compared with basal levels of
Gal4-NCoR or Gal4-SMRT alone (mean ± SE). In the
lower panel, data are presented as the fold enhancement
of activation of luciferase activity in the presence vs.
absence of cotransfected RXR-VP16. E, CV-1 cells were transfected with
1.7 µg UAS reporter, 80 ng of indicated Gal4 construct; and 80 ng of
TRß1-VP16 or RAR -VP16. Data are expressed as relative luciferase
activity compared with basal levels of Gal4 construct alone (mean
± SE).
|
|
As shown in Fig. 8A
(upper panel), transfection of
TRß1-VP16 with Gal4-NCoR causes approximately 80-fold stimulation
over the activity of Gal4-NCoR alone. In contrast, transfection of
RAR
-VP16 with Gal4-NCoR causes only 25- to 30-fold stimulation over
the activity of Gal4-NCoR. Finally, R429Q TRß1-VP16 only minimally
interacts with Gal4-NCoR in the absence of RXR. We next used the
identical paradigm with Gal4-SMRT as the bait (Fig. 8A
, lower
panel). Cotransfection of RAR
-VP16 with Gal4-SMRT caused a
40-fold stimulation over the activity of Gal4-SMRT alone. In contrast,
TRß1-VP16 caused only a 6- to 7-fold stimulation in luciferase
activity. Again, R429Q TRß1-VP16 did not interact well in the absence
of cotransfected RXR. All of the nuclear receptor-VP16 constructs
interact to a similar degree with Gal4-RXR (data not shown), suggesting
that they are expressed at similar levels in cells.
These constructs were then cotransfected with RXR-VP16; as shown
in Fig. 8B
, cotransfection with RXR-VP16 actually decreased the
interactions between TRß1-VP16 and Gal4-NCoR. In contrast, the
interactions of R429Q TRß1-VP16 with Gal4-NCoR was enhanced 18-fold
by the cotransfection of RXR. Although RXR-VP16 decreased interactions
between RAR
-VP16 and Gal4-NCoR (upper panel),
cotransfection of RXR-VP16 enhanced the interactions between this
receptor and Gal4-SMRT (Fig. 8B
, lower panel). Additionally,
our previous data (Figure 3B
) had showed that, in the presence of a
RARE, RXR is important for the binding of RAR to SMRT. In addition,
although R429Q TRß1-VP16 interacted minimally with Gal4-SMRT in the
absence of RXR, the interaction was enhanced more than 20-fold when
RXR-VP16 was cotransfected. In fact, in the mammalian two-hybrid
system, the interaction between TRß1-VP16 and Gal4-SMRT was also
enhanced by the cotransfection of RXR (
6-fold). Although RXR-VP16
itself interacted minimally with Gal4-SMRT (data not shown), it did not
interact to an extent where it could affect the synergistic level of
interactions seen. Thus, SMRT appears to particularly favor receptor
heterodimers. Moreover, R429Q TRß1, a mutant TR, appears to interact
with corepressors mainly as a heterodimer with RXR, both in solution
and on a thyroid hormone response element.
While RXR-VP16 was used to keep the total amount of VP16 moiety
constant in each dimer pair, we next repeated these experiments in the
presence of RXR-pKCR2. As shown in Fig. 8C
, similar results are seen
when RXR-pKCR2 is used instead (although the values are less
pronounced). Again, the presence of RXR is more important in the
context of receptor interactions with SMRT. In addition, cotransfection
of RXR-pKCR2 increased interactions between R429Q TRß1 and the
Gal4-corepressor constructs 5- to 10-fold (data not shown).
While other studies have suggested that RXR can enhance the
interaction between TRß1 and NCoR, these studies used the LBD of the
TR linked to VP16 (29). We therefore used the same paradigm as above in
Fig. 8D
, in the context of the nuclear receptor LBDs. In these
experiments, RXR-VP16 had significant impact on the strength of the
interaction of the R429Q LBD (
117-fold), and, in contrast to the
full-length TR (see above), also stimulated interactions when
cotransfected with the wild-type LBD (although only 9-fold). In
addition, the ability of Gal4-SMRT to recruit both wild-type and R429Q
mutant LBDs was greatly enhanced by the addition of RXR. In contrast to
the interactions seen with Gal4 NCoR, the interaction of the LBD of
R429Q with Gal4 SMRT was enhanced by more than 300-fold by the addition
of VP16-RXR, consistent with the preferences noted on the DR+4 element
in EMSA. Both the absolute luciferase activity (with RXR) and the
fold-enhancement by RXR were greater in these experiments (as opposed
to experiments using full-length nuclear receptors), suggesting that
the amino termini, and potentially the DNA-binding domains, of TR
modulate interactions with corepressors.
To examine the specificity of the individual interacting domains
in cells, constructs containing N2 or S2 were placed downstream of the
Gal4 DNA binding domain, and used in a similar mammalian two-hybrid
system. As shown in Fig. 8E
(upper panel), transfection of
TRß1-VP16 with Gal4-N2 causes approximately 100-fold stimulation. In
contrast, cotransfection with RAR-VP16 results in only about half that
level of activity. While interactions with Gal4-S2 were weaker than
with Gal4-N2, Fig. 8E
(lower panel) shows that Gal4-S2
interacts strongly with RAR-VP16, but only minimally with TRß1-VP16,
consistent with the EMSA data (e.g. Fig. 4B
).
 |
DISCUSSION
|
---|
The identification and cloning of the nuclear
corepressors have allowed for an understanding of the mechanisms by
which members of the NR family regulate gene expression in the absence
of ligand or in the presence of antagonists that recruit either NCoR or
SMRT (35, 36). In addition to the NRs, NCoR and SMRT can also be
recruited by the oncoproteins PML-RAR, PZLF-RAR, and AML-ETO and thus
may be linked to human disease by blocking myeloid differentiation
through their ability to block transcription of target genes (26, 37, 38). The role of the corepressors in NR action in vivo has
not been confirmed, although it is tempting to speculate that, in the
context of thyroid hormone action, the corepressors are important in
the manifestations of hypothyroidism and in the presentation of the RTH
syndromes (39, 40, 41). TR isoforms are able to widely regulate gene
expression in a specific fashion based on their ability to bind to
cognate response elements in the regulatory regions of target genes.
Once bound to the elements, the TRs recruit members of the corepressor
family (NCoR/RIP13, SMRT/TRAC1) in the absence of ligand, which
mediates ligand-independent repression on positive TREs. The presence
of T3 causes the corepressor complex to be
released and the coactivator complex, which includes members of the
p160 family, pCAF, CBP/p300, and possibly other coactivator molecules,
to be recruited. Specificity in the context of cofactor recruitment and
its ramifications for gene expression have not been ascertained. Recent
work in the context of the p160 family suggests that although the
LxxLL-containing motifs (LXDs) mediate specific interactions with
members of the NR family, the surrounding sequences and the spacing
between LXDs are critical for nuclear receptor specificity
(42, 43, 44, 45).
Recent work has suggested that the L/I-x-x-I/V-I motifs in the
corepressor IDs are required for interactions with nuclear receptors
(46, 47, 48). Both NCoR and SMRT contain two separate IDs, which have been
independently identified by a number of separate groups. The more
proximal ID2 of murine and human NCoR shares limited sequence homology
with the homologous region of SMRT (23%), whereas the distal ID 1 of
murine and human NCoR share approximately 50% amino acid homology.
Based on these differences, we and others have hypothesized that the
two corepressors may differentially recruit nuclear receptors. Initial
studies using EMSA demonstrated that murine (m) NCoR and human (h) SMRT
could interact with both the TR and RAR on their respective response
elements (3, 4). However, these studies did not examine specificity in
context of the interactions, nor did they examine the complexes with
which the corepressors may interact. Zamir et al. (49)
demonstrated that both TR isoforms could interact with NCoR and SMRT on
a thyroid hormone response element (TRE) but did not assess
differences in binding, nor did they examine the specificity of the
individual interaction domains. Wong and Privalsky examined
interactions between individual interaction domains and a number of
nuclear receptor isoforms. These data were generated in solution assays
(GST pull-down and mammalian two-hybrid assays) and did not incorporate
the role of DNA binding into corepressor recruitment. However, these
results showed that specificity in corepressor recruitment exists,
especially in the context of RAR isoforms and the amino acid sequences
present in the hinge region (28). More recently, Hu and Lazar showed
that the distal NCoR interacting domain interacts well with RXR,
whereas the proximal domain vastly prefers TR (46). These data
suggested that the corepressor interacting domains might recognize
receptor complexes, which we have examined in this report.
In the present study, we have examined the role of the TRß1 and
RAR
isoforms to recruit nuclear corepressors to TREs and RAREs,
respectively. In addition, we have further evaluated the role of RXR in
corepressor recruitment to ascertain the role of homo- vs.
heterodimerization in corepressor recruitment. Our data suggest that
the TRß1 homodimer preferably recruits NCoR while the RAR
/RXR
heterodimer preferably recruits SMRT on DNA response elements. This
specificity appears to map the more proximal ID 2 region, as shown in
Fig. 4
. Furthermore, chimeric corepressors require the SMRT ID 2 region
(S2N1 or S2S1) to interact with the RAR. In contrast, the ID 2 of NCoR
preferably binds the TRß1 homodimer. These data are consistent with
the marked differences in the ID 2 regions between NCoR and SMRT and
suggest that this region may exert specificity in the recognition of
the TR and RAR.
Given the differences in the ability of the TR and RAR to recruit NCoR
and SMRT, we next investigated the ability of TRß mutants found in
kindreds with RTH to recruit the corepressors to a TRE. Surprisingly,
R429Q TRß1 exhibited altered corepressor specificity in the context
of an underlying TRE. R429Q TRß1 preferred to interact with SMRT
rather than NCoR on a DR+4 element and did not interact with
corepressors well as a homodimer. Previous studies by others using the
R429Q mutant demonstrated an inability to release SMRT as compared with
the wild-type TRß1 isoform but did not examine the preference of the
mutant TR for SMRT (40). Another RTH mutant
337T (30), which forms
strong homodimers, preferably recruits NCoR to the DR+4 element.
The mammalian two-hybrid system also displayed strong differences
between the R429Q mutant and wild-type TRß1. In this assay, the
interactions between the nuclear receptors and corepressors occur in
solution, so the effects of underlying DNA response elements can not be
assessed. However, this assay was used to assess the role of
heterodimerization in corepressor recruitment in cells. In fact, the
addition of RXR enhanced the interaction of R429Q with NCoR and to a
greater degree with SMRT, suggesting that its heterodimer form allows
for the recruitment of SMRT. In contrast, RXR decreased the interaction
of wild-type TRß1 with NCoR, but increased interactions with SMRT in
this context. Interactions between RAR and SMRT were also enhanced by
the presence of RXR. Thus, SMRT appears to prefer to interact with
nuclear receptor-RXR heterodimers. Although SMRT can independently
interact with RXR in solution, the absolute value of this interaction
is weak when compared with the synergy imparted by the addition of RXR
to TR or RAR. Thus, the heterodimer is favorably recognized by SMRT,
while homodimer binding recognizes NCoR. Previous work by Zamir
et al. (22) demonstrated that the orphan receptor RevErb,
which binds DNA only as a homodimer, can recruit only NCoR but not
SMRT; these findings are consistent with the data demonstrated here,
suggesting that the corepressor interacting domains may recognize
complexes as well as specific nuclear receptors.
In addition, the experiments performed with the LBD alone of the
wild-type and mutant receptor fused to VP16 demonstrate that the A/B
and DNA-binding domains influence corepressor recruitment in the
presence or absence of RXR and help explain differences seen by a
number of groups in the role of RXR in corepressor recruitment (28, 29). The A/B domain (33, 34) and DNA-binding domain (50) have been
shown to influence receptor complex formation on TREs. Alterations in
complex formation may explain their influence on TR-corepressor
interactions. Alternatively, portions of the A/B and/or DNA-binding
domains might conceivably interact directly with the nuclear
corepressors.
The data presented here reinforce the need to examine cofactor
interactions in the context of DNA binding, given the likely
restrictions placed on the receptor once it is bound to DNA. Solution
interactions performed by us and others have demonstrated equivalent
interactions between NCoR and SMRT and the TR, whereas the introduction
of a TRE brings out the differences in the isoforms in their ability to
recruit corepressors. Indeed, Wong and Privalsky demonstrated, using
solution interactions, that the TR bound equally well to both of the
NCoR interaction domains, while on the DR+4 response element it is
clear that N2 is preferred over N1. In addition, the S1 domain appears
to act to enhance binding to the heterodimer, suggesting that it plays
a role in identifying complexes when bound to DNA, consistent with its
ability to bind RXR (28, 46).
In summary, the work discussed here demonstrates that both the complex
formed by the nuclear receptor and the DNA response element present in
the responsive promoter can dictate the preference for the corepressor
that is recruited. In addition, the corepressor interacting domains
appear to have preference for specific nuclear receptors, such that the
RAR
isoform prefers to interact with SMRT, which is mediated by the
proximal S2 domain. It will be interesting to discern which portion of
the interacting domains are important in the recognition of nuclear
receptors and how complex formation furthers this specificity.
 |
MATERIALS AND METHODS
|
---|
Plasmids
All GST fusion plasmids 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 hNCoR or hSMRT (see Fig. 2
), which were then placed in
frame downstream of the GST moiety. GST-NCoR includes amino acids (aa)
from human NCoR corresponding to mNCoR, aa 20632300; GST-N2
includes aa 20632142; and GST-N1 includes aa 22392300 (GST-N1'
includes aa 22392453). GST-SMRT includes hSMRTe (7), aa
20982507; GST-S2 includes aa 20982266; and GST-S1 includes aa
22672507. The chimeric N2S1 and S2N1 constructs were made by
amplifying the separate domains with an artificial XhoI site
introduced at the 3'-end of the proximal domain and 5'-end of the
distal domain. The pieces were then ligated into PGEX 4T1 as
EcoRI fragments. GST-N2S1 includes amino acids from hNCoR
corresponding to mNCoR, aa 20632142, and hSMRTe aa 22672507;
GST-S2N1 includes hSMRTe, aa 20982266, and NCoR, aa 21432300. All
GST constructs were confirmed by dideoxy sequencing.
GAL4-NCoR and GAL4-SMRT were created by ligating the interacting
domains of the respective corepressors downstream of the sequence
encoding the GAL4 DNA-binding domain in the SV40-driven expression
vector pECE. The VP-16 TRß1 and RAR
fusions were created by
introducing an in-frame EcoRI site at the 5'-end of the
receptor using PCR and ligating them downstream of the VP16 activation
domain in AASV-VP16. They include aa 1461 of hTRß1, and aa 1462
of RAR. The VP-16 R429Q mutant was made by introducing the mutant
sequence as a PstI-Asp718 fragment into the wild-type
construct. The VP16-RXR
fusion was created by PCR and consists of aa
2462 of the human isoform. IVT NCoR (aa 15792454) and IVT-N2 (aa
15792211) were cloned into pKCR2 and have been described previously
(24, 51). Gal4-N2 was made by placing NCoR, aa 15792211, downstream
of the Gal4 DNA-binding domain. Similarly, Gal4-S2 consists of SMRT, aa
20982266, downstream of GAL4 DBA binding domain. Construct integrity
was confirmed by restriction endonuclease digestion and dideoxy
sequencing.
GST Fusion Proteins
GST fusion proteins were expressed either in DH5
or BL21
Escherichia coli expressing thioredoxin by induction with
0.1 mM
isopropylthio-ß-D-galactosidase at 30 C (24).
The proteins were isolated by lysis with lysozyme and purified on
Sepharose beads. 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 using the Bradford
assay.
EMSA
EMSAs were carried out as previously described with either a
32P-radiolabeled DR+4 or DR+5 probe (52).
GST-corepressor fusion 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. IVT NCoR or IVT N2 was
translated similarly. For each EMSA, 4 µl of IVT TR or RAR were used.
For experiments using RXR, 2 µl were used (or an equivalent amount of
unprogrammed reticulocyte lysate as a control). The amount of GST
protein used in each EMSA was identical and is indicated in each
figure. Incubations were carried out for 20 min, and complexes were
resolved on a 5% nondenaturing gel, followed by auto-
radiography.
Cell Culture and Transfection
All transient transfections were performed in CV-1 cells, which
were maintained as described previously (24). Transient transfections
were performed in six-well plates using the calcium phosphate
technique, with each well receiving 1.7 µg of the upstream activity
sequence (UAS-TK) luciferase reporter and 20 ng of a cytomegalovirus
(CMV) ß-galactosidase expression vector. Each well received 80 ng of
Gal4-corepressor construct, along with 80 ng of VP16-TR or -RAR
construct. The addition of 80 ng VP-16 RXR was controlled for by the
presence of empty vector AASV VP-16 (EV-VP16). Similarly, the addition
of 320 ng pKCR2-RXR was controlled with empty vector pKCR2. Fifteen to
18 h after transfection, the cells were washed in PBS and refed
with 10% steroid hormone- depleted FBS. To remove steroid and
thyroid hormones, FBS was treated with 50 mg/ml activated charcoal
(Sigma, St. Louis, MO) and 30 mg/ml anion exchange resin
(type AGX-8, analytical grade, Bio-Rad Laboratories, Inc.
Richmond, CA), as previously described (24). Forty to 44 h after
transfection the cells were lysed and assayed for luciferase and
ß-galactosidase activity. Experiments were performed two to three
times in triplicate. ß-Galactosidase activity was used to control for
transfection efficiency.
The data shown are the pooled results ± SEM and are
presented as relative or fold luciferase activity. In particular, the
interactions between Gal4-NCoR (or Gal4-SMRT) and nuclear receptor-VP16
constructs are presented relative to luciferase activity in the
presence of Gal4-NCoR (or Gal4-SMRT) alone. Fold luciferase activity
was measured in the presence vs. absence of cotransfected
RXR-VP16 to determine the effect of heterodimerization on interactions
between Gal4-NCoR (or Gal4-SMRT) and nuclear receptor-VP16 constructs.
For each experiment, the total amount of VP16 construct transfected was
held constant with empty vector-VP16 (EV-VP16).
 |
ACKNOWLEDGMENTS
|
---|
We would like to thank R. Evans, C. Glass, N. Moghal, and J.
Safer for plasmids, and A. Takeshita for the thioredoxin-expressing
BL21 E. coli. We would also like to thank B. Kim for helpful
discussions.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Anthony N. Hollenberg M.D., Thyroid Unit, Department of Medicine, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, Massachusetts 02215.
This work was supported by NIH Grants to R. Cohen (DK-02581), F.
Wondisford (DK-53036), and A. Hollenberg (DK-56188 and DK-02354).
Received for publication November 8, 1999.
Revision received February 1, 2000.
Accepted for publication March 6, 2000.
 |
REFERENCES
|
---|
-
Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G,
Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM 1995 The
nuclear receptor superfamily: the second decade. Cell 83:835839[Medline]
-
Seol W, Choi HS, Moore DD 1995 Isolation of proteins that
interact specifically with the retinoid x receptor: two novel
orphan nuclear receptors. Mol Endocrinol 9:7285[Abstract]
-
Horlein AJ, Naar AM, Heinzel T, Torchia J, Gloss B, Kurokawa
R, Ryan A, Kamei Y, Soderstrom M, Glass CK, Rosenfeld MG 1995 Ligand-independent repression by the thyroid hormone receptor mediated
by a nuclear receptor co-repressor. Nature 377:397404[CrossRef][Medline]
-
Chen JD, Evans RM 1995 A transcriptional co-repressor that
interacts with nuclear hormone receptors. Nature 377:454457[CrossRef][Medline]
-
Sande S, Privalsky ML 1996 Identification of TRACs
(T3 receptor-associating cofactors), a family of
cofactors that associate with and modulate the activity of, nuclear
hormone receptors. Mol Endocrinol 10:813825[Abstract]
-
Ordentlich P, Downes M, Xie W, Genin A, Spinner NB, Evans RM 1999 Unique forms of human and mouse nuclear receptor corepressor SMRT.
Proc Natl Acad Sci USA 96:26392644[Abstract/Free Full Text]
-
Park EJ, Schroen DJ, Yang M, Li H, Li L, Chen JD 1999 SMRTe,
a silencing mediator for retinoid and thyroid hormone
receptors-extended isoform that is more related to the nuclear receptor
corepressor. Proc Natl Acad Sci USA 96:35193524[Abstract/Free Full Text]
-
Alland L, Muhle R, Hou HJ, Potes J, Chin L, Schreiber-Agus N,
DePinho RA 1997 Role for N-CoR and histone deacetylase in Sin3-mediated
transcriptional repression. Nature 387:4955[CrossRef][Medline]
-
Heinzel T, Lavinsky RM, Mullen TM, Soderstrom M, Laherty CD,
Torchia J, Yang WM, Brard G, Ngo SD, Davie JR, Seto E, Eisenman RN,
Rose DW, Glass CK, Rosenfeld MG 1997 A complex containing N-CoR, mSin3
and histone deacetylase mediates transcriptional repression. Nature 387:4348[CrossRef][Medline]
-
Nagy L, Kao H-K, Chakravarti D, Lin RJ, Hassig CA, Ayer DE,
Schreiber SL, Evans RM 1997 Nuclear receptor repression mediated by a
complex containing SMRT, mSin3A, and histone deacetylase. Cell 89:373380[Medline]
-
Onate SA, Tsai SY, Tsai M-J, OMalley BW 1995 Sequence and
characterization of a coactivator for the steroid hormone receptor
superfamily. Science 270:13541357[Abstract]
-
Takeshita A, Yen PM, Misiti S, Cardona GR, Liu Y, Chin WW 1996 Molecular cloning and properties of a full-length putative thyroid
hormone receptor coactivator. Endocrinology 137:35943597[Abstract]
-
Voegel JJ, Heine MJS, Zechel C, Chambon P, Grone- meyer H 1996 TIF2, a 160 KD transcriptional mediator for the ligand-dependent
activation function AF-2 of nuclear receptors. EMBO J 15:36673675[Abstract]
-
Cavailles V, Dauvois S, LHorset F, Lopez G, Hoare S, Kushner
PJ, Parker MG 1995 Nuclear factor RIP140 modulates transcriptional
activation by the estrogen receptor. EMBO J 14:37413751[Abstract]
-
Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Lin
SC, Heyman RA, Rose DW, Glass CK, Rosenfeld MG 1996 A CBP integrator
complex mediates transcriptional activation and AP-1 inhibition by
nuclear receptors. Cell 85:403414[Medline]
-
Torchia J, Rose DW, Inostroza J, Kamei Y, Westin S, Glass CK,
Rosenfeld MG 1997 The transcriptional co-activator p/CIP binds CBP and
mediates nuclear receptor function. Nature 387:677684[CrossRef][Medline]
-
Anzick SL, Kononen J, Walker RL, Azorsa DO, Tanner MM, Guan
X-Y, Sauter G, Kallioniemi O-P, Trent JM, Meltzer PS 1997 AIB1, a
steroid receptor coactivator amplified in breast and ovarian cancer.
Nature 277:965968
-
Chen H, Lin RJ, Schlitz RL, Chakravarti D, Nash A, Nagy L,
Privalsky M, Nakatani Y, Evans RM 1997 Nuclear receptor coactivator
ACTR is a novel histone acetyltransferase and forms a multimeric
activation complex with P/CAF and CBP/p300. Cell 90:569580[Medline]
-
Hollenberg AN 1998 Thyroid hormone receptor isoforms, nuclear
corepressors and coactivators and their role in thyroid hormone action.
Curr Opin Endocrinol Diabetes 5:314320
-
Monden T, Wondisford FE, Hollenberg AN 1997 Isolation and
characterization of a novel ligand-dependent thyroid hormone
receptor-coactivating protein. J Biol Chem 272:2983429841[Abstract/Free Full Text]
-
Seol W, Mahon MJ, Lee Y-K, Moore DD 1996 Two receptor
interacting domains in the nuclear hormone receptor corepressor
RIP13/N-CoR. Mol Endocrinol 10:16461655[Abstract]
-
Zamir I, Harding HP, Atkins GB, Horlein A, Glass CK, Rosenfeld
MG, Lazar MA 1996 A nuclear hormone receptor corepressor mediates
transcriptional silencing by receptors with distinct repression
domains. Mol Cell Biol 16:54585465[Abstract]
-
Li H, Leo C, Schroen DJ, Chen JD 1997 Characterization of
receptor interaction and transcriptional repression by the corepressor
SMRT. Mol Endocrinol 11:20252037[Abstract/Free Full Text]
-
Cohen RN, Wondisford FE, Hollenberg AN 1998 Two separate NCoR
interacting domains mediate corepressor action on thyroid hormone
response elements. Mol Endocrinol 12:15671581[Abstract/Free Full Text]
-
Zamir I, Dawson J, Lavinsky RM, Glass CK, Rosenfeld MG, Lazar
MA 1997 Cloning and characterization of a corepressor and potential
component of the nuclear hormone repression complex. Proc Natl Acad Sci
USA 94:1440014405[Abstract/Free Full Text]
-
Lutterbach B, Westendorf JJ, Linggi B, Patten A, Moniwa M,
Davie JR, Huynh KD, Bardwell VJ, Lavinsky RM, Rosenfeld MG, Glass C,
Seto E, Hiebert SW 1998 ETO, a target of t(8;21) in acute leukemia,
interacts with the N-CoR and mSin3 corepressors. Mol Cell Biol 18:71767184[Abstract/Free Full Text]
-
Zhang J, Zamir I, Lazar MA 1998 Proteosomal regulation of
nuclear receptor corepressor-mediated repression. Genes Dev 12:17751780[Abstract/Free Full Text]
-
Wong CW, Privalsky ML 1998 Transcriptional silencing is
defined by isoform- and heterodimer-specific interactions between
nuclear hormone receptors and corepressors. Mol Cell Biol 18:57245733[Abstract/Free Full Text]
-
Zhang J, Zamir I, Lazar MA 1997 Differential recognition of
liganded and unliganded thyroid hormone receptor by retinoid X receptor
regulates transcriptional repression. Mol Cell Biol 17:68876897[Abstract]
-
Refetoff S, Weiss RE, Usala SJ 1993 The syndromes of
resistance to thyroid hormone. Endocr Rev 14:348399[Medline]
-
Flynn TR, Hollenberg AN, Cohen O, Menke JB, Usala SJ, Tollin
S, Hegarty MK, Wondisford FE 1994 A novel C-terminal domain in the
thyroid hormone receptor selectively mediates thyroid hormone
inhibition. J Biol Chem 269:3271332716[Abstract/Free Full Text]
-
Yen PM, Wilcox EC, Hayashi Y, Refetoff S, Chin WW 1995 Studies
on the repression of basal transcription (silencing) by artificial and
natural human thyroid hormone receptor-ß mutants. Endocrinology 136:28452851[Abstract]
-
Hollenberg AN, Monden T, Wondisford FE 1995 Ligand-independent
and -dependent functions of thyroid hormone receptor isoforms depend
upon their distinct amino termini. J Biol Chem 270:1427414280[Abstract/Free Full Text]
-
Hadzic E, Habeos I, Raaka BM, Samuels HH 1998 A novel
multifunctional motif in the amino-terminal A/B domain of T3R
modulates DNA binding and receptor dimerization. J Biol Chem 273:1027010278[Abstract/Free Full Text]
-
Wagner BL, Norris JD, Knotts TA, Weigel NL, McDonnell DP 1998 The nuclear corepressors NCoR and SMRT are key regulators of both
ligand- and 8-bromo-cyclic AMP-dependent transcriptional activity of
the human progesterone receptor. Mol Cell Biol 18:13691378[Abstract/Free Full Text]
-
Jackson TA, Richer JK, Bain DL, Takimoto GS, Tung L, Horwitz
KB 1997 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. Mol Endocrinol 11:693705[Abstract/Free Full Text]
-
Hong SH, David G, Wong CW, Dejean A, Privalsky ML 1997 SMRT
corepressor interacts with PZLF and with the PML-retinoic receptor
(RARa) and PZLF-RARa oncoproteins associated with acute promyelocytic
leukemia. Proc Natl Acad Sci USA 94:90289033[Abstract/Free Full Text]
-
Grignani F, De Matteis S, Nervi C, Tomassoni L, Gelmetti V,
Cioce M, Fanelli M, Ruthardt M, Ferrara FF, Zamir I, Seiser C, Lazar
MA, Minucci S, Pelicci PG 1998 Fusion proteins of the retinoic
receptor-
recruit histone deacetylase in promyelocytic leukemia.
Nature 391:815818[CrossRef][Medline]
-
Yoh SM, Chatterjee VK, Privalsky ML 1997 Thyroid hormone
resistance syndrome manifests as an aberrant interaction between
mutant T3 receptors and transcriptional
corepressors. Mol Endocrinol 11:470480[Abstract/Free Full Text]
-
Clifton-Bligh RJ, deZegher F, Wagner RL, Collingwood TN,
Francois I, Van Helvoirt M, Fletterick RJ, Chatterjee VK 1998 A novel
TRß mutation (R383H) in resistance to thyroid hormone syndrome
predominantly impairs corepressor release and negative transcriptional
regulation. Mol Endocrinol 12:609621[Abstract/Free Full Text]
-
Safer JD, Cohen RN, Hollenberg AN, Wondisford FE 1998 Defective release of corepressor by hinge mutants of the thyroid
hormone receptor found in patients with resistance to thyroid hormone.
J Biol Chem 273:3017530182[Abstract/Free Full Text]
-
Le Douarin B, Nielsen AL, Garnier JM, Ichinose H, Jeanmougin
F, Losson R, Chambon P 1996 A possible involvement of TIF1
and
TIF1ß in the epigenetic control of transcription by nuclear
receptors. EMBO J 15:67016715[Abstract]
-
Lin BC, Hong SH, Krig S, Yoh SM, Privalsky ML 1997 A
conformational switch in nuclear hormone receptors is involved in
coupling hormone binding to corepressor release. Mol Cell Biol 17:61316138[Abstract]
-
McInerney EM, Rose DW, Flynn SE, Westin S, Mullen TM, Krones
A, Inostroza J, Torchia J, Nolte RT, Assa-Munt N, Milburn MV, Glass CK,
Rosenfeld MG 1998 Determinants of coactivator LXXLL motif specificity
in nuclear receptor transcriptional activation. Genes Dev 12:33573368[Abstract/Free Full Text]
-
Darimont BD, Wagner RL, Apriletti JW, Stallcup MR, Kushner PJ,
Baxter JD, Fletterick RJ, Yamamoto KR 1998 Structure and
specificity of nuclear receptor-coactivator interactions. Genes Dev 12:33433356[Abstract/Free Full Text]
-
Hu X, Lazar MA 2000 The CoRNR motif controls the recruitment
of corepressors by nuclear hormone receptors. Nature 402:9396
-
Perissi V, Staszewski LM, McIerney IM, Kurokawa R, Krones, A,
Rose DW, Lambert MH, Milburn MV, Glass CK, Rosenfeld MG 2000 Molecular
determinants of nuclear receptor-corepressor interactions. Genes Dev 13:31983208[Abstract/Free Full Text]
-
Nagy L, Kao H-Y, Love JD, Li C, Banayo E, Gooch JT, Krishna V,
Chatterjee K, Evans RM, Schwabe JWR 2000 Mechanism of corepressor
binding, release from nuclear hormone receptors. Genes Dev 13:32093216[Abstract/Free Full Text]
-
Zamir I, Zhang J, Lazar MA 1997 Stoichiometric and steric
principles governing repression by nuclear hormone receptors. Genes Dev 11:835846[Abstract]
-
Nagaya T, Kopp P, Kitajima K, Jameson JL, Seo H 1996 Second
zinc finger mutants of thyroid hormone receptor selectively preserve
DNA binding and heterodimerization but eliminate transcriptional
activation. Biochem Biophys Res Commun 222:524530[CrossRef][Medline]
-
Hollenberg AN, Monden T, Madura JP, Lee K, Wondisford FE 1996 Function of nuclear co-repressor protein on thyroid hormone response
elements is regulated by the receptor A/B domain. J Biol Chem 271:28516285[Abstract/Free Full Text]
-
Hollenberg AN, Monden T, Flynn TR, Boers M-E, Cohen O, and
Wondisford FE 1995 The human thyrotropin-releasing hormone gene is
regulated by thyroid hormone through two distinct classes of negative
thyroid hormone response elements. Mol Endocrinol 10:540550