Human Immunodeficiency Virus Type 1 Tat Binding Protein-1 Is a Transcriptional Coactivator Specific for TR
Takahiro Ishizuka,
Teturou Satoh,
Tsuyoshi Monden,
Nobuyuki Shibusawa,
Tetsu Hashida,
Masanobu Yamada and
Masatomo Mori
First Department of Internal Medicine, Gunma University School of
Medicine 339-15, Maebashi 371-8511, Japan
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ABSTRACT
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The DNA-binding domain of nuclear hormone receptors functions
as an interaction interface for other transcription factors. Using the
DNA-binding domain of TRß1 as bait in the yeast two-hybrid system, we
cloned the Tat binding protein-1 that was originally isolated as a
protein binding to the human immunodeficiency virus type 1 Tat
transactivator. Tat binding protein-1 has subsequently been identified
as a member of the ATPase family and a component of the 26S proteasome.
Tat binding protein-1 interacted with the DNA-binding domain but not
with the ligand binding domain of TR in vivo and
in vitro. TR bound to the amino-terminal portion of Tat
binding protein-1 that contains a leucine zipper-like structure. In
mammalian cells, Tat binding protein-1 potentiated the ligand-dependent
transactivation by TRß1 and TR
1 via thyroid hormone response
elements. Both the intact DNA-binding domain and activation function-2
of the TR were required for the transcriptional enhancement in the
presence of Tat binding protein-1. Tat binding protein-1 did not
augment the transactivation function of the RAR, RXR, PPAR
, or ER.
The intrinsic activation domain in Tat binding protein-1 resided within
the carboxyl-terminal conserved ATPase domain, and a mutation of a
putative ATP binding motif but not a helicase motif in the
carboxyl-terminal conserved ATPase domain abolished the activation
function. Tat binding protein-1 synergistically activated the
TR-mediated transcription with the steroid receptor coactivator 1,
p120, and cAMP response element-binding protein, although Tat binding
protein-1 did not directly interact with these coactivators in
vitro. In contrast, the N-terminal portion of Tat binding
protein-1 directly interacted in vitro and in
vivo with the TR-interacting protein 1 possessing an ATPase
activity that interacts with the activation function-2 of liganded TR.
Collectively, Tat binding protein-1 might function as a novel
DNA-binding domain-binding transcriptional coactivator specific for the
TR probably in cooperation with other activation function-2-interacting
cofactors such as TR-interacting protein 1.
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INTRODUCTION
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THE NUCLEAR HORMONE receptors (NRs)
are ligand-dependent transcription factors that profoundly
participate in multiple aspects of development, differentiation and
homeostasis in eukaryotes (1, 2, 3). NRs bind to the specific
cis-DNA sequences, so-called hormone response elements in
the vicinity of the promoter of target genes, and stimulate gene
transcription in a ligand-dependent manner (4, 5). NRs
possess a characteristic modular structure conserved through the large
superfamily of NRs that consists of the DNA-binding domain (DBD),
ligand-binding domain (LBD), hinge region between DBD and LBD, and two
separate transcription activation domains termed N-terminal activation
function-1 (AF-1) and C-terminal AF-2 (1, 2, 3). NRs have
recently been shown to function in concert with basal transcription
factors and multiple transcriptional cofactors including corepressors
and coactivators (6, 7, 8, 9). Different portions of NRs
directly interact with these distinct coregulators in ligand-dependent
and independent manners (6, 7, 8, 9). In the case of TR and RAR,
ligand binding causes a conformation change in the LBD allowing release
of corepressors from the hinge region and recruitment of coactivator
complexes to the AF-2 domain (10, 11, 12, 13, 14, 15). The coactivators
themselves and their associated cofactors possess histone
acetyltransferase activity, and targeted hyperacetylation of core
histones by these factors has been postulated to be a key step
responsible for hormone-dependent gene activation by NRs
(16, 17, 18).
The DBD of NRs appears to be mainly involved in DNA binding and
receptor dimerization (5). Recent cumulating evidence
indicates that this domain also serves as a direct interaction
interface for a variety of cellular factors including a tumor
suppressor, an oncoprotein, transcription factors, an RNA-binding
protein, viral proteins, and an enzyme involved in a signal
transduction pathway (19, 20, 21, 22, 23, 24, 25, 26). In some cases, NRs appear
to interfere with the function of these DBD-binding factors independent
of DNA binding (19, 22, 23, 24). Direct evidence that the
DBD-binding factor of NRs is involved in ligand-dependent
transactivation has been shown by Cheng et al.
(27) who reported that the hematopoietic bZIP protein,
p45/NF-E2 interacts with the DBD of TR and RAR and potentiates
ligand-dependent gene activation in cooperation with the cAMP response
element-binding protein-binding protein (CBP) . A novel protein termed
SNURF (small nuclear ring finger protein), which was cloned using the
DBD of AR as bait, can potentiate gene activation mediated by AR, GR,
and PR in a ligand-dependent manner, possibly by functioning as a
bridging factor between these NRs and the TATA-binding protein, a basal
transcription factor (28). A cold-inducible coactivator of
NRs, PGC-1, binds to the DBD and a part of the hinge region of PPAR
(29). The p300/CBP associated factor has a histone
acetylase activity and directly binds to the DBD of NRs to function as
a coactivator (30). Paradoxically, histone deacetylases,
HDAC1 and HDAC2, have also been found to bind the DBD of TR and might
participate in the negative regulation of the TSH ß subunit gene by
thyroid hormone (31). These findings suggest that the
DBD-interacting proteins might be classified as a novel class of
coregulators involved in transcriptional control by NRs.
To gain additional insights into the role of DBD-binding factors
of NRs for ligand-dependent transactivation, we cloned a protein that
binds to the DBD of human TRß1 using the yeast two-hybrid system. The
nucleotide sequence of the cloned cDNA was found to be identical to the
human immunodeficiency virus type 1 (HIV-1) Tat binding protein-1
(TBP-1) that was originally isolated as a protein interacting with the
HIV-1 Tat transactivator (32). TBP-1 potentiated the
TR-mediated, but not other NR-mediated, transactivations in mammalian
cells. Although TBP-1 itself possessed an intrinsic transactivation
function, augmentation of TR-mediated transactivation by TBP-1 required
the intact AF-2 of TR. TBP-1 directly interacted with the
TR-interacting protein 1 (Trip1), a putative transcriptional mediator
for TR that binds to the AF-2 of TR in a ligand-dependent manner
(33, 34). These findings demonstrated that TBP-1 interacts
not only with Tat but also with TR, and TBP-1 might function as a novel
DBD-binding coactivator specifically involved in the TR-mediated gene
activation.
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RESULTS
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Cloning of a Protein That Interacts with the DBD of TRß1
To isolate the proteins that interact with the DBD of TR, we
performed a yeast two-hybrid screen of a Hela cell cDNA library using
the DBD plus a part of the hinge region of human TRß1 as bait (Fig. 1A
). Sixteen positive clones were
obtained, and their partial nucleotide sequences were determined. A
clone containing the identical partial cDNA sequence was also isolated
from a human lung cDNA library. A computer search for data resources
suggested that the partial cDNA sequence isolated from two different
libraries was apparently identical to that of the human TBP-1 that was
previously cloned as a protein interacting with the HIV-1 Tat
transactivator (32). TBP-1 has been reported to be
predominantly localized in the nucleus (32). The reported
TBP-1 was encoded by 404 amino acids and was shown to possess a
putative nucleotide binding motif and an RNA/DNA helicase motif in the
C-terminal conserved ATPase domain (CAD), a characteristic structure of
an ATPase family (Fig. 1B
) (32, 35). It was also noted
that a heptad repeat of hydrophobic amino acids, reminiscent of a
leucine zipper, was located in the N-terminal region of TBP-1 (Fig. 1B
)
(32). A putative NR-box motif (L-X-X-L-L), an NR-binding
site identified in several coactivator proteins (36), was
not found in TBP-1. Importantly, TBP-1 shares significant homology with
Trip1/mouse SUG1 (mSUG1) and MB67-interacting protein (MIP) 224, other
members of the ATPase family that have been isolated as the factors
interacting with the LBD of TR/RAR and an orphan NR, MB67, respectively
(Fig. 1C
) (34, 37, 38). Further sequence analyses of the
cDNA clone isolated in our yeast dual-hybrid screen revealed that the
cDNA contained the full-open reading frame of the originally isolated
TBP-1 (32). Ohana et al. reported the cloning
of a longer version of TBP-1 cDNA that has two additional in-frame ATG
codons 35 and 19 amino acids upstream of the first ATG in the
originally isolated TBP-1 (39). The present clone lacked
the sequence corresponding to the first in-frame 16 amino acids of the
longer version of TBP-1. The third ATG codon was previously established
to be used as an initiation codon (32). Therefore, the
third and probably the second ATG codon might be used in our cloned
TBP-1 cDNA when translated in vitro. For convenience,
numbering of the amino acids of TBP-1 in this manuscript followed that
of the longer TBP-1 cDNA version (39)

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Figure 1. Cloning of a Protein Interacting with the DBD of
TRß1 Using the Yeast Two-Hybrid System
A, Schematic representation of the structure of hTRß1. The portion of
TRß1 used for the yeast two-hybrid screen is shown as bait. B,
Schematic representation of the structure of human TBP-1. The positions
and amino acid alignments of the putative leucine zipper-like
structure, nucleotide binding motif, and helicase motif are indicated.
Underlines indicate the heptad-repeat of hydrophobic
amino acids. The conserved ATPase domain (CAD) is shown as a
shaded box. C, Comparison of the amino acid sequences of
TBP-1, Trip1, and MIP224. The amino acid sequences of Trip1 and MIP224
that were cloned as proteins interacting with NRs (9 31 )
were compared with TBP-1 and are shown as percent sequence identity.
GenBank accession numbers of TBP-1, Trip1, and MIP224 are M34079,
L38810, and U27515, respectively.
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TBP-1 Interacts with the DBD but Not with the LBD of TR
To confirm the interaction between TBP-1 and TR, TRDBD plus
a part of the hinge region (TRD-H) ligated to GAL4DBD (pGBT9TRD-H) and
the full-length TBP-1 ligated to GAL4AD (pGAD10TBP-1) were
cotransformed into yeast containing the lacZ reporter gene
under control of the GAL4-binding site, and the protein-protein
interaction was analyzed by a ß-galactosidase assay. To determine
whether TBP-1 also binds to a domain other than TRDBD, the interaction
of TBP-1 with GAL4DBD separately fused to the DBD alone (pGBT9TRDBD),
and the remainder of the hinge region plus LBD (pGBT9TRLBD) of TRß1
was tested. As shown in Fig. 2
, TBP-1
interacted with TRDBD and TRD-H, but not with TRLBD. GAL4DBD
itself (pGBT9) did not interact with TBP-1 (Fig. 2A
), and GAL4TRDBD did
not show any ß-galactosidase activity in the presence of GAL4AD alone
(data not shown), indicating specificity of the interaction. TRLBD did
not interact with TBP-1 even in the presence of
T3 (Fig. 2A
), whereas the interaction of TRLBD
with Trip1 fused to GAL4AD was T3-dependent in
our yeast system in agreement with a previous study (34)
(data not shown). To next examine whether TBP-1 physically associates
with TR in mammalian cells, CV-1 cells were cotransfected with TR and
FLAG-tagged TBP-1 expression vectors. Protein complexes associated with
TBP-1 were first immunoprecipitated with the anti-FLAG monoclonal
antibody, and the bound proteins were subsequently analyzed by
immunoblotting with an anti-TR-specific antibody. As shown in Fig. 2B
, TR protein was detected in immunoprecipitates from cells transfected
with both TBP-1 and TR expression vectors, but not with the empty FLAG
vector and TR expression plasmid, confirming that TR and TBP-1 are
found as complexes in vivo.

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Figure 2. Interactions between TR and TBP-1 in
Vivo
A, Interactions of TRß1 polypeptides fused to GAL4DBD (pGBT9TR) with
the full-length TBP-1 ligated to GAL4AD (pGAD10TBP-1) in yeast were
measured by ß-galactosidase (ß-gal) activity. The interaction
between pGBT9TRLBD and pGAD10TBP-1 was measured in the absence and
presence of 100 nM T3. B, Nuclear extracts from
CV-1 cells transfected with pKCR2TRß1 (7.5 µg/dish) in the presence
of a vector encoding FLAG-tagged TBP-1 or an empty FLAG vector (7.5
µg/dish) were immunoprecipitated by M2 anti-FLAG monoclonal antibody.
The protein complexes were resolved by SDS-PAGE and transferred to
nitrocellulose membranes. Immunoblotting was performed using an
anti-TRß1 monoclonal antibody. An arrow indicates the
TRß1 protein, and an asterisk indicates a nonspecific
binding.
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Glutathione-S-Transferase (GST) Interaction
Assay
We next assessed the in vitro interaction of
TBP-1 with TR using a GST pull-down assay.
[35S]-labeled TRß1 proteins encoding the A/B
domain (TRA/B), A/B plus DBD (TRA/B-DBD), A/B to a part of the hinge
region (TRA/B-hinge), the remainder of the hinge region plus LBD
(TRLBD), and the full-length TRß1 were synthesized using in
vitro transcription/translation and were incubated with a GST
fused full-length TBP-1 (GST-TBP-1) immobilized to glutathione beads.
After extensive washing, TRA/B-DBD, TRA/B-hinge, and the full-length TR
were retained by GST-TBP-1, but not by GST itself (Fig. 3A
). In contrast, TRA/B and TRLBD did not
bind to GST-TBP-1. The interaction of the full-length TR with TBP-1 was
not T3 dependent as expected from the findings
obtained in the yeast experiment. The binding of TR to GST-TBP-1 was
comparable to that of a GST-fused receptor interaction domain of the
nuclear receptor corepressor (N-CoR) (Fig. 3B
). These findings
confirmed the interaction of TBP-1 with the DBD of TR in
vitro.

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Figure 3. Interactions between TR and TBP-1 in
Vitro
A, [35S]-labeled TRß1 polypeptides were synthesized
using in vitro transcription/translation, and their
interactions with GST alone or GST TBP-1 in the absence or presence of
100 nM T3 were analyzed using a pull-down
assay. The polypeptides encoding the A/B domain (A/B), A/B plus DBD
(A/B-DBD), and A/B to a part of the hinge region (A/B-hinge) of TR were
synthesized as fusion proteins with GAL4DBD for better resolution in a
15% polyacrylamide gel. Interaction of TR LBD and the full-length TR
was analyzed in a 10% gel. The positions of the molecular size markers
are indicated. "Input" represents 10% of input protein. The
percent bound protein is shown below each lane. B,
Binding of [35S]-labeled full-length TR to equivalent
amounts of GST TBP-1 and the GST-fused receptor interaction domain of
N-CoR was compared using a pull-down assay in the presence or absence
of 100 nM T3 (left panel).
SDS-PAGE of GSTTBP-1 and GST-fused receptor interaction domain of N-CoR
is shown in the right panel.
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Mapping the TR-Interacting Domain in TBP-1
To localize the domain necessary for interaction with TR in
TBP-1, GST-fusion proteins of the N-terminal (residues 17176) and
C-terminal portions (residues 176439) of TBP-1 were synthesized, and
their interaction with [35S]-labeled
full-length TRß1 was examined using a GST pull-down assay. As
shown in Fig. 4A
, TR bound to the
N-terminal portion of TBP-1 as well as to the full-length TBP-1. In
contrast, no apparent TR binding to the C-terminal portion of TBP-1 was
observed. T3 again did not affect the interaction
between TR and TBP-1. The interaction of the N-terminal region, but not
the C-terminal portion, of TBP-1 with TR was also confirmed using the
yeast two hybrid-system (Fig. 4B
). These findings indicated that the
N-terminal portion of TBP-1 containing a leucine zipper-like structure
serves as the interaction interface for TRDBD.

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Figure 4. The N-Terminal Portion of TBP-1 Interacts with TR
in Vitro and in Vivo
A, Interactions between [35S]-labeled full-length TR
vis-a-vis GST alone or N-terminal (GST TBP-1N), C-terminal (GST
TBP-1C), and full-length TBP (GST TBP-1) were analyzed using a
pull-down assay in the presence or absence of 100 nM
T3. B, Interactions of the TRDBD plus a part of the hinge
region fused to GAL4DBD (pGBT9TR) or GAL4DBD alone (pGBT9) with TBP-1
polypeptides expressed from pGAD10TBP-1, TBP-1N, and TBP-1C were
measured by ß-gal assay in the yeast two-hybrid system.
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TBP-1 Potentiated the Ligand-Dependent Transactivation by TRß1 in
Mammalian Cells
We next analyzed whether TBP-1 affects the ligand-dependent
transactivation function of the TR in mammalian cells using a transient
transfection assay. In TR-deficient CV-1 cells, the activity of
promoter carrying the palindromic thyroid hormone response
element (TRE) was stimulated by the cotransfected hTRß1 in the
presence of 10 nM of T3. The
cotransfection of TBP-1 augmented the ligand-dependent stimulation of
the promoter activity by TR in a dose-dependent manner (Fig. 5A
). A similar ligand-dependent
augmentation by TBP-1 was observed with a reporter construct carrying
another TRE, DR4, that is direct repeat-type TRE with a spacer of four
nucleotides (Fig. 5B
). In contrast, TBP-1 did not potentiate the
activities of thymidine kinase (TK), Simian virus 40 early and Rous
sarcoma virus (RSV) long terminal repeat (LTR) promoters that lack the
positive TRE in the presence of liganded TR (data not shown). The
transcriptional enhancement by TBP-1 of the palindromic TRE was not
observed when a TR with a mutation in the first zinc finger motif in
DBD (C127S) was cotransfected (Fig. 5C
). Moreover, TBP-1 lacking the
N-terminal leucine zipper-like structure did not potentiate
transcription in the presence of wild-type TR in transient transfection
assays (Fig. 5D
). These findings collectively suggest that TBP-1
potentiated the ligand-dependent transactivation of TR in mammalian
cells, and that TBP-1 functioned in concert with TR that bound to
DNA.

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Figure 5. TBP-1 Potentiates Ligand-Dependent Transcriptional
Activation by TR
A, pKCR2TRß1 (83 ng/well) was cotransfected into CV-1 cells with the
palindromic TRE reporter gene (PAL) (1.7 µg/well) in the absence or
presence of increasing amounts of pKCR2TBP-1 (170, 500, and 830
ng/well). Luciferase activity was measured after 48 h incubation
with or without 10 nM T3. Values represent the
mean ± SE from triplicate determinants. B, pKCR2TRß1 (83
ng/µl) was cotransfected with the DR4 TRE reporter gene (1.7
µg/well) in the presence of pKCR2 or pKCR2TBP-1 (830 ng/well). C, The
wild-type or a mutant TRß1 possessing a substitutive mutation in DBD
(C127S) (83 ng/well) was cotransfected with PAL (1.7 µg/well) in the
absence or presence of pKCR2TBP-1 (830 ng/well). D, The wild-type or
mutant TBP-1 lacking the N-terminal leucine zipper-like structure (830
ng/well) was cotransfected with PAL (1.7 µg/well) in the presence of
pKCR2TRß1 (83 ng/well).
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TBP-1 Selectively Enhances the TR-Mediated Transactivation
TR is known to be encoded by two different genes, c-erb
A
and ß (40, 41). To clarify whether TBP-1 functions
in a TR isoform-specific manner, hTR
1 was cotransfected with TBP-1
into CV-1 cells in the presence of the palindromic TRE reporter gene.
TBP-1 could potentiate transactivation by TR
1 in the presence of
T3 similar to TRß1 (Fig. 6A
). Identical findings were obtained
using the DR4 reporter gene (data not shown). We next examined whether
TBP-1 can potentiate transactivation by other members of NRs in CV-1
cells. As shown in Fig. 6B
, the cotransfected TBP-1 did not
significantly enhance activation of the promoters containing cognate
hormone response elements by RAR, RXR, PPAR
, and ER in the presence
of individual specific ligands. The direct interaction of TBP-1 with
RXR and RAR, known heterodimer partners for TR (5, 42),
was also evaluated using the yeast two-hybrid system. As shown in Fig. 6C
, TBP-1 interacted neither with the full-length RXR in the absence
and presence of 9-cis-retinoic acid (9-cis-RA)
nor the DBD plus the hinge region of RAR. Moreover,
[35S]-labeled full-length RAR, RXR, PPAR, and
ER did not bind to GST-TBP-1 in in vitro pull-down assays
(Fig. 6D
). These findings indicated that TBP-1 selectively potentiates
the transactivation function of TR.

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Figure 6. TBP-1 Specifically Potentiates TR-Mediated
Transactivation
A, The TR 1 or TRß1 expression vector (83 ng/well) was
cotransfected with PAL (1.7 µg/well) in the absence or presence of
pKCR2TBP-1 (830 ng/well). B, Expression vectors for hRAR , RXR ,
PPAR , or ER (83 ng/well) was transfected with the reporter vector
containing retinoic acid response element (DR5), retinoid X response
element (DR1), peroxisome proliferator-activated response
element (DR1) or estrogen response element (1.7 µg/well),
respectively, in the absence or presence of pKCR2TBP-1 (830 ng/well).
One micromolar all-trans-RA (atRA), 1 µM
9-cis-RA, 10 µM troglitazone
(Tz), or 10 nM E2 was used as a specific ligand for
RAR, RXR, PPAR, or ER, respectively. C, Two hybrid interactions of
pGBT9RAR (DBD plus a part of the hinge region) and pGBT9RXR with
pGAD10TBP-1 in yeast were measured using the ß-gal assay. One
micromolar 9-cis-RA was used as a ligand for RXR.
pGBT9TRDBD was used as a positive control. D, Interactions between
[35S]-labeled full-length RAR, RXR, PPAR, and ER
vis-a-vis GST alone or full-length TBP (GST TBP-1) were analyzed using
a pull-down assay in the presence or absence of cognate ligands.
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TBP-1 Possesses an Autonomous Activation Function
To investigate whether TBP-1 possesses an intrinsic
activation function, the N-terminal and C-terminal portions of TBP-1
were ligated to GAL4DBD and cotransfected with the upstream activating
sequence (UAS)-TK reporter gene into CV-1 cells. As shown in Fig. 7
, GAL4VP16 significantly activated the
UAS-TK promoter activity. GAL4TBP-1N did not stimulate the promoter
activity over that of GAL4DBD itself. In contrast, GAL4TBP-1C
significantly stimulated the promoter activity. Identical findings were
obtained using Hela cells (data not shown). These findings indicated
that TBP-1 might possess an intrinsic activation function in the
C-terminal CAD region.

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Figure 7. TBP-1 Possesses an Intrinsic Activation Potential
The N-terminal (residues 7176) or C-terminal portion (residues
176439) of TBP-1 ligated to GAL4DBD or GAL4DBD alone (830 ng/well)
was cotransfected with the UAS-TK reporter gene (1.7 µg/well) into
CV-1 cells. GAL4VP16 (83 ng/well) was used as a positive control.
Luciferase activity was measured as described in Materials and
Methods.
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Mutation of the Potential ATP-Binding Motif in the CAD Abolished
Augmentation of the TR-Mediated Transcriptional Activation by TBP-1
Ohana et al. (39) reported that mutation
or deletion of the putative ATP-binding motif and helicase motif in the
CAD region abolished the intrinsic activation potential of TBP-1.
We, therefore, evaluated whether these motifs in the CAD are necessary
for enhancement of the TR-mediated transactivation by TBP-1 using a
transient transfection assay. As shown in Fig. 8A
, a mutation introduced into the
putative ATP-binding site (K233H) abolished augmentation of the
palindromic reporter activity. In contrast, TBP-1 with a mutation
within the putative helicase motif (D286A) showed significant
enhancement similar to the wild-type TBP-1. These mutant TBP-1s were
similarly bound to TR in a GST pull-down assay (Fig. 8B
). These
findings clearly revealed that the potential nucleotide binding motif,
but not the helicase motif in CAD, was required for augmentation by
TBP-1 of the TR-mediated gene activation.

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Figure 8. Mutation of a Putative ATP-Binding Motif in CAD
Abolished Enhancement by TBP-1 of TR-Mediated Transactivation
A, pKCR2TRß1 (83 ng/well) was cotransfected into CV-1 cells with PAL
(1.7 µg/well) in the absence or presence of the wild-type or mutant
TBP-1 expression vector (830 ng/well) possessing a substitutive
mutation in the ATP-binding motif (K233H) or the helicase motif
(D286A). B, GST-fused wild-type and mutant TBP-1 proteins similarly
bound to [35S]-labeled full-length TRß1 in a pull-down
assay.
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TBP-1 Requires the AF-2 Domain of TR for Transcriptional
Enhancement
In addition to its intrinsic activation potential, it was unclear
whether TBP-1 requires interaction with other transcriptional
regulators to augment ligand-dependent transactivation by TR. To assess
this possibility, we first examined whether TBP-1 could enhance the
transactivation function of an AF-2 mutant TR (E457A). E457A has been
shown to bind to TRE and possesses an affinity for
T3 similar to the wild-type receptor, but lacks
the ligand-dependent transactivation function because of its impaired
ability for interacting with coactivators (43). As shown
in Fig. 9
, TBP-1 did not potentiate the
promoter activity carrying the palindromic TRE in the presence of
liganded E457A, although TBP-1 could directly bind to E457A in a
pull-down assay. This indicated that the potentiation by TBP-1 of
transactivation by TR might be dependent on the AF-2 domain of TR.

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Figure 9. TBP-1 Requires the AF-2 of TR for Potentiation of
T3-Dependent Gene Activation
The wild-type or an AF-2 mutant TRß1 (E457A) expression vector (83
ng/well) was cotransfected into CV-1 cells with PAL (1.7 µg/well) in
the absence or presence of pKCR2TBP-1 (830 ng/well) (left
panel). Interaction between [35S]-labeled mutant
TR (E457A) and GST-fused TBP-1 was analyzed using a GST pull-down assay
(right panel).
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TBP-1 and Coactivators Synergistically Stimulate TR-Mediated
Transactivation
The AF-2 of TR has recently been demonstrated to directly interact
with multiple protein complexes containing the coactivators and
cointegrators (6, 7, 8, 9). We, therefore, examined whether
TBP-1 influences the enhancement of TR-mediated transactivation by
coactivators using a transient transfection assay. As shown in Fig. 10A
, cotransfection of three
coactivators of TR, steroid receptor coactivator 1 (SRC-1), p120, and
CBP enhanced the ligand-dependent stimulation by TR of the palindromic
TRE in a magnitude similar to the cotransfected TBP-1. When TBP-1 was
simultaneously transfected with these coactivators, the promoter
activity in the presence of liganded TR was strongly augmented (Fig. 10A
). We next examined whether TBP-1 can directly interact with these
coactivators using the in vitro pull-down assay. As shown in
Fig. 10B
, GST-TBP-1 did not interact with in vitro
translated SRC-1, p120, and CBP. These findings suggest that TBP-1
could synergistically function with the AF-2-interacting coactivators
in the absence of direct interaction.

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Figure 10. TBP-1 Synergistically Stimulates TR-Mediated
Transactivation with Coactivators in the Absence of Direct Interaction
A, pKCR2TRß1 (83 ng/well) was cotransfected into CV-1 cells with PAL
(1.7 µg/well) in the absence or presence of TBP-1, SRC-1, p120, or
CBP expression vector (830 ng/well). B, GST TBP-1 did not bind
in vitro translated SRC-1, p120, and CBP in a pull-down
assay.
|
|
TBP-1 Directly Interacts with Trip1 in Vivo and
in Vitro
TBP-1 has been shown to be able to form a homodimer with TBP-1
itself and to weakly form a heterodimer with TBP7, a putative ATPase
highly homologous to MIP224 (38), possibly through its
N-terminal leucine zipper-like motif (39). These findings
suggested that the homo- or heterodimer formation of TBP-1 might be
important for its function and led us to evaluate the interaction
between TBP-1 and Trip1, a member of the ATPase family that binds to
the AF-2 of TR in a ligand-dependent manner (34). As shown
in Fig. 11A
, [35S]-labeled Trip1 bound to GST-TBP-1, but not
to GST itself. Moreover, Trip1 interacted with the N-terminal, but not
the C-terminal, region of TBP-1 (Fig. 11A
). Synthesis of TBP-1 proteins
of appropriate molecular weights were confirmed by SDS-PAGE analyses
(Fig. 11B
). In agreement with the previous findings (39),
[35S]-labeled TBP-1, in addition to Trip1,
bound to GST-TBP-1 in a pull-down assay (data not shown). The
interaction of the N-terminal, but not the C-terminal, region of TBP-1
with Trip1 was also confirmed using the yeast two-hybrid system (Fig. 11C
). We next examined the effect of transfection of Trip1 on
TR-mediated transcription in the presence of cotransfected TBP-1 using
a transient transfection into CV-1 cells. Transfection of Trip1 did not
enhance the palindromic TRE-driven promoter activity in the presence of
cotransfected TR in agreement with previous findings (34, 37). In contrast, cotransfection of both TBP-1 and Trip1 further
augmented the promoter activity in the presence of
T3 when compared with that in the presence of
TBP-1 alone (Fig. 11D
). These in vitro and in
vivo findings suggest that TBP-1 binding to the DBD of TR might
cooperate with Trip1, which interacts with the AF-2 via a
protein-protein interaction.

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Figure 11. TBP-1 Interacts with Trip1 in Vivo
and in Vitro
A, Interactions of the GST-fused full-length, N-terminal, and
C-terminal portions of TBP-1 with [35S]-labeled
full-length Trip1 were examined using a pull-down assay. B, SDS-PAGE
analysis of the GST-fused TBP-1 proteins used in panel A. C,
Interactions of the N-terminal and C-terminal portions of TBP-1 in
pGBT9 with pGAD10Trip1 and pGAD10 were examined using the yeast
two-hybrid system. ß-gal assay was performed as describe above. D,
Increasing amounts of pKCR2TBP-1 (170, 500, and 830 ng/well) were
transfected with pKCR2TRß1 (17 ng/well) into CV-1 cells in the
absence or presence of pKCR2Trip1 (170 ng/well). Luciferase activities
of PAL in the absence or presence of T3 were measured and
were expressed as light units/µg protein.
|
|
Overexpression of TBP-1 Did Not Alter TR Protein Levels
TBP-1 has previously been identified as a component of the
19S regulatory subunit of the 26S proteasome that degrades
ubiquitinated proteins (35, 44, 45). Recent studies
demonstrated that several NRs including ER, vitamin D receptor (VDR),
and promyelocytic leukemia-RAR fusion protein were degraded via the
proteasome system (46, 47, 48). It was, therefore, anticipated
that overexpression of the single component of the proteasome complex
in transient transfection assays might disrupt the normal processing
function of the protein degradation system, thereby increasing the
available TR proteins, resulting in potentiation of the promoter
activity. To clarify whether levels of the TR protein are affected by
overexpression of TBP-1, we quantitated the TRß1 protein levels in
the absence or presence of cotransfected TBP-1 using Western blot
analysis. As shown in Fig. 12
, the
cellular levels of TRß1 protein were not altered in the absence or
presence of TBP-1 transfected in conditions identical to that used in
transient transfection experiments. These findings suggest that TBP-1
might not potentiate the TR-mediated gene activation by increasing
TR protein levels.

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|
Figure 12. Overexpression of TBP-1 Did Not Alter the Levels
of TR Proteins in CV-1
Cells CV-1 cells in 100-mm2 dishes were transfected
with pKCR2TRß1 (5 µg/dish) and TBP-1 (10 µg/dish) using a calcium
phosphate precipitation method, and nuclear extracts were prepared
after incubation for 48 h with 10 nM T3 or
vehicle alone. Forty micrograms of nuclear proteins were resolved by
SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with an
anti-TRß1 monoclonal antibody. Levels of Tß1 protein were
visualized as described in Materials and Methods.
|
|
 |
DISCUSSION
|
---|
TBP-1 was originally isolated as a protein interacting with the
HIV-1 Tat transactivator and was reported to inhibit the TAT-mediated
activation of the HIV-1 LTR promoter (32). Proteins highly
homologous to the human TBP-1 have subsequently been cloned from
different species indicating that TBP-1 might have some evolutionary
conserved functions (49, 50, 51, 52). The TBP-1 gene is mapped to
human chromosome 11p (53, 54) and is ubiquitously
expressed in a variety of human cell lines and in rodent tissues
(32, 55, 56). However, the physiological importance of
TBP-1 in the regulation of the transcription of specific cellular genes
remains to be elucidated. The present study, therefore, provides the
first evidence that TBP-1 is involved in transcriptional activation of
the thyroid hormoneresponsive gene.
TBP-1 belongs to a protein family possessing the CAD named AAA family
(ATPases associated with a variety of cellular activities)
(35), and the CAD region in TBP-1 shares significant
homology with those of Trip1, mSUG1, and other members of the ATPase
family (34, 35, 37). When compared with Trip1/mSUG1,
several unique features of TBP-1 as a transcriptional cofactor for TR
were observed in the present study. First, TBP-1 constitutively bound
to TR in the presence and absence of T3, whereas
Trip1/mSUG1 binds to the receptors in a ligand-dependent manner
(34, 37). Second, TBP-1 interacted with the DBD of TR,
whereas Trip1/mSUG1 interacts with the LBD of TR (34, 37).
Third, overexpression of TBP-1 strongly potentiated the
ligand-dependent transactivation by TR in mammalian cells. In contrast,
cotransfected Trip1 did not enhance the TR-mediated transactivation in
transient transfection assays (Fig. 11D
and Ref. 34).
Lastly, TBP-1 appeared to selectively function with TR, whereas
Trip1/mSUG1 has been shown to interact not only with TR, but also with
RAR, RXR, VDR, and ER (34, 37, 57).
In addition, TBP-1 has a characteristic function that requires the AF-2
domain of TR to potentiate the TR-mediated transcription. The AF-2 of
NRs was recently shown to interact with multiprotein complexes
containing coactivators and cointegrators in the presence of ligand
(6, 7, 8, 9). In the present study, we showed that TBP-1 was
able to directly interact and synergistically function with Trip1, one
of the AF-2 interacting cofactors that bind to TR in a ligand-dependent
manner. Moreover, the present transfection study revealed that TBP-1
could synergistically enhance the TR-mediated transactivation with a
subset of coactivators such as SRC-1, p120, and CBP that interact with
the AF-2 of TR, although TBP-1 did not interact with these coactivators
in vitro. These findings collectively suggest that TBP-1
might function in cooperation with other transcriptional cofactors
interacting with the AF-2 of TR, such as Trip1, to augment the
ligand-dependent gene activation.
In agreement with a previous finding reported by Ohana et
al. (39), we could detect an intrinsic activation
function of TBP-1 using a mammalian one-hybrid assay and showed that
the activation domain resided within the C-terminal CAD region. TBP-1
lacked the consensus L-x-x-L-L sequence, a characteristic NR-binding
site in many coactivators that possess intrinsic histone
acetyltransferase activity (36). Mutation of the potential
ATP-binding motif, but not the helicase motif, abolished the
enhancement of TR-mediated transactivation by TBP-1 in the present
study. The recombinant rat SUG1 has been established to possess an
RNA-dependent ATPase activity (58). Moreover, mSUG1 has
been shown to have a bona fide helicase activity (59) and
interacts with the p89/XPB (xeroderma pigmentosum B) subunit of TFIIH
(60). The DEAD (the predicted ATPase B motif)-box proteins
such as the eukaryotic translation initiation factor 4A and p68 exhibit
an RNA-unwinding activity as well as RNA-stimulated ATPase activity
(60, 61). Endoh et al. (62)
recently reported that p68 stimulates the ER-mediated gene activation
via a protein-protein interaction with the AF-1 of ER and showed that
the intrinsic helicase activity was dispensable for the coactivator
function of p68. In conjunction with these findings, the present study
indicates that the putative ATPase activity of TBP-1 is responsible for
enhancement of the ligand-dependent gene activation by TR.
TBP-1 and Trip1/mSUG1 are the integral components of the 19S regulatory
subunit of the 26S proteasome that degrades the ubiquitinated and
nonubiquitinated proteins contributing to a variety of cellular
regulatory processes (35, 44, 45). It was, therefore,
anticipated that ectopic expression of TBP-1 might alter the levels of
TRß1 protein, thereby affecting the TR-mediated transactivation.
However, TBP-1 selectively enhanced the TR-mediated, but not other
NR-mediated, activation in the present transfection studies. Moreover,
TBP-1 overexpressed in a condition identical to that used in
transfection studies did not alter the steady-state levels of TRß1
protein. Makino et al. recently reported that the complex of
proteasomal ATPases could directly interact with the TATA-binding
protein, and that TBP-1 was present in the complex as well as mSUG1,
SUG2, TBP7, MSS1, S4, and a novel transcription regulatory factor, TIP
(TBP-interacting protein)120 (63). These findings taken
together suggest that the proteasomal function of TBP-1 was likely to
be dispensable for augmentation of the TR-mediated gene transcription.
TBP-1, like other proteasomal ATPases, might be a multifunctional
protein directly involved in the regulation of gene transcription as
well as in protein degradation.
The HIV-1 Tat is a powerful activator of viral gene expression
from the integrated LTR (64). TR has previously been
demonstrated to directly interact with Tat through its DBD and has been
shown to stimulate activity of the HIV-1 LTR promoter in the presence
of T3 (22). HIV-1 Tat has recently
been shown to directly recruit the histone acetyltransferases p300/CBP
and p300/CBP associated factor to the viral RNA (65).
These findings suggested that the Tat/liganded TR complex on the HIV-1
LTR promoter might facilitate recruitment of the host transcriptional
coregulators to activate the viral gene expression in vivo.
As mentioned above, TBP-1 inhibits activation of the Tat-mediated HIV-1
LTR promoter in vivo (32). Taken together with
the present findings, TBP-1 might compete for binding of Tat to the DBD
of TR, thereby, in part, suppressing the Tat-mediated activation of the
HIV-1 promoter. Further studies are required to ascertain this
possibility.
 |
MATERIALS AND METHODS
|
---|
Plasmids
Expression vectors for human (h)TRß1, TR
1, RAR
, RXR
,
PPAR
, and ER in a pKCR2 expression vector were described previously
(43, 66, 67, 68). Two mutant TRs (C127S and E457A) in pKCR2
were described previously (43). Firefly luciferase
reporter plasmids (pA3Luc) carrying the palindromic or DR4 TRE were
previously described (43). Luciferase reporter vectors
containing the direct repeat-type (DR5) retinoic acid response element
or direct repeat-type (DR1) retinoid X response element/peroxisome
proliferator-activated response element upstream of the TK gene
promoter were described previously (43, 68). A reporter
plasmid carrying the estrogen response element was previously described
(43). pA3Luc driven by the TK gene promoter (TK109) and
RSV long terminal repeat (RSV400) were described previously (67, 69). The Simian virus 40 early promoter was excised from
pGL3-promoter vector (Promega Corp., Madison, WI) using
HindIII and SmaI and inserted into the
HindIII/SmaI cloning site in the promoter-less
pSV0Luc (69). An expression vector for TBP-1 was
constructed by ligating the cloned TBP-1 cDNA into pKCR2 (pKCR2TBP-1).
Mutant TBP-1 expression vectors (K233H and D286A) (39)
were created using a sequential PCR method as described previously
(70). A mutant TBP-1 cDNA that lacked the N-terminal
leucine zipper-like structure (residues 69439) was amplified by PCR
and subcloned into the pKCR2 expression vector (pKCR2delta N-TBP-1).
Expression vectors for SRC-1, p120, Trip1, and CBP in pKCR2 were
described previously (43). The cDNA fragments encoding A/B
domain (residues 1106), A/B domain plus DBD (residues 1180), and
from A/B domain to a part of the hinge region (residues 1205) of
TRß1 were amplified by PCR using the peA101 plasmid (40)
as the template and ligated into pKCR2GAL4DBD (43) to
create pKCR2TRA/B, A/B-DBD, and A/B-hinge, respectively. The
PCR-amplified cDNA fragment encoding DBD (residues 106- 180) of TRß1
was inserted into pGAD10 (CLONTECH Laboratories, Inc.,
Palo Alto, CA) (pGAD10TRDBD). A cDNA fragment encompassing a part
of the hinge and LBD of TRß1 (residues 204 to 461) was amplified by
PCR and inserted to pKCR2 (pKCR2TRLBD) and pGAD10 (pGAD10TRLBD).
pGBT9TBP-1, pGBT9TBP-1N, and pGBT9TBP-1C were constructed by inserting
the full-length and the PCR-amplified N-terminal (residues 17176) and
C-terminal (residues 176439) portions of TBP-1 cDNA, respectively,
into pGBT9 (CLONTECH Laboratories, Inc.). pGBT9RARDBD and
pGBT9RXR were constructed by ligating a PCR-amplified cDNA fragment
encoding the DBD plus a part of the hinge region of hRAR
(residues
85175) and the full-length hRXR
into pGBT9. pGAD10TBP-1 and
pGAD10Trip1 were created by ligating the full-length TBP-1 and Trip1
cDNA (43), respectively, into pGAD10.
Yeast Two-Hybrid System
A yeast two-hybrid screen of Hela cell and human lung MATCHMAKER
cDNA libraries (CLONTECH Laboratories, Inc.) was performed
to identify the clones that interacted with TRDBD. A cDNA fragment
encoding the DBD and a part of the hinge region of hTRß1 (residues
106205) was amplified by PCR, ligated in-frame into the
EcoRI site of pGBT9, and used as bait. The library, with
randomly primed size-selected cDNA fragments in pGAD10, used the GAL4
activation domain (GAL4AD) as a transcriptional activator. The yeast
strain HF7c (MATa, ura352, his3200, lys2801, ade2101,
trp1901, leu23, 112, gal4542, gal80538, LYS2 : : GAL1-HIS3,
URA3 : : (GAL4 17-mers)3-CYC1-LacZ) was used for cotransformation.
Transformants were plated onto synthetic complete media plates lacking
histidine, leucine, and tryptophan. Approximately 1 x
106 transformants were screened for interaction
with TRDBD. After 34 d, viable colonies were dissolved in 40 µl of
Z buffer (0.06 M
Na2HPO4, 0.04
M
NaH2PO4, 1
mM MgSO4, 10
mM KCl). After two rounds of freezing and
thawing, ß-galactosidase activity was measured by a chemiluminescent
assay (Tropix, Bedford, MA). Identification of positive clones was
performed by culturing His+, LacZ+ transformant yeast colonies in 3 ml
of liquid synthetic media (Trp+, Leu). Isolation of yeast DNA was
performed as described previously (43). The resulting
plasmid DNA was transformed by electroporation into HB101
Escherichia coli containing a leuB mutation to allow for
selection of the pGAD10 plasmid.
Sequence Analysis
The plasmids isolated from positive colonies were amplified on a
large scale, and the nucleotide sequences of both strands were
determined using an autosequencer (PRISMTM 310, PE Applied Biosystems, Tokyo, Japan). All the PCR-amplified cDNA
fragments used for plasmid construction were verified by direct
sequencing.
GST Pull-Down Assay
[35S] methionine-labeled full-length and
partial proteins of hTRß1 were synthesized by in vitro
transcription/translation from pKCR2TRA/B, TRA/B-DBD, TRA/B-hinge,
TRLBD, and the full-length TR using T7 RNA polymerase and a TNT-coupled
reticulocyte lysate system (Promega Corp.).
[35S]-labeled full-length RAR, RXR, PPAR
,
and ER proteins were synthesized in vitro from the pKCR2
expression vectors. Synthesis of proteins of expected molecular weights
was confirmed by SDS-PAGE analyses. The full-length and PCR-amplified
partial fragments of TBP-1 cDNA were ligated in-frame into pGEX4T1
(Pharmacia Biotech, Piscataway, NJ) to yield GST fusion
proteins in E. coli DH5
. A cDNA fragment encoding the
receptor interaction domain of N-CoR was amplified by PCR using
pKCR2N-CoRI (43) as a template and subcloned in flame into
pGEX4T1. The GST fusion proteins were purified on glutathione-agarose
beads (Sigma, St. Louis, MO) and analyzed by SDS-PAGE.
Interaction assays and autoradiographies were performed as described
previously (43). Bound protein was quantified by Molecular
Imager FX (Bio-Rad Laboratories, Inc., Hercules, CA).
Cell Culture, Transfection, and Luciferase Assay
CV-1 and Hela cells were grown in DMEM supplemented with 10%
FBS, as described previously (67, 68). Twenty-four hours
before transfection, cells were split into six-well plates in
subconfluency. Transient transfection was performed using a calcium
phosphate precipitation method, as described previously (67, 68). The total amounts of transfected plasmids were adjusted by
adding an empty expression vector in all experiments. Luciferase assay
was performed and the luciferase activity was normalized by the protein
concentration, as described previously (67, 68). All the
transfection experiments were repeated at least twice with triplicate
determinants. T3, all-trans-RA,
9-cis-RA, and E2 were purchased from Sigma.
Troglitazone was from Sankyo Co., Ltd.
(Tokyo, Japan).
Immunoprecipitation and Immunoblotting
The full-length TBP-1 cDNA was ligated in flame into a
pFLAG-CMV-2 expression vector (Sigma) and transfected into
CV-1 cells cultured in 100 mm2 dishes in the
presence of pKCR2TRß1 using a calcium phosphate precipitation method.
Forty-eight hours after transfection, nuclear extracts were prepared as
previously described (70). After immunoprecipitation with
an anti-FLAG M2 monoclonal antibody (Sigma), the samples
were resolved on 10% polyacrylamide gels under denaturing conditions
and transferred onto nitrocellulose membranes (Hybond ECL,
Amersham Pharmacia Biotech, Arlington Heights, IL). After
incubation in a blocking buffer [20 mM Tris-HCl (pH 7.6),
137 mM NaCl and 5% skimmed milk] for 1 h at room
temperature, the membranes were incubated overnight at 4 C with an
anti-TR antibody (J52), which recognizes the human TRß1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After extensive
washing, the membranes were incubated with a sheep antimouse Ig
antibody, and TR proteins were visualized with the ECL Plus Western
blotting detection kit (Amersham Pharmacia Biotech).
Mammalian One-Hybrid Assay
The cDNAs corresponding to the N-terminal (residues 17176) and
C-terminal portions (residues 176439) of TBP-1 were amplified by PCR,
ligated in frame into the EcoRI site of pMGAL4DBD
(CLONTECH Laboratories, Inc.), and transfected with UAS-TK
luciferase reporter (43) into CV-1 and Hela cells as
described above. The herpes simplex virus VP16 ligated to GAL4DBD
(GAL4VP16) (CLONTECH Laboratories, Inc.) was used as a
positive control. Luciferase activities were measured as described
above.
Statistical Analysis
Statistical analyses were performed using Duncans multiple
range test among multiple groups. The level of significance was set at
P < 0.05.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Ronald M. Evans (Howard Hughes Medical Center, The
Salk Institute of Biological Studies, La Jolla, CA) and Dr. William M.
Wood (University of Colorado Health Science Center, Denver, CO) for
providing materials.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Teturou Satoh, M.D., Ph.D., First Department of Internal Medicine, Gunma University School of Medicine, 339-15 Showa-machi, Maebashi 371-8511 Japan. E-mail:
tsato{at}showa.gunma-u.ac.jp
This work was supported by grants-in-aid for scientific research from
the Ministry of Education, Science, Sports and Culture of Japan (to
M.M. and to T.S.) and from the Ministry of Health and Welfare (to
M.Y.).
Abbreviations: CAD, conserved ATPase domain; CBP,
cAMP-response element-binding protein; 9-cis-RA,
9-cis-retinoic acid; DBD, DNA binding domain; GST,
glutathione-S-transferase; HIV, human immunodeficiency
virus; LBD, ligand binding domain; LTR, long terminal repeat; MIP,
MB67-interacting protein; N-CoR, nuclear receptor corepressor; NR,
nuclear receptor; RSV, rous sarcoma virus; SRC-1, steroid receptor
coactivator-1; TBP-1, Tat-binding protein-1; TK, thymidine kinase; TRE,
thyroid hormone response element; Trip1, TR-interacting protein 1; UAS,
upstream activating sequence.
Received for publication October 3, 2000.
Accepted for publication March 22, 2001.
 |
REFERENCES
|
---|
-
Lazar MA 1993 Thyroid hormone receptors: multiple forms,
multiple possibilities. Endocr Rev 14:184193[Medline]
-
Ribeiro RC, Kushner PJ, Baxter JD 1995 The nuclear hormone
receptor gene superfamily. Annu Rev Med 46:443453[CrossRef][Medline]
-
Perimann T, Evans RM 1997 Nuclear receptors in Sicily: all in
the famiglia. Cell 90:391397[Medline]
-
Freedman LP, Luisi BF 1993 On the mechanism of DNA binding by
nuclear hormone receptors: a structural and functional perspective.
J Cell Biochem 51:140150[Medline]
-
Glass CK 1994 Differential recognition of target genes by
nuclear receptor monomers, dimers, and heterodimers. Endocr Rev 15:391407[Medline]
-
Beato M, Sanchez-Pacheco A 1996 Interaction of steroid
hormone receptors with the transcription initiation complex. Endocr Rev 17:587609[Medline]
-
Ayer DE 1999 Histone deacetylases: transcriptional repression
with SINers and NuRDs. Trends Cell Biol 9:193198[CrossRef][Medline]
-
McKenna NJ, Lanz RB, OMalley BW 1999 Nuclear receptor
coregulators: cellular and molecular biology. Endocr Rev 20:321344[Abstract/Free Full Text]
-
Glass CK, Rosenfeld MG 2000 The coregulator exchange in
transcriptional functions of nuclear receptors. Genes Dev 14:121141[Free Full Text]
-
Chen JD, Evans RM 1995 A transcriptional co-repressor that
interacts with nuclear hormone receptors. Nature 377:454457[CrossRef][Medline]
-
Horlein AJ, Naar AM, Heinzel T, et al. 1995 Ligand-independent
repression by the thyroid hormone receptor mediated by a nuclear
receptor co-repressor. Nature 377:397404[CrossRef][Medline]
-
Onate SA, Tsai SY, Tsai MJ, OMalley BW 1995 Sequence and
characterization of a coactivator for the steroid hormone receptor
superfamily. Science 270:13541357[Abstract]
-
Hong H, Kohli K, Garabedian MJ, Stallcup MR 1997 GRIP1, a
transcriptional coactivator for the AF-2 transactivation domain of
steroid, thyroid, retinoid, and vitamin D receptors. Mol Cell Biol 17:27352744[Abstract]
-
Chakravarti D, LaMorte VJ, Nelson MC, et al. 1996 Role of
CBP/P300 in nuclear receptor signaling. Nature 383:99102[CrossRef][Medline]
-
Kamei Y, Xu L, Heinzel T, et al. 1996 A CBP integrator complex
mediates transcriptional activation and AP-1 inhibition by nuclear
receptors. Cell 85:403414[Medline]
-
Yang XJ, Ogryzko VV, Nishikawa J, Howard BH, Nakatani Y 1996 A
p300/CBP-associated factor that competes with the adenoviral
oncoprotein E1A. Nature 382:319324[CrossRef][Medline]
-
Chen H, Lin RJ, Louis Schiltz R, et al. 1997 Nuclear receptor
coactivator ACTR is a novel histone acethytransferase and forms a
multimeric activation complex with P/CAF and CBP/p300. Cell 90:569580[Medline]
-
Korzus E, Torchia J, Rose DW, et al. 1998 Transcription
factor-specific requirements for coactivators and their
acetyltransferase functions. Science 279:703707[Abstract/Free Full Text]
-
Caelles C, Gonzalez-Sancho JM, Munoz A 1997 Nuclear hormone
receptor antagonism with AP-1 by inhibition of the JNK pathway. Genes
Dev 11:33513364[Abstract/Free Full Text]
-
Desai-Tajnik V, Hadzic E, Modlinger P, Malhotra S, Gechlik G,
Samuels HH 1993 Interaction of thyroid hormone receptor with the human
immunodeficiency virus type 1 (HIV-1) long terminal repeat and the
HIV-1 Tat transactivator. J Virol 69:51025112
-
Lee Y, Nadel-Ginard B, Mahdavi V, Izumo S 1997 Myocyte-specific enhancer factor 2 and thyroid hormone receptor
associate and synergistically activate the
cardiac myosin
heavy-chain gene. Mol Cell Biol 17:27452755[Abstract]
-
Pflitzner E, Kirfel J, Becker P, Polke A, Schule R 1998 Physical interaction between retinoic acid receptor and the oncoprotein
Myb inhibits retinoic acid-dependent transactivation. Proc Natl Acad
Sci USA 95:55395544[Abstract/Free Full Text]
-
QI JS, Desai-Yajnik V, Yuan Y, Samuels HH 1997 Constitutive
activation of gene expression by thyroid hormone receptor results from
reversal of p53-mediated repression. Mol Cell Biol 17:71957207[Abstract]
-
Yap N, Yu CL, Cheng SY 1996 Modulation of the transcriptional
activity of thyroid hormone receptors by the tumor suppressor p53. Proc
Natl Acad Sci USA 93:42734277[Abstract/Free Full Text]
-
Andrew Powers C, Mathur M, Raaka BM, Ron D, Samuels HH 1998 TLS (translocated-in-liposarcoma) is a high-affinity interactor for
steroid, thyroid hormone, and retinoid receptors. Mol Endocrinol 12:418[Abstract/Free Full Text]
-
Miyamoto T, Kakizawa T, Hashizume K 1999 Inhibition of nuclear
receptor signaling by poly(ADP-ribose) polymerase. Mol Cell Biol 19:26442649[Abstract/Free Full Text]
-
Cheng X, Reginato MJ, Andrews NC, Lazar MA 1997 The
transcriptional integrator CREB-binding protein mediates positive cross
talk between nuclear hormone receptors and the hematopoietic bZip
protein p45/NF-E2. Mol Cell Biol 17:14071416[Abstract]
-
Milanen AM, Poulla H, Karvonen U, Hakli M, Janne OA, Palvimo
JJ 1998 Identification of a novel RING finger protein as a coregulator
in steroid receptor-mediated gene transcription. Mol Cell Biol 18:51285139[Abstract/Free Full Text]
-
Puigserver P, Wu Z, Park CW, Graves R, Wright M, Spiegelman BM 1998 A cold-inducible coactivator of nuclear receptors linked to
adaptive thermogenesis. Cell 92:829839[Medline]
-
Blanco C, Jorge G, Minucci S, et al. 1999 The histone
acetylase PCAF is a nuclear receptor coactivator. Genes Dev 12:16391651
-
Sasaki S, Lesoon-Wood LA, Dey A, et al. 1999 Ligand-induced
recruitment of a histone deacetylase in the negative-feedback
regulation of the thyrotropin ß gene. EMBO J 18:53895398[Abstract/Free Full Text]
-
Nelbock P, Dillon PJ, Perkins A, Rosen CA 1990 A cDNA for a
protein that interacts with the human immunodeficiency virus Tat
transactivator. Science 248:16501653[Medline]
-
Lee JW, Choi H, Gyurist J, Brent R, Moore DD 1995 Two classes
of proteins dependent on either the presence or absence of thyroid
hormone for interaction with the thyroid hormone receptor. Mol
Endocrinol 9:243254[Abstract]
-
Lee JW, Ryan F, Swaffield JC, Johnston SA, Moore DD 1995 Interaction of thyroid-hormone receptor with a conserved
transcriptional mediator. Nature 374:9194[CrossRef][Medline]
-
Patel S, Latterich M 1998 The AAA team: related ATPases with
diverse function. Trends Cell Biol 8:6571[CrossRef][Medline]
-
Herry DM, Kalkhoven E, Hoare S, Parker MG 1997 A signature
motif in transcriptional co-activators mediates binding to nuclear
receptors. Nature 387:733736[CrossRef][Medline]
-
vom Bauer E, Zechel C, Heery D, et al. 1996 Differential
ligand-dependent interactions between the AF-2 activating domain of
nuclear receptors and the putative transcriptional intermediary factors
mSUG1 and TIF1. EMBO J 15:110124[Abstract]
-
Choi H, Soel W, Moore DD 1996 A component of the 26S
proteasome binds an orphan member of the nuclear hormone receptor
superfamily. J Steroid Biochem Mol Biol 56:2330[CrossRef][Medline]
-
Ohana B, Moore PA, Ruben SM, Southgate CD, Green MR, Rosen CA 1993 The type-1 human immunodeficiency virus Tat binding protein is a
transcriptional coactivator belonging to an additional family of
evolutionary conserved genes. Proc Natl Acad Sci USA 90:138142[Abstract]
-
Weinberger C, Thompson CC, Ong ES, Lebo R, Gruol DJ, Evans RM 1986 The c-erb-A gene encodes a thyroid hormone receptor. Nature 324:641646[Medline]
-
Sap J, Munoz A, Damm K, et al. 1986 The c-erb-A protein is a
high-affinity receptor for thyroid hormone. Nature 324:635640[Medline]
-
Yen PM, Sugawara A, Chin WW 1992 Triiodothyronine (T3)
differentially affects T3-receptor/retinoic acid receptor and
T3-receptor/retinoid X receptor heterodimer binding to DNA. J Biol
Chem 267:2324823252[Abstract/Free Full Text]
-
Monden T, Wondisford FE, Hollenberg AN 1997 Isolation and
characterization of a novel ligand-dependent thyroid hormone
receptor-coactivating protein. J Biol Chem 272:2983429841[Abstract/Free Full Text]
-
Ciechanover A 1994 The ubiquitin-proteasome proteolytic
pathway. Cell 79:1321[Medline]
-
Peters JM 1994 Proteasomes: protein degradation machines of
the cell. Trends Biochem Sci 19:377382[CrossRef][Medline]
-
Masuyama H, MacDonald PN 1998 Proteasome-mediated degradation
of the vitamin D receptor (VDR) and a putative role for SUG1
interaction with the AF-2 domain of VDR. J Cell Biol 71:429440[CrossRef]
-
Nawaz Z, Lonard DM, Dennis AP, Smith CL, OMalley BW 1999 Proteasome-dependent degradation of the human estrogen receptor. Proc
Natl Acad Sci USA 96:18581862[Abstract/Free Full Text]
-
Yoshida H, Kitamura K, Tanaka K, et al. 1996 Accelerated
degradation of PML-retinoic acid receptor alpha (PML-RARA) oncoprotein
by all-trans retinoic acid in acute polymyelocytic leukemia: possible
role of the proteasome pathway. Cancer Res 56:2945294850[Abstract]
-
Goyer C, Lee HS, Malo D, Sonenberg N 1992 Isolation of a yeast
gene encoding a protein homologous to the human Tat-binding protein
TBP-1. DNA Cell Biol 11:579585[Medline]
-
Kitabayashi H, Toriyama K 1997 Expression of a gene for a
protein similar to HIV-1 Tat binding protein 1 (TBP1) in floral organs
of Brassica rapa. Plant Cell Physiol 38:966969[Medline]
-
Makino Y, Yogosawa S, Kanetani M, et al. 1996 Structures of
the rat proteasome ATPases: determination of highly conserved
structural motifs and rules for their spacing. Biochem Biophys Res
Commun 220:10491054[CrossRef][Medline]
-
Suzuku I, Yanagawa Y, Yamazaki K, Ueda T, Nakagawa H,
Hashimoto J 1998 Biochemical and immunological characterization of rice
homologues of the human immunodeficiency virus-1 Tat binding protein
and subunit 4 of human 26S proteasome subunits. Plant Mol Biol 37:495504[CrossRef][Medline]
-
Hoyle J, Tan KH, Fisher EM 1997 Localization of genes encoding
two human one-domain members of the AAA family: PSMC5 (the thyroid
hormone receptor-interacting protein, TRIP1) and PSMC3 (the Tat binding
protein, TBP1). Hum Genet 99:285288[CrossRef][Medline]
-
Tanahashi N, Suzuki M, Fujiwara T, et al. 1998 Chromosomal
localization and immunological analysis of a family of human 26S
proteasomal ATPases. Biochem Biophys Res Commun 243:229232[CrossRef][Medline]
-
Nakamura T, Tanaka T, Takagi H, Sato M 1998 Cloning and
heterogeneous in vivo expression of Tat binding protein-1
(TBP-1) in the mouse. Biochim Biophys Acta 30:93100
-
Nakamura T, Tanaka T, Nagano T, Yoneda T, Takagi H, Sato M 1998 Distribution of mRNA encoding Tat-binding protein-1(TBP-1), a
component of 26S proteasome, in the rat brain. Mol Brain Res 53:321327[Medline]
-
Masuyama H, Brownfield CM, St-Arnaud R, MacDonald PN 1997 Evidence for ligand-dependent intramolecular folding of the AF-2 domain
in vitamin D receptor-activated transcription and coactivator
interaction. Mol Endocrinol 11:15071517[Abstract/Free Full Text]
-
Makino Y, Yamano K, Kanemaki M, et al. 1997 SUG1, a component
of the 26S proteasome, is an ATPase stimulated by specific RNAs. J
Biol Chem 272:2320123205[Abstract/Free Full Text]
-
Frazer RA, Rossignol M, Heard DJ, Egly JM, Chambon P 1997 SUG1, a putative transcriptional mediator and subunit of the PA700
proteasome regulatory complex, is a DNA helicase. J Biol Chem 272:71227126[Abstract/Free Full Text]
-
Weeda G, Rossignol M, Traser RA, et al. 1997 The XPB subunit
of repair/transcription factor TFIIH directly interacts with SUG1, a
subunit of the 26S proteasome and putative transcription factor.
Nucleic Acids Res 25:22742283[Abstract/Free Full Text]
-
Hirling H, Scheffner M, Restel T, Stahl H 1989 RNA helicase
activity associated with the human p68 protein. Nature 339:562564[CrossRef][Medline]
-
Endoh H, Maruyama K, Masuhiro Y, et al. 1999 Purification and
identification of p68 RNA helicase acting a transcriptional coactivator
specific for the activation function 1 of human estrogen receptor
.
Mol Cell Biol 19:53635372[Abstract/Free Full Text]
-
Makino Y, Yoshida T, Yogosawa S, Tanaka K, Muramatsu M, Tamura
TA 1999 Multiple mammalian proteasomal ATPases, but not proteasome
itself, are associated with TATA-binding protein and a novel
transcriptional activator, TIP120. Genes Cells 4:529539[Abstract/Free Full Text]
-
Jeang K, Xiao H, Rich EA 1999 Multifaceted activities of the
HIV-1 transactivator of transcription, Tat. J Biol Chem 274:2883728840[Free Full Text]
-
Benkirane M, Chun RF, Xiao H, et al. 1998 Activation of
integrated provirus requires histone acetyltransferase. J Biol
Chem 273:2489824905[Abstract/Free Full Text]
-
Monden T, Kishi M, Hosoya T, et al. 1999 p120 acts as a
specific coactivator for 9-cis-retinoic acid receptor (RXR)
on peroxisome proliferator-activated receptor-
/RXR heterodimers. Mol
Endocrinol 13:16951703[Abstract/Free Full Text]
-
Satoh T, Yamada M, Iwasaki T, Mori M 1996 Negative regulation
of the gene for the preprothyrotropin-releasing hormone from the mouse
by thyroid hormone requires additional factors in conjunction with
thyroid hormone receptor. J Biol Chem 271:2791927926[Abstract/Free Full Text]
-
Satoh T, Ishizuka T, Monden T, et al. 1999 Regulation of the
mouse preprothyrotropin-releasing hormone gene by retinoic acid
receptor. Endocrinology 140:50045013[Abstract/Free Full Text]
-
Wood WM, Kao MY, Gordon DF, Ridgway EC 1989 Thyroid hormone
regulates the mouse thyrotropin ß-subunit gene promoter in
transfected pituitary thyrotropes. J Biol Chem 264:1484014847[Abstract/Free Full Text]
-
Ren Y, Satoh T, Yamada M, et al. 1998 Regulation of the mouse
preprothyrotropin-releasing hormone gene by epidermal growth factor.
Endocrinology 139:195203[Abstract/Free Full Text]