From the George Whipple Laboratory for Cancer
Research Departments of Pathology, Urology, Radiation Oncology, and
Cancer Center, University of Rochester Medical Center, Rochester, New
York 14642, the § Department of Surgery, Beijing Institute
for Cancer Research, Beijing Cancer Hospital, Peking University 100036 Beijing, China, and the ¶ Department of Surgery, First Hospital,
Peking University, 100034, Beijing, China
Received for publication, July 16, 2002, and in revised form, December 16, 2002
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ABSTRACT |
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Although many co-activators have been identified
for various nuclear receptors, relatively fewer co-repressors have been
isolated and characterized. Here we report the identification of a
novel testicular orphan nuclear receptor-4 (TR4)-associated protein (TRA16) that is mainly localized in the nucleus of cells as a repressor
to suppress TR4-mediated transactivation. The suppression of
TR4-mediated transactivation is selective because TRA16 shows only a
slight influence on the transactivation of androgen receptor, glucocorticoid receptor, and progesterone receptor. Sequence
analysis shows that TRA16 is a novel gene with 139 amino acids in an
open reading frame with a molecular mass of 16 kDa, which did
not match any published gene sequences. Mammalian two-hybrid system and co-immunoprecipitation assays both demonstrate that TRA16 can interact
strongly with TR4. The electrophoretic mobility shift assay suggests
that TRA16 may suppress TR4-mediated transactivation via decreased
binding between the TR4 protein and the TR4 response element on the
target gene(s). Furthermore, TRA16 can also block the interaction
between TR4 and TR4 ligand-binding domain through interacting
with TR4-DNA-binding and ligand-binding domains. These unique
suppression mechanisms suggest that TRA16 may function as a novel
repressor to selectively suppress the TR4-mediated transactivation.
Steroid hormones play important physiological roles in cellular
differentiation, development, and homeostasis, which function through
binding to the specific receptors that belong to the nuclear receptor
superfamily (1-5). The superfamily members may act as transcriptional
activators or repressors for the modulation of gene expression in a
wide variety of biological processes via binding to the hormone
response element that is located mainly on the 5' promoter of target
genes (2, 6, 7).
The human testis orphan receptor
(TR4),1 a member of the
nuclear receptor superfamily, was initially isolated from human
prostate, testis, and hypothalamus cDNA libraries (8). Sequence
analysis shows that TR4 has 615 amino acids with a calculated molecular mass of 67 kDa and has a very high homology with TR2 orphan receptor (9, 10). Northern blot analysis and in situ hybridization indicate that TR4 was expressed in a variety of tissues, including the
central neural system (habenula, hippocampal pyramidal cell, and
granule cells of both hippocampus and cerebellum) and peripheral organs
(most abundantly expressed in spermatocytes of testis, with lower
amounts in adrenal cortex, spleen, thyroid, prostate, and pituitary
gland) (8, 11-13). TR4 may function as a transcriptional factor to
regulate many signal transduction pathways. For example, it has been
shown that TR4 can function as repressor to suppress the retinoic acid
receptor-, retinoid X receptor-, vitamin D receptor-, androgen
receptor (AR)-, and estrogen receptor-mediated transactivation (14-17). A recent report (18) demonstrated that TR4 could suppress the
expression of the steroid 21-hydroxylase gene, which belongs to the
cytochrome P450 superfamily and is one of the key enzymes in
biosynthesis of adrenal steroid hormones, leading to the production of
cortisol and aldosterone. On the other hand, TR4 also can function as
enhancer to induce the ciliary neurotrophic factor receptor- Here we show the isolation and characterization of a novel protein,
TR4-associated protein (TRA16), localized in the nucleus that may
function as a repressor for suppression of TR4-mediated transactivation
via decreased binding between TR4 protein and TR4 response element
(TR4RE) and blockage of TR4 dimerization.
Screening of Human Testis cDNA Library by Yeast Two-hybrid
System--
The Cyto Trap yeast two-hybrid system (Stratagene) and a
pMyr plasmid library (Stratagene) that consists of a DNA sequence encoding the myristylation membrane localization signal, which is fused
to human testis cDNA, were used in the yeast two-hybrid screening.
The pSos vector containing hSos gene was fused with the full length of
TR4 cDNA using the BamHI cloning site as bait. As
previously described (22), the library was screened by transformation with the pSos-TR4 bait constructor, and then the transformed yeast cells were selected for growth on both synthetic dropout/glucose minus
uronolactone ( Rapid Amplification of cDNA Ends (RACE-PCR) for Full Length
of TRA16--
The missing 5'-coding region was isolated by RACE-PCR
technology (8). The gene-specific antisense primer used for 5' RACE-PCR was 5'-TCACACCTTCTCCCCAAGCACCCGCAGGTGGTA-3'. The PCR
reaction condition was 95 °C for 2 min; 30 cycles of 95 °C
for 20 s, 58 °C for 30 s, and 72 °C for 30 s; and
then 72 °C for 7 min. The PCR product was sequenced for confirmation.
Northern Blot Analysis--
A human multiple tissues RNA blot,
purchased from Clontech (catalog number 7760-1) was
hybridized against a TRA16 cDNA probe labeled with
[ Large Scale Expression and Purification of TR4-DL and
TRA16--
Plasmids TR4-DL containing DBD and LBD or the full length
of TRA16 cDNAs were constructed into the pET expression system, and
then large scale expression and purification were carried out from
transformed Escherichia coli BL21 (DE3) bacteria following the manufacturer's procedures (Qiagen, Chatsworth, CA). Both proteins of TR4-DL and TRA16 were then confirmed directly on 15% SDS-PAGE followed by Coomassie Blue staining.
Production of Polyclonal Antibody against Human TRA16--
The
large amount of TRA16 using pET expression system was purified from
transformed E. coli BL21 (DE3) bacteria and used directly for immunizing rabbits.
The polyclonal TRA16 antibody was obtained from Cocalico Biologicals,
Inc. (Reamstown, PA). It was diluted 1:100 in phosphate-buffered saline
for immunofluorescence assay and 1:1000 for Western blot assay.
Immunocytofluorescence Assay--
Human cancer cell lines,
H1299, MCF-7, and LNCaP, and monkey kidney cell line COS-1 were seeded
on two-well Lab Tek Chamber slides (Nalge) for 48 h.
Immunostaining was performed by incubating with anti-TRA16 polyclonal
antibody or with anti-TR4 monoclonal antibody (antibody 17), followed
by incubating with either fluorescein-conjugated goat anti-rabbit or
anti-mouse antibodies. Coverslips were fixed on the glass slides with a
drop of DAPI to stain the nucleus. The slides were observed
under 40-fold magnification using a fluorescence microscope or confocal
fluorescence microscope. The green signal represents the localization
of TRA16, the red signal represents the localization of TR4, and the
blue signal represents the DAPI-stained nucleus.
Cell Culture, Transient Transfection, and Reporter Gene
Assay--
H1299, DU145, and COS-1 cells were cultured with
Dulbecco's minimal essential medium containing penicillin (25 units/ml), streptomycin (25 µg/ml), and 10% fetal calf serum.
Transfection was performed by modified calcium phosphate precipitation
as previously described (22) or using SuperFect according to the
manufacturer's procedures (Qiagen). The dual luciferase (LUC) reporter
assay system was conducted according to the manufacturer's
instructions (Promega). The Renilla luciferase reporter
plasmid-simian virus 40 (pRL-SV40) was used as an internal control. The
chloramphenicol acetyltransferase (CAT) assay was performed as
described previously (23). The Mammalian Two-hybrid Assay--
COS-1 cells were transiently
co-transfected with 3 µg of reporter plasmid pG5-LUC and 4 µg of
both the GAL4DBD and VP16-hybrid expression plasmids as described
previously (22). The pRL-SV40 plasmid (10 ng) was co-transfected for
normalization of transfection efficiency. The LUC assay was performed
24 h after transfection.
Coupled in Vitro Transcription and Translation--
Plasmids
containing the full-length TR4 or TRA16 cDNAs were in
vitro transcribed and translated directly by a TNT-coupled reticulocyte lysate system (Promega) as previously described (23). The
in vitro translated products were then analyzed directly by SDS-PAGE.
The in Vitro or in Vivo Co-immunoprecipitation of TR4, TR4-N, or
TR4-DL and TRA16--
For in vitro co-immunoprecipitation,
the TR4, TR4-N terminus, DL (containing DBD and LBD), TRA16, and
pcDNA4c-His control proteins, transcribed and translated by the
TNT-coupled reticulocyte lysate system, were purified as instructed by
the manufacturer (Promega). For immunoprecipitation (IP) of each
mixture, 5 µl of each in vitro translated
35S-labeled proteins were incubated for 1 h at
30 °C in various combinations as indicated and then incubated with
an antibody bound to protein G-agarose beads (Santa Cruz Biotechnology)
at 4 °C for another 2 h. After washing each mixture four times,
each complex was loaded onto 15% SDS-PAGE gel and visualized by
autoradiography. For in vivo co-IP, 10 µg of
pcDNA4c-His or pcDNA4c-His-TRA16 was transiently transfected in
H1299 cells for 48 h, and the lysates were harvested. Then
anti-His probe antibody and protein G-agarose beads were added to 500 µg of protein lysate for 2 h for IP of pcDNA4c-His-TRA16 and
endogenous TR4 complex. After washing each mixture four times, 15%
SDS-PAGE gel was used to separate the complex. After transferring to
the membrane, the expression was detected by anti-TR4 antibody or
anti-His probe antibody. Each cell lysate input was loaded to show the
expression of TR4 and pcDNA4c-His-TRA16.
Stable Transfection of COS-1 Cells--
COS-1 cells, which are
TR4-negative and show little expression of TRA16, were transfected with
pBig or pBig-TRA16 using SuperFect (Qiagen). The cells were then
selected using 100 µg/ml hygromycin B (24), and a single colony was
chosen, amplified, and confirmed by reporter gene assay.
Nuclear Extract Preparation--
The nuclear protein extraction
was prepared as described previously (25). Briefly, the cells were
collected and pelleted with 10 s of centrifugation and then
resuspended in 5 volumes of cold Buffer A. The samples were put on ice
for 10 min, vortexed for 10 s, and centrifuged for 10 s, and
the supernatant fraction was separated (cytosol protein). The pellet
was resuspended in 100 µl of cold Buffer C and incubated on ice for
20 min for high salt extraction, centrifugation for 2 min at 4 °C
was used to remove the cellular debris, and then the supernatant
fraction containing the nuclear protein was used for experimentation.
Electrophoretic Mobility Shift Assay (EMSA)--
The EMSA was
performed as described previously (19, 21). Briefly, the reaction was
performed by incubating the 32P-labeled DR1-TR4RE probe
with 10 µg nuclear extracts of different cells or only with the
purified proteins of TR4-DL and TRA16 from bacteria. For the antibody
supershift analyses, 1 µl of anti-TR4 antibody (antibody 15 or C-16)
was added to the reaction, DNA-protein complexes were resolved on
a 5% native gel for electrophoresis, and the gel was dried and then
analyzed by autoradiography.
Identification of Orphan Receptor TR4-associated Protein,
TRA16--
To further understand the function and mechanism of TR4, a
human TR4 was used as bait with the yeast two-hybrid system to fish out
the interacting proteins from testis cDNA library. One clone, which
can interact with TR4, was isolated and named human TRA16 (Fig.
1). Using a specific primer
(5'-TCACACCTTCTCCCCAAGCACCCGCAGGTGGTA-3'), we applied the RACE-PCR
technology with the isolated cDNA insert as template to amplify the
full-length human TRA16 from the Marathon human testis cDNA
library. Sequence analysis revealed that TRA16 is a novel protein whose
sequence did not match any known published genes. The open reading
frame between the first ATG and terminal TGA encodes 139 amino acids
with the predicted molecular mass of 16 kDa (GenBankTM
accession number AY101377). Genomic sequence analysis indicates that
human TRA16 is located at human chromosome 19p12.
Distribution of TRA16 and TR4--
Northern blot analysis
indicated that TRA16 was expressed as a mRNA transcript of about 1 kb in several human tissues, such as heart, placenta, liver, skeletal
muscle, kidney, and pancreas as shown in Fig.
2A. Interestingly, the
distribution of TRA16 was found to be relatively higher in heart,
skeletal muscle, and pancreas. In contrast, the expression of TRA16 was
lowest in normal human brain and lung. To further check the TRA16 at
protein level, we generated the anti-TRA16 antibody (Cocalico
Biologicals, Inc.) to stain TRA16 protein. As shown in Fig.
2B, both the endogenous TRA16 and transiently transfected
pcDNA4c-TRA16 expressed TRA16 are recognized by anti-TRA16
antibody. Furthermore, we determine the subcellular localization of the
TRA16 in several cell lines using immunocytofluorescence assay. As
shown in Fig. 2C, we found that TRA16 was located mainly in
the nucleus detected by anti-TRA16 antibody.
For comparison, we also showed the expression of TR4 in various cell
lines with Western blot assay. As shown in Fig. 2D, the expression of TR4 protein in H1299, PC-3, 293T (human kidney cell line), LNCaP, and CWR22 cell lines (lanes 1, and 5-8) is
higher compared with the expression in DU145, HTB-14, and COS-1 cell lines, which expressed little TR4 (lanes 2-4).
The in Vivo and in Vitro Interaction between TR4 and
TRA16--
To further confirm that the interaction between TR4 and
TRA16 in yeast cells may also occur in mammalian cells, we applied the
immunocytofluorescence assay to test whether both TR4 and TRA16 are
localized in the same cells. As shown in Fig.
3A, the majority of TRA16
signal (panel 2, green) could be detected
together with TR4 signal (panel 1, red) on the
nuclear membrane (panel 3, yellow). This
observation provided strong in vivo evidence that TRA16
co-localizes with TR4 on the nuclear membrane. Using the mammalian
two-hybrid system to assay their direct interaction in COS-1 cells, we
also found that the GAL4-TRA16 can interact strongly with VP16-TR4
(Fig. 3B, lane 4). We then used co-IP with in vitro translated TR4 and His tag fused TRA16 to assay
their interaction. As shown in Fig. 3C (lane 3),
the precipitated complex containing in vitro translated
35S-labeled TR4 and in vitro translated
35S-labeled pcDNA4c-His was immunoprecipitated with the
polyclonal anti-His tag antibody. The TR4 protein could be clearly
detected in the immunoprecipitated complex of both in vitro
translated 35S-labeled TR4 and in vitro
translated 35S-labeled TRA16 (Fig. 3C,
lane 4). To further test that the endogenous TR4 could
interact with the exogenous TRA16, the whole cell extracts from H1299
cells only transfected with TRA16 were immunoprecipitated with
polyclonal anti-His tag antibody, and the results show that TR4 and
TRA16 can definitely co-immunoprecipitate (Fig. 3D).
Together, the results from Fig. 3 (A-D), using four
different interaction assays, demonstrate that TR4 can interact with
TRA16 in mammalian cells.
TRA16 Represses TR4-mediated Transactivation--
The
potential effects of TRA16 on TR4-mediated transactivation were
tested through LUC and CAT reporter assays. The full-length TRA16 and
TR4 in eukaryotically expressed vectors (pSG5-TRA16, or
pcDNA4c-TRA16 and pCMX-TR4) were co-transfected with a luciferase reporter HCR-1-LUC containing a
TR4RE2 in COS-1 cells. As
shown in Fig. 4A, the
TR4RE-LUC activity induced by pCMX-TR4 could be significantly
suppressed by TRA16 in a dose-dependent manner. Using H1299
cells, which express stronger endogenous TR4 protein levels (shown in
Fig. 2D), LUC reporter assays confirm that exogenously
transfected TRA16 can suppress TR4-mediated transactivation (Fig.
4B). To reduce any potential artifact effect related to any
particular TR4RE reporter assay, we also replaced TR4 HCR-1-LUC reporter with other reporters containing different TR4REs, such as
CpFL4-LUC and DR4-TK-CAT (16). As shown in Fig.
5A and B, addition
of TRA16 can repress TR4-mediated transactivation in all reporter
assays in a dose-dependent manner.
To avoid the effect of transiently transfected TRA16, we then stably
transfected COS-1 cells with pBig-TRA16 using a doxycycline-inducible system. As shown in Fig. 5C, treatment of the stably
transfected pBig-TRA16 COS-1 cells with doxycycline results in
significant suppression of TR4-mediated transactivation. In contrast,
COS-1 cells stably transfected with the pBig vector shows little effect on TR4-mediated transactivation in the presence of doxycycline.
To test the specificity of TRA16 suppressive effect on other nuclear
receptor transactivation, we used mouse mammary tumor virus (MMTV)-LUC
reporter (22) to determine the influence of TRA16 on the
transactivation of AR, progesterone receptor, and glucocorticoid
receptor. Interestingly, in contrast to suppressing TR4-mediated
transactivation, TRA16 has only a slight modulation effect on these
three classic steroid receptors transactivation in COS-1 cells (Fig.
6). Together, these data demonstrate
clearly that the suppression of TR4-mediated transactivation by TRA16 is selective.
The Potential Mechanism for the TRA16 to Suppress TR4-mediated
Transactivation--
We applied Western blot analysis to see whether
the addition of TRA16 could influence the TR4 expression at the protein
level. As shown in Fig. 7 (A
and B), the addition of TRA16 at different doses shows
little influence on either the transfected TR4 protein level in COS-1
cells or the endogenous TR4 protein level in H1299 cells. We then
assayed the potential influence of TR4 nuclear translocation by the
addition of TRA16. As shown in Fig. 7C, there is little
influence in the endogenous TR4 protein in whole H1299 cell extract in
the presence or the absence of TRA16 (lane 2 versus lane 1), which is consistent with results in Fig.
7B. Furthermore, the amount of TR4 protein remains
relatively consistent in either nuclear fraction or cytosol fraction
after the addition of TRA16 (Fig. 7C, lane 4 versus lane 3 and lane 6 versus lane
5). Together, the results from Fig. 7C suggest that
TRA16 has little influence on the nuclear translocation of TR4.
To test the hypothesis that suppression of TR4-mediated transactivation
by TRA16 might involve the modulation of histone deacetylase (HDAC)
activity, we first examined the effect of trichostatin A (TSA), a
specific inhibitor of HDACs (26), on the TRA16 suppression of
TR4-mediated transactivation. As shown in Fig. 7D, the
addition of TSA can dramatically enhance TR4-mediated transactivation
in a dose-dependent manner (lanes 2 versus
lanes 6 and 10). However, the addition of TSA
cannot reverse significantly the TRA16 suppressed TR4-mediated
transactivation in COS-1 cells (Fig. 7D, lane 6 versus lanes 7 and 8 or lane 10 versus lanes 11 and 12), suggesting that HDAC activity may not play major roles in the TRA16 suppressed TR4-mediated transactivation.
We then focused on the influence of TRA16 on the TR4 binding to the
TR4RE of the target genes. Using 32P-labeled DR1-TR4RE
(AGGTTAAAGGTCT) as probe, we applied the EMSA to see whether adding
TRA16 can influence the TR4 binding to DR1-TR4RE. As shown in Fig.
8A, TR4 binds specifically to
DR1-TR4RE, and this specific binding (open arrow,
lanes 3 and lane 4) can be supershifted by adding
anti-TR4 antibody (solid arrow, lane 4). In
contrast, TR4 failed to bind to mutant DR1-TR4RE
(AGGTTAAATGACT), even when adding anti-TR4
antibody (lanes 1 and 2). The addition of
different doses of TRA16 can then reduce the binding between TR4 and
DR1-TR4RE as shown in Fig. 8B (lane 1 versus
lanes 2 and 3), even if supershifted by adding
anti-TR4 antibody (Fig. 8B, lane 4 versus
lanes 5 and 6). Then we demonstrated that the
addition of TRA16 could reduce the binding between DR1-TR4RE and
endogenous TR4 expressed in H1299 cells as shown in Fig. 8C
(lane 2 versus lane 3 and lane 4 versus lane 5). To avoid any potential effects involved
in the DNA binding assay, we also purified two proteins of TR4-DL
(containing DBD and LBD of TR4) and TRA16 from E. coli strain DE3 and then checked with EMSA. As shown in Fig. 8D,
TRA16 definitely can decrease TR4 binding to its target gene, even with the addition of anti-TR4 antibody (lane 3 versus lane
4 and lane 5 versus lane 6). Together, the results from
Fig. 8 clearly demonstrate that TRA16 may suppress TR4-mediated
transactivation via the interruption of the binding between TR4 and
TR4RE on its target gene.
We also checked whether dimerization of TR4 might play any role in the
TRA16 suppression of TR4 transactivation. We first demonstrated that
TR4 can form the dimers via the interaction between TR4 and TR4-LBD
(amino acids 224-615) in the mammalian two-hybrid assay as shown in
Fig. 9A (lane 4).
Interestingly, the addition of TRA16 could then suppress the
interaction between TR4 and TR4-LBD significantly (Fig. 9, lane
5). In contrast, the addition of AR showed little influence on the
interaction between TR4 and TR4-LBD (Fig. 9A, lane
6). Our early report suggested that AR could also function as a
repressor to suppress TR4 transactivation (16). These contrasting
effects between TRA16 and AR strongly suggest that different TR4
repressors may go through different mechanisms to suppress TR4-mediated
transactivation. To determine which region of TR4 dimerizes with
TR4-LBD and mediates the protein-protein interaction with TRA16, the
TR4 N terminus (amino acids 1-125) and TR4-DL (amino acids 125-615)
including DBD and LBD of TR4 were constructed as shown in Fig.
9B. With the mammalian two-hybrid assay, we found that
TR4-DL can interact with TR4-LBD as shown in Fig. 9B
(lane 6). In vitro co-IP experiments were
performed to demonstrate that TRA16 could be co-immunoprecipitated with TR4DL but not TR4-N (Fig. 9C), which was a competitor to
interfere with the interaction between TR4-DL and TR4-LBD.
Nuclear hormone receptors comprise a huge family of
transcriptional factors that represent important regulators in cellular proliferation, differentiation, development, and homeostasis (7, 27).
The transcriptional activity of nuclear hormone receptors has been
generally recognized to be modulated by co-activators or co-repressors
(28, 29). To date, many co-activators have been identified for various
nuclear receptors, but relatively fewer co-repressors have been
isolated and characterized.
In general, nuclear receptor co-activators (such as p160/SRC, p300/CBP,
and P/CAF) act through an inherent histone acetyltransferase activity
to increase the level of histone acetylation and enhance the
transcriptional activity of ligand bound receptors (30-34). The
molecular studies demonstrated that the helical motifs containing the
LXXLL core consensus in co-activators play important roles for the interaction with nuclear receptors (35, 36). The nuclear receptor co-repressor (N-CoR) and the silencing mediator for retinoid and thyroid hormone receptors (SMRT) also contain three repeated receptor interaction domains that include the conserved hydrophobic core motif (I/L)XXII, which can interact with unliganded
nuclear receptors, such as retinoic acid receptor On the other hand, there are some co-repressors that can exert their
suppression on nuclear receptors via interfering with the binding
between nuclear receptors and their DNA response elements. For example,
a TR uncoupling protein may inhibit TR and retinoic acid
receptor-mediated transactivation via binding to the TR hinge region
and the N-terminal portion of the ligand-binding domain (50). Another
co-repressor, calreticulin, was suggested to be able to inhibit the
transactivation of glucocorticoid receptor and AR via interruption of
the binding between receptors and DNA response elements (51, 52).
Cyclin D1, a cell cycle regulating protein that functions as an AR
co-repressor, may rely on its cell cycle regulating function to
suppress AR (53). There are also several proteins, such as a small
ubiquitous nuclear co-repressor that may function as nuclear receptor
co-repressor via forming complexes with N-CoR (54). Finally, Mathur
et al. (55) found that polypyrimidine tract-binding
protein-associated splicing factor could suppress TR and retinoid
X receptor mediated transcription through a novel polypyrimidine
tract-binding protein-associated splicing factor/Sin3-mediated pathway
to recruit HDACs to the receptor DBD.
For the suppression of TR4-mediated transactivation, early reports
suggested that AR could also function as repressor to interrupt the
binding between TR4 and TR4 DNA response elements (16). Here we report
the cloning and characterization of a novel TR4 repressor, TRA16, whose
sequence was not previously reported. The amino acid sequence
comparison also shows that TRA16 lacks the classic hydrophobic core
motif (I/L)XXII of other repressors. The other interesting
characteristic was the distribution of TRA16. We also detected the
expression of TRA16 mRNA level in different cell lines with
Northern blot assay (data not shown) with the same result as
immunocytofluorescence assays. TRA16 expression in human lung cancer
cell line H1299 cells was found to be higher than in normal human lung
tissue, suggesting that TRA16 could play roles in some kinds of cancer
tissues. TSA showed little effect on the TRA16-mediated suppression of
TR4 transactivation, suggesting that HDAC activity may not play any
roles for TRA16. Instead, we demonstrated that TRA16 may suppress TR4
transactivation via either interruption of the interaction between TR4
and its DNA response element or as competitor to block the interaction between TR4-DL and TR4-LBD. Together, our data suggest that TRA16 may
function as a novel repressor to suppress TR4 function.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
thyroid hormone receptor (TR) (12, 19). A recent report (20) has shown
that the TR4 is an important regulator of myeloid progenitor cell
proliferation and development. The hormone response element for the TR4
is AGGTCA with a variety of direct repeats (DRs) (16, 21). The detailed
mechanisms of how TR4 can differentially modulate its target genes,
however, remain unclear. Whether some of those mechanisms may involve
the association of co-repressors also remains unclear.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
UL) agar and synthetic dropout/galactose (
UL) agar
plates and cultured at 25 and 37 °C. The positive clones grow at
25 °C both on synthetic dropout/glucose (
UL) and synthetic dropout/galactose (
UL) plates but at 37 °C grow on synthetic dropout/galactose (
UL) plates and not on synthetic dropout/glucose (
UL) plates. One positive clone was selected that encodes about 700 bp of an unknown cDNA sequence and was called fragment TRA16. PMyr-TRA16 fragment and pSos-TR4 were co-transformed into the yeast to
verify the interaction between these two proteins. All of the positive
and negative controls were set up following the instruction manual of
the system.
-32P]dCTP. The
-actin was used as a control for
normalization, and the results were analyzed by autoradiography.
-galactosidase expression gene was
co-transfected to normalize the transfection efficiency. A
PhosphorImager visualized the CAT activity. For all of the
transfections, the total amount of plasmids in each transfection was
adjusted to be equal by the addition of backbone vectors.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
cDNA sequences and deduced amino acid
sequences of human TRA16. The sequence data have been deposited
into GenBankTM (GenBankTM accession number
AY101377). The nucleotide sequences and the deduced amino acid
sequences of TRA16 are presented and numbered on the
left.
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Fig. 2.
Distribution of endogenous TRA16 and
TR4. A, Northern blot analysis of TRA16 mRNA levels
in human normal tissues. Human multiple tissue Northern blot
(Clontech, catalog number 7760-1) was used to
determine the expression of TRA16 in different human tissues, including
heart, whole brain, placenta, lung, liver, skeletal muscle, kidney, and
pancreas. The mRNA species migrating ~1 kb of TRA16 was detected.
The -actin expression was used an equal loading control.
B, Western blot assay of endogenous TRA16 only and
transfected TRA16 protein level. H1299 cells were transiently
transfected either with 10 µg of pcDNA4c-His or
pcDNA4c-His-TRA16 as indicated for 24 h. After harvesting, 50 µg
of whole cell lysates were run in the 15% SDS-PAGE gel. The anti-TRA16
antibody or anti-His tag antibody was used to immunoblot the
membrane to detect the expression of TRA16.
-Actin expression level
was used as a loading control. C, immunocytofluorescence
detection of TRA16 in different cell lines. H1299, COS-1, MCF-7, and
LNCaP cells were prepared for immunostaining with anti-TRA16 polyclonal
antibody, followed by incubation with fluorescein-conjugated goat
anti-rabbit (ICN). The green signal represents the
localization of TRA16, and the blue signal represents the
DAPI-stained nucleus. D, Western blot analysis of endogenous
TR4 protein from various cell lines. The cells indicated were cultured
in 10-cm dishes and harvested, and 50 µg of total protein was run in
a 10% SDS-PAGE gel. After transferring to the membrane, the anti-TR4
antibody was used to immunoblot the membrane to detect the expression
of TR4. The
-actin expression level was used as a loading
control.
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Fig. 3.
Interaction between TR4 and
TRA16. A, immunocytofluorescence detection of
co-localization of TR4 and TRA16 in H1299 cells. The H1299 cells were
prepared for immunostaining with anti-TRA16 polyclonal antibody or
anti-TR4 monoclonal antibody (antibody 17) and then followed by
incubating with either fluorescein-conjugated goat anti-rabbit or
anti-mouse antibodies. The green signal represents the
localization of TRA16, the red signal represents the
localization of TR4, the yellow signal represents
co-localization of the both TRA16 and TR4, and the blue
signal represents the DAPI-stained nucleus. B, TRA16 can
interact with TR4 in mammalian two-hybrid system. COS-1 cells in 60-mm
dishes were transiently co-transfected with 3 µg of reporter plasmid
pG5-LUC and 4 µg each of GAL4DBD, VP16, VP16-TR4, and GAL4-TRA16 in
various combinations as indicated. The luciferase assay was performed
24 h after transfection. All of the values represent the
means ± S.D. of three independent experiments. C,
in vitro co-IP of TR4 and TRA16. 5 µl of each complex
in vitro translated 35S-labeled TR4, TRA16, or
pcDNA4c-His control proteins (mock) described under "Experimental
Procedures" were loaded onto 15% SDS-PAGE gel as indicated and
visualized by autoradiography. D, 10 µg of pcDNA4c-His
or pcDNA4c- His-TRA16 was transiently transfected in H1299 cells
as indicated for 48 h, the lysates were harvested, and then
anti-His probe antibody and protein G-agarose beads were added to 500 µg of protein lysate for 2 h for IP of pcDNA4c-His-TRA16 and
endogenous TR4 complex. 15% SDS-PAGE gel was used to separate the
complex. After transferring to the membrane, the expression was
detected by anti-TR4 antibody or anti-His sprobe antibody. Each cell
lysate input was loaded to show the expression of TR4 and
pcDNA4c-His-TRA16.
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Fig. 4.
TRA16 can suppress TR4 target gene expression
in a dose-dependent manner. A, COS-1 cells
were transiently co-transfected with 3 µg of reporter plasmid
HCR-1LUC, 1 µg of TR4, and increasing amounts of TRA16 expression
plasmid as indicated for 24 h. B, H1299 cells were
transiently co-transfected with 3 µg of reporter plasmid HCR-1LUC and
increasing amounts of TRA16 expression plasmid for 24 h. All of
the values represent the means ± S.D. of three independent
experiments.
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Fig. 5.
TRA16 suppresses TR4-mediated transactivation
with different reporter assay and in TRA16 stably transfected COS-1
cells. A, TRA16 repression of TR4-mediated CpFL-LUC
transcriptional activity comparison with HCR-1LUC. COS-1 cells were
co-transfected with 3 µg of reporter plasmid CpFL-LUC with both
ratios of pCMX-TR4 and pSG5-TRA16 (1:3 and 1:6). B, COS-1
cells were transiently co-transfected with 3 µg of reporter plasmid
DR4-TK-CAT, 1 µg of TR4, and increasing amounts of TRA16 expression
plasmid for 24 h. A PhosphorImager visualized the CAT activity.
C, COS-1 (pBig-TRA16) and COS-1 (pBig) stably transfected
cells were transiently co-transfected with 3 µg of HCR-1 LUC reporter
plasmid, 10 ng of SV40-pRL internal control plasmid, and 1 µg of TR4
for 16 h. The cells were then treated with 6 µl of
Me2SO (as negative control) or 6 µg/ml doxycycline for
another 24 h and then harvested for luciferase assay. All of the
values represent the means ± S.D. of three independent
experiments.
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Fig. 6.
The suppression of TR4-mediated
transactivation by TRA16 is selective. COS-1 cells were
transiently co-transfected with 3 µg of reporter plasmid
MMTV-LUC and 0.5 µg of different steroid receptors (AR,
progesterone receptor, or glucocorticoid receptor) and doses (1.5 and 3 µg) of pSG5-TRA16. After 24 h of transfection, the cells were
treated with 10 nM of synthetic steroids
(dihydrotestosterone for AR, progesterone for progesterone receptor or
dexamethasone for glucocorticoid receptor) and then harvested after
24 h for luciferase assay. All of the values represent the
means ± S.D. of three independent experiments.
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Fig. 7.
TRA16 effects on TR4 protein expression,
localization, and abrogation through HDAC activity. A,
COS-1 cells were transiently co-transfected with 1 µg of TR4 and two
doses (6 and 12 µg) of TRA16 for 24 h, and then the medium was
changed for another 16 h. After harvesting, 50 µg of whole cell
lysate from each sample was run in the 10% SDS-PAGE gel and
transferred to the membrane, and the anti-TR4 antibody was used to
immunoblot the membrane to detect the expression of TR4. Anti- -actin
antibody was used to detect
-actin expression level as a loading
control. B, H1299 cells were transiently transfected with
increasing amounts of TRA16 (2-6 µg) for 38 h. After
harvesting, 50 µg of whole cell lysate from each sample was run on
the 10% SDS-PAGE gel and transferred to the membrane, and the anti-TR4
antibody was used to immunoblot the membrane to detect the expression
of TR4. C, H1299 cells were transiently transfected
without/with 12 µg of TRA16. After harvesting, the nuclear protein
extraction was prepared as described under "Experimental
Procedures," and cytosol protein and whole cell lysate were prepared.
20 µg of different samples as indicated were run in the 15% SDS-PAGE
gel and transferred to the membrane, and the anti-TR4 and anti-His-tag
antibodies were used to immunoblot the membrane to detect the
expression of TR4 and TRA16. D, COS-1 cells were transiently
co-transfected with 3 µg of reporter plasmid HCR-1LUC with both
ratios of TR4 and TRA16 (1:3 and 1:6) for 24 h. The cells were
treated with 50 or 100 nM of TSA or ethanol for another
24 h and then harvested for the luciferase assay. All of the
values represent the means ± S.D. of three independent
experiments.
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Fig. 8.
Inhibition of TR4 DNA binding by TRA16.
A, DU145 cells were transiently transfected with 1 µg of
TR4 for 24 h. Then the medium was changed for another 16 h,
and the cells were harvested; the nuclear protein extraction was
prepared as described under "Experimental Procedures." EMSA was
performed using the 32P-radiolabeled wild-type DR1-TR4RE
(AGGTTAAAGGTCT) (wt) or mutated DR1-TR4RE
(AGGTTAAATGACT) (mt)
oligonucleotides as probe with 10-µg nuclear extracts isolated from
DU145 cells as indicated. Addition of the TR4 (antibody 15)
monoclonal antibody produced a TR4-DNA supershift band (solid
arrow) in lane 4 compared with DR1-TR4RE-binding
proteins (open arrow) in lane 3. B,
COS-1 cells were transiently co-transfected with 1 µg of TR4 and
doses of TRA16 (6 and 12 µg), and nuclear protein extraction was
prepared as described under "Experimental Procedures." EMSA was
performed using the 32P-radiolabeled DR1-TR4RE
oligonucleotides with 10 µg of nuclear extracts. Addition of the TR4
(antibody 15) monoclonal antibody produced a TR4-DNA supershift band
(solid arrow) in lanes 4-6 and separated TR4
from the rest of DR1-TR4RE-binding proteins (open arrow) in
lanes 1-3. C, H1299 cells were transiently
transfected with 12 µg of TRA16 only, and nuclear protein extract was
prepared as described under "Experimental Procedures." EMSA was
performed using the 32P-radiolabeled DR1-TR4RE
oligonucleotides with 10 µg of nuclear extracts isolated from H1299
cells. Addition of the TR4 (antibody 15) monoclonal antibody produced a
TR4-DNA supershift band (solid arrow) in lanes 4 and 5 and separated TR4 from the rest of DR1-TR4RE-binding
proteins (open arrow) in lanes 2 and
3. 32P-Labeled DR1-TR4RE oligonucleotides probe
only was used as a loading control in lane 1. D,
EMSA was performed using the 32P-labeled DR1-TR4RE
oligonucleotides with both TR4-DL containing DBD and LBD of TR4, and
TRA16 purified from the E. coli strain DE3 bacteria (1:5).
Addition of the TR4 (antibody C-16) polyclonal antibody produced a
TR4-DNA supershift band (solid arrow) in lanes 5 and 6 and separated TR4 from the rest of DR1-TR4RE-binding
proteins (open arrow) in lanes 3 and
4. 32P-Labeled DR1-TR4RE oligonucleotides probe
with mock or mock and TR4 antibody was used as negative control in
lanes 1 and 2.
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Fig. 9.
TRA16 blocks the TR4 dimerization in
mammalian two-hybrid assay. A, COS-1 cells were
transiently co-transfected with 3 µg of reporter plasmid pG5-LUC and
3 µg each of GAL4DBD, VP16, VP16-TR4, GAL4-TR4-LBD, pcDNA4c,
pcDNA4c-TRA16, and pSG5-AR in various combinations as indicated.
The luciferase assay was performed 24 h after transfection. All of
the values represent the means ± S.D. of three independent
experiments. B, diagram of each construct and localization
of the interaction domain within TR4. COS-1 cells were transiently
co-transfected with 3 µg of reporter plasmid pG5-LUC and 3 µg each
of GAL4DBD, VP16, VP16-TR4-N, VP16-TR4-DL, and GAL4-TR4-LBD in various
combinations as indicated. The luciferase assay was performed 24 h
after transfection. All of the values represent the means ± S.D.
of three independent experiments. C, in vitro
co-IP to detect which region of TR4 interacts with TRA16. 5 µl of
each complex in vitro translated 35S-labeled
TR4-N, TR4-DL, or TRA16 immunoprecipitated with anti-TR4 antibodies
(antibody 15 is recognized by TR4-N; antibody c-16 is recognized by
TR4-DL) described under "Experimental Procedures" was loaded onto a
15% SDS-PAGE gel as indicated and visualized by autoradiography.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, thyroid hormone
receptor, and the orphan receptor, chicken ovalbumin upstream
promoter-transcription factor I (37-43). Both N-CoR and SMRT proteins
function as co-repressors with HDACs activity that can recruit a
complex containing Sin3, HDACs, and several additional proteins
(44-48). Lavinsky et al. (49) reported that both N-CoR and
SMRT might interact with ER in the presence of the antagonist
trans-hydroxytamoxifen, but forskolin or epidermal growth
factor, which can change trans-hydroxytamoxifen function from
antagonist to agonist, can decrease the ER/N-CoR interaction via
phosphorylation of ER at amino acid Ser118. Their data
suggested that multiple signal transduction pathways regulate the
actions of both N-CoR and SMRT.
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ACKNOWLEDGEMENT |
---|
We thank Karen Wolf for preparation of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant DK56784 and a George Whipple Professorship Endowment.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. E-mail:
chang@urmc.rochester.edu.
Published, JBC Papers in Press, December 16, 2002, DOI 10.1074/jbc.M207116200
2 E. Kim and C. Chang, submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are:
TR4, testicular
orphan receptor-4;
TRA16, TR4 associated protein;
AR, androgen
receptor;
TR, thyroid hormone receptor;
TR4RE, TR4 response element;
TR4-N, TR4-N terminus;
TR4-DL, TR4 DNA binding domain (DBD) and ligand
binding domain (LBD);
DR, direct repeat;
HDACs, histone deacetylases;
TSA, Trichostatin;
CAT, chloramphenicol acetyltransferase;
EMSA, electrophoretic mobility shift assay;
LUC, luciferase;
UL, minus
uronolactone;
RACE, rapid amplification of cDNA ends;
IP, immunoprecipitation;
N-CoR, nuclear receptor co-repressor;
SMRT, silencing mediator for retinoid and thyroid hormone receptor;
DAPI, 4'6-diamidino-2-phenylendole;
MMTV, mouse mammary tumor
virus.
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