Gene Silencing by Chicken Ovalbumin Upstream Promoter-Transcription Factor I (COUP-TFI) Is Mediated by Transcriptional Corepressors, Nuclear Receptor-Corepressor (N-CoR) and Silencing Mediator for Retinoic Acid Receptor and Thyroid Hormone Receptor (SMRT)
Hirotaka Shibata,
Zafar Nawaz,
Sophia Y. Tsai,
Bert W. OMalley and
Ming-Jer Tsai
Department of Cell Biology, Baylor College of Medicine,
Houston, Texas 77030
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ABSTRACT
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Chicken ovalbumin upstream promoter-transcription
factors (COUP-TFs) are orphan receptors that belong to the
steroid/thyroid hormone receptor (TR) superfamily and can repress the
transcriptional activity of several target genes; however, the precise
mechanism of this repression is unknown. Transfection of a Gal4
DNA-binding domain fused to the putative ligand-binding domain of
COUP-TFI (Gal4-COUP-TFI) significantly represses the basal
transcriptional activity of a reporter gene containing Gal4-binding
sites. Cotransfection of COUP-TFI can relieve the Gal4-COUP-TFI
repression in a dose-dependent manner. In contrast, COUP-TFI
35,
which lacks the repressor domain (the C-terminal 35 amino acids), fails
to relieve this repression. This finding suggests that the repressor
domain of COUP-TFI may squelch a limiting amount of corepressor in HeLa
cells. In addition, increasing concentrations of TRß also can relieve
the COUP-TFI repression in a hormone-sensitive manner. Similarly,
overexpression of increasing concentration of COUP-TFI, but not
COUP-TFI
35, can squelch the silencing activity of the unliganded
TRß. Collectively, these results indicate that COUP-TFI and TRß
share a common corepressor(s) for their silencing activity. To
determine which corepressor is involved in the COUP-TF-silencing
activity, we used a yeast two-hybrid and in vitro GST
pull-down assays to demonstrate that COUP-TFI can interact with the
fragment of N-CoR (nuclear receptor-corepressor) encoding amino acids
921-2453 and the fragments of SMRT (silencing mediator for retinoic
acid receptor and TR) encoding amino acids 29564 and 565-1289,
respectively. Interestingly, the fragment of SMRT encoding amino acids
11921495, which strongly interacts with TRß, interacts very weakly
with COUP-TFI. Furthermore, overexpression of N-CoR or SMRT potentiates
the silencing activity of COUP-TFI and can relieve the
COUP-TFI-mediated squelching of Gal4-COUP-TFI activity. Therefore, our
studies indicate that N-CoR and SMRT act as corepressors for the
COUP-TFI silencing activity.
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INTRODUCTION
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Steroid/thyroid hormone receptors are ligand-dependent
transcription factors that regulate diverse aspects of growth,
development, and homeostasis by binding as monomer, homodimers, or
heterodimers to their cognate DNA response elements to modulate
transcription of target genes (1, 2, 3). Thyroid hormone (TR) and retinoic
acid receptors (RAR) bind to cognate DNA response elements and repress
basal promoter activity in the absence of ligand (3, 4, 5). The region
important for repression may represent a unique interaction surface for
contacting a particular target within the basal transcriptional
machinery. Highly charged regions are a common feature of other
repression motifs. These charged regions consist of basic residues (6)
or acidic residues (7, 8). In contrast to these well-defined small
repression motifs, the homologous repression regions of TR and RAR are
large and appear to include multiple regions within the ligand-binding
domains (LBDs) and hinge regions. Many interacting proteins have been
identified and proposed to be important for mediating the repressor
function. The identified molecular targets of repressors can be
categorized into three groups (9): basal transcription factors,
activators, and coregulators, such as coactivators and
corepressors.
Silencing activity of unliganded TR and RAR requires limiting factors
termed corepressors (5). Recently, two corepressors that bind to
unliganded TR and RAR have been cloned using a yeast two-hybrid screen:
N-CoR (nuclear receptor-corepressor) (10, 11) and SMRT (silencing
mediator for RAR and TR) (12, 13, 14, 15). Binding of these corepressors is
necessary for unliganded receptors to silence the activity of target
promoters. Prevailing evidence suggests that binding of hormone changes
the conformation of the receptors, which results in the release of the
corepressor and recruitment of coactivators, thereby abolishing their
silencing activity. These two corepressors exhibit significant sequence
homologies in their receptor-interacting domains, suggesting the
existence of a corepressor gene family. COUP-TFs (chicken ovalbumin
upstream promoter-transcription factors) belong to the steroid receptor
superfamily and are classified as orphan receptors because their ligand
has yet to be defined. There are two COUP-TF genes in mammals, COUP-TFI
and COUP-TFII. COUP-TFs have been implicated in neurogenesis,
organogenesis, and cell fate determination (16, 17, 18, 19). COUP-TFs can form
stable homodimers and bind to a variety of hormone response elements
recognized by other members of the steroid receptor superfamily, such
as RAR, retinoid X receptor (RXR), TR, vitamin D receptor (VDR),
peroxisome proliferator-activated receptor, and hepatocyte nuclear
factor 4. COUP-TFs can thereby inhibit transcriptional activities of
these receptors on both consensus and natural response elements (17, 20). Four mechanisms have been proposed to address COUP-TFs ability
to inhibit the transactivation function of other members of the steroid
receptor superfamily. First, COUP-TFs repress the hormone-dependent
transactivation of target genes by VDR, TR, and RAR through direct
competition for occupancy of their response elements (21, 22, 23). Second,
COUP-TFs heterodimerize with RXR to reduce the available concentration
of RXR for heterodimerization with TR, VDR, RAR, and peroxisome
proliferator-activated receptor and thus indirectly interfere with
these receptors to transactivate their target genes (21, 22, 24, 25, 26, 27).
Third, COUP-TFs can tether to DNA via LBD-LBD interactions with TR,
RAR, and RXR to transrepress their activities (27). Finally, COUP-TFs
have been shown to repress basal and activator-dependent
transcriptional activities of various promoters when their binding site
is placed upstream or downstream of these promoters (27). The silencing
domain in COUP-TFs was localized to the C terminus of the putative LBD,
which can be transferred to the heterologous Gal4 DNA-binding domain
(DBD). Activators that can be repressed by COUP-TFs include acidic
(Gal4-RII), glutamine-rich (Gal4-ftzQ), proline-rich (Gal4-CTF1P), and
Ser/Thr-rich (Gal4-ZenST) transactivators (27). Because COUP-TF can
repress such a diverse group of transactivators, it is unlikely that
COUP-TFs repression is through direct quenching of these
transactivators or by interfering with their respective targets. It is
rather likely that COUP-TFs interact with a common target, a putative
corepressor(s) that mediates their repression.
To substantiate this hypothesis, we address two major questions in this
paper; 1) Does COUP-TFI-mediated repression require corepressors? and
2) If it does, does COUP-TFI share corepressors with other receptors,
such as TR and RAR? To answer these questions, we first examined
whether cofactors are involved in COUP-TFI-mediated repression using
either self-squelching or TR/COUP-TF-mediated mutual squelching
experiments. Next, we examined whether COUP-TFI can interact with N-CoR
or SMRT. Finally, we examined whether N-CoR or SMRT can function as a
corepressor in COUP-TFI-mediated repression. Results from these
experiments indicate that both N-CoR and SMRT can function as
corepressors for COUP-TFI-mediated repression of target genes.
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RESULTS
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COUP-TFI-Mediated Transcriptional Repression Requires
Corepressor(s)
The putative LBD of the COUP-TFI (amino acid 184423) has been
previously shown to contain a transcriptional repression domain. To
repress transcription, it is generally believed that the COUP-TFI
interacts with the basal transcriptional machinery either directly or
indirectly through other cofactor molecules. To determine whether a
cofactor(s) is required for this repressor activity, we carried out
transfection experiments. Transfection of a Gal4 DBD fused to the
COUP-TFI (amino acid 184423) cDNA, designated as Gal4-COUP-TFI,
significantly repressed basal promoter activity of the 17 mer x
4-thymidine kinase promoter-linked luciferase reporter DNA as shown
previously (Fig. 1
). This repression activity can be
reversed by the exogenously expressed LBD of COUP-TFI (184423), which
does not bind to the reporter construct, in a dose-dependent manner.
The observed release of repression cannot be attributed to the
nonspecific effect of overexpression of COUP-TFI on Gal DNA-binding
activity because overexpression of COUP-TFI had little effect on the
basal promoter activity when cotransfected with the Gal4 DBD alone
(27). In contrast, COUP-TFI mutant, COUP-TFI
35 (amino acid
184388), which does not contain a repressor domain, cannot reverse
Gal4-COUP-TFI-mediated repression. Overexpression of COUP-TFI or
COUP-TFI
35 does not affect the basal promoter activity of Gal4 (data
not shown). These results clearly indicate that a limiting factor,
termed corepressor(s), is required for the activity of
COUP-TFI-mediated repression. These results also suggest that the
C-terminal 35 amino acids are required for interaction with the
putative corepressor(s).

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Figure 1. COUP-TFI Is Able to Squelch the Repressor Activity
of Gal4-COUP-TFI
HeLa cells were transfected with 0.1 µg Gal4-COUP-TFI cDNA, 0.3 µg
17 mer x 4-tk-luciferase reporter DNA, and increasing amounts of
COUP-TFI or COUP-TFI 35 expression plasmid (0.2, 0.4, and 1.0 µg).
Gal4 cDNA (0.1 µg) was transfected instead of Gal4-COUP-TFI in lane
1.
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COUP-TFI Shares Common Corepressor(s) with TRß
To elucidate whether COUP-TFI shares common corepressors with
TRß, squelching experiments were performed. Overexpression of TRß
in the absence of T3 can reverse the basal repression
activity of Gal4-COUP-TFI by squelching endogenous corepressors in a
dose-dependent manner. In contrast, the liganded TRß cannot squelch
the repression (lines 38, Fig. 2
). Similarly,
overexpression of TR mutant, TR168456(V174A/D177A), which loses its
silencing activity but retains hormone-dependent transactivation (28),
cannot relieve the basal repression activity of Gal4-COUP-TFI (lines
911, Fig. 2
). These findings strongly suggest that TRß- and
COUP-TFI-mediated repression share the same corepressor(s).

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Figure 2. Unliganded TRß, but Not Liganded TRß or
TR168456 (V174A/D177A), Is Able to Squelch Repressor Activity of
Gal4-COUP-TFI
HeLa cells were transfected with 0.1 µg Gal4-COUP-TFI cDNA, 0.3 µg
17 mer x 4-tk-luciferase reporter DNA, and increasing amounts of
TRß or TR168456 (V174A/D177A) expression plasmids (0.2, 0.5, and
1.0 µg). Cells were treated with 10-7 M
T3 for 24 h in lanes 68. Gal4 cDNA (0.1 µg) was
transfected instead of Gal4-COUP-TFI in lane 1.
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In a reverse experiment, we examined whether COUP-TFI can squelch the
repression activity of Gal4-TRß. As shown in Fig. 3
, transfection of Gal4-TRß into HeLa cells significantly repressed the
basal promoter activity of the reporter construct. Overexpression of
COUP-TFI can reverse this repression in a dose-dependent manner. As
expected, we observe no reversal of TRß-mediated repression by
overexpression of the repression-defective COUP-TFI mutant,
COUP-TFI
35. These results further suggest that common corepressors
are shared by COUP-TFI and TRß for their repression activity.

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Figure 3. COUP-TFI, but Not COUP-TFI 35, Is Able to Squelch
the Repressor Activity of Gal4-TRß
HeLa cells were transfected with 0.1 µg Gal4-TRß cDNA, 0.3 µg 17
mer x 4-tk-luciferase reporter DNA, and increasing amounts of
COUP-TFI or COUP-TFI 35 (0.2, 0.4, and 1.0 µg). Gal4 cDNA (0.1
µg) was transfected instead of Gal4-COUP-TFI in lane 1.
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Both N-CoR and SMRT Interact with COUP-TFI
Using the LBD of TRß as a bait in a yeast two-hybrid screen, the
TR-interacting protein, SMRT, has been cloned (12, 14, 15). Similarly,
Hörlein et al. (10) have cloned another TR-interacting
protein, N-CoR, which has sequence similarity to SMRT. Both N-CoR and
SMRT have been identified as corepressors for TR and RAR. We asked
whether these two corepressors can function as corepressors for
COUP-TFI. First, we examined whether COUP-TFI can interact specifically
with these authentic corepressors. Using a yeast two-hybrid assay, we
confirmed that these corepressors can interact with TRß in a
hormone-dependent manner. As shown in Fig. 4
, A and B, a
fragment of N-CoR encoding amino acids 921-2453 and fragments of SMRT
encoding amino acids 565-1495 and 11921495 can interact with TRß in
a hormone-dependent manner as previously described (10, 12, 14). It
should be pointed out that, to our surprise, a larger fragment of N-CoR
encoding amino acids 190-2453, which contains a fragment of N-CoR
encoding amino acids 921-2453, cannot interact with TRß in a yeast
two-hybrid assay. Although the reason for this discrepancy is not clear
at present, it is possible that the N-terminal repression domain of
N-CoR encoding amino acids 190920 is very potent and overrides the
activation function of Gal4 in a yeast two-hybrid assay. This notion is
further supported by our observation that a fragment of N-CoR encoding
amino acids 190-2453 can interact with TRß in vitro (data
not shown).

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Figure 4. Interaction of TRß or COUP-TFI with N-CoR or SMRT
in Yeast Two-Hybrid Assays
A, Various fragments of SMRT and N-CoR constructs. The fragments of
SMRT encoding amino acids 29564, 565-1289, 565-1495, and 11921495
were constructed. The fragment of SMRT encoding amino acids 11921495
was obtained by yeast two-hybrid screen as a bait of the LBD of TRß.
The fragments of N-CoR encoding amino acids 190-2453 and 921-2453 were
constructed. In N-CoR, there are two repression domains (RD1 and RD2)
as shown in the N-terminal region. The acidic-basic motif (AB) and the
serine-glycine-rich domain (SG) in the central region are indicated.
Glutamine-rich regions (Q) and predicted amphipathic -helix (H)
regions are also indicated. The most N-terminal region of SMRT (amino
acids 1483) contains four putative repeated motifs, and it has 44%
identity with N-CoR. A series of deletion mutants of N-CoR or SMRT
(illustrated in Fig. 4A ) were created and tested for interaction in the
yeast two-hybrid system with TRß (B) or COUP-TFI (C). For the
experiments presented here, Gal4 DBD-TR168456 or Gal4 DBD-COUP-TFI
constructs were coexpressed with Gal4 AD-N-CoR or SMRT deletion
mutants. Each two-hybrid pair was transformed into the yeast y190, the
transformants were propagated, and the ß-galactosidase activity was
determined. The effects of T3 on the TR/N-CoR or SMRT
interaction in the presence of 1 µM T3 in the
culture medium was also tested. Cotransformation of Gal4 DBD-COUP-TFI
and Gal4 AD-TFIIB was used as a positive control. At least two
independent yeast transformants were analyzed, and the results were
determined as mean ± SEM.
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Next, we examined the interaction between COUP-TFI and N-CoR or SMRT.
We first demonstrated that no interaction between Gal4DBD-COUP-TFI
(amino acids 184423) fusion and Gal4 activation domain (AD) (empty
vector) was shown as a negative control (lane 1 in Fig. 4C
). In
contrast, as a positive control, we showed a strong interaction between
Gal4DBD-COUP-TFI and Gal4AD-TFIIB (lane 7 in Fig. 4C
). We then
examined the interaction of various deletion fragments of N-CoR or SMRT
with COUP-TFI (Fig. 4C
). In a yeast two-hybrid assay system, the
interaction of COUP-TFI with N-CoR or SMRT was detected when COUP-TFI
was bound to DNA response element. In the absence of DNA response
element, no interaction was observed (data not shown). A fragment of
N-CoR encoding amino acids 921-2453 strongly interacts with COUP-TFI;
however, a fragment of N-CoR encoding amino acids 190-2453 does not
interact with COUP-TFI in a yeast two-hybrid assay. Similar to the
TRß interaction study (Fig. 4B
), the repression domains of N-CoR
encoding amino acids 190920 may interfere with the Gal4 activation
domain function in a yeast two-hybrid assay; thus, a fragment of N-CoR
encoding amino acids 190-2453 is not able to activate ß-galactosidase
activity of the reporter. However, both N-CoR fragments encoding amino
acids 190-2453 and 921-2453 were found to interact with COUP-TFI in an
in vitro glutathione S-transferase (GST)
pull-down assay because these in vitro interactions are
presumably not influenced by the repressor function of N-CoR. Thus, a
fragment of COUP-TFI encoding amino acids 56423 can interact
specifically with N-CoR in yeast and in vitro. On the other
hand, fragments of SMRT encoding amino acids 29564 and 565-1495
strongly interact with COUP-TFI (Fig. 4C
). In contrast, a fragment of
SMRT encoding amino acids 11921495, which interacts well with TR,
interacts only weakly with COUP-TFI in a yeast two-hybrid assay (Fig. 4C
). These results suggest that the COUP-TFI-interacting domain of SMRT
is located mainly at amino acids 29564 and 565-1191. These
interaction data in yeast are further supported by the in
vitro protein-protein interactions as shown in Fig. 5B
. Both fragments of SMRT encoding amino acids 29564
and 565-1289 are able to interact with GST-COUP-TFI, whereas a fragment
of SMRT encoding amino acids 11921495 fails to interact with
GST-COUP-TFI.

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Figure 5. Interaction between COUP-TFI and N-CoR or SMRT in
in Vitro GST Pull-Down Assays
Radiolabeled N-CoR, SMRT, or TFIIB were tested for their abilities to
bind to an immobilized GST-COUP-TFI fusion protein or to GST control
protein. GST or GST-COUP-TFI fusion protein were expressed in yeast and
purified by adsorption to a glutathione-Sepharose beads. A, The
fragments of N-CoR encoding amino acids 190-2453 and 921-2453 and
TFIIB, translated in vitro, were incubated with the GST
control protein (lanes 2, 5, and 8) or GST-COUP-TFI (lanes 3, 6, and 9)
for 3060 min at 4 C with rocking. The GST or GST-COUP-TFI Sepharose
beads were then extensively washed, and radiolabeled corepressor
proteins (lanes 3 and 6) or TFIIB (lane 9) bound to the matrix were
eluted with SDS loading buffer and analyzed by SDS-PAGE and
autoradiography. Lanes 1, 4, and 7 represent each input protein. B, The
fragments of SMRT encoding amino acids 29564 (lanes 13), 565-1285
(lanes 46), and 11921495 (lanes 79) were radiolabeled and tested
for their abilities to bind to an immobilized GST-COUP-TFI (lanes 3, 6,
and 9 or to GST control protein (lanes 2, 5, and 8), while Lanes 1, 4,
and 7 represent each input protein. The molecular weight of each
protein is indicated.
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N-CoR and SMRT Potentiate the Basal Repression by Gal4-COUP-TFI
From the above studies, we have shown that N-CoR and SMRT can
interact with COUP-TFI specifically. We then asked whether these two
corepressors are required to mediate COUP-TFI repression activity in
mammalian cells. As shown in Fig. 6
, neither N-CoR nor
SMRT have a significant effect on transcription activity of the
reporter construct in the absence of cotransfected Gal4-COUP-TFI.
However, both N-CoR and SMRT can potentiate the repressor function of
Gal4-COUP-TFI in a dose-dependent manner in HeLa cells. These data
suggest that both N-CoR and SMRT can function as corepressors of
COUP-TFI in HeLa cells. Thus, it is likely that they are the endogenous
corepressors of COUP-TFI in HeLa cells.

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Figure 6. N-CoR and SMRT Can Potentiate Basal Repression
Activity of Gal4-COUP-TFI
HeLa cells were transfected with 0.1 µg Gal4 or Gal4-COUP-TFI cDNA,
0.3 µg 17 mer x 4-tk-luciferase reporter DNA, and increasing
amounts of N-CoR or SMRT expression plasmid (0.5 and 1.0 µg).
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N-CoR and SMRT Can Reverse the Squelching of COUP-TFI-Mediated
Repression
As shown earlier (Fig. 1
), overexpression of COUP-TFI can squelch
the repressor activity of Gal4-COUP-TFI. If N-CoR and SMRT are the
limiting factors required for COUP-TFI-mediated repression, we expect
that overexpression of N-CoR or SMRT should reverse this squelching
activity. As shown in Fig. 7
, overexpression of N-CoR or
SMRT was able to relieve the squelching effects of COUP-TFI on
Gal4-COUP-TFI repression function in a dose-dependent manner. On the
other hand, overexpression of N-CoR or SMRT had no significant effect
on transcription activity of the reporter construct in the absence of
cotransfected Gal4-COUP-TFI (data not shown). Therefore, both N-CoR and
SMRT can substitute for the endogenous corepressor(s) in mediating
COUP-TFIs repressor function.

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Figure 7. Reversal of COUP-TFI-Mediated Squelching of Basal
Repression Activity of Gal4-COUP-TFI
HeLa cells were transfected with 0.1 µg Gal4 or Gal4-COUP-TFI cDNA,
0.3 µg 17 mer x 4-tk-luciferase reporter DNA, 1.0 µg COUP-TFI
cDNA, and increasing amounts of N-CoR or SMRT expression plasmid (50,
100, and 200 ng).
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DISCUSSION
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We have shown that both N-CoR and SMRT can function as
corepressors for COUP-TFI-mediated gene silencing. Since COUP-TFI has
been shown to interact with TFIIB in vitro (29, 30), it is
proposed that COUP-TFI can interact with TFIIB in a nonproductive
manner to interfere with the basal transcription. However, many
coactivators and corepressors have been identified as positive and
negative cofactors in the transcriptional control of target genes by
members of the steroid/TR superfamily (15, 31). Therefore, it is
tempting to speculate that some of the cofactors may also be involved
in gene-silencing mechanisms of orphan receptors, such as COUP-TFI.
Initial evidence for mediators of nuclear receptor transactivation came
from the finding that transiently transfecting increasing amounts of an
ER-expressing plasmid into cultured cells results in a bell-shaped
curve of transcriptional activation and was attributed to the
squelching of a coactivator(s) by the excessive amounts of estrogen
receptor (32). By analogy with that, we demonstrated that a putative
corepressor(s) may be involved in COUP-TFI-mediated basal repression
because overexpression of COUP-TFI, but not COUP-TFI
35 (which has
lost its repression domain), can squelch the repression activity of
Gal4-COUP-TFI. Previously, we showed that a 15-amino-acid deletion from
the C terminus (Gal4-COUP-TF
15) had little effect on Gal4-COUP-TF
activity, whereas a 25-amino-acid (Gal4-COUP-TF
25) or a
35-amino-acid (Gal4-COUP-TF
35) deletion largely impaired its active
repression function (27). Therefore, these data suggested that the
C-terminal border of the major active repression function of COUP-TF is
located within amino acids 408 and 398, and it is this region that may
interact with the corepressor(s). In addition, the repressor domain of
COUP-TFI may directly target the basal transcriptional machinery.
To examine whether a corepressor(s) can be shared between TRß and
COUP-TFI, squelching experiments were performed. As shown in Fig. 2
, unliganded but not liganded TRß can squelch the basal repression of
Gal4-COUP-TFI. The evidence that the TR mutant, TR168456
(V174A/D177A) (28), which does not interact with the putative
corepressors, cannot squelch the basal repression of Gal4-COUP-TFI
further supports the notion that common corepressors may be involved in
basal repression between TRß and COUP-TFI. Furthermore,
overexpression of COUP-TFI, but not COUP-TFI
35, can squelch the
basal repression activity of Gal4-TRß, also suggesting that common
corepressors can function for both COUP-TFI and TRß. Theoretically,
the target of transcriptional interference may be either a basal
transcription factor(s) or a corepressor(s). Transfection of COUP-TFI
or TRß did not change the basal transcription of a PRE-tk-luciferase
reporter lacking a proper response element (data not shown), suggesting
that squelchers are unlikely to interfere directly with the function of
any basal transcriptional machinery. Therefore, the target(s) of
interference between COUP-TF and TRß is likely to be one or more
transcriptional corepressor protein(s).
Because COUP-TFI can form homodimers by itself or form heterodimers
with TRß (27), it is possible that the squelchers can form
non-DNA-binding heterodimers with the repressors used in Figs. 1
, 2
, and 3
(Gal4-COUP-TFI/COUP-TFI for Fig. 1
, Gal4-COUP-TFI/TRß for Fig. 2
, and Gal4-TRß/COUP-TFI for Fig. 3
). If this is the case, inhibition
of repressor activity may be due to inhibition of DNA binding rather
than to squelching of a limiting corepressor(s). However, several lines
of evidence suggest that this is not the case. First, both COUP-TFI and
TRß have the ability to bind specifically to corepressors. Second, a
mutant form of TRß (TR168456 (V174A/D177A)), which loses its
ability to interact with corepressor but retains its intact
dimerization domain, is not able to inhibit the repression activity of
Gal4-COUP-TFI. Third, COUP-TF mutant (COUP-TFI
35), which loses its
ability in silencing, also loses its ability to squelch repressor
activity of TRß. Finally and most importantly, we have demonstrated
directly that both N-CoR and SMRT can mediate repressor activity of
COUP-TFI (Fig. 6
) and reverse the self-squelching activity (Fig. 7
).
Therefore, inhibition of repression activity by squelchers in Figs. 1
, 2
, and 3
indeed results from squelching of a limiting
corepressor(s).
Because squelching experiments showed that COUP-TFI may share a
corepressor(s) with TRß, we examined protein-protein interaction
between COUP-TFI and N-CoR or SMRT in a yeast two-hybrid assay and in
in vitro GST pull-down assays as shown in Figs. 4
and 5
. The
amino acid 921-2453 fragment of N-CoR interacts with COUP-TFI. This
result is consistent with what has been observed in TR interaction
studies (10, 33). Interaction between COUP-TFI and the amino acid
190-2453 fragment of N-CoR was not detected in a yeast two-hybrid
assay, but was detected in an in vitro GST pull-down assay.
This discrepancy is likely due to the potent repressor domain of the
N-terminal portion of N-CoR, which suppresses the Gal4 activation
function, resulting in interference with ß-galactosidase expression.
Consistent with this interpretation, Seol et al. (33) also
recently demonstrated that the repressor domain of N-CoR strongly
interferes with the mammalian two-hybrid assay to study the
protein-protein interaction between N-CoR and TR or RAR. On the other
hand, the amino acid 29564 and amino acid 565-1289, but not amino
acid 11921495, fragments of SMRT strongly interact with COUP-TF in
yeast and in vitro assays. Based on these data, we concluded
that there are at least two COUP-TFI-interacting domains within the
SMRT molecules. The lack of interaction between COUP-TFI and SMRT
(11921495) is surprising because this region interacts very strongly
with TRß. Thus, different repressors may interact differentially with
SMRT. Finally, the corepressor-interacting region in COUP-TFI is
localized in the extreme C terminus, which is quite different from the
region of TR and RAR where hinge and N-terminal portions of LBD are
involved in this interaction. Thus, repression function of COUP-TFs may
act in a different way from that of TR and RAR (Fig. 8
).

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Figure 8. Structure Domains of Repressors and Corepressors
A, Functional domains of COUP-TFI, TRß, and RevErb A. A/B, C, D, E/F
are functional domains of receptors (3). CID (corepressor-interacting
domain) is a sequence important for repressor function and contains a
region that interacts with corepressors. The CoR box is a conserved
region of human TR , TRß, RAR , and RevErb A. The CoR box of
TRß is a repressor domain that interacts with corepressors; however,
CIDs of RevErb A are different from the region of CoR box. B,
Receptor-interacting domains of SMRT and N-CoR. Receptor-interacting
domains of corepressors are indicated below each scheme
of SMRT and N-CoR. In N-CoR, there are two repression domains (RD1 and
RD2) as shown in the N-terminal region. The most N-terminal region of
SMRT (amino acids 1483) contains four putative repeated motifs and it
has 44% identity with N-CoR. The acidic-basic motif (<1108>) and the
serine-glycine-rich domain (SG) in the central region are indicated.
Glutamine-rich regions (Q) and predicted amphipathic -helix (H)
regions are also indicated.
|
|
In support of this observation, N-CoR has been shown to function as a
corepressor for RevErb A (34). However, a region homologous to the CoR
box in the RevErb A, which is necessary for TR and RAR to interact with
N-CoR, is not required for RevErb A-mediated repression. N-CoR contains
two adjacent but distinct interaction domains, amino acids 20402239
and 22392296. The former fragment of N-CoR strongly interacts with
both TR and RevErb A; however, the latter fragment of N-CoR interacts
weakly and preferentially with RevErb A. Taken together, these data
suggest that different nuclear receptors, utilizing different amino
acid sequences, repress transcription by interacting with different
region of N-CoR or SMRT as shown in Fig. 8
.
Recently, an activator for COUP-TFII has been identified using a yeast
two-hybrid screen, designated ORCA (orphan receptor coactivator) (35),
and this factor is identical to a recently described ligand (p62) of
tyrosine kinase- signaling molecule p56lck, suggesting that
ORCA may link COUP-TFII and cell surface signal transduction pathways.
This integrating role may be similar to what is observed with cAMP
response element-binding protein CBP and the related protein p300. CBP
plays a role in integrating cAMP second messenger and nuclear hormone
receptor signal transduction pathways. Of interest, ORCA/p62 shares a
small region of homology with CBP, suggesting a potential similarity in
their mechanism of action. However, based on their data, ORCA/p62 does
not bind directly to COUP-TFII-binding sites and COUP-TFII/ORCA complex
is not detected in gel retardation assays. Therefore, it is possible
that ORCA/p62 may function directly or indirectly by phosphorylating
COUP-TFII or ORCA/p62 may overcome the function of a specific
COUP-TFII-associated corepressor. Because ORCA/p62 can convert
COUP-TFII into a transcriptional activator in a ligand-independent
manner in mammalian cells, it is possible that COUP-TFI can also be
activated by a coactivator(s) in a ligand-independent manner. Our
preliminary data showed that overexpression of hSRC-1a (36), which is a
general coactivator for members of steroid receptors, cannot relieve
the repressor activity of Gal4-COUP-TFI in HeLa cells (data not
shown).
In conclusion, we demonstrated that corepressors are involved in the
mechanisms of COUP-TFI-mediated gene silencing, and that both N-CoR and
SMRT can function as corepressors for COUP-TFI in mammalian cells.
Therefore, orphan receptors such as COUP-TFI and RevErb A can function
as a repressor in vivo by utilizing corepressors that are
common for members of the TR and RAR subfamily.
 |
MATERIALS AND METHODS
|
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Mammalian Cell Culture, Transient Transfections, and Luciferase
Assays
HeLa cells were routinely maintained in DMEM supplemented with
10% FBS. Twenty-four hours before transfection, 2 x
105 cells were plated per well on a six-well dish in DMEM
containing 5% dextran-coated charcoal-stripped serum. Cells were
transfected with the indicated DNAs using Lipofectin (Life
Technologies, Grand Island, NY) according to the manufacturers
guidelines. Usually, 1.8 µg of total DNA including 0.3 µg of
reporter and 0.05 to 1.0 µg of expression vector were used per 35-mm
diameter dish. Within 24 h, the DNA/Lipofectin mixture was removed
and cells were fed with DMEM containing 5% stripped serum and the
indicated hormones; cells were harvested 24 h later. Cells
extracts were assayed for luciferase activity using the Luciferase
Assay System (Promega, Madison, WI). Data are presented as an average
of three separate experiments ± SEM. All
transfections were performed at least three times in triplicate.
Cloning of SMRT by Yeast Two-Hybrid System
Yeast strains used are as follows. y190:
MATa, leu23, 112,
ura352, trp1901, his2-D200, ade2101,
gal4
gal180
URA3 GAL-lacZ, LYS GAL-HIS3,
cyhr. y187: MAT
, gal4, gal80,
his3, trp1901, ade2101, ura352, leu23, -112, URA3 GAL-lacZ
met-. BJ2168: MATa, prc1407, Prb11122,
pep43, leu2, trp1, ura 352. Yeast-selective media and plates
were prepared according to Guthrie and Fink (37). The yeast strain y190
containing pAS1cyh2-TR168456 was transformed with a human brain cDNA
library in pGAD10 (Clontech, Palo Alto, CA) and plated on synthetic
complete medium lacking tryptophan, leucine, and histidine (containing
25 mM 3-aminotriazole) as described by Durfee et
al. (38). His+ colonies exhibiting ß-galactosidase
activity using the filter lift assay were further characterized.
ß-Galactosidase activity was determined using chlorophenol red
ß-D galactopyranoside as described (38). To recover the
library plasmids, total DNA from yeast was isolated and used to
transform Escherichia coli (HB101) which lacks
leu2 gene. Transformants were identified on minimal medium
lacking leucine and containing ampicillin. To ensure that the correct
cDNAs were identified, library plasmids isolated were retransformed
into y190 containing pAS1cyh2-TR168456, and ß-galactosidase
activity was determined. The specificity of the interaction of cor 10.1
(SMRT 11921495), one of the 12 positive clones, with TR was
determined by mating y190 containing pGAD10-cor 10.1 with the strain
y187 containing either pAS1-SNF, pAS1-cdk2, pAS1-p53, or pAS1-lamin.
The ß-galactosidase activity of these diploids was examined using the
filter lift and chlorophenyl red ß-D galactopyranoside
methods. The cor 10.1 clone was identical to recently identified
corepressor SMRT. The yeast two-hybrid system was also used to
determine protein-protein interaction between COUP-TFI and SMRT or
N-CoR.
Protein-Protein Interaction by GST-Pulldown Assay
GST-COUP-TFI (pCBGST1-COUP-TFI) fusion protein was expressed and
extracted in yeast strain BJ2168 as described previously (39).
GST-pulldown assay was performed as described with modifications (4, 39): 30 µl glutathione-Sepharose beads stored in NENT buffer (500
mM NaCl, 1 mM EDTA, 20 mM Tris, pH
8.0, 0.5% NP-40, 1 mM dithiothreitol, 6 mM
MgCl2, and 8% glycerol) were incubated with yeast extracts
containing GST-fusion proteins in a 1:1 vol ratio together with NENT
buffer for 3060 min at 4 C. Preparation of yeast extracts containing
GST-fusion protein was described previously (39). Subsequently, the
supernatant was removed and the beads were washed twice with 1 ml NENT
buffer and twice with 1 ml transcription washing buffer (60
mM NaCl, 1 mM EDTA, 20 mM Tris, pH
8.0, 0.05% NP-40, 1 mM dithiothreitol, 6 mM
MgCl2, and 8% glycerol). In vitro-translated and
-radiolabeled proteins were obtained using TNT Coupled Reticulocyte
Lysate Systems (Promega). Five to 10 µl crude lysate were incubated
with the beads in 200 µl transcription washing buffer for 2 h at
4 C. Finally, the beads were washed five times with 1 ml NENT buffer
and proteins were solubilized in SDS loading buffer and analyzed on
SDS-PAGE. The input lane contains 10% of the labeled protein used for
binding.
Plasmids
Mammalian Expression Vectors
The expression plasmids pABgal147, pAB
gal, pABgalTRß,
pAB
galTRß, pAB
gal-TR168456 (V174A/D177A), pRSV-COUP-TFI,
pRSV-COUP-TFI
35, and pRSVgalCOUP-TFI were described previously (5, 20, 21, 27, 28, 40). pCMX-N-CoR (10) and pCMX-SMRT (12) were generous
gifts from Dr. A. J. Hörlein and Dr. J. D. Chen, respectively.
The 17 mer x 4-tk-LUC reporter gene contains four copies of a
17-mer upstream activating sequence located upstream of the thymidine
kinase promoter and luciferase gene. pCR3-SMRT5651289 was constructed
by TA Cloning (Invitrogen) of PCR-amplified product with primers
5'-AGCTGACGTCGACGCCTCGTG-3' and 5'-CTGCACCGCCTGGCTTCTAT-3' in which
template cDNA was made by reverse transcription of human skeletal
muscle mRNA (Clontech) with primer 5'-GCTGGCATGTTCCTGCACCG-3'.
pCR3-SMRT5651495 was constructed by inserting the EcoRI
(filled)-BglII fragment of pGAD10-SMRT11921495 into the
BglII-EcoRV site of pCR3-SMRT5651289.
pCR3-SMRT29564 was constructed by TA Cloning (Invitrogen) of
PCR-amplified product with primers 5'-AAGATTCCGAGCTCTGCTAC-3' and
5'-CACGAGGCGTCGACGTCAGC-3' in which template cDNA was made by reverse
transcription of human skeletal muscle mRNA (Clontech) with primer
5'-GTGCGGGGACTTGGCGATCT-3'. pCR3-SMRT291495 was constructed by
inserting the SalI fragment of pCR3-SMRT29564 into the
SalI site of pCR3-SMRT5651495. pAB
galSMRT291495 was
constructed by inserting the SalI (partial)-XbaI
fragment into the PvuII site of pAB
gal. All PCR generates
clones were sequenced to ensure that no mutation occurred during PCR
reactions.
Yeast Vectors
The Gal4 DBD-TRß168456 yeast expression plasmid
(pAS1cyh2-TR168456) was constructed by inserting the
HindIII-SmaI blunt-ended fragment of
pABgalTR168456 into the SmaI site of pAS1cyh2 (38).
The Gal4 DBD-COUP-TFI56423 yeast expression plasmid
(pAS1cyh2-COUP-TFI) was constructed by inserting the
EcoRI-SmaI (filled) fragment of
pGEM7Zf(+)-COUP-TFI into the SmaI site of pAS1cyh2. Yeast
expression plasmid, pCBGST1-COUP-TFI56423, was constructed by
inserting the EcoRI-SmaI (filled) fragment (amino
acids 56423) of pGEM7Zf(+)-COUP-TFI into the SmaI site of
pCBGST1 (39). Yeast expression plasmid, pACTII-N-CoR1902453, was
constructed by inserting the PvuI-SalI (filled)
fragment of pCMX-N-CoR into the NcoI-XhoI site
(filled) of pACTII (38). Yeast expression plasmid,
pACTII-N-CoR9212453, was constructed by inserting the
HincII-SalI (filled) fragment of pCMX-N-CoR into
the SmaI site of pACTII. pACTII-SMRT29564 was constructed
by inserting the SalI fragment (filled) of pCR3-SMRT29564
into the NcoI (filled) site of pACTII. pACTII-SMRT5651495
was constructed by inserting the SalI-XhoI
fragment of pCR3-SMRT5651495 into the NcoI (filled) site
of pACTII. Yeast expression plasmid, pGAD10-SMRT11921495, was
recovered from a yeast two-hybrid screen using pAS1cyh2-TR168456 as a
bait. Yeast expression plasmid, pACTII-TFIIB, was constructed by
inserting the NcoI (partial)-EcoRI fragment of
pGST-TFIIB (4) into the NcoI-EcoRI sites of
pACTII.
In Vitro Transcription and Translation Vectors
pT7-N-CoR1902453 was constructed by inserting the
PvuI-SalI fragment of pCMX-N-CoR into the
NcoI site (filled) of pT7ßSal (41). PT7-N-CoR9212453 was
constructed by inserting the HincII-SalI fragment
of pCMX-N-CoR into the AccI site (filled) of pT7ßSal.
pT7-SMRT29564 was constructed by inserting the SalI
fragment (filled) of pCR3-SMRT29564 into the
NcoI-EcoRI (filled) site of pT7ßSal.
pT7-SMRT5651289 was constructed by inserting the
SalI-EcoRV fragment of pCR3-SMRT5651289 into
the NcoI-EcoRI (filled) site of pT7ßSal.
PT7-SMRT11921495 was constructed by inserting the EcoRI
fragment (filled) of pGAD10-SMRT11921495 into the HincII
site of pT7ßSal. pT7-TFIIB was constructed by inserting the
NcoI (partial)-EcoRI fragment (filled) of
pGST-TFIIB (4) into the AccI site (filled) of pT7ßSal.
 |
ACKNOWLEDGMENTS
|
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We thank members of our laboratories for critically reading this
manuscript. We thank Drs. A. J. Hörlein and J. D. Chen for
providing pCMX-N-CoR and pCMX-SMRT, respectively. We also thank Dr.
Stephen Elledge for providing the yeast two-hybrid system.
 |
FOOTNOTES
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Address requests for reprints to: Ming-Jer Tsai, Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030.
This work was supported by NIH Grants (DK-45641 to M.J.T. and HD-08188
to B.W.O.).
Received for publication January 15, 1997.
Accepted for publication February 14, 1997.
 |
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