Silencing Mediator for Retinoid and Thyroid Hormone Receptors
Interacts with Octamer Transcription Factor-1 and Acts as a
Transcriptional Repressor*
Tomoko
Kakizawa,
Takahide
Miyamoto
,
Kazuo
Ichikawa,
Teiji
Takeda,
Satoru
Suzuki,
Jun-ichiro
Mori,
Mieko
Kumagai,
Koh
Yamashita, and
Kiyoshi
Hashizume
From the Department of Aging Medicine and Geriatrics, Shinshu
University School of Medicine, Matsumoto 390-8621, Japan
Received for publication, September 18, 2000, and in revised form, December 18, 2000
 |
ABSTRACT |
Octamer transcription factor-1 (Oct-1) is
a member of the POU (Pit-1, Oct-1,
unc-86) family of transcription factors and is involved in
the transcriptional regulation of a variety of gene expressions related
to cell cycle regulation, development, and hormonal signals. It has
been shown that Oct-1 acts not only as a transcriptional activator but
also as a transcriptional repressor for certain genes. The mechanism of
the repressive function of Oct-1 has not been well understood. Here we
demonstrate by using the glutathione S-transferase
pull-down assays and coimmunoprecipitation assays that the POU domain
of Oct-1 directly interacts with a silencing mediator for retinoid and
thyroid hormone receptors (SMRT). The interaction surfaces are located
in the C-terminal region of SMRT, which are different from previously
described silencing domains I and II or receptor interacting domains I
and II. In transient transfection assays in COS1 cells, overexpression of SMRT attenuated the augmentation of Oct-1 transcriptional activity by OBF-1/OCA-B, activator for Oct-1. In pull-down assays, increasing amounts of SMRT could compete the binding of OCA-B to Oct-1 POU domain.
The activity of Oct-1 could be determined by a regulated balance
between SMRT and OCA-B. Furthermore, cotransfected unliganded thyroid
hormone receptor enhanced the transactivation by Oct-1, and addition of
3,3',5-tri-iodo-L-thyronine obliterated the
stimulatory effects. Consequently, in the presence of cotransfected
thyroid hormone receptor, the octamer response element acts as an
element negatively regulated by
3,3',5-tri-iodo-L-thyronine. The results suggest that the
transcriptional activity of Oct-1 can be modulated by interaction
through its POU domain by a silencing mediator SMRT resulting in the
cross-talk between Oct-1 and nuclear receptors.
 |
INTRODUCTION |
Octamer transcription factor-1
(Oct-1)1 activates the
octamer motif containing gene promoters that are ubiquitously as well as tissue-specifically expressed genes such as histone H2B, the small
nuclear RNA, and Ig (1-3). Oct-1 is a member of a family of
transcription factors characterized by the presence of a bipartite DNA-binding domain (POU domain). The POU domain consists of two conserved regions, a POU-specific domain and a POU homeodomain (4, 5).
The both subdomains have a helix-turn-helix motif, acting not only as a
DNA-binding domain but also as a protein-protein interaction domain. A
number of transcription factors have been identified to interact with
the POU domains of Oct-1 such as TBP, TFIIB, HMG2, and Oct-binding
factor 1 (OBF-1) also referred to as Oct-1-associated coactivator
(OCA-B) (6-11). It has been shown that Oct-1 interacts with nuclear
hormone receptors such as retinoid X receptor, thyroid hormone receptor
(TR), and glucocorticoid receptor and influences their transcriptional
activity (12-14).
Oct-1 possesses not only transactivation function but also repression
function; von Willebrand factor promoter (15), prolactin gene promoter
(16), or rGH promoter (14) was shown to be down-regulated by Oct-1.
However, the mechanism of the bifunctional transcriptional activity of
Oct-1 was not fully understood. OCAB/OBF-1 has been shown to be
involved in the transcriptional activation by Oct-1, whereas the factor
mediating the repressor function has not been isolated (15, 16).
Silencing mediator for retinoid and thyroid hormone receptors (SMRT)
and the nuclear hormone receptor corepressor (N-CoR) were identified as
interacting proteins for unliganded nuclear receptors that repress the
basal transcriptional activity of target genes (17, 18). These
corepressors associate with hinge domain of nuclear receptors. It has
been demonstrated that SMRT and N-CoR directly interact with mSin3 and
recruit the histone deacetylases (HDAC1/Rpd3) to form a multisubunit
complex to modify the chromatin template of target genes (19).
SMRT/N-CoR functions as an adapter to link unliganded receptor
heterodimers with mSin3A and HDAC1 to create a hormone-sensitive
multimeric repressor complex that lead the chromatin to be
transcriptionally inactive (20). Interestingly, it has been
recently shown that SMRT/N-CoR interacts with various nonreceptor
transcription factors, such as oncoprotein PLZF (21, 22), acute myeloid
leukemia-associated protein ETO (23-25), activating protein 1 (AP-1),
nuclear factor-
B (26), and TBL1 (27). Furthermore, previous studies
demonstrated that several homeodomain proteins such as Pit-1 and Pbx1
functionally interact with N-CoR/SMRT resulted in a formation of the
repressor complex (28, 29). These results suggested that N-CoR/SMRT
possesses transcriptional repression activity for multiple
transcriptional factors.
In this study, we have shown that SMRT physically interacts with POU
domain of Oct-1 and is involved in transcriptional repression by Oct-1.
Our results illustrate a novel mechanism by which Oct-1 regulates genes
both negatively and positively. These results indicate that SMRT and
N-CoR are involved in a wide array of biological processes and
signaling pathways.
 |
EXPERIMENTAL PROCEDURES |
Plasmid Constructions--
The Oct-1 expression vector
pcDNA3HA Oct-1 was a gift from Dr. H. Singh (30). The in
vitro transcription and translation vector for Oct-1 p6His Oct-1
and OCA-B expression vector pRc OCA-B were from Dr. R. G. Roeder
(11, 31). The OBF-1 expression vector pcDNA OBF-1 was a gift from
Dr. P. Matthias (10). The pBS Oct-1+ was a gift from Dr. W. Herr (2).
The eukaryotic GST expression plasmid for Oct-1 POU domain, POU
homeodomain, and POU-specific domain was a gift from Dr. P. C. van
der Vliet (32). The SMRT cDNA was a gift from Dr. R. M. Evans
(17). To construct the bacterial expression vector for GST fusion
proteins, polymerase chain reaction amplified fragments were cloned
into EcoRI and XhoI or SalI
restriction sites of pGEX-6P1 vector (Amersham Pharmacia Biotech).
Following oligonucleotides were used to amplify the full-length SMRT:
forward primer, '-gaattc ATGGAGGCATGGGACGCC-3'; reverse primer,
5'-ctcgagCTCGCTGTCGGAGAGTGT-3'; SMRT I: forward primer, 5'-gaattc
ATGGAGGCATGGGACGCC-3'; reverse primer, 5'-gtcgac CTCGTAGCAGGCACGTT-3';
SMRT II: forward primer, 5'-gaattcGAGAGCCTGAAGAGCCGGC-3'; reverse
primer, 5'-ctcgagGTGGCGGCTGCTGAAGGGC-3'; SMRT III: forward primer,
5'-gaattcCAGCCCTTCAGCAGCCG-3'; reverse primer, 5'-ctcgag CGAGGGCTGGCTCTCAG-3'; and SMRT IV: forward primer,
5'-gaattcGTGTCCCCACCGGAGGG-3'; reverse primer,
5'-ctcgagCTCGCTGTCGGAGAGTGT-3'. TR
1 expression vector pCDM
TR
1 was described previously (33). The octamer sites containing
reporter plasmids 8xOcta-Luc and 8xOcta/mut-Luc were gifts from Dr. P. Matthias (34). The pGL3-IgH promoter -luciferase reporter was a gift
from Dr. R. G. Roeder (35).
Cell Culture and Transient Transfection and Reporter
Assays--
COS1 cells were grown in Dulbecco's modified Eagle's
medium supplemented with 10% fetal calf serum, 100 units/ml
penicillin, and 0.25 mg/ml streptomycin at 37 C in 5% CO2.
Transfection was done in COS1 cells using the standard calcium
phosphate procedure. Briefly, cells were plated in 24-well plates
6 h prior to transfection. Luciferase reporter (0.25 µg) was
cotransfected with 0.1 µg of the indicated expression vectors. After
12 h of incubation, the medium on the cells was replaced with
fresh medium. 10
7 M of TSA (see Fig. 4) or
10
7 M of T3 (see Fig. 5) was added to the
medium when indicated. Cells were harvested after 24 h for
reporter assays. Luciferase activity was determined by the PicaGene
Luciferase Assay System (Toyo Inki, Tokyo) using Lumat LB9501 (Berthold
Japan K.K., Tokyo, Japan) and expressed as relative light units
normalized to the amounts of protein. Each transfection was conducted
in triplicate, and data represent the means ± S.D. of more than
three individual experiments.
In Vitro Transcription and Translation--
Coupled
transcription and translation of Oct-1, SMRT were carried out using a
T7 TNT in vitro transcription/translation kit (Promega)
according to the manufacturer's instructions.
Expression of Recombinant Proteins--
Overnight cultures of
Escherichia coli BL21 carrying the recombinant GST fusion or GST
control plasmid was diluted 100-fold, cultured for 5-6 h, and then
induced with 0.1 mM isopropyl
-D-thiogalactopyranoside. After 3 h of induction,
bacteria were collected and washed with PBS. Pellets were suspended in
PBS containing 1% (v/v) Triton X-100 and sonicated. Debris was removed
by centrifugation. The fusion protein or the GST control protein was
bound to glutathione-Sepharose (Amersham Pharmacia Biotech) and
extensively washed with PBS containing 1% (v/v) Triton X-100.
Matrix-bound proteins were used for interaction experiments.
Interaction Experiments--
In vitro translated
35S-labeled proteins (1-2 µl) were incubated for 20 min
at room temperature with glutathione-Sepharose (10 µl) beads
containing 2-5 µg of GST recombinant proteins in 250 µl of binding
buffer (20 mM Tris-Cl, pH 7.8, 0.1% Triton X-100, 100 mM NaCl, 10% glycerol, 1 mM dithiothreitol, 1 mM EDTA, 1 mM phenylmethanesulfonyl fluoride, 1 mM leupeptin, 1 mM pepstatin, 2 mg/ml
aprotinin). Ethidium bromide (50 µg/ml) was included in the binding
reaction. After extensive washing with binding buffer, bound proteins
were eluted in 25 µl of Laemmli sample buffer and resolved by
SDS-polyacrylamide gel electrophoresis (PAGE 10%). The results
of the in vitro reactions and the amount of
35S-labeled proteins bound by GST fusions were visualized
and quantified using a Phosphor Imager (Fuji BAS 1500).
Preparation of Nuclear Extracts, Communopresipitation, and
Western Blotting--
COS1 cells were plated in 10-cm plates 6 h
prior to transfection at a density of 2 × 106/plate.
The amount of transfected DNA was kept constant (20 µg) by addition
of appropriate amounts of the parental empty expression vector. Cells
were transfected with 10 µg of expression vectors for SMRT along with
Oct-1 or control vector using standard calcium-phosphate method. 12 hours later, cells were washed with PBS and refed with Dulbecco's
modified Eagle's medium containing 10% fetal calf serum and incubated
for 24 h. Nuclear extracts were prepared according to the method
previously described (36). Briefly, after washing two times with cold
phosphate-buffered saline, cells were lysed by adding 5 ml of ice-cold
SMT buffer (0.32 M sucrose, 1 mM
MgCl2, 1% Triton X-100, 10 mM Tris-Cl, pH 7.5, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin) to the plates. After nuclear fractions were
recovered by centrifugation at 4,000 rpm for 10 min, pellets were
resuspended in 40 µl of buffer A (20 mM Tris-Cl, pH 7.5, 1 mM MgCl2, 400 mM NaCl, 1 mM EDTA, 25% glycerol, 1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml
aprotinin, 0.5% Triton X-100), followed by a 30-min incubation at
4 °C. Nuclear extracts were spun down at 12,000 rpm for 5 min. Immunoprecipitations were then performed with the anti-Oct-1 antibody for 2 h at 4 C on a rotator, followed by the addition of 40 µl of protein A-Sepharose for 1 h at 4 °C. Immunoprecipitates were washed three times with buffer A. The bound proteins were solubilized in SDS-PAGE sample buffer, resolved by SDS-polyacrylamide gel electrophoresis, and transferred to PVDF (Millipore Immobilon-P) at 100 mA for 1 h at 4 C with a MiniTransblot system (Bio-Rad). Sheets of
PVDF containing transferred protein from entire gels were incubated
first in TBS-T (10 mM Tris, pH 8.0, 150 mM
NaCl, 0.05% Tween 20) containing 5% nonfat dry milk for 1-3 h to
block nonspecific binding of antibody, followed by 2 h of
incubation in primary antibody (anti-SMRT antibody). The sheets were
then washed in TBS-T and incubated for 1 h with an appropriate
horseradish peroxidase-conjugated secondary antibody diluted 1:2000 in
TBS-T. After washing two times in TBS-T, bound antibody was detected with the ECLTM system (Amersham Pharmacia Biotech)
according to the manufacturer's protocols. PVDF blots were reprobed
with additional primary antibodies after stripping away the first
antibody. This was accomplished by incubating the PVDF sheets in 2%
SDS, 100 mM
-mercaptoethanol, 50 mM
Tris, pH 6.9, for 60 min at 70 C.
 |
RESULTS |
POU Domain of Oct-1 Interacts with SMRT--
To examine the
interaction between Oct-1 and SMRT, we used the matrix-bound fusion
protein of glutathione-S-transferase with Oct-1 (GST-Oct-1) for
in vitro pull-down assays. As shown in Fig. 1A,
[35S]methionine-labeled in vitro translated
SMRT interacted with GST-Oct-1 POU domain (lane 3) but not
GST alone (lane 2), and GST-Oct-1 did not retain any of the
in vitro-translated control luciferase protein (lane
7-10). The matrix-bound GST-POU-specific domain (lane
4) and GST-POU homeodomain (lane 5) retained
considerable amounts of [35S]methionine-labeled SMRT.
Because the binding mixture included ethidium bromide to destroy the
nonspecific DNA-protein interaction, it was concluded that the
association of SMRT to matrix-bound Oct-1 was not due to the presence
of contaminating DNA. Reciprocal pull-down experiment was performed to
confirm the interaction between Oct-1 and SMRT. We examined the
specific domains in SMRT that interact with Oct-1. Series of deletion
mutants of GST fusion proteins representing overlapping portions of
SMRT (Fig. 1B) were expressed in bacteria, purified, and
used to bind 35S-labeled full-length Oct-1. As shown in
Fig. 1C, the third domain of SMRT (SMRT III) was required
for the interaction. The interacting surface of SMRT with Oct-1 is
distinct from the nuclear receptor interacting domains (RID) or
silencing domains (SD-1 and SD-2), which are known to interact with
mSin3. As control experiments, the nuclear receptor interactions with
SMRT were also determined. [35S]Methionine-labeled TR was
incubated with matrix-bound GST fusions of SMRT (lanes
8-12). In agreements with previous reports (17), significant
associations of TR were detected with GST-SMRT-IV and GST-SMRT-III+IV,
whereas no association with GST-SMRT-III was observed. To
further confirm the interaction between Oct-1 and SMRT within the
cells, coimmunopresipitation experiments were performed using COS1
cells cotransfected with SMRT and/or Oct-1 expression vector. As shown
in Fig. 2, SMRT was coimmunoprecipitated by anti-Oct-1-specific antibody in the presence of transfected Oct-1,
suggesting the physical interaction between Oct-1 and SMRT within the
cells. In the absence of transfected Oct-1, because of the limiting
amounts of endogenous Oct-1, interaction with SMRT could not be
detected in this assays.

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Fig. 1.
Oct-1 interacts with SMRT in
vitro. A, 35S-labeled SMRT or
luciferase was synthesized in vitro translation and
incubated separately with matrix-bound GST (lanes 2 and
7), GST-Oct-1 POU domain (lanes 3 and
8) GST-Oct-1 POU-specific domain (lanes 4 and
9), and GST-Oct-1 POU homeodomain (lane 5 and
10). 10% of input 35S-labeled proteins were
indicated (lanes 1 and 6). B, series
of deletion mutants of SMRT used in pull-down experiments are shown:
SMRT I (amino acids 1-483), SMT II (amino acids 484-750), SMRT III
(amino acids 744-1093), SMRT and (amino acids 1087-1495). The nuclear
receptor interacting domains (RID-I and RID-II) and silencing domains
(SD-1 and SD-2) are as indicated. C,
[35S]methionine-labeled in vitro translated
Oct-1, TR, or luciferase was incubated with matrix-bound GST-SMRT
deletion mutants. Matrix-bound GST was used as a control (lanes
2, 9, and 16) and 10% of input
35S-labeled protein was indicated (lanes 1,
8, and 15). Associated proteins were analyzed by
10% SDS-PAGE and visualized by BAS 1500 (Fuji, Tokyo).
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Fig. 2.
In vivo association between SMRT
and Oct-1 proteins. COS1 cells were transfected with 10 µg of
expression vectors for SMRT along with Oct-1 or control vector using
standard calcium-phosphate method. Nuclear cell extracts (50 µg) were
immunoprecipitated (IP) with monoclonal antibody
(Ab) directed against Oct-1 protein, separated by SDS-PAGE,
transferred to a PVDF filter, and probed with polyclonal antibody
directed against SMRT proteins.
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SMRT Represses the Transcriptional Activity of Oct-1--
To test
the possible role of SMRT in Oct-1 transcriptional activity, we
performed transient transfection experiments in COS1 cells. Full-length
Oct-1 expression vectors or empty expression vectors were cotransfected
with luciferase reporter plasmids containing eight copies of the Oct-1
response element (8xOcta) into COS1 cells. As shown in Fig.
3A, cotransfection of SMRT
repressed the Oct-1-activated reporter activity by ~50%. When
OCA-B/OBF-1 was cotransfected, significant stimulation of the
Oct-1-dependent transcription was observed in agreement
with previous reports (10, 11). Even in the presence of cotransfected
OBF-1/OCA-B, SMRT still repressed the transcriptional activation by
Oct-1. In contrast, expression of Oct-1 and/or SMRT did not affect the mutant reporter activity, suggesting that repression by SMRT was mediated through Oct-1 element. Next we examined the effects of SMRT on
a naturally occurring Oct-1 response element using Ig heavy-chain (BCL1
IgH) promoter in COS1 cells. As shown in Fig. 3B,
coexpression of Oct-1 and OBF-1/OCA-B stimulated the BCL1 IgH promoter.
The expression of SMRT inhibited the Oct-1 transcriptional activity on
IgH promoter in either the presence or absence of OBF-1/OCA-B but had
no effect on the control reporter. These results strongly suggested
that SMRT could function as a corepressor for Oct-1-dependent transcription, indicating physiological
relevance of the interaction of SMRT with Oct-1.

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Fig. 3.
Effect of SMRT on transcriptional activity of
Oct-1. Control vector or Oct-1 (0.1 µg) expression vector was
cotransfected into COS1 cells with 0.25 µg of 8xOcta or 8xOcta-mutant
luciferase reporter (A) or BCL1 IgH promoter or BCL1
luciferase reporter (B) and 0.1 µg of SMRT or OBF-1/OCA-B
expression vector. Relative luciferase activities are presented after
being normalized by the amounts of protein. Each transfection was
conducted in triplicate, and data represent the means ± S.D. of
more than three individual experiments.
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TSA Releases the Transcriptional Repression by Oct-1 through
SMRT--
SMRT has been shown to recruit histone deacetylases (HDAC1
and HDAC2) through direct interaction with mSin3. These histone deacetylases are thought to be involved in the trans-repression function of unliganded nuclear receptors. To clarify whether HDACs mediated the trans-repression by Oct-1 through SMRT, we next examined the effects of TSA, a specific HDAC inhibitor, on the transcriptional activity of Oct-1 (37). As shown in Fig.
4, treatment of COS1 cells with
10
7 M TSA abolished the trans-repression by
cotransfected Oct-1 and SMRT. Because of the presence of endogenous
Oct-1 and SMRT, TSA increased the promoter activity in some degree even
in the absence of cotransfected Oct-1 and SMRT. In contrast, TSA had no
effect on the control promoter. These results suggested that histone deacetylase system would be involved in the Oct-1-mediated
transcriptional repression.

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Fig. 4.
Effect of TSA on transcriptional activity of
Oct-1. Oct-1 (0.1 µg) and SMRT (0.05 or 0.15 µg) expression
vectors were cotransfected into COS1 cells with 0.25 µg of 8xOcta
luciferase reporter. Luciferase activities in the absence (solid
bar) or presence (shaded bar) of TSA (10 7
M) are presented. Each transfection was conducted in
triplicate, and data represent the means ± S.D. of more than
three individual experiments.
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T3-dependent Repression of Oct-1-responsive Element
Containing Promoter--
Because SMRT interacts with TR and acts as a
nuclear receptor corepressor, we assume that coexpression of TR may
enhance the transcriptional activity of Oct-1 by sequestration of the
limiting amount of SMRT from Oct-1. If this is the case, ligand-induced dissociation of SMRT from TR increases the amounts of SMRT that can be
recruited to Oct-1, resulting in the transcriptional repression of
Oct-1 element. Therefore, we next examined the effects of
overexpression of TR and addition of T3 on Oct-1 function. As shown in
Fig. 5A, as we assumed,
unliganded TR enhanced the transcriptional activation of 8xOcta by
Oct-1 and addition of T3 obliterated the enhancement, indicating that
the promoter regulated by Oct-1 might act as a negative T3-responsive
promoter through the mechanism that involves SMRT. To further confirm
the mechanism that unligand TR can sequester SMRT from Oct-1, we next
performed the pull-down assay. As shown in Fig. 5B,
unliganded TR decreased the amount of SMRT that interacted with Oct-1
POU domain. Addition of T3, which releases the SMRT from TR and
increases the number of SMRT available for the interaction with Oct-1,
restored the intraction between SMRT and Oct-1.

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Fig. 5.
T3-dependent repression of Oct-1
response element. A, Oct-1 (0.1 µg) and SMRT (0.1 µg) expression vectors were cotransfected into COS1 cells with 0.25 µg of 8xOcta luciferase reporter with or without 0.05 µg of TR
expression vector. Luciferase activities in the absence (solid
bar) or presence (hatched bar) of T3 (10 7
M) are presented. Each transfection was conducted in
triplicate, and data represent the means ± S.D. of more than
three individual experiments. B, in the upper
panel, 35S-labeled SMRT was synthesized in
vitro translation and incubated with matrix-bound GST (lane
2) or GST-Oct-1 POU domain (lanes 3-5). The in
vitro translated TR was included in lanes 4 and
5. T3 (10 7 M) was added to
lane 5. 10% of input 35S-labeled SMRT was
indicated (lane 1). The lower panel shows the
relative intensity of each lane.
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SMRT Influences the Interaction of Oct-1 with
OCA-B/OBF-1--
SMRT and OCA-B interacts with the same POU domain;
therefore the balance of the amounts of SMRT and OCA-B may regulate the bifunctional transcriptional activity of Oct-1. To determine whether SMRT and OCA-B/OBF-1 bound to Oct-1 competitively, the effect of SMRT
on the interaction between Oct-1 and OCA-B were analyzed using
pull-down assays. As shown in Fig. 6,
addition of increasing amounts of SMRT results in diminished binding of
OCA-B to Oct-1. These data suggested that SMRT and OCA-B/OBF-1 actually
compete for binding to Oct-1 POU domain.

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Fig. 6.
SMRT influences the interaction of Oct-1 with
OCA-B/OBF-1. 35S-Labeled OCA-B was incubated with
matrix-bound GST (lane 2), GST-Oct-1 POU domain (lanes
3-7) in the presence of Oct-1 elements. Unprogrammed lysate
(lane 4) and increasing amounts of in vitro
translated SMRT (lanes 5 and 6) were added to the
reaction. 10% of input 35S-labeled OCA-B was indicated
(lane 1).
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 |
DISCUSSION |
In this study, we have examined the interaction between SMRT and
POU domain of Oct-1. Our results indicated that Oct-1 interacted with
SMRT by direct protein-protein interaction and led to the recruitment
of histone deacetylases complex to the promoter, resulting in the
transcriptional repression. Furthermore, as shown in Fig. 6, SMRT and
coactivator OCA-B/OBF-1 could competitively interact with Oct-1 POU
domain. When Oct-1 recruits OCA-B/OBF-1, it activates the transcription
of Oct-1 response element. Dominant recruitment of SMRT switches the
Oct-1 from transcriptional activator to repressor. It was previously
reported that the POU transcription factor Pit-1 or the homeobox
proteins Pbx-Pdx can interact with both CREB-binding protein containing
coactivator complex and N-CoR/SMRT containing corepressor complex and
regulate the transcriptional activity by balance between these
complexes. Similarly we proposed the hypotheses that the activity of
Oct-1 is also determined by a regulated balance between SMRT and
OCA-B/OBF-1. The SMRT interaction surface was located in POU domain of
Oct-1. The POU domain has been shown to be involved in protein-protein
interaction as well as sequence-specific DNA binding. A number of
transcription factors have been identified to interact with the POU
domains of Oct-1, e.g. TBP, TFIIB, HMG2, OBF-1/OCA-B, and
nuclear hormone receptors such as TR, retinoid X receptor, and
glucocorticoid receptor, androgen receptor (6-11, 38). Various
transcription factors interact with Oct-1 POU domain and influence
mutually divergent actions of Oct-1.
SMRT, which was originally cloned as a factor mediating the
transcriptional repression of unliganded nuclear receptors, directly interacts with hinge domain of receptors through its C-terminal region
called RID resulting in the recruitment of the histone deacetylases
(HDAC1/Rpd3) to the receptors. This RID was also reported to be
involved in the interaction with homeodomain transcription factors,
Pit-1 and Pbx-1 (28, 29). However, our results demonstrated that SMRT
uses a different domain than the RID for the interaction with Oct-1,
suggesting the possibility that there might be certain interface in
SMRT for each interacting factors. In this study we showed that
coexpression of unliganded TR led to the enhancement of transactivation
by Oct-1, and this effect was abolished by addition of T3, supporting
the hypothesis that SMRT recruited to the DNA-bound Oct-1 could be
competed by unliganded TR. The pull-down assays demonstrated that
limiting amounts of SMRT were titrated from Oct-1 by unliganded thyroid
hormone receptors, and addition of T3 redelivered the SMRT to Oct-1.
These data indicated that the Oct-1 element might work as a negative
TRE when TR coexist with Oct-1.
Previously we and others have reported that nuclear receptors such as
TR, retinoid X receptor, vitamin D receptor, glucocorticoid receptor,
progesterone receptor, and androgen receptor interact with POU domain
of Oct-1 and modulate the ligand-dependent transcriptional activity of nuclear receptors (12, 38). Taken together, these results
suggest that SMRT mediates the cross-talk between Oct-1 and nuclear
receptors, resulting in a formation of complex transcriptional networks.
 |
ACKNOWLEDGEMENTS |
We thank Dr. W. Herr, Dr. H. Singh, for
providing Oct-1 cDNA. We thank Dr. P. C. van der Vliet for
providing expression plasmids for Oct-1 POU domain, POU homeodomain,
and POU-specific domain. We also thank Dr R. M. Evans for
providing SMRT cDNA, Dr. P Matthias for 8xOcta-Luc and OBF-1
cDNA, and Dr. R. G. Roeder for pGL3-IgH Luc and OCA-B cDNA.
 |
FOOTNOTES |
*
This work was supported in part by a grant from the Research
Fellowship of the Japan Society for the Promotion of Science for Young
Scientists and the Ministry of Education, Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Aging
Medicine and Geriatrics, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, 390-8621 Japan. Tel.: 81-263-37-2686; Fax:
81-263-37-2710; E-mail: miyamoto@hsp.md.shinshu-u.ac.jp.
Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M008531200
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ABBREVIATIONS |
The abbreviations used are:
Oct-1, octamer
transcription factor-1;
TR, thyroid hormone receptor;
SMRT, silencing
mediator for retinoid and thyroid hormone receptors;
N-CoR, nuclear
hormone receptor corepressor;
GST, glutathione S-transferase;
T3, 3,3',5tri-iodo-L-thyronine;
POU, Pit-1, Oct-1, unc-86;
TSA, trichostatin A;
OBF-1, Oct-binding factor 1;
OCA-B, Oct-1-associated
coactivator;
PBS, phosphate-buffered saline;
PAGE, polyacrylamide gel
electrophoresis;
PVDF, polyvinylidene difluoride;
RID, receptor
interacting domain;
SD, silencing domain.
 |
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