Two Separate NCoR (Nuclear Receptor Corepressor) Interaction Domains Mediate Corepressor Action on Thyroid Hormone Response Elements
Ronald N. Cohen,
Fredric E. Wondisford and
Anthony N. Hollenberg
Thyroid Unit, Department of Medicine Beth Israel Deaconess
Medical Center and Harvard Medical School Boston, Massachusetts
02215
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ABSTRACT
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The nuclear corepressor (NCoR) binds to the
thyroid hormone receptor (TR) in the absence of ligand. NCoR-TR
interactions are mediated by two interaction domains in the C-terminal
portion of NCoR. Binding of NCoR to TR results in ligand-independent
repression on positive thyroid hormone response elements. The
interactions between NCoR interaction domains and TR on DNA response
elements, however, have not been well characterized. We have found that
both interaction domains are capable of binding TR on thyroid hormone
response elements. In addition, the NCoR interaction domains interact
much more strongly with the TR than those present in the silencing
mediator of retinoic acid and TRs (SMRT). Furthermore, deletion of
either NCoR interaction domain does not significantly impair
ligand-independent effects on positive or negative thyroid hormone
response elements. Finally, both NCoR interaction domains appear to
preferentially bind TR homodimer over TR-retinoid X receptor
heterodimer in electrophoretic mobility shift assays. These data
suggest that either NCoR interaction domain is capable of mediating the
ligand-independent effects of TR on positive and negative thyroid
hormone response elements.
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INTRODUCTION
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The thyroid hormone receptor (TR), a member of the nuclear hormone
receptor (NHR) superfamily, is a transcription factor that possesses
transcriptional activity both in the presence and absence of its
ligand, T3 (1). The TR mediates its ligand-independent and
dependent actions by initially interacting with thyroid hormone
response elements (TREs) either as a monomer, homodimer, or as a
heterodimer with the retinoid X receptor (RXR) (2, 3, 4). Once bound to a
TRE, the TR then interacts with accessory proteins and members of the
basal machinery to mediate its transcriptional effects. In the absence
of ligand, the TR is a potent silencer of gene expression when bound to
certain positive TREs. This ligand-independent activity is mediated by
a family of proteins termed nuclear corepressors. To date, two such
family members have been identified NCoR/RIP13 (nuclear receptor
corepressor) (5, 6) and SMRT (the silencing mediator of RARs and TRs)
(7, 8). In the presence of ligand, TR releases corepressors and
recruits coactivators such as SRC-1 (9, 10), TIF2 (GRIP1) (11), RIP 140
(12), CBP (13, 14), and p120 (15), to mediate ligand-dependent
activation.
Full-length NCoR is a ubiquitously expressed 270 kDa protein (6). A
second shorter isoform, RIP13a, of unknown significance also exists
(16). NCoR is a modular protein and likely possesses four separate
N-terminal repressing domains, two of which appear to interact with
mSin3 and the histone deacetylase RPD3, HDAC1, to mediate repression
(17, 18). It has been proposed that repression results from changes in
chromatin structure mediated by this complex.
NCoR binds a portion of the TR hinge region termed the CoR box (6).
Mutations in this region block the ability of unliganded TR to mediate
repression. Two C-terminal NCoR domains, termed the interaction domains
(ID I and ID II), appear to be involved in TR binding (16, 19). NCoR
also appears to mediate repression by the orphan receptor RevErb (19).
Protein interaction assays revealed that the NCoR interaction domains
that mediate NCoR-RevErb binding are similar to those that mediate
NCoR-TR effects; however, the domain of RevErb that binds NCoR is not
homologous to the CoR box. The NCoR interaction domains have been
identified using yeast two-hybrid and mammalian two-hybrid systems, but
their functional significance on native TREs has not yet been defined.
These interactions are critical as NCoR may be able to bind a number of
NHRs in solution [peroxisome proliferator-activated receptors (PPARs)
and RXR] but the presence of DNA response elements appears to
functionally limit the interactions between NCoR and NHRs (20).
SMRT (TRAC2) is also a modular protein. It shares close to 40%
amino-acid-identity with NCoR (21). It also contains
N-terminal-repressing domains that appear to function through mSin3,
and C-terminal interacting domains that interact with the TR, retinoic
acid receptor (RAR), and RXR in a yeast two-hybrid system (7, 17). The interacting domains also share significant homology with those
of NCoR. Recently, an endogenous inhibitor of SMRT, TRAC1, has been
identified (8). It appears to be an alternatively spliced variant that
contains the interacting domains but lacks the repressing domains.
We have identified a human homolog of NCoR that contains the C-terminal
IDs, but not the N-terminal repressor domains (22). In transient
transfection experiments, this construct inhibits endogenous NCoR
function and has thus been termed NCoR inhibitor, or NCoRI. This
truncated inhibitor of NCoR, which may or may not be expressed
endogenously, provides an ideal tool to study the functional
significance of the NCoR IDs on endogenous TREs. In this study,
deletion constructs of hNCoRI were made that lack one or both of the
known IDs. The functional characteristics of these constructs were
assessed in transient transfection experiments using both positive
(direct repeat, palindromic, and inverted palindromic) and negative
(TRH) reporter elements. Electrophoretic mobility shift assays were
also performed to characterize the binding of NCoRI deletion constructs
to TR on DNA response elements and to compare structural binding with
functional interactions.
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RESULTS
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A Truncated Homolog of NCoR Serves as an Antirepressor
Human NCoR inhibitor (hNCoRI), a human homolog of NCoR, is an
approximately 130 kDa protein, which was cloned using the yeast
two-hybrid system (22). It has over 93% amino acid homology with the
comparable region of mouse (m)NCoR (Fig. 1
). It contains the C-terminal
interacting domains of NCoR, but lacks the N-terminal-repressing
domains (6). Recently, others have shown that a region present in NCoRI
may, in fact, bind mSin3 and repress (18). However, we have
demonstrated that hNCoRI has little repressing activity when linked to
the Gal4 DNA-binding domain (data not shown). In transient transfection
experiments, cotransfection of hNCoRI reverses ligand-independent
repression on direct repeat (DR+4), palindromic (PAL), and inverted
palindromic (LYS) reporters, presumably by interfering with endogenous
NCoR function (22). To ensure that NCoRI possesses separate activity
from the full-length moiety, we analyzed the function of NCoRI and NCoR
on a positive and negative reporter in an identical transfection
paradigm. As is shown in Fig. 2
, NCoRI
inhibits ligand-independent repression on the palindromic positive TRE,
while full-length NCoR functions to enhance ligand-independent
repression. In contrast, on the negatively regulated TRH reporter NCoRI
is a strong activator of ligand-independent activation while NCoR has
only a slight effect. However, the activation function on the negative
reporter appears to be located downstream of the repressing domains as
NCoRI is far more effective than NCoR. Thus, NCoRI is a potent
antirepressor on both the positive and negative reporters. NCoRI has no
effect on reporter gene activity in the absence of cotransfected TRß1
(data not shown).

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Figure 1. hNCoRI Deletion Constructs
A, mNCoR and hNCoRI are aligned schematically. Amino acid numbering is
per mNCoR sequence. hNCoRI has 93% homology when compared with the
comparable region of mNCoR. B, hNCoRI constructs lacking ID I and/or ID
II were constructed, as described in Materials and
Methods, and placed into the expression vector pKCR2. Amino
acids corresponding to NCoRI sequence are identified with the
corresponding sequence designations of mNCoR noted in
parentheses.
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Figure 2. NCoR and NCoRI Have Separate Actions in CV-1 Cells
CV-1 cells were cotransfected with 1.7 µg of the indicated reporter
construct and 80 ng of pKCR2-TR with either 330 ng of NCoR, NCoRI, or
empty expression vector. The data are quantified as relative luciferase
activity (mean ± SE) with basal activity in the
absence of TR set as 1.
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NCoR Prefers to Interact with the TR
The two C-terminal IDs of mNCoR, ID I and ID II, have been
identified using yeast and mammalian two-hybrid systems (16) with the
ligand-binding domain of the TR as bait. Similarly, two putative
interacting domains have been identified in SMRT using solution
interaction assays (23). It is unclear, however, what role these
domains play on hormone response elements or in vivo.
Alignment of the NCoR interacting domains with the SMRT amino acid
sequence reveal considerable homology (53%) between NCoR ID I and an
analogous region in SMRT and only 25% homology between NCoR ID II and
the analogous region in SMRT (Fig. 3A
).
In contrast, the NCoR interacting domains are very well conserved
between species as is shown in Fig. 3B
.

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Figure 3. Sequence Alignments of the NCoR ID Sequences Differ
from SMRT and Each Other, but Are Conserved across Species
A, The amino acid sequences of ID I and ID II from human NCoRI were
aligned with the hSMRT AA sequence and the area of identity was shown.
B, The ID I and ID II sequences from hNCoRI were aligned with the
analogous sequences from mNCoR (6 ) and each other.
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Given the differences in the amino acid sequences of the NCoR and SMRT
interacting domains, we tested whether NCoR or SMRT preferred to
interact with TRß1. We initially performed glutathione
S-transferase (GST) pull-down assays and electrophoretic
mobility shift assays (EMSA). In solution the ligand-binding domain of
the TR appears to be able to interact equally well with both SMRT and
NCoR (Fig. 4A
); however, when equal
amounts of the full-length protein are used in EMSA on a DR+4 element,
NCoR is preferred by the TR, and little SMRT binds to either the TR
homo or heterodimer (Fig. 4B
). In addition, neither NCoR nor SMRT
interacts with DNA in the absence of TR. These data suggest that either
the DNA-binding region or other regions of the TR outside its
ligand-binding domain alter the interaction between NCoR and SMRT.
Further analysis of the interaction between the TR and NCoR and SMRT
was performed in CV-1 cells. Using a mammalian two-hybrid system, where
the ligand-binding domain of TRß1 was fused to the GAL4 DNA-binding
domain and either corepressor to the VP-16 activation domain, we found
that the interaction between TR and NCoR (Fig. 4C
) was extremely strong
and in fact stronger than the interaction between TR and RXR (data not
shown). The interaction between TR and SMRT was much weaker in
comparison. In contrast, Gal4-RAR
preferred to interact with SMRT
rather than NCoR, ensuring that the VP-16 SMRT construct was adequately
expressed and localized to the nucleus. Finally, we compared the
ability of a dominant inhibitor of SMRT activity termed SMRTI (TRAC 1)
(8) and NCoRI to function as antirepressors on the DR+4 reporter in
CV-1 cells as shown in Fig. 4D
. SMRTI was a consistently weaker
inhibitor at all amounts transfected, in keeping with its poorer
ability to interact with the TR. Unlike what has been reported for
full-length mNCoR (24), hNCoRI acts as a potent antirepressor at all
concentrations examined on the DR+4 reporter construct and other
positive TREs (Fig. 4D
).

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Figure 4. NCoR Prefers to Interact with the TR
A, GST-TR was used to pull down radiolabeled NCoR or SMRT. I, 20%
input; G, GST alone. The concentration of T3 used was
10-6 M. B, EMSA was performed using in
vitro translated proteins and a radiolabeled DR + 4 probe. The
resolved complexes are indicated. C, CV-1 cells were transfected with
1.7 µg of the UAS reporter with 80 ng of the indicated GAL4 construct
and 80 ng of the indicated VP-16 vector. The data are quantified as
fold activation in the presence of empty VP-16 expression vector. D,
CV-1 cells were transfected with increasing amounts of either NCoRI or
SMRTI on the DR+4 reporter. The data are shown as fold repression in
the presence of TR alone.
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While both NCoR interacting domains appear to favor interactions with
the TR, little is known about the ability of the individual NCoR IDs to
interact with and function on endogenous TREs. Thus, deletion
constructs of hNCoRI were made that lack ID I and/or ID II (Fig. 1
) so
that their function could be ascertained both functionally and
structurally on endogenous TREs. Sequence alignment between these two
regions in the hNCoRI protein disclosed only remote similarity (Fig. 3B
).
Both IDs Reverse Ligand-Independent Repression on Positive TREs
To assess the function of the interacting domains on the
ligand-independent activity of the TR, hNCoRI deletion constructs and
TRß1 were transfected into CV-1 cells at a 1:1 DNA ratio. As shown in
Fig. 5A
, when cotransfected with two
copies of the LYS element linked to the luciferase reporter,
transfection of TRß1 causes approximately 3-fold repression of
basal activity. Cotransfection of hNCoRI fully reverses this
repression. Likewise, when a construct lacking ID I (hNCoRI
ID I) is
cotransfected, there is also full reversal of ligand-independent
repression. Similarly, hNCoRI
ID II reverses basal repression when
cotransfected with TRß1. In contrast, hNCoRI
ID I/II, which lacks
both IDs, does not reverse ligand-independent repression on the LYS
element. Fig. 5B
shows similar data, using the palindromic reporter.
Again, hNCoRI reverses basal repression, as do constructs lacking
either ID. In contrast to the LYS reporter, however, deletion of either
ID I or ID II did impair somewhat the ability of the construct to
reverse TR-mediated repression. In addition, the construct lacking both
IDs again did not reverse repression.

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Figure 5. Both IDs Are Capable of Functionally Interacting
with the TR on Positive TREs
CV-1 cells were cotransfected with 1.7 µg of LYS (A) PAL (B), or DR+4
(C) reporter plasmid and 80 ng of pKCR2-TR and/or 80 ng of pKCR2 and/or
80 ng of pKCR2-hNCoRI deletion construct. The data are quantified as
fold repression (mean ± SE). D, CV-1 cells were
transfected with GFP alone (mock) or the indicated NCoR construct and
subjected to fluorescence microscopy.
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On the DR+4 reporter (Fig. 5C
), hNCoRI reverses basal repression, as do
constructs lacking ID I or ID II. Unlike the PAL and LYS elements,
however, hNCoRI
ID I/II is not fully deficient in its ability to
reverse basal repression, as there is still 2-fold repression. Deletion
of another 168 amino acids (hNCoR 1302), which removes a portion of
the mSin3-binding site (18) [mNCoR amino acids (AA) 18291940; hNCoRI
AA 240351], causes virtually all ligand-independent activity to be
lost.
To rule out defective nuclear localization when IDs were deleted from
NCoR, hNCoRI and the dual ID deletion construct were placed into a
green fluorescent protein (GFP) expression vector and transfected into
CV-1 cells. The cells were viewed under fluorescence microscopy 24
h after transfection to identify cellular localization of the fusion
proteins. GFP vector alone exhibits diffuse staining in both the
nuclear and cytoplasmic compartments. In contrast, hNCoRI
ID
I/II-GFP was restricted to the nucleus (Fig. 5D
), suggesting that lack
of functional activity in transient transfection assays was not the
result of impaired nuclear localization. In addition, hNCoRI-GFP also
exhibited nuclear localization.
NCoR IDs Bind TRß1 on TREs and Preferentially Bind TR
Homodimer
We next tested the ability of these hNCoRI constructs to interact
with the TR on DNA response elements using EMSAs. As shown in Fig. 6A
, wild-type TRß1 homodimer interacts
strongly with hNCoRI. The addition of RXR
(or RXRß) causes NCoRI
binding to be reduced without altering the position of the band. The
addition of an RXR
antibody supershifts the TR-RXR heterodimer. The
NCoR-TR complex is not supershifted by the anti-RXR
antibody,
however, suggesting that this complex does not contain RXR. In
contrast, an anti-TR antibody supershifts all complexes, including
TR-NCoR (Fig. 6B
). An antibody against C/EBP
does not shift any
complex, indicating that these antibody supershift assays are specific.
These data suggest that the NCoRI complex contains TR, but not RXR, and
that NCoRI preferably binds to the TR homodimer over the TR-RXR
heterodimer in this assay. Similar studies were performed using
bacterially expressed NCoR-interacting domains and, even in the
presence of limiting TRß1, the homodimer preferred to interact with
NCoR (Fig. 6C
). When the amount of GST-NCoR was increased by up to
50-fold (20 ng to 1000 ng) there was no difference found in the
preference of binding.


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Figure 6. hNCoRI Preferentially Binds TR Homodimer
A gel mobility shift assay was carried out using in
vitro translated proteins from rabbit reticulocyte lysate,
except where indicated, and a labeled DR+4 oligonucleotide probe. After
a 20-min room temperature incubation, 1 µl anti-RXR (A), anti-TRb
(B), or anti-C/EBP (B) antisera were added. Incubations were then
carried out for an additional 20 min at room temperature. C, A
gel-mobility shift assay was carried out using 20 ng of GST-NCoR with
increasing amounts of TRß1 (shown as microliters of reticulocyte
lysate) with the amount of RXR held constant in the presence of a
labeled DR+4 oligonucleotide probe. D and E, CV-1 cells were
transfected with the indicated constructs. The data are expressed as
relative luciferase activity with the activity of GAL4-TR set as 1.
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To further study the interactions between the TR and NCoR, we employed
the mammalian two-hybrid assay. We first tested the ability of a
GAL4-RXR
construct to affect both silencing by GAL4-TR and its
interactions with NCoR. Cotransfection of GAL4-RXR with GAL4-TR will
cause a heterodimer to form on the upstream activating sequence (UAS)
response element in contrast to the TR homodimer when GAL4-TR is
transfected alone. As is shown in Fig. 6D
, cotransfected GAL4-RXR
causes both a loss of silencing (GAL4-TR alone causes between 5- and
10-fold repression of basal activity) and diminishes considerably the
ability of the TR to interact with the VP-16 NCoR construct. These
results are consistent with the GAL4-TR homodimers avidly binding
corepressor to mediate silencing. As the TR homodimer is shifted by
NCoR in EMSA, we next investigated whether overexpressed NCoR could
promote homodimerization of the TR in solution. Collingwood et
al. (25) have previously shown that TR homodimers do not form in
solution in the mammalian two-hybrid system. This is demonstrated in
Fig. 6E
as GAL4-TR is unable to interact with a VP-16-TR chimeric
protein in CV-1 cells. We next asked whether increased amounts of NCoR
could enhance the homodimer interaction. Indeed, cotransfected NCoRI
allowed the TR homodimer to form in solution, suggesting that the
homodimer may be stabilized by corepressor binding. In contrast,
cotransfected SMRTI had no affect on TR homodimer formation, again
supporting the preference of the TR for NCoR.
Since NCoRI appears to preferentially interact with the TR homodimer,
the ability of the NCoRI deletion constructs to bind TRß1 on DNA
response elements was assessed principally using the homodimer in EMSA.
As shown in Fig. 7A
, on the LYS element,
TRß1 bound well as a homodimer. The addition of hNCoRI results in a
supershift, representing TRß1-hNCoRI binding. Constructs lacking
either ID alone bind TR on the LYS element, although these complexes
migrate slightly further than TR-NCoRI, due to their smaller size. In
contrast, constructs lacking both IDs do not bind TRß1 on the LYS
element. However, on the PAL (Fig. 7B
) and DR+4 (Fig. 7C
) elements,
although either interacting domain promoted TR binding, there appeared
to be a preference for the ID II-containing construct. Constructs
lacking either ID were unable to bind these elements.

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Figure 7. Both IDs Mediate Binding of NCoR to TR on DNA
Response Elements
Gel mobility shift assays were carried out using 34 µl of in
vitro translated proteins from rabbit reticulocyte lysate and
32P-radiolabeled LYS (A) PAL (B), or DR+4 (C)
oligonucleotide probes. The labeled probe was incubated with in
vitro translated TRb1 and hNCoRI deletion construct, or
unprogrammed reticulocyte lysate as indicated.
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To confirm that the presence of a heterodimer did not alter these
results, EMSA was next performed on DR+4 in the context of RXR
as
discussed above and shown in Fig. 8A
.
Similar results are seen with constructs lacking either ID I or ID II,
suggesting that these IDs do not differentially bind TR-RXR
vs. TR-TR on DNA response elements. Fig. 8B
shows similar
data in the context of 10 nM T3. As expected,
there is loss of NCoR binding to TR in the
presence of ligand, as well as a decrease in the homodimeric complex
(26, 27). There is no significant binding of NCoRI to TR-RXR
heterodimers in the presence of T3.

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Figure 8. NCoR IDs Bind TR Homodimer
The DR+4 probe was incubated with TRß1 and RXR and/or hNCoRI
deletion construct, in the absence (A) or presence (B) of 10
nM T3.
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NCoR IDs Functionally Interact with TRß1 on Negative TREs by
Enhancing Ligand-Independent Activation
To examine the function of NCoR IDs on genes negatively regulated
by T3, constructs were cotransfected with the negatively
regulated TRH promoter, linked to the luciferase reporter (28). In
contrast to positive TREs, TR stimulates gene expression in the absence
of T3 on negative TREs (28, 29, 30, 31). As seen in Fig. 9
, cotransfection of hNCoRI with TRß1
resulted in an approximately 7- to 8-fold increase in
ligand-independent activation. Cotransfection of hNCoRI
ID I or
ID II resulted in a similar stimulation of activity. In contrast,
cotransfection of hNCoR
ID I/II only stimulated activity 2-fold
above baseline. These data suggest that either ID is able to
functionally interact with TR on negative TREs.

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Figure 9. hNCoRI Deletion Constructs Enhance
Ligand-Independent Activation on a Negative TRE
TRH-pA3Luc (1.7 µg) was cotransfected with 80 ng pKCR2-TR and/or 80
ng pKCR2 alone and/or pKCR2-hNCoRI deletion construct. Each well was
also transfected with 20 ng pCMV-ßGAL to control for transfection
efficiency. The data are quantified as relative fold activation, where
1 represents activation in the presence of pKCR2-TR, but not
pKCR2-hNCoRI, corrected for ß-galactosidase activity.
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DISCUSSION
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Nuclear corepressors silence transcription by interacting with
unliganded TRs and RARs bound to their cognate response elements
(5, 6, 7, 8). Once bound, corepressors silence through their ability to
recruit RPD3 (HDAC1), which causes histone deacetylation of active
chromatin and represses transcription (17, 18). Activation of
transcription by ligand occurs through the reversal of repression (the
release of corepressors), and the recruitment of coactivators, some of
which contain endogenous histone acetylase activity (32, 33, 34). While
coactivators appear to interact with the AF-2 domain of NRs on DNA
through a leucine-rich motif (LxxLL) (35), it is unclear what allows
the corepressor to bind to the CoR box of the TR in the absence of
ligand. Furthermore, while SMRT and NCoR contain two separate TR IDs
that have been previously been shown to function independently in
solution (16, 19), they are dissimilar in sequence, and their function
on native positive and negative TREs has not been addressed previous to
this study.
We have initially compared the ability of the TR to
interact with each of the corepressors. Our data demonstrate that the
TR prefers to interact with NCoR on TREs and in mammalian cells. The
EMSA data suggest that conformation on a DNA-response element may be
important for this specificity. While others have shown interaction
between SMRT and TR homodimers in EMSA, these studies were performed in
the presence of significantly greater amounts of corepressor (20). The
contrast between the GST-TR pull-down results, which showed equal
binding to SMRT and NCoR, and the mammalian two-hybrid experiments,
which showed an overwhelming preference by the TR for NCoR, is striking
as both systems employed only the TRß1 ligand-binding domain. These
experiments suggest that other nuclear proteins may influence the
interactions between nuclear receptors and corepressors. Given the
recent cloning of a novel corepressor, SunCoR (36), it is reasonable to
hypothesize that other such proteins may influence the formation of
corepressor complexes in cells. The preference of the TR for NCoR is
supported by recent antibody microinjection studies, which demonstrate
that inhibition of NCoR blocks silencing by the TR while anti-SMRT
antibodies had no effect on TR-mediated silencing (37).
In transient transfection experiments, NCoR enhances the repressing
function of TR. However, when increasing amounts of NCoR are
transfected, paradoxical activation has been reported to occur with
localization of transfected NCoR, but not endogenous NCoR, to nuclear
dot structures (24). This complexity was addressed in the present study
using NCoRI, which inhibits endogenous NCoR function, and thus provides
an ideal tool for examining the function of the NCoR-interacting
domains. Moreover, NCoRI progressively reversed ligand-independent
repression on a positive TRE without any paradoxical effects on TR
action. Furthermore, full-length NCoR did not activate when transfected
in an identical paradigm.
When tested in cotransfection experiments, deletion of either
interacting domain in the context of NCoRI had a negligible effect on
the inhibitory activity of NCoRI on each of the three positive TREs
tested. Furthermore, constructs containing a single ID retained the
ability to bind to the TR on these TREs in EMSA, although ID II
appeared to promote a stronger interaction. Comparison of the NCoR- and
SMRT-interacting domains shows that ID I is quite similar while ID II
is more dissimilar. Thus, the increased avidity of ID II for the TR is
consistent with the preference of NCoR for the TR. Functional activity
of NCoRI was abolished on the PAL and LYS reporters when both IDs were
deleted. This NCoRI construct (hNCoRI
ID I/II) localized to the
nucleus when fused to GFP, implying that this functional defect was not
due to defective nuclear transport. When examined in EMSA, hNCoRI
ID
I/II was unable to bind to TR on any of the response elements studied.
Therefore, the lack of functional activity of this construct on the PAL
and LYS elements results from its inability to bind to the TR on DNA.
In contrast, hNCoRI
ID I/II retained a small but consistent
inhibitory function on the DR+4 reporter without evidence of DNA
binding, raising the possibility that the region remaining of NCoR may
interact with TR to mediate repression on DR+4, or that the
mSin3-binding site present in this construct may be important in
mediating repression on this TRE. In fact, deletion of a portion of the
mSin3 ID resulted in further loss of NCoRI function on the DR+4
element.
In contrast to ligand-independent repression on positive TREs, the TR
causes ligand-independent activation in tissue culture on negative
TREs. While the mechanism underlying negative regulation has not been
defined, it is clear that TR-binding sites exist in the regulatory
regions of genes negatively regulated by the TR (28). We have
previously demonstrated that cotransfected NCoRI enhances
ligand-independent activation on the TRH promoter in the presence of
TRß1 and TR
1 (22). Deletion of either interacting domain does not
impair the ability of hNCoRI to activate the TRH promoter. However,
once both interacting domains are deleted, enhancement of
ligand-independent activation is all but lost. Interestingly
full-length NCoR also slightly activated the TRH promoter, as has been
reported previously (38), but the effect was far less than that seen
with NCoRI. These differences may be the result of the different cell
types used as well as the amounts of cotransfected NCoR or NCoRI.
Further studies will be needed to define the mechanism by which the
corepressor influences negative regulation. The data presented here
indicate that a direct interaction between the TR and NCoR is needed to
enhance ligand-independent activity.
In the present study, we have also addressed the ability of hNCoRI to
interact with either the TR homodimer of TR/RXR heterodimer. It is
unclear in vivo what is the preferred TR configuration in
the absence of ligand. Previous work by Zamir et al. (20)
has demonstrated that the TR homodimer and TR/RXR heterodimer can both
bind nuclear corepressor on a DR+4 element (20). These studies did not
address the relative preference of homo- or heterodimer for the
corepressors. In addition, it is also clear from another study (39)
that RXR/TR
1 heterodimers can interact in cells with NCoR. The data
presented here also show an interaction between NCoR and RXR/TRß1
heterodimers in the mammalian two-hybrid assay. However, this
interaction is considerably weaker than the interaction with TR
homodimers. Furthermore, in the mammalian two-hybrid system, RXR-TR
heterodimers also silence less well. In addition, NCoRI appears to
allow the TR homodimer to form in solution through its interacting
domains. Further support for the preference of the homodimer for NCoR
comes from gel-mobility shift assays on TREs, which demonstrate that
the TRß1 homodimer binds in vitro translated NCoR and
bacterially expressed NCoR, while the heterodimer demonstrates little
avidity for NCoR. These data support the concept that the homodimer may
be able to play a role in thyroid hormone action through its strong
interaction with nuclear corepressors. While our data do not rule out
that the heterodimer is an active silencer, they suggest that the
homodimer preferentially interacts with each of the NCoR interacting
domains. An alternative explanation could be that the gel-mobility
shift assay is too insensitive to detect in vivo
interactions between the TR/RXR heterodimer and NCoR and that
interaction studies are cell specific. Further work will be necessary
to clearly define the role of the in vivo configuration of
TR when ligand-independent repression occurs and to determine the exact
stoichiometry of the homodimer and heterodimer interactions.
In summary, we have demonstrated that the two separate NCoR domains
interact with the TR on both positive and negative TREs. Furthermore,
individual IDs can mediate dominant inhibition of endogenous NCoR
activity, suggesting that the separate IDs are powerful mediators of
ligand-independent interaction with the TR. The IDs are remarkably
conserved across species; however, the two IDs themselves share only
remote homology with each other. While the two IDs may be present to
ensure strong interactions with TR, our data suggest that either ID
alone allows NCoR-TR interactions to occur on native TREs. Further work
on these modular domains will help delineate the specific amino acids
necessary for ligand-independent interactions with the TR and will help
identify the important differences that allow the TR to interact much
more strongly with NCoR. This may allow for the identification of other
such proteins that can interact with the TR to mediate its
ligand-independent functions.
 |
MATERIALS AND METHODS
|
---|
Plasmids
The positive TRE constructs each consist of two copies of DR+4
palindromic, or F2LYS elements upstream of the TK109 promoter in the
vector pA3Luc (22, 40). The human TRH promoter from -900 to +55 was
also cloned into pA3Luc (28). Human TRß1 was cloned into the
SV40-driven expression vector pKCR2, as previously described (40). A
868-AA NCoR fragment, termed hNCoRI, was cloned into pKCR2 as
previously described (22). Full-length NCoR (a gift of C. Glass) was
cloned as a NotI fragment into pKCR2. SMRTI was cloned as a
SalIEcoRI fragment of SMRT (a gift of R. Evans)
into pKCR2. SMRTI was also cloned into AASVP-16 (a gift of S.
Weissman), using PCR from full-length SMRT, to encompass AA 657-1495.
The NCoRI construct lacking ID I was made by deleting AA 625701 and
creating an in-frame stop codon with a HindIII deletion. The
construct lacking both ID I and ID II was made by PCR amplification of
a portion of NCoRI from AA 1470 inserted next to the novel stop codon
created by the HindIII deletion. The construct lacking ID II
was created by PCR amplification of AA 1464 and AA 557868 joined by
a novel Sac-1 site created next to AA 464. NCoR 1302 was obtained by
a BglII deletion of the C-terminal portion of NCoRI in
pKCR2. AASVP16-NCoRI was created by ligating AA 215868 of NCoRI into
AASVP16. The Gal4 and UAS-TKLuc constructs have been described
previously (15). Gal4-TR was created by ligating a
PstIEcoRI fragment from hTRß1 encompassing AA
203461 into the Gal4 vector. GAL4 RXR
encompasses AA 201462 of
human RXR
in frame with the GAL4 DNA-binding domain. GST-TR and
AASV-TR were created by ligating the ligand-binding domain of the TR
from Gal4-TR in frame with the GST moiety in PGEX4T1 and the VP-16
moiety in AASV, respectively. GST-NCoR was created by ligating the two
interacting domains of hNCoR (AA 20632300) into PGEX 4T1. Constructs
were cloned into pKCR2 for transient transfection experiments and pEGFP
(CLONTECH, Palo Alto, CA) for cellular localization studies. The
integrity of each plasmid was confirmed by restriction endonuclease
digestion and dideoxy sequencing. All plasmids for transfection were
purified using column (Qiagen, Chatsworth, CA) purification.
Cell Culture and Transient Transfection
CV-1 cells were maintained in DMEM supplemented with
L-glutamine, 10% FCS, 100 µg/ml penicillin, 0.25 µg/ml
streptomycin, and amphotericin.
Transient transfections using NCoRI alone were performed using the
calcium phosphate technique in six-well plates with each well receiving
1.7 µg of reporter and 80 ng of pKCR2-TR and/or equal amounts of
pKCR2 alone and/or equal amounts of pKCR2-NCoRI construct, except as
noted. Each well also received 20 ng of a ß-galactosidase expression
vector to control for transfection efficiency. Studies involving the
full-length murine NCoR included 330 ng of expression vector per well.
Fifteen to 18 h after transfection, the cells were washed in PBS,
and refed with DMEM with 10% steroid hormone-depleted FBS. To remove
steroid and thyroid hormones, FBS was treated for 24 h at 4 C with
50 mg/ml activated charcoal (Sigma Chemical Co., St. Louis, MO) and 30
mg/ml anion exchange resin (type AGX-8, analytical grade, Bio-Rad,
Richmond, CA). Forty to 44 h after transfection, the cells were
harvested in extraction buffer and assayed for both luciferase and
ß-galactosidase (Tropix, Bedford, MA) activity. Experiments were
performed in triplicate and repeated two to three times. The data are
the pooled results ± SEM.
Transient transfections to assess cellular localization of hNCoR
constructs were performed in 100-mm tissue culture dishes. Twenty
micrograms of pEGFP-hNCoRI construct or pEGFP alone were transfected
using the calcium phosphate technique. Twenty four hours later, cells
were washed in PBS and refed with DMEM. Cells were then visualized
under fluorescence microscopy.
EMSAs
Gel-mobility shift assays were performed with proteins derived
either from a coupled in vitro translation/transcription
reaction in reticulocyte lysate (Promega, Madison WI) or from
Escherichia coli as GST-fusion proteins. Either TRß1 or
RXR
(0.5 to 4 µl) with equal amounts of NCoRI or SMRTI construct
were used, as well as 50,000150,000 cpm of a
32P-radiolabeled DR+4 (5'-AGGTCACAGGAGGTCA-3'), palindromic
(5'-AGGTCATGACCT-3'), F2-LYS (5'-TGACCCCAGCTGAGGTCA-3'),
oligonucleotide probe. When employed, 201000 ng of GST-NCoR were
used. Incubations were carried out at room temperature, and complexes
were resolved on 5% nondenaturing acrylamide gels, followed by
autoradiography. The concentration of T3 used was 10
nM. EMSA with antibody supershifts (anti-RXR
, Santa Cruz
Biotechnology, Santa Cruz, CA; anti-TR, Affinity Bioreagents, Inc.,
Golden, CO; anti-C/EBP, Santa Cruz Biotechnology) were carried out
similarly, except that in vitro translated proteins and
probe were incubated at room temperature for 20 min, followed by
addition of 1 µl antibody. The resulting complexes were incubated for
another 20 min at room temperature and then resolved on a 5%
nondenaturing gel, followed by autoradiography.
GST-Protein Binding Assays
GST-TR was expressed in BL21 E. coli expressing
thioredoxin (41) by induction with 0.1 MM
isopropylthio-ß-D-galactosidase (IPTG) at 30 C. GST-NCoR
was expressed in DH5
E. coli under similar conditions.
The proteins were isolated by lysis with lysozyme and purified on
Sepharose beads. Verification of protein synthesis was obtained on
SDS-PAGE. GST-TR (50 µl) and equivalent amounts of GST alone were
incubated with 6 µl of [35S]methionine-labeled in
vitro translated NCoRI or SMRTI. The concentration of
T3 used was 10-6 M. After
extensive washing, the bound proteins were eluted by boiling in
SDS-PAGE 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 the thioredoxin-expressing BL21 E.
coli.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Anthony Hollenberg, Thyroid Unit, Department of Medicine, Beth Israel-Deaconess Medical Center and Harvard Medical School, 330 Brookline Avenue, Boston, Massachusetts 02215. E-mail: thollenb{at}bidmc.harvard.edu
This work was supported by NIH Grants to A.N.H. (5DK 02354) and
F.E.W. (3DK49126).
Received for publication April 28, 1998.
Revision received June 9, 1998.
Accepted for publication July 10, 1998.
 |
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