The Orphan Nuclear Receptor TR2 Interacts Directly with Both Class I and Class II Histone Deacetylases
Peter J. Franco,
Mariya Farooqui,
Edward Seto and
Li-Na Wei
Department of Pharmacology (P.J.F., M.F., L.-N.W.) University
of Minnesota Medical School, Minneapolis, Minnesota 55455; and
Molecular Oncology Program (E.S.), H. Lee Moffitt Cancer Center and
Research Institute, University of South Florida, Tampa, Florida
33612
Address all correspondence and requests for reprints to: Dr. Li-Na Wei, Department of Pharmacology, University of Minnesota, 6-120 Jackson Hall, 321 Church Street S.E., Minneapolis, Minnesota 55455. E-mail: weixx009{at}maroon.tc.umn.edu
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ABSTRACT
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A combination of in vivo and in vitro
assays was employed to describe the ligand-independent interaction of
the orphan nuclear receptor TR2 and histone deacetylase proteins. The
repressive effect of TR2 on transcription of a luciferase reporter
driven by a promoter containing a direct repeat-5 (DR5) derived from
the human RARß gene was suppressed by the addition of the histone
deacetylase inhibitor trichostatin A. Immunoprecipitation with
FLAG-epitope (MDYKDDDDK)-tagged histone deacetylase proteins was used
to demonstrate that TR2 and histone deacetylases 3 or 4 are present in
the same immunoprecipitated complex. Deacetylase activity was
demonstrated for these coimmunoprecipitates, further confirming the
in vivo interaction of TR2 and histone deacetylases.
Immunoprecipitation with anti-TR2 antibody was used to demonstrate
interaction of TR2 with endogenously expressed histone deacetylases 3
and 4 in COS-1 cells. Dissection of TR2 domains showed that the DNA
binding domain of the receptor was responsible for interaction
with both histone deacetylases 3 and 4 in
glutathione-S-transferase pull-down assays, while the
ligand binding domain did not interact. The pull-down data were
confirmed with far Western blots that also showed a direct interaction
between labeled histone deacetylase proteins and TR2. It is suggested
that repression mediated by unliganded TR2 is mediated, in part, by a
direct interaction of this receptor with histone deacetylase
proteins.
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INTRODUCTION
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NUCLEAR RECEPTORS COMPRISE a superfamily of
transcription factors that regulate gene expression by binding to DNA
target sequences through a zinc-finger type DNA binding domain (DBD)
(1, 2, 3, 4, 5). While some of these nuclear receptors are known
hormone receptors, a large number of cloned nuclear receptors remain as
orphan members. Despite the lack of identified ligands, several orphan
receptors have been shown to play important roles in animal physiology
as demonstrated in genetic knockout studies (6, 7, 8, 9).
The mouse orphan receptor TR2, isolated from an E8.5 embryonic
cDNA library (10) is the mouse homolog of human TR2 that
was cloned from a human prostrate cDNA library (11). The
biological activity of TR2 was demonstrated as repressive in several
heterologous reporter systems. These included reporters driven by a DR4
hormone response element derived from the mouse cellular retinoic acid
binding protein I gene promoter (12), a DR1 type
retinoic acid response element (RARE) derived from the cellular retinol
binding protein II gene promoter (13), and a DR5 derived
from the RARß2 promoter (14). As TR2 was able to bind to
these promoters with high affinity, it was proposed that the
suppressive activity of TR2 in these reporter systems is mediated by
competition with other receptors at DNA binding sites
(14, 15, 16). By using a DR5 reporter as a model system, the
functional characteristics of TR2 suppression have been examined. The
molecular features of TR2 required for full suppressive activity
included the DBD, the ability to dimerize, the ligand binding domain
(LBD), as well as two adjacent glutamate residues (positions 553/554),
and three adjacent leucine residues (positions 537539) that are
required for efficient DNA binding (14). In addition, TR2
was able to heterodimerize with orphan receptor TR4 (17)
and to recruit nuclear receptor interacting protein 140
(18).
Recently, the regulatory activity of nuclear receptors on gene
promoters has been demonstrated to be mediated by a large number of
associate proteins called coactivators, corepressors, or coregulators
(19, 20, 21, 22, 23). Current models of nuclear receptor action
involve chromatin modification such as alteration in the acetylation
status of histone proteins brought about by associated proteins of
nuclear receptors (24). The repressive activity of
apo-receptors is mediated by interaction with corepressors such as
nuclear receptor corepressor/silencing mediator of retinoid and thyroid
hormone receptor that recruit histone deacetylases (HDACs) to
nuclear receptor complexes, thereby deacetylating histone proteins in
the regulatory region of target genes (25). Upon ligand
binding to the LBD, nuclear receptors undergo a conformational change
causing the release of corepressors. The holo-receptors are then able
to interact with coactivator proteins, mainly the p160 family, which
encode intrinsic histone acetyltransferase activity. The resulting
acetylation of histones at target genes is believed to relax chromatin
structure such that the transcription machinery is able to efficiently
activate gene expression (26). Two major classes of HDACs
have been cloned in higher eukaryotes. The yeast Rpd-3 homologs belong
to class I HDACs (HDACs 13, and 8) and the yeast Hda1 homologs belong
to class II HDACs (HDACs 47) (27, 28, 29, 30).
In the current study, we examine whether the ability of the orphan
nuclear receptor TR2 to act as a repressor involves recruitment of
HDACs. We chose HDAC3 as a representative of class I HDACs and HDAC4 as
a representative of class II HDACs. We have found that TR2 possesses
the ability to interact directly with both of these HDAC proteins. This
is a property that has not been reported for other orphan receptors or
hormone receptors such as the RARs. The HDAC interacting domain was
localized to the DBD portion of the receptor that encompasses the two
zinc fingers present in the receptor. The repressive activity of TR2
was also found to be suppressed by the HDAC inhibitor trichostatin A
(TSA). In addition, immunoprecipitation of both HDAC3 and HDAC4 yielded
complexes that included TR2, and immunocomplexes precipitated with
anti-TR2 antibody encoded HDAC activity. The combination of in
vivo and in vitro data supporting a direct interaction
between TR2 and HDACs suggests a role for HDACs in mediating TR2
repression.
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RESULTS
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In Vivo Association of TR2 and HDAC
Coimmunoprecipitation.
Previously we, and others have demonstrated a strongly suppressive
activity of TR2 on reporter gene expression driven by several direct
repeat sequences (e.g. DR1, DR4, and DR5)
(12, 13, 14). To investigate whether HDACs, enzymes
responsible for deacetylating histone proteins, and the TR2 suppressive
activity are related, we first examined the possibility that TR2 and
HDAC proteins are present in the same immunocomplex. COS-1 cells were
cotransfected with TR2 and FLAG-epitope (MDYKDDDDK)-tagged HDAC3 or
FLAG-tagged HDAC4. An anti-FLAG monoclonal antibody
(Sigma, St. Louis, MO) was then used to immunoprecipitate
the transfected HDAC and associated proteins from the cotransfected
cells. The anti-FLAG immunoprecipitates were resolved using SDS-PAGE
and transferred to polyvinylidene difluoride (PVDF) membranes.
The membranes were then probed with anti-TR2 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) to detect the presence of
TR2 in the HDAC immunoprecipitates. A 70-kDa band corresponding to TR2
is clearly shown for COS-1 cells cotransfected with TR2 and either
FLAG-HDAC3 or FLAG-HDAC4 (Fig. 1A
, lanes
7 and 9). In contrast, cells transfected with TR2, FLAG-HDAC3, or
FLAG-HDAC4 alone showed no TR2 band (Fig. 1A
, lanes 5, 6, and 8,
respectively) when precipitation was done with the anti-FLAG antibody.
In addition, as a negative control, a VP16-TR2 fusion construct
containing only the TR2 LBD (18) was cotransfected with
FLAG-HDAC3. In the anti-FLAG precipitated immunocomplex from these
cells, the TR2-LBD could not be detected with the anti-TR2 antibody
(Fig. 1A
, lane 10). To ensure that transiently transfected cells were
efficiently expressing TR2 or the TR2-LBD, lysate from cells
transfected with TR2 alone (Fig. 1A
, lane 1), TR2 and FLAG-HDAC3 (Fig. 1A
, lane 2), TR2 and FLAG-HDAC4 (Fig. 1A
, lane 3), or TR2-LBD and
FLAG-HDAC3 (Fig. 1A
, lane 4) were probed with an anti-TR2 antibody. We
were able to detect TR2 or the TR2-LBD in all of these samples. These
results indicated that TR2 can be coprecipitated with a member of class
I and class II HDAC proteins.

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Figure 1. In Vivo Interaction of TR2 and HDACs
A, COS-1 cells were transfected with either hemagglutinin-TR2 (lanes 1
and 5), FLAG-HDAC3 (lane 6), FLAG-HDAC3 and TR2 (lanes 2 and 7),
FLAG-HDAC4 (lane 8), FLAG-HDAC4 and TR2 (lanes 3 and 9), or TR2-LBD and
FLAG-HDAC-3 (lanes 4 and 10). Whole cell lysates were
immunoprecipitated with anti-FLAG monoclonal antibody (lanes 510) and
pulled out with Protein-G beads. Samples were then separated on 10%
SDS-PAGE and immunoblotted with anti-TR2 antibodies. Lanes 14 show
whole-cell lysates immunoblotted with anti-TR2 antibody to indicate the
input level of TR2 and TR2-LBD for the immunoprecipitated samples. B,
To test for interaction of TR2 with endogenously expressed HDACs, COS-1
cells were transfected with TR2. Whole-cell lysates were then
immunoblotted with antibody against HDAC3 or HDAC4 (lane 1). Samples of
the same lysates were immunoprecipitated with TR2 antibody followed by
immunoblotting with antibody against HDAC3 or HDAC4 to test whether TR2
coprecipitates the endogenously expressed HDAC proteins (lane 2).
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Coimmunoprecipitation of TR2 and Endogenous HDACs.
To strengthen the data showing an interaction between TR2 and HDACs, we
showed the presence of endogenous HDAC3 and HDAC4 in COS-1 cells and
subsequently detected an interaction between TR2 and these proteins. To
show the presence of endogenous HDACs, COS-1 cells were transfected
with TR2 and cell lysates were collected. These lysates were analyzed
by Western blot using antibodies directed against either HDAC3 or
HDAC4. The first column of Fig. 1B
shows strong bands for HDAC3
(top panel) and HDAC4 (lower panel) indicating
good levels of endogenous HDAC expression in COS-1 cells. To then
demonstrate that TR2 interacts with these endogenous HDACs, lysates
from the same TR2 transfected sample were immunoprecipitated with
anti-TR2 antibody followed by probing of the precipitated immunocomplex
with antibodies against HDAC3 or HDAC4. The second column of Fig. 1B
shows that the immunoprecipitated TR2 complex contains bands
corresponding to both HDAC3 (top panel) and HDAC4
(lower panel).
Inhibition of TR2-Mediated Repression by the HDAC Inhibitor
Trichostatin A
In previous studies we have demonstrated that TR2 is able to
repress activity of reporters containing a DR5 type RARE from the human
RARß promoter (14). In this study, we have used the DR5
reporter (RARE-tk-luc), which responds well to retinoic acid (RA)
induction, to test whether TR2-mediated repression from this reporter
can be blocked by the HDAC inhibitor TSA. If TR2 repression involves
the recruitment of HDACs, either directly or indirectly, it is expected
that TSA should ameliorate this repression to some extent. In the
experiment of Fig. 2
, COS-1 cells were
transiently transfected with an internal LacZ control and the
RARE-tk-luc reporter. The addition of RA (1 µM) to these
cells activates endogenous RAR and RXR to drive expression from the
luciferase reporter. Each column in Fig. 2A
represents the ratio of
normalized luciferase activity in the presence of RA to the normalized
luciferase activity in the absence of RA (see Materials and
Methods), reported as the fold-RA induction. Column 1 shows that
addition of RA caused an 11-fold increase in normalized luciferase
activity. However, when TR2 was cotransfected with the reporter, this
activation is repressed by 51% (Fig. 2A
, compare columns 1 and 2). If
the TR2-mediated repression involves recruitment of HDACs, either
directly or indirectly, it is expected that TSA should block this
repression to some extent. To test this notion, the experiment was
repeated with the addition of 100 nM TSA (Fig. 2A
, columns 3 and 4) or 200 nM TSA (Fig. 2A
, columns 5 and 6). The addition of TSA in the absence of TR2 was found
to cause a reduction in the fold-RA induction (Fig. 2A
, compare column
1 with columns 3 and 5; see below for explanation). When both TSA and
TR2 are present in the COS-1 cells, the repression of reporter activity
mediated by TR2 is reduced significantly. Comparing the difference in
fold-RA induction between columns 1 and 2 with the difference
between columns 3 and 4 of Fig. 2A
, it appears that
TR2-mediated repression is reduced from 51% with no TSA, to 27%
with 100 nM TSA (Fig. 2B
). When the concentration
of TSA is increased to 200 nM, TR2 repression is
almost absent at just 5% (compare the difference between columns 1 and
2 to the difference between columns 4 and 5 in Fig. 2A
). The
amelioration of TR2 repression caused by the addition of TSA suggests
that the repressive activity of TR2 involves recruitment of HDAC. When
these experiments were repeated with exogenously added RAR and RXR,
similar results were obtained (data not shown).

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Figure 2. TSA Blocks TR2-Mediated Repression of RA Induction
of the RARE-tk-luc Reporter
A, The RA induction of the RARE-tk-luc reporter in COS-1 cells was
assayed in the absence and presence of TR2 with 0, 100 nM,
and 200 nM TSA present in the media as described in
Materials and Methods. The fold-RA induction was
determined by comparing activity in the presence of RA to activity
without added RA. B, Percent TR2- mediated repression of fold-RA
induction of the RARE-tk-luc reporter in COS-1 cells calculated from
the data in panel A.
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The reduction in fold-RA induction with the addition of TSA is
explained by an increase in RARE-tk-luc reporter activity in the
absence of RA (data not shown), caused presumably by a general relief
of transcriptional repression. However, TSA effects on the reporter in
the presence of RA were less dramatic. It is possible that histone
acetylation has already contributed to the induction of the RARE-tk-luc
reporter in the presence of RA, and inhibition of histone
deacetylation, by TSA, does not afford further enhancement of RA
induction.
Measurement of Deacetylase Activity in TR2/HDAC Immunocomplexes
To confirm the in vivo interaction between TR2 and
HDACs, and to determine enzymatic activities of these complexes, the
TR2/HDAC cotransfected cell lysates were immunoprecipitated with
anti-TR2 antibody and assayed for deacetylase activity. Anti-TR2
antibody was incubated with COS-1 cell lysates to pull out TR2 and any
associated proteins. The anti-TR2 complex was then bound to Protein-G
beads, washed extensively with HDAC buffer, and incubated with
3H-labeled H4 histone peptide. The released
3H-acetic acid was extracted with ethyl acetate
and quantitated using liquid scintillation. For basal level controls,
untransfected (Fig. 3
, column 1) and
TR2-transfected (Fig. 3
, column 2) COS-1 cells were precipitated with
anti-TR2 antibody and tested for deacetylase activity. The activity
detected in the TR2-transfected cells (Fig. 3
, column 2) above the
activity found in untransfected cells (Fig. 3
, column 1) is attributed
to the presence of endogenously expressed HDACs pulled down with TR2
(see Fig. 1B
, lane 2). A low but significant increase in deacetylase
activity above that found in the control samples was detected in the
immunoprecipitates of cells that had been cotransfected with TR2 and
HDACs (Fig. 3
, columns 3 and 4). The demonstration of deacetylase
activity in the TR2 coimmunoprecipitates provides evidence for a
biologically active complex that includes TR2 and HDAC enzymes.

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Figure 3. HDAC Activity Is Associated with TR2 Protein
Complex in Vivo
COS-1 cells were transfected as described in Fig. 1 . COS-1 cells were
transfected with TR2 (column 2), HDAC3 and TR2 (column 3), or HDAC4 and
TR2 (column 4), and cell lysates were immunoprecipitated with anti-TR2
antibody. The immunoprecipitates were then bound to Protein-G beads and
assayed directly for deacetylase activity. Deacetylase activity is
expressed as counts per min of 3H-acetic acid released. The
deacetylase activity was normalized to the activity found in
untransfected COS-1 cells precipitated with the TR2 antibody (column
1). The values for deacetylase activity are the average of five
separate experiments with duplicate measurements done for each
experiment.
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Mammalian Two-Hybrid Test
As a further test of the interaction of HDAC3 and TR2, a mammalian
version of the two-hybrid test was done. In this experiment, the TR2-f
(full-length) and the TR2-t (containing an intact DBD but lacking the
LBD, Fig. 4
, upper panel)
clones were fused to the VP16 activation domain, and HDAC3 was fused to
the GAL4 binding domain. The reporter construct was a tk-Luciferase
reporter containing five copies of the GAL4 binding domain (Fig. 4
, upper panel) (17). COS-1 cells were
cotransfected pairwise with the GAL4-BD-HDAC3 fusion and the VP16-TR2
fusions, together with the reporter and a cytomegalovirus (CMV)-LacZ
internal control. A basal activity level was measured by testing
GAL4-BD-HDAC3 with VP16 (Fig. 4
, column 1). The relative luciferase
activity was calculated by normalizing luciferase units to LacZ
activity. The (-fold) relative luciferase activity was then calculated
by normalizing to the basal level of activity found for the
GAL4-BD-HDAC3/VP16 cotransfection. Figure 4
shows interaction
between HDAC3 and full-length TR2 results in an approximately 3-fold
higher reporter activity (column 2), whereas interaction with TR2-t
resulted in approximately 2.5-fold higher reporter activity (column 3).
Although this test did not show a dramatic increase in reporter
activity, it should be considered that recruitment of HDAC3 to the
reporter might attenuate a more vigorous response. In any case, the
two-hybrid test showed a low but significant increase in luciferase
reporter activity for both the TR2-full length and truncated constructs
in this assay. These results suggest that an in vivo
interaction between TR2 and HDAC3 exists that does not require the TR2
LBD.

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Figure 4. Interaction of HDAC3 and TR2 in Mammalian Cells
A mammalian version of the two-hybrid system was used to examine
interaction between HDAC3 and TR2 in COS-1 cells. The upper
panel shows a luciferase reporter with five copies of the GAL4
binding site and the expression vectors for HDAC3 and TR2 clones. The
GAL4-BD-HDAC3 fusion and VP16-TR2 fusions were cotransfected pairwise
in COS-1 cells along with the reporter and a CMV-LacZ internal control.
Thirty to 40 h after transfection, cells were harvested and
luciferase and LacZ activities were determined. Relative RLU was
calculated as described in Materials and
Methods.
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In Vitro Interaction of TR2 and HDACs
Glutatione-S-Transferase (GST) Pull-Down Assay.
To test the interaction between class I and class II HDACs and the
mouse orphan receptor TR2, a GST pull-down assay was employed.
Initially, the full-length TR2 receptor, the TR2-LBD, and the TR2-t
coding regions were ligated to a GST expression vector, expressed in
Escherichia coli, and bound to glutathione agarose beads.
The bound GST-TR2 samples were then incubated with in vitro
translated 35S-labeled HDAC3 (class I) or HDAC4
(class II). Interaction with HDACs was observed with full-length TR2
(Fig. 5B
, TR2-f lane) and a region that
includes the TR2 DBD (Fig. 5B
, TR2-t lane) but not the LBD region of
the receptor (Fig. 5B
, LBD lane). The N-terminal and DBD regions of the
protein were further dissected by construction of several additional
recombinant GST-TR2 fusion proteins (Fig. 5A
). To verify the binding of
GST-TR2 fusion proteins to the glutathione agarose beads, samples of
beads from the binding of each recombinant protein were resolved using
SDS-PAGE, and the gel was stained with Coomassie blue (Fig. 5C
).
The overlapping region between the original full-length and TR2-t
constructs, amino acids 166219 (Fig. 5B
, TR2-D lane), was found to be
negative for interaction with both HDACs, as was the A domain, which is
comprised of amino acids 150 (Fig. 5B
, A lane). The C domain of the
TR2 receptor (amino acids 99166), which contains two zinc finger
motifs located between amino acids 99133 (ZF-1) and 133166 (ZF-2),
showed interaction with both HDACs (Fig. 5B
, lane C). When each zinc
finger was expressed separately, both were also found to pull down
HDAC3 and HDAC4 (Fig. 5B
, lanes ZF-1 and ZF-2). In addition, those
constructs that contain only a portion of the C domain, but encompass
either zinc finger, showed interaction (Fig. 5B
, lanes A/B/C', B/C',
and ZF-2/D).

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Figure 5. Recombinant GST-TR2 Constructs and HDAC Pull-Down
Data
A, Schematic representation of GST-TR2 recombinant protein constructs.
The full-length TR2 construct, TR2-f, includes delineation of nuclear
receptor domains. Numbers on the left represent the
first amino acid encoded by TR2 in each construct and numbers on
the right represent the last TR2 amino acid. B, GST-TR2
recombinant proteins pull down HDAC3 and HDAC4. Recombinant GST-TR2
protein samples were bound to glutathione agarose and incubated with
35S-labeled HDAC3 or HDAC4. After extensive washing,
specific interacting protein was eluted and analyzed by SDS-PAGE and
autoradiography. Input control (lane 1), GST control (lane 2), TR2-f
(lane 3), TR2-LBD (lane 4), TR2-t (lane 5), TR2-A/B/C' (lane 6),
TR2-C/D (lane 7), TR2-A (lane 8), TR2-B/C' (lane 9), TR2-C (lane 10),
TR2-D (lane 11), TR2-ZF-2/D (lane 12), TR2-ZF-1 (lane 13), and TR2-ZF-2
(lane 14). C, Protein sample input for GST pull down. Recombinant
GST-TR2 protein samples were bound to glutathione agarose, washed,
separated using SDS-PAGE (10% acrylamide gel), and stained with
Coomassie brilliant blue. The same amount of sample material used in
the pull-down experiments was loaded on this gel. Samples include
protein marker in 10-kDa increments (lane 1); lanes 214 are as
described in panel B. Asterisks mark the GST-TR2 bands
of interest.
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Far Western.
To verify a direct interaction between TR2 and HDACs, a far Western
assay was employed. In this assay the GST-TR2 fusion clones including
TR2-f, TR2-LBD, TR2-t, and TR2-C/D were partially purified using
glutathione agarose beads, resolved on a 10% polyacrylamide gel using
SDS-PAGE, and transferred to PVDF membranes. The membranes were then
probed with in vitro translated,
35S-labeled HDAC3 or HDAC4. If the HDAC proteins
interact with TR2, autoradiography should produce bands of the
appropriate size for the various GST-TR2 fusion proteins. Figure 6
shows the results of the far Western
blot. Of the clones tested, bands of appropriate size were detected for
the full-length TR2, TR2-t, and TR2-C/D clones (Fig. 6
, A and B, lanes
2, 3, and 5, respectively). No bands were detected for the negative
control (GST sample) or the TR2-LBD (Fig. 6
, A and B, lanes 1 and 4,
respectively). This result is in agreement with the GST pull-down assay
that showed the LBD of TR2 did not interact with either HDAC3 or HDAC4
and confirms that the TR2 DBD domain mediates HDAC interaction.

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Figure 6. Far Western Blot Showing Direct Interaction between
TR2 and HDACs
GST-TR2 fusion proteins were partially purified by binding to
glutathione agarose. After extensive washing, bound protein was eluted
with glutathione buffer. Protein samples were subjected to SDS-PAGE and
transferred to PVDF membrane. Membranes were probed with
35S-labeled HDAC3 or 35S-labeled HDAC4. A,
Autoradiogram showing interaction of TR2 and HDAC3. GST control (lane
1), TR2-f (lane 2), TR2-t (lane 3), TR2-LBD (lane 4), TR-C/D (lane 5).
Asterisks indicate the interacting TR2 bands. B,
Autoradiogram showing interaction of TR2 and HDAC4. Lanes 15 are as
described in panel A. C, Coomassie-stained gel showing input for
far Western samples in panels A and B. Protein marker in 10-kDa
increments (lane M); lanes 15 are as described in panel A.
Asterisks mark the bands for GST (lane 1) and TR2
samples (lanes 25).
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Interaction of the TR2 DBD and HDAC Requires Zinc
After identifying the TR2 DBD as the region of TR2 that
interacts with HDACs, it was of interest to determine whether the
three-dimensional structure of this domain was required to promote HDAC
binding to TR2. To investigate this possibility, the GST-TR2-C clone,
which contains the two zinc fingers present in TR2 (Fig. 5A
), was used
in a pull-down assay after chelation of zinc. In the experiment of Fig. 7
, GST-TR2-C was bound to glutathione
agarose followed by extensive washing and overnight incubation in the
presence of 100 mM EDTA. GST alone was included as a
negative control. After chelation of zinc with EDTA, the bound TR2-C
was then incubated with in vitro translated
35S-labeled HDAC3 or HDAC4 in the presence or
absence of 100 mM ZnCl2. In
the case of HDAC3 the chelation of zinc caused a near complete
disruption of binding that was restored with the addition of zinc (Fig. 7A
, compare lanes 4 and 5, upper panel). For HDAC4, binding
to TR2-C still occurred in the absence of zinc, but there was a
distinct enhancement of this binding with the addition of zinc (Fig. 7A
, compare lanes 4 and 5, lower panel). The binding signals
of HDAC3 and HDAC4 in the presence and absence of zinc observed in Fig. 7
were consistent over several trials of the experiment. The addition
of zinc to bound GST did not enhance interaction with HDACs (Fig. 7A
, compare lanes 2 and 3). These results support the notion that the TR2
DBD is the domain that contacts HDACs and indicate that proper folding
of the zinc finger domains of TR2 is required for interaction with
HDAC3 and enhances interaction of TR2 with HDAC4.

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Figure 7. GST Pull-Down of HDACs after Chelation of Zinc
GST and the GST-TR2-C recombinant clone, containing the two zinc
fingers of the TR2 DBD, were bound to glutathione agarose, washed
extensively, and incubated overnight in the presence of 100
mM EDTA. The bound proteins were then incubated with
35S-labeled HDAC3 or HDAC4 in the presence and absence of
100 mM ZnCl2. After extensive washing,
interacting protein was eluted and analyzed by SDS-PAGE and
autoradiography. Input control (lane 1), GST (lane 2), GST +
ZnCl2 (lane 3), TR2-C (lane 4), TR2-C + ZnCl2
(lane 5). B, Protein sample input for GST pull down. GST and
recombinant TR2-C protein samples were bound to glutathione agarose,
washed, and separated using SDS-PAGE (10% acrylamide gel) and stained
with Coomassie brilliant blue. The same amount of sample material used
in the pull-down experiments was loaded on this gel. Protein marker in
10-kDa increments (lane 1); lanes 25 are as described in panel A.
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DISCUSSION
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Recently, identification of HDAC proteins has provided
support for the long held belief that the acetylation status of
histones influences transcriptional activity (28, 31, 32, 33, 34, 35, 36).
Histone deacetylation is thought to repress transcription by inducing
changes in chromatin structure that disrupt transcription, or by
blocking assembly of the transcription initiation complex
(25). Consistent with a role for HDACs in repression,
corepressor complexes have been found to include HDAC proteins
(20, 23, 34, 35, 36, 37). A direct link between HDAC activity and
transcriptional repression was recently established when point
mutations were introduced in the deacetylation domains of HDAC 5 and 7
(38). The resulting HDAC mutants lost the ability to
deacetylate histones, no longer could interact with silencing mediator
of retinoid and thyroid hormone receptor, and could no longer
efficiently repress basal transcription from a heterologous
promoter.
In the present study we addressed the questions of
whether TR2-mediated repression involves HDACs, and whether there is a
direct interaction between this orphan receptor and the HDAC3 and HDAC4
proteins. A functional connection between TR2-mediated repression and
HDAC activity was implied when we were able to demonstrate that the
HDAC inhibitor TSA blocks TR2 repression. We were then able to show
that TR2 and HDAC3 or HDAC4 are present in the same immunocomplex. When
a TR2 antibody was used to immunoprecipitate protein complexes from
cells cotransfected with TR2 and either of these HDACs, HDAC activity
could be demonstrated. These results gave a clear indication that TR2
and HDACs can be found in the same immunocomplex and that these HDACs
are active. Further evidence for interaction was provided by showing
that TR2 coprecipitates endogenous HDAC3 and HDAC4 from COS-1
cells.
After demonstrating an in vivo association of TR2
and HDAC3 and HDAC4, we then tested the possibility that these proteins
interact directly. GST pull-downs showed that both HDAC3 and HDAC4 were
able to interact with the wild-type TR2 receptor. Further dissection of
TR2 revealed that the interaction domain includes the DBD but excludes
the LBD of the receptor. To verify that a direct interaction between
HDACs and TR2 was mediated by the DBD, we used a far Western blot. The
far Western has the advantage of showing interaction specifically with
bands corresponding to the TR2 constructs. This helped us to rule out
the possibility that impurities in the GST-TR2 fusion protein
preparations were responsible for interaction with labeled HDACs in the
GST pull-down assay. The results of the far Western blots were
consistent with those of the pull-downs. The DBD of TR2 is a region
highly conserved among nuclear receptors, which contains two zinc
finger binding motifs. The three-dimensional structure of these domains
within TR2 appears to be important for interaction with HDACs as
chelation of zinc from these domains completely disrupted the
interaction with HDAC3 and to a lesser extent with HDAC4.
We conclude that repression mediated by the mouse orphan
receptor TR2 involves recruitment of both class I (HDAC3) and class II
(HDAC4) HDACs. While it is likely that this deacetylase activity can be
exerted through binding of a corepressor complex, we have provided
evidence for a second level of transcriptional regulation through
direct interaction of TR2 and HDAC proteins. As a conserved region of
nuclear receptors mediates the direct interaction of HDACs and TR2, it
is possible that this type of interaction is common to other receptors
as well. In support of this notion, the TRß receptor has been shown
to bind HDAC-2 directly and that this binding is mediated by the TRß
DBD (39). Presently, we are testing the DBD of several
nuclear receptors for direct interaction with both classes of HDAC
proteins.
 |
MATERIALS AND METHODS
|
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Construction of Expression Vectors
To construct GST-TR2 expression vectors, coding regions from TR2
were subcloned into the pGEX-2T (Pharmacia Biotech,
Piscataway, NJ) vector such that the fusion proteins produced would
have GST fused to the amino-terminal end of the various TR2 constructs.
The full-length TR2 construct (TR2-f, amino acids 1590) was made by
releasing a BglII/XbaI fragment from the TR2 cDNA
(40) and ligating to the BamHI/XbaI
sites of pGEX-2T. The TR2-LBD region (amino acids 166590) was
released from the pBD-DEF construct (18) with
EcoRI/XbaI and ligated to the same sites in
pGEX-2T. The TR2-t (amino acids 1219) clone was released by digestion
with EcoRI and XhoI of a cDNA encoding a
truncated isoform of TR2 (16) that includes an intact DBD
but lacks the LBD. This fragment was then ligated to the
EcoRI/SalI sites of the pGEX-2T expression
vector. TR2-A/B/C' (amino acids 1138) and TR2-A (amino acids 150)
constructs were created by releasing
EcoRI/HindIII fragments from pM vector constructs
containing these coding regions (41). These fragments were
then subcloned into the EcoRI/HindIII sites of
pGEX-2T. The TR2-B/C' (amino acids 50138) coding region was released
from the full-length cDNA using BamHI and subcloned into the
same site in pGEX-2T. The remaining TR2 constructs: TR2-C (amino acids
99166), TR2-D (amino acids 166219), TR2- C/D (amino acids 99219),
TR2-ZF-1 (amino acids 99133), TR2-ZF-2 (amino acids 133166), and
TR2-ZF-2/D (amino acids 133219) were PCR amplified from the TR2 cDNA
such that EcoRI and HindIII sites were introduced
at the 5'- and 3'-ends of the PCR products, respectively. These TR2
fragments were then ligated to the EcoRI/HindIII
sites of pGEX-2T.
The RARE-tk-luc reporter construct, containing a DR5, was kindly
provided by Dr. R. Evans of the Salk Institute, San Diego, CA
(42). The FLAG-epitope-tagged HDAC3 (37) and
FLAG-tagged HDAC4 (28) vectors were as described. For the
mammalian two-hybrid test, HDAC3 was fused to the GAL4 DBD by cloning
into the pM vector (CLONTECH Laboratories, Inc.), and
full-length TR2 (TR2-f clone) and the isoform TR2-t (lacking the LBD)
were fused to the VP16 activation domain by cloning into the pVP16
vector (CLONTECH Laboratories, Inc.). The
GAL4-tk-lucerifase reporter was as described previously
(17).
Preparation of Protein Samples
GST and GST-TR2 fusion proteins were produced in the E.
coli BL21(DE3)/pLysS strain. Cells were grown at 37 C to an
OD600 of 0.50.6, followed by induction with 0.1
mM isopropyl
ß-D-thiogalactopyranoside for 35 h. Cells were
harvested by centrifugation, washed once in PBS, and suspended
in lysis buffer [50 mM Tris-Cl (pH 8), 50
mM NaCl, 5 mM EDTA, 1%
Triton X-100, 0.1% ß-mercaptoethanol, and protease inhibitor
cocktail). Cells were sonicated three times for 30 sec followed by two
passes in a French Pressure cell at 16,000 psi. Samples were then
centrifuged at 20,000 x g for 1 h. The
supernatant was separated from insoluble material, passed through a
0.22-µm filter, and used as the source of fusion protein. The TR2-f
and TR2-t constructs were not soluble and had to be extracted from the
insoluble material. Sample pellets for these constructs were
solubilized in extraction buffer [10 mM Tris-Cl
(pH 7.5), 8 M guanidine-HCl, 5
mM EDTA, 5 mM DTT] for
1 h on ice. The solubilized samples were then dialyzed for 16
h against a urea buffer [1 M urea, 50
mM Tris-Cl (pH 7.5), 250 mM
NaCl, 1 mM EDTA, 1 mM DTT,
protease inhibitor cocktail]. Samples were then centrifuged at
12,000 x g for 30 min and passed through a 0.22 µm
filter. All samples were stored at -80 C.
TR2 Repression Assay
COS-1 cells were maintained in DMEM supplemented with 10% FBS
treated with charcoal. To determine the effect of TSA on the
suppressive activity of TR2, the reporter RARE-tk-luc, the receptor
vector, and an internal control (CMV-LacZ) were cotransfected into
COS-1 cells by calcium phosphate coprecipitation. For cotransfection,
0.1 µg of TR2 expression vector, 0.3 µg of the reporter, and 0.05
µg of internal control plasmid were used. For control transfections
in which TR2 DNA was not added, the concentration of DNA was made up
with sheared salmon sperm DNA. Cells were plated at a density of 5
x 104/well in 24-well plates and incubated
overnight before transfection. For induction, RA (1 µM)
was added to the medium 68 h following transfection. TSA was added
(0200 nM) to the samples at the same time as RA addition.
Thirty to 40 h after transfection, total cell extracts were
collected and assayed for luciferase activity, LacZ activity, and
total protein concentration. Luciferase activity, determined with a
commercial assay system (Promega Corp., Madison, WI), was
normalized to the internal control LacZ activity determined using
orthonitrophenyl-ß-D-galactopyranoside
(Sigma, St. Louis, MO) as the substrate, and represented
as relative luciferase unit (RLU). The reporter activity was normalized
to total cell protein in each sample by calculating the ratio of RLU/mg
total protein. This was done to correct for cell death caused by
expression of TR2 and addition of TSA. All transfections were done in
the presence and absence of RA, and the ratio of [RLU/mg protein +
RA]/[RLU/mg protein - RA] is reported as the fold
RA-induction. Two independent experiments were carried out with three
replicate cultures measured for each condition.
Mammalian Two-Hybrid Test
COS-1 cells were maintained in DMEM supplemented with 10% FBS
at 37 C in 5% CO2. To test the interaction of
TR2 and HDAC3, cells were plated at a density of 5 x
104/well in 24-well plates and cotransfected with
pM-HDAC3 (0.1 µg) and pVP16-TR2-f (0.1 µg) or pVP16-TR2-t (0.1
µg), along with a GAL4-tk-luc (0.5 µg) reporter and a CMV-LacZ
internal control (0.05 µg). Thirty to 40 h after transfection,
total cell extracts were collected and tested for luciferase and LacZ
activity. RLU was calculated as described in the preceding paragraph.
The fold-relative luciferase activity was calculated by normalizing RLU
activity found in experimental samples to the basal level RLU activity
found in the pM-HDAC3/pVP16 control cotransfection. Reported values are
an average of three experiments with triplicate measurements taken in
each experiment.
Coimmunoprecipitation
COS-1 cells were maintained in DMEM supplemented with 10% FBS
at 37 C in 5% CO2. Cells were plated at a
density of 1 x 105/10-cm dish and
cotransfected with hemagglutinin (HA)-tagged TR2 (7.5 µg) and
FLAG-tagged HDAC3 (7.5 µg) or FLAG-tagged HDAC-4 (7.5 µg)
expression vectors or TR2-LBD (7.5 µg) and FLAG-tagged HDAC-3 (7.5
µg). Forty eight hours after transfection, cells were harvested and
resuspended in lysis buffer [20 mM Tris-Cl (pH 8.0), 100
mM NaCl, 1 mM EDTA, 0.1% Nonidet P-40, 1
mM DTT, 2 µg PMSF, 10% glycerol, protease inhibitor
cocktail]. Cells were sonicated twice in 20-sec pulses on ice. Lysates
were clarified by centrifugation at 10,000 x g for 10
min, and supernatant was used as the whole cell extract. For
coimmunoprecipitation, 150200 µl of cell extract were incubated
with anti-FLAG monoclonal antibody (Sigma) at 4 C for
3 h, followed by addition of 20 µl of Protein-G agarose resin
(Sigma). The samples were rocked for 1 h at 4 C,
followed by extensive washing with lysis buffer to remove unbound
proteins. The beads were then suspended in SDS-PAGE buffer for Western
blot analysis. Samples were separated on a 10% gel and transferred on
to PVDF membrane (Amersham Pharmacia Biotech, Arlington
Heights, IL). The blot was incubated with rabbit anti-TR2 antibody for
2 h at 4 C, followed by washing in 1x PBS, 0.1% Tween-20.
Secondary antibody, antirabbit horseradish peroxidase, was then added
to the blot for an additional 1.5 h. The signal was then detected
with enhanced chemiluminescence (ECL) (Amersham Pharmacia Biotech).
For determining the interaction of TR2 with endogenous HDAC3 or HDAC4
in COS-1 cells, lysates of TR2-transfected cells were incubated
with anti-TR2 antibody. The immunocomplex was precipitated by adding
Protein-G beads, separated on 10% SDS-PAGE and immunoblotted with
rabbit anti-HDAC3 or HDAC4 antibodies as detailed above. The anti-HDAC3
and -HDAC4 antibodies were also used to show the presence of
endogenously expressed HDAC3 and HDAC4 in the whole-cell lysate of
TR2-transfected COS-1 cells by Western blot analysis.
HDAC Activity
Deacetylase activity was measured in the samples using a kit
(Upstate Biotechnology, Inc., Lake Placid, NY) according
to the manufacturers instructions.
GST Pull-Down Assay
GST and GST fusion proteins were partially purified by binding
to 60 µl of glutathione-agarose beads (Sigma). Due to
differences in binding affinity for the various GST-TR2 constructs,
preliminary binding studies were done to determine the amount of each
sample preparation that would yield bands of approximately equal
intensity on a Coomassie-stained SDS-PAGE (10% gel). After sample
binding, the beads were washed twice with 20 vol of 1x PBS and once
with binding buffer [20 mM HEPES (pH 7.5), 100
mM NaCl, 0.5 mM EDTA, 0.1% Triton X-100, 10%
Glycerol]. 35S-labeled HDAC3 or HDAC4 (2 µl
per GST-TR2 fusion sample) prepared in TNT reactions (Promega Corp.) was then added to each GST-TR2 sample in 300 µl of
binding buffer supplemented with protease inhibitor cocktail. The
samples were allowed to incubate at 4 C for 90 min followed by three
washes with 20-bead volumes of binding buffer to remove unbound
proteins. The beads were collected by centrifugation and suspended in
binding buffer (20 µl) and 4x SDS sample buffer (20 µl). Binding
to GST was included as a negative control. Samples were divided, and an
equal amount was resolved using SDS-PAGE (10% gel) on two separate
gels. One gel was stained with Coomassie and the second gel was fixed,
dried, and exposed to a PhosphoImager screen (Molecular Dynamics, Inc., Sunnyvale, CA) overnight to detect labeled HDAC
proteins.
Zinc Chelation with GST Pull Down
The pull-down procedure described above was repeated using GST
(negative control) and the GST-TR2-C clone with the following
modifications. After sample binding, beads were washed three times with
20 vol of 1x PBS, 0.1% NP-40 (Sigma), followed by two
washes with the same buffer containing 100 mM EDTA. Beads
were then washed overnight in the 1x PBS, 0.1% NP-40, 100
mM EDTA buffer to chelate zinc from the two zinc finger
domains present in the TR2-C construct. The samples were then washed
three more times in 1x PBS, 0.1% NP-40 and once in binding buffer.
35S-labeled HDAC3 or HDAC4 (2 µl per sample)
was then added to each sample in 300 µl of binding buffer (containing
10 mM EDTA) in the presence or absence of 100
mM ZnCl2. To remove unbound proteins,
samples were washed three times with binding buffer lacking EDTA. The
beads were collected and suspended in 20 µl of 4x SDS sample buffer.
Samples were analyzed as described in the preceding paragraph.
Far Western
GST-TR2 fusion protein samples were partially purified by
binding to glutathione agarose as described above, followed by
extensive washing with PBS to remove unbound protein. Bound protein was
then eluted from the beads with a glutathione buffer (10 mM
Tris-Cl, 10 mM reduced glutathione). Protein samples
(1030 µg) were resolved on a 10% polyacrylamide gel using SDS-PAGE
and subsequently transferred to PVDF membrane. The far Western protocol
was modified from that of Guichet et al. (43)
omitting the denaturation/renaturation steps. Briefly, after transfer,
membranes were washed twice in PBS and once in binding buffer followed
by overnight blocking in binding buffer/5% BSA. A solution composed of
binding buffer/5% BSA/1 mM DTT and
35S-labeled HDAC3 or HDAC4 prepared in TNT
reactions was then used to probe the membranes for 35 h. GST (50
µg) was added to the probe mix to block nonspecific binding.
Membranes were then washed once with binding buffer/5% BSA/1
mM DTT and five times with binding buffer. All
washing and probe steps were carried out at 4 C. The membranes were
dried and exposed to a PhosphoImager screen.
 |
ACKNOWLEDGMENTS
|
---|
The HDAC4 cDNA was kindly provided by S. L. Schreiber
(Department of Chemistry and Chemical Biology, Harvard University,
Cambridge, MA). We thank S.C. Tsai (Molecular Oncology Program, H. Lee
Moffit Cancer Center and Research Institute, University of South
Florida, Tampa, FL) for construction of the HDAC4 expression
vectors.
 |
FOOTNOTES
|
---|
This work was supported by grants from the NIH (DK-54733), the United
States Department of Agriculture (98-35200-6264), and the
American Cancer Society (RPG-99-237).
Abbreviations: CMV, cytomegalovirus; DBD, DNA binding domain;
FLAG-epitope, MDYKDDDDK; GST, glutathione-S-transferase;
HDAC, histone deacetylase; LBD, ligand binding domain; PVDF;
polyvinylidene difluoride; RA, retinoic acid; RARE, retinoic acid
response element; RLU, relative light unit; TSA, trichostatin A.
Received for publication October 19, 2000.
Accepted for publication May 8, 2001.
 |
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