Identification of Mouse TRAP100: a Transcriptional Coregulatory Factor for Thyroid Hormone and Vitamin D Receptors
Jiachang Zhang and
Joseph D. Fondell
Department of Physiology University of Maryland School of
Medicine Baltimore, Maryland 21201
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ABSTRACT
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Nuclear hormone receptors (NRs) regulate
transcription in part by recruiting distinct transcriptional
coregulatory complexes to target gene promoters. The thyroid hormone
receptor (TR) was recently purified from thyroid hormone-cultured HeLa
cells in association with a complex of novel nuclear proteins termed
TRAPs (thyroid hormone receptor-associated proteins)
ranging in size from 20 to 240 kDa. The TRAP complex markedly enhances
TR-mediated transcription in vitro, suggesting a
coactivator role for one or more of the TRAP components. Here we
present the mouse cDNA for the 100-kDa component of the TRAP complex
(mTRAP100). The mTRAP100 protein contains seven LxxLL motifs thought to
be potential binding surfaces for liganded NRs, yet surprisingly fails
to interact with TR and other NRs in vitro. By contrast,
mTRAP100 coprecipitates in vivo with another component of
the TRAP complex (TRAP220), which directly contacts TR and the vitamin
D receptor in a ligand-dependent manner. Our findings thus
suggest that TRAP100 is targeted to NRs in association with TRAP
complexes specifically containing TRAP220. Transient overexpression of
mTRAP100 in mammalian cells further enhances ligand-dependent
transcription by both TR and the vitamin D receptor, revealing a
functional role for mTRAP100 in NR-mediated transactivation. The
presence of an intrinsic mTRAP100 transactivation function is suggested
by the ability of mTRAP100 to activate transcription constituitively
when tethered to the GAL4 DNA-binding domain. Collectively, these
findings suggest that TRAP100, in concert with other TRAPs, plays
an important functional role in mediating transactivation by specific
NRs.
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INTRODUCTION
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Members of the nuclear hormone receptor (NR) superfamily are
potent regulators of gene expression in response to their cognate
physiological ligands which include steroids, retinoids, thyroid
hormone, and vitamin D3 (1, 2, 3). NRs have a modular
structure consisting of three separable domains: 1) a highly conserved
DNA-binding domain containing two zinc finger motifs (2, 3); 2)
a poorly conserved amino-terminal domain which, at least for some NRs,
contains an autonomous activation function (AF-1) (2, 3, 4, 5); and 3) a
carboxy-terminal ligand-binding domain containing a dimerization
surface and an additional activation function (AF-2), which is
essential for ligand-dependent activation (2, 3, 6, 7, 8, 9). Importantly,
the AF-2 core domain comprises a highly conserved amphipathic
-helical motif (2, 3) that is present in all known transcriptionally
active members of the NR superfamily (3, 6, 7, 8, 9). Both AF-1 and AF-2
activities depend on promoter and tissue type (4, 5), and both activate
transcription in yeast (which do not express endogenous NRs), thus
indicating that the molecular mechanisms of NR gene regulation have
been conserved during evolution (10, 11).
A great wealth of evidence implicates the involvement of specific
coregulatory factors in NR-mediated gene regulation (for reviews see
Refs. 12, 13). Remarkably, yeast two-hybrid screens using NR-AF-2
regions as bait have led to the identification of a wide array of
putative NR-coactivators including TRIP1 (14), SRC-1 (15), ARA70 (16),
TIF1 (17), GRIP1 (18), RAC3/ACTR/AIB1/TRAM-1 (19, 20, 21, 22), p120 (23),
TRIP230 (24), PGC-1 (25), and PBP (26). Similarly,
conventional expression library screens have identified other
presumptive coactivators including p160 (renamed NCoA-1) (27), RIP140
(28), TIF2 (29), RAP46 (30), and p/CIP (31). Most of these proteins
display one or more copies of a consensus
-helical motif of repeated
leucines (LxxLL), which are thought to be necessary for
ligand-dependent interactions with the AF-2 domain of NRs (31, 32). Of
note, SRC-1/p160, TIF2/GRIP1, RAC3/ACTR/AIB1/TRAM-1, and p/CIP all
share sequence homologies suggesting a novel family of SRC-like
NR-coactivators.
Recent studies demonstrated that the pleiotropic coactivator CBP/p300
forms a ternary complex with NRs and various SRC-like cofactors and is
functionally required for ligand-dependent activation (20, 27, 33).
Given that CBP/p300 is a histone acetyltransferase (HAT) (34), and a
known correlation exists between hyperacetylated histones and
derepressed chromatin (35), these findings are consistent with a NR
role in chromatin remodeling. Interestingly, intrinsic HAT activity has
also been attributed to some of the SRC-like coactivators (20, 33). On
the basis of these and other studies, a model has been proposed in
which liganded NRs are thought to activate gene expression by
promoter-specific recruitment of HAT activity (20, 27, 31, 33). Other
studies, however, using chromatin-based transcription assays indicate
that ligand-induced disruption of chromatin by NRs may be insufficient
for transcriptional activation (36, 37). The chromatin studies thus
suggest that additional accessory factors and additional steps may be
necessary for the liganded-NR to productively interface with the basal
transcription apparatus and activate gene transcription.
As an alternative method of identifying specific nuclear factors that
interact with NRs and possibly potentiate their function, a
HeLa-derived cell line was generated that stably expresses an
epitope-tagged thyroid hormone receptor (TR) (38). When immunopurified
from T3-treated cells, TR is specifically associated with a
distinct set of novel nuclear proteins termed TRAPs (TR-associated
proteins) ranging in size from 20 to 240 kDa. Cell-free transcription
assays demonstrated that the TRAP complex dramatically enhances
TR-mediated transcription from thyroid response element
(TRE)-linked promoter templates (38). It was therefore hypothesized
that specific TRAPs, either collectively or individually, might
function as TR-specific transcriptional coactivators. Evidence
supporting a more common TRAP coactivator role for other NRs comes from
the recent purification of an apparently identical complex of proteins
(termed DRIPs) that specifically interact with the vitamin D
receptor (VDR) and enhance VDR transactivation in vitro
(39). Cognate human cDNAs for the 220-kDa and 100-kDa components of the
TRAP complex (hTRAP220 and hTRAP100) have been reported (40). Sequence
homologies show that hTRAP100 is nearly identical to the 100-kDa
component of the human DRIP complex (DRIP100) (39) and that TRAP220 is
the probable human ortholog of the mouse-derived PPAR-binding protein
(26). On the basis of its ability to interact with TR and other NRs in
an avid ligand-dependent fashion (40), TRAP220 has been proposed to act
as an anchoring factor, possibly serving to target other TRAP
components to a liganded NR during TRAP complex-mediated coactivation.
In contrast, the functional role played by the TRAP100 component is ill
understood.
In this study, we present the cDNA sequence of the mouse TRAP100
(mTRAP100) gene. We provide evidence that mTRAP100 enhances
ligand-dependent transcriptional activation by both TR and VDR when
transiently overexpressed in cultured mammalian cells. Although
mTRAP100 fails to exhibit strong direct interactions with various NRs,
the findings presented here indicate that TRAP100 is targeted to a
liganded NR through TRAP protein complexes specifically containing the
NR-interacting factor TRAP220. When tethered to a heterologous
DNA-binding domain, mTRAP100 activates transcription constituitively,
possibly revealing intrinsic transactivation functions. We suggest that
the TRAP100 component of the TRAP coactivator complex may play an
important functional role in facilitating TRAP-mediated enhancement of
NR- signaling pathways.
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RESULTS
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Identification and Structure of the mTRAP100 Gene
The recent cloning of the mouse homolog for hTRAP220 demonstrated
that specific components of the TRAP coactivator complex are conserved
among mammals (26). To determine whether the TRAP100 component was also
conserved in mice, we screened a mouse-expressed sequence tag (EST)
data base (41) with random peptide sequences derived from the hTRAP100
protein (40). Several mouse cDNA clones with significant homology were
identified, obtained (from American Type Culture Collection, Manassas, VA) and completely sequenced. The largest
cDNA clone contains an open reading frame of 2868 bp flanked by 5'- and
3'-untranslated regions and encodes a polypeptide of 956 amino acids
with a predicted molecular mass of 106 kDa (Fig. 1A
). The ATG start codon was defined by
the presence of several upstream in-frame stop codons within the
putative 5'-untranslated sequence. In vitro translation of
the open reading frame containing or lacking the 5'-untranslated region
generates proteins of equal molecular mass, thus confirming the coding
sequence start site (data not shown). The relatively short
3'-untranslated region (298 bp) contains a consensus AAUAAA sequence 12
bp upstream of the poly(A) tail, further defining the 3'-end of the
cDNA (data not shown). Sequence comparison of the translated open
reading frame reveals a 91% identity and 95% similarity to the
hTRAP100 protein (Fig. 1B
). We have thus termed the protein encoded by
this cDNA as mTRAP100. To verify that the cloned mTRAP100 cDNA
corresponds to the 100-kDa component of the TRAP coactivator complex,
polyclonal antibodies were raised against a portion of the expressed
mTRAP100 protein and used to probe the purified hTRAP complex by
Western blot. As shown in Fig. 1C
, anti-mTRAP100 antibodies
specifically recognize a 100-kDa protein in a crude Hela cell nuclear
extract as well as in the purified TR/TRAP coactivator complex.
Although highly homologous to its human counterpart, mTRAP100 lacks the
amino-terminal 50 amino acids found on the hTRAP100 protein (Fig. 1B
).
Interestingly, mTRAP100 contains seven LxxLL motifs (Fig. 1A
)
previously implicated as signature NR-interaction protein surfaces (31, 32). Six of the signature motifs are conserved in both mTRAP100 and
hTRAP100 in the same relative locations (Fig. 1B
), whereas the seventh
additional LxxLL motif found in mTRAP100 is located at positions
699703 (Fig. 1
, A and B). The mTRAP100 protein also contains a
consensus ATP/GTP-binding site motif or P-loop at positions 407 to 414
and a presumptive leucine zipper motif at positions 734 to 755 (Fig. 1
, A and B). Both the P-loop and leucine zipper are highly conserved
within the hTRAP100 protein (Fig. 1B
), possibly indicating that these
motifs facilitate important functional or regulatory roles for the
TRAP100 proteins. To examine whether expression of the mTRAP100 gene
exhibits tissue specificity, we performed Northern blot analysis using
poly-A+ RNA from eight different mouse tissues (Fig. 1D
). The mTRAP100
mRNA displays relatively low expression in the spleen, lung, and
skeletal muscle, whereas the mRNA is abundantly expressed in the
testes, heart, and brain (Fig. 1D
).
mTRAP100 and TRAP220 Interactions with Nuclear Receptors
The simple LxxLL signature motif is thought to provide a binding
surface for liganded NRs, and its presence within some transcriptional
coactivators is both necessary and sufficient for functional NR
interactions (31, 32). The presence of seven such signature motifs
within the mTRAP100 protein (Fig. 1A
) therefore suggested that mTRAP100
might be a direct target for NR interactions. To test this hypothesis,
we expressed full-length mTRAP100 as glutathione
S-transferase (GST)-fusion protein in
Escherichia coli and used the purified protein in GST
pull-down assays. Surprisingly, mTRAP100 failed to bind either TR
or
TRß (Fig. 2A
) in either the presence or
absence of T3. Furthermore, mTRAP100 failed to bind the
retinoic acid receptor-
(RAR
), the rat vitamin D receptor (rVDR),
and the progesterone receptor (PR) (Fig. 2A
) regardless of the presence
or absence of ligand. These findings suggest that mTRAP100 is not a
direct target for ligand-dependent interactions with NRs and that
recruitment of mTRAP100 to NR-regulated promoters presumably requires
other TRAP components.

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Figure 2. mTRAP100 and TRAP220 Interactions with Nuclear
Receptors in Vitro
A, mTRAP100 fails to interact with liganded nuclear receptors in
vitro. GST-mTRAP100 fusion protein (1 µg) was incubated with
[35S]methionine-labeled hTR , hTRß, rVDR, hRAR ,
and hPR in the presence or absence of ligand as indicated (see
Materials and Methods). B, TRAP220 strongly interacts
with TR and VDR in a hormone-dependent manner. GST (1 µg) or
GST-TRAP220-RBD fusion protein (1 µg) containing the TRAP220
receptor-binding domain (a.a. 622701) (40 ) was incubated with
[35S] methionine-labeled hTR , hTRß, hVDR, and rVDR
in the presence or absence of ligand as indicated. In each panel, 25%
of the labeled input is indicated for each reaction.
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LxxLL motifs have also been identified within the TRAP220 component of
the TRAP complex and were recently implicated in mediating the observed
hormone-dependent interactions between TRAP220 and various NRs (40). In
stark contrast to our results with mTRAP100, we found that a GST fusion
protein containing only one TRAP220 LxxLL motif effectively binds to
both TR and VDR in a convincing ligand-dependent manner (Fig. 2B
). This
finding is consistent with TRAP220s proposed role as an intermediary
factor that presumably targets other TRAPs to liganded NRs. Moreover,
and in light of mTRAP100s inability to contact NRs despite the
presence of multiple consensus LxxLL motifs, these data support the
notion that specific amino acid residues flanking the consensus LxxLL
motifs of NR-coregulatory factors are critical determinants in
modulating the affinity of the NR interaction (42, 43).
mTRAP100 Coprecipitates with TRAP220 in Vivo
Given that TRAP100 was first identified as a component of the
multimeric TR/TRAP complex (Fig. 1C
) (38, 40) yet fails to directly
contact TR (Fig. 2A
) (40), it is conceivable that TRAP100 is targeted
to TR (and possibly other NRs) indirectly through interactions with
other components of the TRAP coactivator complex. TRAP220 would appear
to be the most plausible candidate to fulfill this role in view of its
strong ligand-dependent interactions with various NRs. In support of
this supposition, we did find that mTRAP100 directly (albeit weakly)
contacts the full-length hTRAP220 protein in vitro as
determined by the GST pull-down assay (data not shown). However,
further attempts to elucidate direct mTRAP100/TRAP220 interactions
in vitro using other protein-binding assays were largely
unsuccessful, possibly implying that the mTRAP100/TRAP220 association
is indirect or that additional TRAP components are required to
effectively facilitate or stabilize this association.
To examine the association of mTRAP100 and TRAP220 under more
physiologically relevant conditions, both TRAP220 and a
FLAG-tagged mTRAP100 were overexpressed in COS cells and assayed
for association by coimmunoprecipitation. Protein complexes containing
mTRAP100 were immunoprecipitated from transfected cells with anti-FLAG
antibodies coupled to agarose beads, fractionated by SDS/PAGE, and
subsequently probed by Western blot with antibodies generated against
TRAP220. Figure 3
(lane 10) clearly shows
TRAP220 associates with mTRAP100 when both proteins are overexpressed
in COS cells. This result suggests that TRAP220 and mTRAP100 are in the
same protein complex that ultimately binds to TR rather than each
protein forming a separate complex with TR. Taking into account the
likely presence of other TRAP components endogenously expressed in COS
cells, these experiments also support the notion that other TRAP
subunits may interact with TRAP100 and TRAP220 and possibly stabilize
or facilitate formation of TRAP100/TRAP220-containing protein
complexes. Taken collectively, the findings in Figs. 2
and 3
are
consistent with a TRAP complex recruitment model in which TRAP220
functions as an intermediary protein, interacting with other TRAP
components as well as TRAP100 and subsequently targeting them to a NR
in a ligand-dependent fashion. The ability of a liganded NR to
recognize specific hormone response elements would ultimately target
the entire TRAP coactivator complex to gene-specific regulatory regions
where TRAPs presumably interface with the basal transcription
apparatus.

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Figure 3. mTRAP100 Associates with TRAP220 in
Vivo
COS cells were transfected with pSG5-FLAG-mTRAP100 or pSG5-TRAP220,
either separately or together as indicated above the
lanes. To verify TRAP100 and TRAP220 coexpression, whole-cell extract
from transfected cells (lanes 2, 4, and 5) or mock transfected cells
(lanes 1 and 3) was fractionated by SDS/PAGE, transferred to a
nitrocellulose membrane, and probed by Western blot with rabbit
polyclonal antisera against TRAP220 ( -220), mTRAP100 ( -100), or
mouse monoclonal antisera against the FLAG epitope ( -FLAG). The weak
220-kDa and 100-kDa bands in lanes 1 and 3 presumably reveal endogenous
TRAP proteins. In lanes 710, whole-cell extract prepared from
transfected and mock transfected cells was first incubated with
anti-FLAG antibodies coupled to a resin (M2-affinity resin;
Eastman Kodak Co.). The resulting immuno-protein complexes
were then precipitated and washed, fractionated by SDS/PAGE,
transferred to a nitrocellulose membrane, and probed by Western blot
with -220. The portion of membrane corresponding to lane 10 was
stripped and reprobed with -100 (lane 11) to verify the presence of
TRAP100 in the immunoprecipitated complex. HeLa nuclear extract (30
µg) was used in lane 6 as a positive control for the -220
antisera.
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mTRAP100 Enhances Ligand-Dependent Transcription by both TR and
VDR
We next sought to determine the functional significance of TRAP100
in affecting T3-dependent transcription by TR. Toward this
end, CV-1 cells were transiently cotransfected with mTRAP100 and TR
expression vectors and grown in either the presence or absence of
T3. Transcription was then measured from a luciferase
reporter containing two T3-response elements
(2xT3RE-tk-Luc) (Fig. 4A
). In the absence
of T3, mTRAP100 modestly reduced the basal level of
expression from the TRE-containing reporter. In the presence of
T3, however, mTRAP100 enhances TR-mediated activation
3-fold (Fig. 4A
) consistent with a transcriptional coactivator function
for the mTRAP100 protein. Given the presence of the hTRAP100 homolog
(DRIP100) in a VDR-associated coactivator complex (39), we further
asked whether mTRAP100 might enhance VDR-mediated transcription.
Accordingly, NIH3T3 cells were transiently transfected with both
mTRAP100 and VDR expression vectors, and transcription was measured
from a cotransfected luciferase reporter gene containing four vitamin D
response elements (4xVDRE-Ld-Luc) (Fig. 4B
). Similar to the
case with TR, mTRAP100 overexpression enhances VDR-mediated activation
greater than 3-fold in the presence of vitamin D3 (Fig. 4B
). The ability of overexpressed mTRAP100 to modestly inhibit basal
transcription from both the 2xT3RE-tk-Luc and 4xVDRE-Ld-Luc
reporters in the absence of ligand may reflect mTRAP100 sequestration
of other commonly required nuclear cofactors. Nonetheless, these
findings suggest that the TRAP100 component of the TRAP coactivator
complex plays an important functional role in facilitating
TRAP-mediated enhancement of ligand-dependent transactivation by both
TR and VDR.

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Figure 4. mTRAP100 Enhances Ligand-Dependent Transcription by
TR and VDR
A, Transient overexpression of mTRAP100 enhances TR-mediated
transactivation from a TRE-reporter. CV-1 cells were transfected with
0.33 µg p2xT3RE-tk-Luc reporter, 0.33 µg pRSV-TRß, 0.15 µg of
the internal control plasmid pSV-ß-gal, and 1 µg of either
pSG5-mTRAP100 or the empty pSG5 vector. TRIAC (10-7
M final) was added as indicated. B, Transient
overexpression of mTRAP100 enhances VDR-mediated transactivation from a
VDRE-reporter. NIH3T3 cells were transfected with 0.66 µg
p4xVDRE-Ld-Luc, 0.33 µg pSG5-hVDR, 0.15 µg of the
internal control plasmid pSV-ß-gal, and 1.33 µg of either
pSG5-mTRAP100 or the empty pSG5 vector. 1,25-(OH)2D3
(2.5 x 10-8 M final) was added as
indicated. Relative luciferase activities were determined from three
independent transfections in the absence (open bars) or
presence of ligand (hatched bars), although similar
results were obtained with multiple transfections. The data are
presented as the mean ± SD of the triplicated
transfections.
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Autonomous Transactivation by mTRAP100
To examine whether mTRAP100 might directly activate
transcription when recruited to a specific promoter, we generated a
chimeric protein vector in which the full-length mTRAP100 protein is
fused to the DNA-binding domain of the yeast transcription factor GAL4
(GAL4-mTRAP100). The GAL4-mTRAP100 vector was then transfected into COS
cells, and transcription was measured from a luciferase reporter
containing five GAL4-binding sites (Fig. 5
). While GAL4 alone does not efficiently
activate transcription, the GAL4-mTRAP100 protein exhibits moderate
transcriptional stimulation from the luciferase reporter (Fig. 5
).
These results indicate that mTRAP100 may contain an intrinsic
transactivation domain that functions autonomously in mammalian cells
and thus suggest a transcription-enhancing functional role for the
TRAP100 protein during TRAP-mediated coactivation. Presumably, higher
levels of transcriptional activation are achieved when TRAP100
functions in concert with the other components of the TRAP coactivator
complex.

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Figure 5. Autonomous Transactivation by mTRAP100
COS cells were transiently transfected with 0.15 µg p5xGAL4-tk-Luc,
0.15 µg pSV-ß-gal, and 0.33 µg of either pSG5 (empty expression
vector), pSG424 (GAL4 expression vector), or pSG424-mTRAP100
(GAL4-full-length mTRAP100 fusion protein expression vector) as
indicated. Relative luciferase activities were determined from three
independent transfections. The data are presented as the mean ±
SD of the triplicated transfections.
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DISCUSSION
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The TRAP coactivator complex was first identified as a large
multimeric complex of novel proteins that copurify with TR from
T3-cultured HeLa cells (38). Subsequent
T3-dependent transcription assays clearly demonstrated a
pronounced TRAP complex enhancement of TR-mediated activation in
vitro (38), thereby revealing a novel NR-coactivator pathway or
activation step (see below) apparently distinct from those involving
SRC-family members and CBP/p300. In this study we have identified the
mouse cDNA for the 100-kDa component of the TRAP complex (mTRAP100)
and, in an effort to better understand how the TRAP coactivator complex
works, we have functionally characterized the mTRAP100 protein. We have
shown that overexpression of mTRAP100 in cultured mammalian cells
significantly enhances ligand-dependent transcription by both TR and
VDR and that mTRAP100 can activate transcription autonomously when
tethered to a heterologous DNA-binding domain. Based on these data, we
suggest that TRAP100 facilitates an important functional role during
TRAP complex-mediated enhancement of ligand-dependent transcription by
specific NRs.
A striking feature of the mTRAP100 protein is the presence of
seven LxxLL signature motifs previously shown in other NR coactivators
to be binding surfaces for liganded NRs (32). In spite of the multiple
signature motifs, however, mTRAP100 failed to interact in
vitro with any of the NRs tested here (VDR, RAR, PR, and TR), thus
suggesting that TRAP100 is not recruited to a hormone-responsive
promoter via direct contacts with a liganded NR. The question then
arises as to how TRAP100 is specifically targeted to NR-regulated
genes. In this report, we have demonstrated that mTRAP100 associates
with the 220-kDa component of the TRAP complex (termed TRAP220)
in vivo. Importantly, TRAP220 is the only TRAP subunit
demonstrated thus far to directly and convincingly contact TR and VDR
(as well as other NRs) in a ligand-dependent manner. Hence, our data
are consistent with a TRAP complex recruitment model in which TRAP220
acts as nucleation surface for the assembly of TRAP100 and other TRAP
components into a holo-complex and subsequently targets their
respective transactivation functions to specific NRs in a
ligand-dependent fashion. Whether TRAP100 directly contacts TRAP220 or
alternatively requires other TRAP components to indirectly associate
with TRAP220 is currently unresolved. However, given the sheer size of
the TRAP complex (
1.5 MDa), the number of putative subunits, and the
potential for multiple protein-protein interactions, it appears highly
probable that other TRAP components are involved in facilitating this
association.
In light of the myriad of putative NR accessory proteins recently
discovered (12, 13), it is puzzling as to why a highly conserved
superfamily of ligand-induced transcription factors would functionally
require such a large and diverse group of accessory factors including
the TRAP coactivator complex. One explanation might be that different
NR cofactors represent alternative transcriptional regulatory pathways
available to a given NR within different cell types. In this manner,
type of ligand (agonist, antagonist, or none) might dictate
differential usage of NR cofactors within different tissues. A second
possibility is that multiple NR-coregulatory factors may be a conserved
mechanism of integrating multiple cellular signal pathways into
hormone-induced NR pathways. Third, different NR cofactors may reflect
different (and perhaps sequential) transcriptional regulatory steps
during a hormone-induced NR pathway (35, 36). For instance, NRs may
initially require chromatin remodeling factors (i.e. the HAT
activity associated with SRC-related factors and CBP/p300) to penetrate
target genes within condensed chromatin. Subsequently, other
coregulatory factors (i.e. TRAPs/DRIPs) may be required in
order for the NR to effectively communicate with the basal
transcription apparatus.
The presence of TRAP100 within two apparently identical
coactivator complexes specifically associated with TR and VDR suggests
that TRAP-mediated transcriptional coactivation may be commonly used by
other members of the NR superfamily, particularly those that
heterodimerize with RXR (44). Indeed, the recent cloning of the
mTRAP220 homolog as a ligand-dependent PPAR-interacting protein
suggests that the TRAP coactivator complex may be involved in PPAR
pathways (26). In the context of TRAP220s proposed role as a bridging
factor between other TRAP components and liganded NRs, the ability of
TRAP220 to interact with a diverse array of NRs suggests that TRAPs may
be involved in other pathways as well. A more complete understanding of
how the holocomplex operates and the functional roles fulfilled by the
other TRAP components await the identification and characterization of
their respective cDNAs.
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MATERIALS AND METHODS
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Identification of the mTRAP100 cDNA
Mouse cDNAs encoding putative mTRAP100 sequences were identified
by screening short polypeptide sequences derived from the hTRAP100
protein (40) against the data base of mouse expressed sequence tags
(dbEST) at the NCBI (http://www.ncbi.nlm.nih.gov/dbEST). cDNA clones
with significant homology were identified, obtained from the
American Type Culture Collection (ATCC,
Manassas, VA) and sequenced (University of Maryland School of
Medicine Biopolymer Laboratory). The mouse EST clone encoding the
full-length mTRAP100 cDNA was deposited by the I.M.A.G.E. Consortium
(I.M.A.G.E. ID 615136) and distributed by the ATCC (ATCC
ID 773544). The mTRAP100 sequence has been deposited in Genbank
(accession number AF126543).
Northern Blot Analysis
A mouse multiple tissue Northern blot (CLONTECH Laboratories, Inc., Palo Alto, CA) was probed with a
32P-labeled DNA fragment corresponding to amino acid
residues 817956 of the mTRAP100 cDNA. Northern blot hybridization and
membrane washes were performed as recommended by the manufacturer.
Plasmid Construction
The GST-mTRAP100 (full) and GST-mTRAP100 (152) plasmids were
constructed by first PCR amplifying either full-length mTRAP100 or
mTRAP100 amino acids 152 with primers creating BamHI and
EcoRI restriction sites at the 5'- and 3'-ends of the cDNA,
respectively. The PCR fragments were then ligated into pGEX-2TK
(Pharmacia Biotech, Piscataway, NJ) predigested
with BamHI and EcoRI, thus generating the
in-frame GST fusion protein expression vectors. The
GST-TRAP220-receptor binding domain (GST-220-RBD[622701]) (40) and
GST-TRAP220 (14011484) plasmids were generated in a similar fashion
by first PCR amplifying hTRAP220 amino acids 622701 and 14011484,
respectively, with primers creating BamHI and
EcoRI restiction sites at the 5'- and 3'-ends followed by
ligation into the BamHI/EcoRI sites of pGEX-2TK.
The pFLAG-hTR
expression plasmid has been described previously (38).
The pSG5-mTRAP100 mammalian expression vector was generated by
subcloning the EcoRI/BamHI fragment of IMAGE
clone 615136 (containing the full-length mTRAP100 cDNA) into the
EcoRI/BamHI sites of pSG5
(Stratagene, La Jolla, CA). The pSG5-FLAG-mTRAP100
expression vector was created by first generating BglII and
BamHI sites at the 5'- and 3'-ends of the mTRAP100 cDNA by
PCR and then cloning the fragment into the BamHI site of
pFLAG-(s). Subsequently, the FLAG-mTRAP100 fragment was liberated by
EcoRI/BamHI digestion and subcloned into the
EcoRI/BamHI sites of pSG5. pSG5-TRAP220 was
generated by liberating the SmaI/SacI TRAP220
cDNA from pGEM-HA-TRAP220 (provided by R. G. Roeder, Rockefeller
University, New York, NY) and subsequently blunt-end ligating the
full-length hTRAP220 cDNA (40) into the BamHI site of pSG5.
The pSG424-mTRAP100 (GAL4-mTRAP100) mammalian expression vector was
generated by first creating EcoRI sites on both the 5'- and
3'-ends of the mTRAP100 cDNA by PCR followed by an in-frame ligation
into the EcoRI site of the plasmid pSG424 (45). The
pBK-CMV-FLAG-hTR
expression vector was generated by subcloning the
BglII/EcoRI fragment of pFLAG-hTR
into the
BamHI/EcoRI sites of pBK-CMV
(Stratagene). The pFLAG-hTRß plasmid was generated by
first PCR-creating an NdeI site at the translation start
codon of the hTRß cDNA within the pRSV-TRß expression vector
(provided by H. Samuels, New York University, New York, NY).
Subsequently, the NdeI-BamHI fragment spanning
the full-length hTRß cDNA was inserted into the
NdeI/BamHI sites of pFLAG(s)-7 (46). pGEM-rVDR
was created by EcoRI digestion of pSV40-rVDR (provided by H.
DeLuca, University of Wisconsin, Madison, WI) and subcloning the
full-length rVDR fragment into the EcoRI site of pGEM-4Z
(Promega Corp., Madison, WI). The NR expression vectors,
pCMX-hRAR
, pT7ß-hPRA, and pSG5-hVDR, were kindly provided by R.
Evans (Salk Institute, San Diego, CA), M.-J. Tsai (Baylor College of
Medicine, Houston, TX), and K. Ozato (The National Institute of Child
Health and Human Development, Bethesda, MD), respectively. The
luciferase reporter plasmids, p2xT3RE-tk-Luc,
p4xVDRE-Ld-Luc, and p5xGAL4-tk-Luc, were kindly provided by
C. Glass (University of California, San Diego, CA), K. Ozato, and M.
Privalsky (University of California, Davis, CA), respectively.
Antibody Production
The GST-mTRAP100 (152) and GST-TRAP220 (14011484) proteins
were expressed in and purified from E. coli strain
BL21(DE3)pLysS and injected into rabbits (Covance Research Products,
Inc., Denver, PA). Anti-FLAG epitope mouse monoclonal antibodies
and anti-FLAG antibodies coupled to agarose (M2-affinity resin) were
obtained commercially (Eastman Kodak Co., Rochester,
NY).
GST Pull-Down Assay
The GST-mTRAP100 and GST-TRAP220-RBD (622701) proteins were
expressed in and purified from E. coli strain
BL21(DE3)pLysS. In general, 20 µl of a 50% GST-protein/glutathione
Sepharose (Pharmacia Biotech) bead slurry were resuspended
in 200 µl binding buffer [20 mM HEPES (pH 7.9), 100
mM KCl, 0.5 mM EDTA, 5 mM
MgCl2, 1 mM dithiothreitol, 10% glycerol,
0.05% NP-40, 0.5% milk] together with 5 µl of in vitro
translated, [35S]methionine-labeled NRs generated from
the pFLAG-hTR
, pFLAG-hTRß, pCMX-hRAR
, pT7ß-hPRA, pCMX-mERß,
pGEM-rVDR, and pSG5-hVDR templates using a kit (TNT, Promega Corp.). The reactions were incubated for 1 h at 4 C on a
rocker. Protein complexes were isolated by pelleting the beads and
washing three times in binding buffer followed by resuspension in
SDS-sample loading buffer. After SDS-PAGE fractionation, bound
[35S]-labeled NRs were visualized by autoradiography. The
ligands TRIAC (1 µM final), retinoic acid (1
µM final), 1,25-dihydroxyvitamin D3
[1,25-(OH)2D3] (0.5 µM final),
and progesterone (1 µM final) were added to the reactions
as indicated in Fig. 2
.
Transient Transfections
CV-1, COS, and NIH3T3 cells were routinely maintained in DMEM
(high glucose) supplemented with 10% FBS. One day before transfection,
cells were seeded in 12-well plates: NIH3T3 cells at a density of
8 x 104 cells per well in DMEM containing 10%
charcoal/dextran-stripped FBS (HyClone Laboratories, Inc.,
Logan, UT); CV-1 cells at a density of 8 x 104 cells
per well in DMEM containing 10% dialyzed FBS (Gibco BRL,
Gaithersburg, MD); COS cells at a density of 5 x 104
cells per well in DMEM containing 10% FBS. For NIH3T3 cells, a DNA
mixture containing 0.66 µg p4xVDRE-Ld-Luc, 0.33 µg
pSG5-hVDR, 0.15 µg of the internal control plasmid pSV-ß-gal
(Promega Corp.), 1.66 µg sonicated salmon sperm DNA, and
1.33 µg of either pSG5-mTRAP100 or the empty pSG5 vector was added to
each well using the calcium phosphate transfection method. For CV-1
cells, a DNA mixture containing 0.33 µg p2xT3RE-tk-Luc, 0.33 µg
pRSV-TRß, 0.15 µg pSV-ß-gal, 2 µg sonicated salmon sperm DNA,
and 1 µg of either pSG5-mTRAP100 or the empty pSG5 vector was added
to each well as above. For COS cells, a DNA mixture containing 0.15
µg p5xGAL4-tk-Luc, 0.15 µg pSV-ß-gal, 1.66 µg sonicated salmon
sperm DNA, and 0.33 µg of either pSG5 (empty expression vector),
pSG424 (GAL4 alone), or pSG424-mTRAP100 (GAL4-mTRAP100) were added to
each well. The precipitate from each set of transfections was removed
after 16 h with PBS and replaced with fresh DMEM containing either
10% charcoal/dextran-stripped FBS (NIH3T3), 10% dialyzed FBS (CV-1),
or 10% FBS (COS) together with vehicle alone or vehicle plus the
ligands TRIAC (10-7 M final) or
1,25-(OH)2D3 (2.5 x 10-8 M
final) as stated in the figure legends. After 3648 h, transfected
cells in each well were harvested with a cell lysis buffer supplied in
a kit (Luciferase Assay System, Promega Corp.), and
luciferase activity was determined by adding a commercial assay
solution according to the manufacturers instructions (Promega Corp.) and then measuring in a Lumat LB 9507 luminometer
(EG&G Wallac, Inc., Gaithersburg, MD). The
ß-galactosidase activity of the lysed transfected cells (as above)
was determined using a kit (ß-galactosidase Enzyme Assay System,
Promega Corp.) according to the manufacturers
instructions. The luciferase activity was normalized to the ß-gal
activity and expressed as relative luciferase light units. For
coimmunoprecipitation experiments, COS cells were seeded in 10-cm
plates containing DMEM/10% FBS at a cell density of 5 x
105 cells per plate 1 day before transfection. The plates
were transfected essentially as above with 5 µg pSG5-FLAG-mTRAP100 or
5 µg pSG5-TRAP220, either separately or together in subsets (see
legend to Fig. 3
) along with pBS-KSII
(Stratagene) as carrier DNA to a total of 40 µg.
Nontransfected COS cells were used as negative controls for the
immunoprecipitation assays (see below).
Coimmunoprecipitation
COS cells in 10-cm culture dishes were collected for harvesting
by gentle scraping in 1 ml ice-cold PBS and pelleting by centrifigution
at 1200 rpm at 4 C. The PBS was aspirated and the cell pellet
(106 cells) resuspended in 0.5 ml ice-cold buffer A [50
mM Tris-Cl (pH 7.4), 150 mM NaCl, 5
mM EDTA, 0.5% NP-40, 1 mM phenylmethylsulfonyl
fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin]. The lysis
mixture was rotated 360o for 30 min at 4 C and then cleared
by centrifugation at 12,000 x g for 10 min at 4 C.
Anti-FLAG antibodies (20 µl) coupled to agarose beads (M2 Affinity
Resin; Eastman Kodak Co.) were added per 1 ml of lysate,
and the mixture was rotated slowly overnight at 4 C. The beads were
then pelleted by gentle centrifugation and washed three times with 1 ml
buffer A. After the final wash, the precipitated protein complexes were
resuspended in SDS-sample loading buffer, fractionated by SDS-PAGE, and
transferred to a nitrocellulose membrane by Western blot. Membranes
were screened with relevant antibodies and developed by enhanced
chemiluminescence (ECL system, Amersham, Arlington
Heights, IL) according to the manufacturers instructions.
Nuclear Extract Preparation and Purification of the TR/TRAP
Complex
HeLa cells and the HeLa-derived constituitively expressing
FLAG-hTR
cell line
-2 (38) were routinely maintained in DMEM/10%
FBS and DMEM/10% dialyzed FBS, respectively. Preparation of nuclear
extract from both cell lines and the subsequent immunopurification of
the TR/TRAP coactivator complex from
-2 cell nuclear extracts was
essentially as described (38).
 |
ACKNOWLEDGMENTS
|
---|
The authors wish to thank Hector DeLuca, Ron Evans, Chris Glass,
Bert OMalley, Keiko Ozato, Martin Privalsky, Herb Samuels, and
Ming-Jer Tsai for plasmids. The authors acknowledge R. G. Roeder
for plasmids originated in his laboratory and Zhao-jun Ren for the
construction of some of the expression vectors used here. We also thank
Dhan Kalvakolanu for critically reading the manuscript and helpful
discussions.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Joseph D. Fondell, Department of Physiology, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, Maryland 21201-1559.
This work was supported in part by Grant IRG97153-01 from the
American Cancer Society.
Received for publication December 28, 1998.
Revision received February 12, 1999.
Accepted for publication March 1, 1999.
 |
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