From the Department of Biosciences at Novum, Karolinska Institute,
S-14157 Huddinge, Sweden, the ¶ Department of Developmental
Biology, Tampere University Medical School and Department of
Pathology, Tampere University Hospital, P. O. Box 607, Fin-33101 Tampere, Finland, and the Department of Cell and
Molecular Biology, Medical Nobel Institute, Karolinska Institute,
S-17177 Stockholm, Sweden
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ABSTRACT |
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Transcriptional activation by nuclear receptors
(NRs) involves the concerted action of coactivators, chromatin
components, and the basal transcription machinery. Crucial NR
coactivators, which target primarily the conserved ligand-regulated
activation (AF-2) domain, include p160 family members, such as TIF2, as
well as p160-associated coactivators, such as CBP/p300. Because these coactivators possess intrinsic histone acetyltransferase activity, they
are believed to function mainly by regulating
chromatin-dependent transcriptional activation. Recent
evidence suggests the existence of an additional NR coactivator
complex, referred to as the thyroid hormone receptor-associated protein
(TRAP) complex, which may function more directly as a bridging complex
to the basal transcription machinery. TRAP220, the 220-kDa NR-binding
subunit of the complex, has been identified in independent studies
using both biochemical and genetic approaches. In light of the
functional differences identified between p160 and TRAP coactivator
complexes in NR activation, we have attempted to compare interaction
and functional characteristics of TIF 2 and TRAP220. Our findings imply
that competition between the NR-binding subunits of distinct
coactivator complexes may act as a putative regulatory step in
establishing either a sequential activation cascade or the formation of
independent coactivator complexes.
Nuclear hormone and orphan receptors
(NRs)1 comprise a large
family of transcription factors and participate in multiple aspects of
development and homeostasis of higher eucaryotic organisms but also in
deregulation of normal cellular functions (for review, see Refs. 1 and
2). They can be categorized into different subfamilies according to
characteristics such as nature of ligand, DNA response element, or
oligomerization status. Usually, steroid hormone receptors, which
mainly form homodimers, are distinguished from a large diverse
subfamily of receptors for nonsteroid ligands, such as thyroid hormone
(TR), retinoids (retinoic acid receptor and retinoid X receptor (RXR)),
and eicosanoids (peroxisome proliferator-activated receptor (PPAR)), as
well as many orphan receptors, for which ligands have not been
identified yet or do not exist. Unlike steroid receptors, most of these
NRs function as heterodimers with RXR and thus represent highly dynamic
transcription factor complexes due to the association of two receptor
subunits with distinct structural and functional features (3-6).
The majority of NRs utilizes two distinct domains for transcription
activation, located in the N and C termini, respectively: a
constitutive AF-1 and a ligand-regulated AF-2 as part of the multifunctional ligand-binding domain (LBD). NRs function in concert with multiple transcriptional cofactors, including basal transcription factors, corepressors, and coactivators (for review, see Refs. 7 and
8). Substantial progress in structural and functional analysis has
allowed a more detailed understanding of interactions between the AF-2
domain and associated cofactors and further revealed a conserved
mechanism for NR activation upon ligand binding (9-14). Briefly,
ligand activation is associated with structural rearrangements within
the LBD, causing the dissociation of corepressors and permitting the
recruitment of coactivators or other cofactors with regulatory functions (e.g. RIP140 (15)). In agreement with the
structural conservation of the coactivator interaction surface (10,
14), most AF-2 domain-binding proteins contain short conserved
LXXLL interaction motifs, referred to as the NR box (16,
17).
NR coactivators are envisaged to function within larger multiprotein
complexes (see, for example, Refs. 18-20). Multiple evidence suggests
the existence of at least two distinct coactivator complexes for the
ligand-regulated AF-2 domain referred to as the p160 complex and the
TRAP complex. The p160 complex is believed to integrate p160 family
members (e.g. SRC-1, TIF2, and ACTR/p/CIP), CBP/p300, and
PCAF (Refs. 17, 18, 21-26 and references therein). The composition of
the complex has been proposed on basis of the direct and functional
interconnection of all subunits, although the existence in
vivo has yet to be demonstrated. Because all these coactivators possess intrinsic histone acetyltransferase (HAT) activity and/or function in complex with other acetyltransferases, whereas corepressors apparently function in complex with deacetylases, functional
connections between NR activation and the histone acetylation status
have been proposed (for reviews, see Refs. 27 and 28).
A different NR coactivator complex, the TRAP complex, has been
biochemically identified as a TR-associated multiprotein complex and
demonstrated to function as coactivator in in vitro
transcription systems (20), suggesting a direct bridging function to
the basal transcription machinery. This complex consists of at least
nine different subunits and apparently does not contain p160/CBP
coactivators. The AF-2 domain-binding 220-kDa subunit of the TRAP
complex, referred to as TRAP220, was recently cloned (29) and found to
be identical to a putative NR coactivator named PBP/TRIP2, which we and
others independently have isolated in genetic two-hybrid screenings
using TR and PPAR, respectively (Refs. 30 and 31 and this study). To
get more insights into the relationship between TRAP220 and p160
coactivators, we have attempted to compare in this study the
interaction characteristics of TRAP220 and the p160 family member TIF2
in the context of the TR AF-2 domain, as well as in the context of the
TR/RXR heterodimer. We describe novel features of TRAP220 and suggest a
competition model that may also have relevance for the recruitment of
p160 and TRAP coactivator complexes to NRs and may further help to
integrate these different complexes into current NR activation models.
Plasmid Constructs--
All constructs were generated using
standard cloning procedures and verified by restriction enzyme analysis
and DNA sequencing. Details of each construction are available upon
request. For yeast expression, GAL4 DNA-binding domain (aa 1-147)
fusions to PPAR Cloning of TRAP220 cDNA--
Yeast two-hybrid screenings for
rPPAR In Situ Hybridization--
Adult male and female Sprague-Dawley
rats (n = 10) were decapitated, and the tissues were
excised and frozen on dry ice. Embryonic rats (days 12-21 of embryonic
development (e12-e21)) were excised from pregnant females and frozen.
Tissue section and in situ hybridization was carried out
essentially as described previously (37) using two different
oligonucleotide probes directed against the TRAP220 mRNA. The
probes produced similar results when used separately and were usually
combined to intensify the hybridization signal. Several probes of
nonrelated mRNAs with known expression patterns, similar length,
and similar GC content were used as controls to verify the specificity
of the hybridizations.
Antibody Production, Western Analysis, and
Immunoprecipitations--
Purified His-tagged TRAP220 protein (aa
503-718) was used to immunize rabbits (Zeneca). Affinity-purified
polyclonal antibodies (aP9) were prepared using a GST-TRAP220 protein
covalently coupled to cyanogen bromide-activated Sepharose 4B (Amersham
Pharmacia Biotech) according to the manufacturer's instructions.
Antibody specificity was tested using recombinant proteins and whole
cell extracts. For Western analysis, whole cell extracts were prepared as described fractionated by SDS-PAGE, and proteins were transferred onto a nitrocellulose filter (Amersham Pharmacia Biotech). Filters were
blocked with 5% milk powder in PBS-Tween 80 and incubated with a
1:1000 dilution of anti-TRAP220 (aP9) or anti-His6 (Santa Cruz) in PBS/Tween80 for 60 min at room temperature. After washing, the
filters were incubated with horseradish peroxidase-conjugated secondary
anti-IgG antibody (Amersham Pharmacia Biotech) at a dilution of 1:2000
for 60 min. After washing, the proteins were visualized with x-ray film
using an enhanced chemiluminescence system (ECL, Amersham Pharmacia
Biotech). For immunoprecipitations, whole cell extracts (usually 0.2 mg) prepared from untransfected 293 cells in radioimmune precipitation
buffer (50 mM Tris-HCl, 150 mM NaCl, 1%
Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, pH 8.0) supplemented with
protease inhibitors ("Complete" Boehringer Mannheim) were
precleared (30 min at 4 °C) with preimmune serum and
immunoprecipitated (2 h at 4 °C) using anti-TRAP220 serum or
purified antibody (aP9) and protein A-Sepharose (Amersham Pharmacia Biotech). Immnuoprecipitates were washed three times and subjected to
Western analysis.
Protein-Protein Interaction Assays in Solution and on
DNA--
Purification of GST- and His-tagged proteins and GST
pull-down assays were essentially performed as described previously
(15, 36) and as indicated in the legends to Figs. 4, 6, and 8.
DNA-dependent interaction assays were carried out according
to the method of Kurokawa et al. (4) with minor
modifications. Approximately 50 µg of COS-1 cell extract transiently
expressing TR Surface Plasmon Resonance (SPR) Analysis of Direct
Protein-Protein Interactions in the BIAcore--
SPR analysis was
performed using a BIAcore 2000 system (Amersham Pharmacia Biotech)
according to the BIAcore instruction manual. Purified proteins were
diluted in running buffer HBS (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, and 0.05% Tween-20, pH
7.4). The flow rate was 5 µl/min. Anti-GST antibody was immobilized
on research grade CM5 sensor chips using the amine coupling kit and the
GST capture kit provided by the manufacturer. The immobilization
procedure was as follows: 30 µl of NHS/EDC mixture was injected to
activate the surface, followed by 40 µl of diluted anti-GST antibody,
and finally 35 µl of ethanolamine was injected to block unreacted groups on the chip. After immobilization, GST, GST-TRAP220 (aa 425-718), or GST-TIF2 (aa 594-766) was bound to individual flow cell
surfaces. 50 µl of purified His-tagged TR
To look for competition between TRAP220 and TIF2, GST or GST-TRAP220
was captured as described above. 70 µl of TR Electrophoretic Mobility Shift Assays (EMSAs)--
EMSAs were
performed under previously described conditions (15, 36) with the
following modification: end-labeled doubled-stranded oligonucleotides
containing DR4-TRE were incubated with either TR Mammalian Cell Culture and Transient Transfections--
All cell
lines except 293 cells were maintained in Dulbecco's modified Eagle's
medium supplemented with 10% fetal calf serum (Life Technologies,
Inc.). 293 cells were cultured in Ham's F-12 nutrient mixture and
Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
supplemented with 10% fetal calf serum and 0.5% nonessential amino
acids. Both media contained 100 µl/ml penicillin and 100 µl/ml
streptomycin. Transfections were performed as previously described (15)
using phenol red-free medium with 1 µg of reporter plasmid UAS-tk-luc
(6) and the indicated amounts of expression vectors in the absence or
presence of ligands.
Yeast Coactivation Assay--
pYEX-based cofactor expression
plasmids were introduced into the Saccharomyces cerevisiae
reporter strain Y187 (MAT Cloning of TRAP220
Partial cDNA clones encoding AF-2 domain-interacting fragments
of TRAP220/PBP (aa 503-754) and TIF2/GRIP1 (aa 320-1119) were isolated in two-hybrid protein-protein interaction screenings using the
rat PPAR
INTRODUCTION
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Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
and TR
have been previously described (15).
GAL4-TRAP220 was generated by insertion of a partial EcoRI
fragment (aa 425-973) into pGBT9 or pAS2-1
(CLONTECH). For yeast coactivation studies, TRAP220
(aa 425-973), mTIF2/GRIP1 (aa 322-1121), and hRIP140 (aa 1-1158)
were cloned into the NLS-vector pYEX (15). For mammalian expression,
pSG5-GAL4-TRAP220 was derived from pYEX. TRAP220 constructs were made
by insertion of original cDNA fragments (TRAP220, aa 222-1582;
dominant negative TRAP220, aa 579-667) into pSG5 (Stratagene) or have
been described before (31). mTIF2 and hRIP140 expression plasmids have
been previously described (15, 32). Nuclear receptors for mammalian
expression and in vitro translation have been expressed from
the following previously described plasmids: pGEM/pCMVhTR
/
,
pBKCMV rPPAR
/
(15), pCMV/pGEM rRXR
, and pGAL4-hTR
(6). The
luciferase reporter construct GAL4-UAS-tk-luc has been previously
described (6).The following plasmids have additionally been used as
templates for in vitro translation of wild-type cofactors
and polymerase chain reaction-generated partial clones: pBSK-mCBP (33),
pE1A13S (34), pcDNA-ADA2 (35), pGEX-hTBP, and pGEX-hTFIIB (15). For
bacterial expression, GST fusions were created by insertion of
polymerase chain reaction fragments into the following pGEX vectors
(Amersham Pharmacia Biotech): pGEX-hTR
(aa 122-410), pGEX-rPPAR
(aa 175-475) (15), pGEX-TRAP220 (aa 425-718 and aa 425-973),
pGEX-TIF2 (aa 594-767), pGEX-CBP (C1, aa 1678-2441; C2, aa
1678-1868; C3, aa 2058-2170). His-tagged TRAP220 (aa 503-718) is
derived from pET15B (Novagen).
interacting proteins using a mouse embryo day 17.5 cDNA
library (CLONTECH) were essentially performed as
described previously (15, 36). RXRs were detected using specific
primers by polymerase chain reaction directly from the yeast colonies
in more than 50% of the growth positives. Library plasmid DNA from 26 positive non-RXR clones was isolated, and inserts were sequenced. To
obtain full-length cDNAs,
-ZAP cDNA libraries from rat
thymus (Stratagene) or rat brown adipose tissue (a kind gift of H. M. Gardiola-Diaz) were screened using the partial mPIP9/TRAP220
two-hybrid clone. Overlapping clones were assembled to the full-length
TRAP220 cDNA encoding an putative open reading frame of 1582 aa,
which is more than 95% identical to the reported sequence of the mouse
homologue PBP (31).
was incubated with 1 µg of double-stranded
biotinylated DR4 oligonucleotides. The complex was immobilized on
streptavidin MagneSphere paramagnetic beads (Promega) and used to
analyze binding of [35S]methionine-labeled in
vitro translated proteins. After washing, the radiolabeled
proteins were eluted, separated by SDS-PAGE, and detected by
fluorography. To detect TRAP220 proteins from cell extracts, bound
proteins were separated by 7.5% SDS-PAGE and subjected to Western
analysis using the TRAP220-specific antibody aP9.
LBD (aa 197-456; 0.1 mg/ml HBS containing 1 µM TR ligand T3), kindly provided
by S. Nilsson (KaroBio), was injected over each surface (multichannel mode). The amount of TR protein that associated with the captured GST
proteins was quantified by measurement of the SPR signal in resonance
units (RU).
(0.1 mg/ml) was
injected over the two surfaces. Finally, 60 µl of TIF2 (0.1 mg/ml),
which was obtained by thrombin cleavage of the GST-TIF2 protein, was
injected. Additional experimental details are described under
"Results" and in the legend to Fig. 5.
/RXR
for 10 min
at room temperature in the absence or presence of ligands. Purified
cofactor or control proteins (0.1-1.0 µg/reaction) were added last,
and binding was allowed to proceed for 20 min.
, ura3-52, his 3-200, ade2-101,
trp1-901, leu2-3, 112, gal4-542, gal80.538, URA3::GAL1-lacZ) bearing various GAL4 constructs.
Quantitative liquid
-galactosidase assays and all standard yeast
manipulations were essentially as described (15, 36).
RESULTS
as bait and a mouse embryo cDNA library (for details,
see under "Experimental Procedures" and Ref. 36). To isolate the
TRAP220 full-length cDNA, we screened two
cDNA libraries (derived from rat thymus or brown adipose tissue mRNA,
respectively). Alignment of six independent overlapping cDNA clones
revealed a putative open reading frame of 1582 aa with a predicted
molecular mass of 156 kDa. Cloning of the full-length rat TIF2 (1464 aa) has been described previously (36). A schematic illustration of the
structural organization and functional characteristics of the two
coactivators is shown in Fig. 1. Except
for the central NR interaction domain containing two (TRAP220) or three
(TIF2) LXXLL motifs (NR box), TRAP220 and TIF2 are not
structurally related to each other. TIF2 contains an N-terminal
bHLH-PAS domain, possibly serving oligomerization and DNA binding
functions. Other characteristics are two transcription activation
domains (AD1 and AD2, respectively) located in the C terminus. Although
AD1, at least in part, may function through the recruitment of
CBP/p300, specific target proteins for AD2 are not known (26). Except
for the NR interaction domain, several putative nuclear localization
signals as well as multiple putative phosphorylation and
N-glycosylation sites, no other domain indicating specific
functions could be recognized in TRAP220. Compositional sequence
analysis identified serine as the dominant amino acid residue (277 aa
or 17.7% of the protein). The TRAP220 C terminus, which includes a
serine-rich region followed by a mixed charged cluster, has been
suggested to be responsible for oligomerization, DNA binding (although
unspecific), and binding to the tumor suppressor protein p53 (38).
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Fig. 1.
Structural organization of TRAP220 and
TIF2. Highlighted are the NR interaction domains containing
conserved NR box motifs; also highlighted are, in case of TRAP220, a
serine-rich region and a charged cluster containing alternating
positively and negatively charged residues, and in case of TIF2, the
putative transcriptional activation domains AD1 and AD2 separated by a
glutamine-rich region, as well as an N-terminal region with homology to
basic helix-loop-helix PAS proteins (for details, see under
"Results").
Expression of TRAP220 in Tissues and Cell Lines
TRAP220 mRNA-- To study the expression of TRAP220 mRNA, in situ hybridizations on rat embryo and adult tissue sections were performed. As shown in Fig. 2, TRAP220 mRNA was widely expressed during embryonic development. At day 12 of embryonic development (e12), high expression could be seen in neural epithelium of the neural tube and in limb buds, whereas lower expression was seen in placenta and visceral yolk sac (Fig. 2A). At later stages of development (e18) (Fig. 2B), TRAP220 mRNA appeared to be highly expressed in thymus, brown adipose tissue, kidney, bladder, the central nervous system (especially the forebrain), and the epithelium of the oral cavity, esophagus, stomach, and intestine. In lung, the expression was stronger in the bronchial epithelium than in surrounding mesenchyme. The expression in liver was moderate, whereas heart and the large blood vessels showed low expression. In adult rat, the expression of TRAP220 mRNA was more restricted than during embryonic development. High expression was observed both in male and female rat genital organs. In testis, seminiferous tubules exhibited a strong signal, and the expression in separate tubules varied, showing that TRAP220 is expressed in a stage specific manner during spermatogenesis (Fig. 2C). In dipped sections, TRAP220 mRNA could be detected in primary spermatocytes, whereas all other testicular cell types were nonlabeled (Fig. 2D). In ovary, the strongest signal was obtained in secondary and tertiary follicles, whereas the more mature follicles had lower expression (Fig. 2E). Also, the corpora lutea had moderate expression. In dipped sections, TRAP220 mRNA was abundant in oocytes and granular cells (Fig. 2F). In lymphoid organs, strong expression was observed in thymus and lymph nodes, whereas lower expression could be seen in white bulb of spleen (Fig. 2, G-I). In endocrine glands, a strong signal was evident in the pituitary, adrenal, and parathyroid glands, whereas the thyroid gland had low levels of TRAP220 mRNA (Fig. 2, J-L). In pituitary, the anterior and intermediate lobes were labeled, and the posterior lobe was nonlabeled. In adrenal gland, the cortex was strongly positive, and the medulla had low expression. In cortex, the zona glomerulosa and zona reticulata exhibited a higher expression of TRAP220 mRNA than the zona fasciculata. In the central nervous system, high expression was present in olfactory bulb, hippocampus, and cerebellar cortex, whereas other areas exhibited low levels of TRAP220 mRNA. In the peripheral nervous system, the expression was low in sympathetic and sensory ganglia. Additional support for the relatively broad expression of TRAP220 mRNA came from reverse transcription polymerase chain reaction and Northern blot analysis. TRAP220 mRNA was detected in all tissues analyzed, with apparently higher levels in brown and white adipose tissue, brain, and reproductive organs and apparently lower levels in liver and kidney (data not shown), consistent with the levels reported for the human transcript in these tissues (29, 38).
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TRAP220 Protein-- To detect the endogenous TRAP220 protein and to determine its native size, specific polyclonal antibodies were raised against the purified NR interaction domain (aa 503-718; see under "Experimental Procedures"). In Western blot experiments, a single 220-kDa protein could be identified using whole cell extracts and nuclear extracts from various mammalian cell lines of different origin (Fig. 3A) or rat tissues (data not shown). Additionally, endogenous TRAP220 could be immunoprecipitated specifically using anti-TRAP220 serum or purified IgG, but not using preimmune serum (Fig. 3B), confirming the identity of the protein encoded from the rat TRAP220 cDNA with a cellular, probably nuclear, 220-kDa protein. The obvious discrepancy with the predicted molecular mass of 156 kDa suggests protein modification or aberrant mobility, probably due to the high serine content, and has also been noticed for the human TRAP220/RB18A (29, 38). In conclusion, TRAP220 seems rather ubiquitously expressed both at the mRNA level and at the protein level.
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Recently, a vitamin D receptor-interacting protein (DRIP) complex, which is probably identical to the previously identified TRAP complex based on its interaction with NRs, and its protein composition has been identified using a pull-down approach (39). Far-Western experiments aimed at defining the protein(s) that directly contacts the receptor identified TRAP220 in case of the TRAP complex but a 180-kDa protein in case of the DRIP complex (29, 39). These seemingly contradictory results may have the following explanation: in some cell extract preparations (Fig. 3C, lane 5) or when cell extracts were incubated under pull-down conditions (see Fig. 4A), the TRAP220 antibody recognized a second band of approximately 170-180 kDa in addition to the 220 kDa band. Subsequent control experiments using cell extracts (Fig. 3C) or in vitro translated protein (data not shown) incubated in the presence or absence of protease inhibitors strongly suggest that p180 is a breakdown product of TRAP220. The use of different cell lines and different experimental conditions to isolate the TRAP and the DRIP complexes, respectively, may help to explain the varying ratio between wild-type TRAP220 and the 180-kDa fragment. These results further imply that part of the protein fraction in the range of 160-180 kDa, designated p160, detected in biochemical interaction assays with ligand-activated NRs (4, 40-43), in addition to p160 coactivators, perhaps contains degradation products of larger proteins, such as TRAP220.
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Competitive Interactions between TRAP220 and TIF2 with TR
TRAP220 was originally isolated using yeast two-hybrid screenings
with either TR (as hTRIP2 (30)) or PPAR (as mPBP (31) or mPIP9 (this
study)). Obvious discrepancies were observed between the two NRs with
regard to the ligand dependence of their interactions with TRAP220 in
yeast (data not shown), which we believe is due to (ligand-) activated
PPAR in this system (15, 36). Therefore, we wished to compare the
interaction with both receptors in vitro, using the GST
pull-down approach. As seen in Fig. 4A (lanes
1-6), full-length translated TRAP220 interacted equally strongly
and ligand-dependently with either TR or PPAR
AF-2
domain. To assess whether endogenous TRAP220 from whole cell extracts
would bind the AF-2 domain in a similar fashion, we used the same
GST-receptor fusion proteins as affinity matrix to enrich interacting
proteins from a 293 cell extract (Fig. 4A, lanes 7-11).
After washing, bound proteins were eluted and subjected to SDS-PAGE and
Western blot analysis using the TRAP220 antibody. Endogenous TRAP220
could be specifically enriched in the presence of ligands, but not in their absence, indicating that endogenous cellular TRAP220 and TRAP220
synthesized by in vitro translation exhibit identical interaction characteristics. Additional pull-down assays, performed in
a reciprocal arrangement using a GST-TRAP220 fusion protein (aa
425-718) and in vitro translated TR, confirmed that the
interaction was stable and ligand-dependent
(i.e. over a range of salt concentrations up to 1 M NaCl), irrespective of the source of either protein or
the ratio between receptor and cofactor (data not shown).
These and previous interaction studies (29, 31) strongly suggest that
TRAP220 exhibits interaction characteristics resembling those of p160
coactivators, including TIF2 (Ref. 36 and references therein).
Furthermore, considering the likelihood that TRAP220 and TIF2 are
coexpressed together in tissues and cell lines, we wanted to examine
the interaction between both cofactors and TR in more detail. The first
set of experiments was designed to elucidate whether TRAP220 and TIF2
bind to the same site on the TR AF-2 domain and involved in
vitro competition experiments based on the GST pull-down assay
(see under "Experimental Procedures"). As evident from the data
presented in Fig. 4B, purified TRAP220 NR interaction domain
(aa 503-718) efficiently inhibited binding of either in
vitro translated TIF2, SRC-1, or CBP to a purified GST-TR fusion
protein. Competition could further be demonstrated in the reciprocal
arrangement using purified TIF2 NR interaction domain (aa 594-766) and
in vitro translated wild-type TRAP220 (data not shown).
These experiments indicate that TRAP220 and TIF2 bind mutually
exclusively and competitively to the TR AF-2 domain. As an important
control, binding of the heterodimerization partner RXR was not
affected, consistent with the separation of dimerization and
coactivator-binding surfaces within the LBD/AF-2 domain and with the
formation of ternary complexes on RXR heterodimers (see below).
In the next set of experiments, we analyzed direct binding and
competition using stoichiometrical amounts of purified proteins on a
BIAcore instrument by SPR analysis (for details, see under "Experimental Procedures" and the legends to Fig. 5). Fig.
5A illustrates the binding of
liganded TR to the NR interaction domains of either TRAP220 (aa
425-718) or TIF2 (aa 594-766). Although the binding kinetics will be
subject of further experimentation, the SPR sensorgram (for
explanation, see the legend to Fig. 5) indicated comparable binding of
liganded TR to both GST-TRAP220 and GST-TIF2. For the control,
virtually no binding was observed to GST control protein under similar
conditions (data not shown). The experiment shown in Fig. 5B
analyzed direct competition between TRAP220 and TIF2. Specifically,
increasing amounts of free TIF2 interaction domain (not fused to GST)
could efficiently dissociate the preformed TR-TRAP220 complex, strongly
suggesting competition between the two cofactors for binding to a
common binding site on the TR AF-2 domain. Importantly, competition was
not observed using a TIF2 protein carrying interaction-deficient NR box
point mutations (data not shown and Ref. 36).
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Binding studies of NR coactivator interaction with DNA-bound wild-type NR dimers may reflect the situation in vivo much better than experimental designs using only the LBD/AF-2 domain, and the last set of experiments was thus designed to investigate whether competition between TRAP220 and TIF2 also occurred on the DNA-bound TR/RXR heterodimer. First, we ascertained the interaction of TRAP220 or TIF2 with the TR/RXR heterodimer bound to a DR4 response element using an EMSA. As seen in Fig. 6A, addition of purified TRAP220 or TIF2 interaction domain, but not of GST control protein, to the binding reaction supershifted the TR/RXR complex, indicating the formation of a ternary complex. Note that ligand for either receptor alone or in combination could efficiently promote formation of always the same ternary complex. Ternary complex formation with either cofactor was also observed with other heterodimers, for example with PPAR/RXR or OR1/RXR (data not shown). To support the relevance of EMSA experiments utilizing partial cofactor fragments for the interaction characteristics of the wild-type cofactors, we compared the interactions of wild-type TRAP220 or TIF2 with DNA-bound receptor dimers using the following DNA-dependent protein-protein interaction assay (Fig. 6B): TR/RXR dimers from cell extracts were assembled onto biotinylated response elements, immobilized on streptavidin beads, and incubated with either in vitro translated TRAP220 or TIF2 in the absence or presence of ligand. Subsequent fluorography revealed ligand-dependent association of both in vitro translated TRAP220 or TIF2 with the TR dimer on DNA.
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These results suggest that ternary complex formation of both TRAP220 and TIF2 with DNA-bound NR heterodimers may follow similar stoichiometrical principles. Furthermore, assuming that both cofactors bind to each receptor subunit in the TR/RXR heterodimer with comparable affinities, competition should occur in the context of the dimeric receptor-DNA complex. This is shown in Fig. 6C: TRAP220 and TIF2, which form distinguishable ternary complexes with different mobility, clearly bind competitively and mutually exclusive to TR/RXR, as indicated by the absence of higher order or intermediary complex formation under any conditions. Although the stoichiometry of the coactivator-dimer complex remains to be established by other methods, the competition, as well as the mobility, of the ternary complexes described here is compatible with each of two alternative binding models for NR box-containing cofactors, i.e. binding of only one or two molecules of cofactor to dimeric receptors (see under "Discussion").
TRAP220 and TIF2 Function as AF-2 Coactivators in Yeast and in Mammalian Cells
Recent studies have established a general coactivator role for TIF2 both in the context of wild-type receptors as well as in the context of the AF-2 domain alone (26, 32, 43). Transient transfection studies in mammalian cells have demonstrated that also TRAP220 can serve as a moderate coactivator for PPAR or TR (29, 31), which is in line with our own results obtained using TR/RXR and retinoic acid receptor/RXR heterodimers (data not shown). Because both TRAP220 and TIF2 are thought to act mainly as coactivators for the ligand-regulated AF-2 domain, we decided to directly assess the coactivation potential of both cofactors in the AF-2 context in both yeast and mammalian cells. In yeast (Fig. 7A), a GAL4-TR AF-2 fusion did not efficiently activate transcription of the integrated lacZ reporter gene, most likely due to the lack of specific AF-2 coactivators such as p160 or TRAPs. However, coexpression of either TRAP220 (aa 425-973) or TIF2 (aa 320-1190), but not of the negative AF-2 coregulator RIP140 (15), led to a substantial increase in AF-2 activity in the presence of ligand. In addition to TR, TRAP220 and TIF2 coactivated the AF-2 of other GAL4 receptor fusions such as those with PPAR (data not shown).
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In mammalian cells (Fig. 7B), a similar GAL4-TR fusion protein exhibited transcriptional activity in response to ligand, as expected from the presence of AF-2 coactivators in cell lines (Fig. 3A). Nevertheless, coexpression of TRAP220 could potentiate the transcriptional activity 2-3-fold, which is similar to the coactivation level previously observed for wild-type TR (29). Moreover, the magnitude of coactivation seen with TRAP220 was comparable to that observed with TIF2, although we cannot exclude differences in expression or stability of both coactivators. In contrast, RIP140 down-regulated AF-2 activation, consistent with the idea that it might act as a dominant negative coregulator by competition with endogenous coactivators (15). Similar dominant-negative effects were obtained when expressing the TRAP220 NR interaction domain (aa 579-667) alone, supporting the relevance of the competition results demonstrated above in vitro (see Fig. 6). Taken together, these experiments reflect the capability of TRAP220 to function as a coactivator for the AF-2 domain of NRs, comparable to the activity seen with TIF2 under similar experimental conditions.
TRAP220 Contains Intrinsic Transcriptional Activity and Interacts with CBP, ADA2, and TBP
To investigate whether TRAP220, as implicated by the yeast coactivation experiment, would activate transcription when tethered to DNA directly, we fused the TRAP220 coactivator fragment (aa 425-973) to GAL4 and assessed its transcriptional activity in yeast and in mammalian COS-1 cells (Fig. 8A). In both systems, we observed appreciable intrinsic activity compared with GAL4 alone. Consistent with the coactivation data, the intrinsic activity of TRAP220 was comparable to that of the TIF2 coactivator fragment (aa 322-1121), whereas RIP140 only had negligible activity under these conditions (15). In view of the hypothesis that NR-specific coactivators recruit other coactivators and coactivator complexes, basal transcription factors or TBP, we tested several potential target proteins by analyzing their in vitro binding to GST-TRAP220 (aa 425-973). Using that approach we identified interactions with the CBP C terminus (aa 1678-2441), ADA2, and TBP but not with TFIIB (Fig. 8B). Furthermore, because the CBP C terminus contacts and the p160 coactivators (including TIF2), and the adenovirus E1A protein, which recently has been implicated to negatively regulate NR activity (18), we examined the interaction of three C-terminal CBP fragments (C1-C3) fused to GST with TRAP220 or with E1A. As it is evident from the pull-down experiment shown in Fig. 8C, TRAP220 and E1A displayed a similar interaction profile and interacted strongly with CBPC2 (aa 1678-1868), but not with the p160 interaction domain CBPC3 (aa 2058-2170). These data indicate that TRAP220 contains activation domains, which in turn may contact putative activation domain targets, such as CBP, ADA2, and TBP (Ref. 35 and references therein). Although TIF2 contacts CBP (albeit a different subdomain) through its AD1, targets for the second activation domain AD2 have not been identified (26). Thus, TRAP220 and TIF2 may essentially function as coactivators utilizing both identical (CBP/p300) but also different putative transcription targets, which is consistent with the lack of any sequence homology between the cofactors within their putative activation domain(s) and which is further consistent with the envisaged differential roles of p160 and TRAP coactivator complexes in NR activation (see under "Discussion").
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DISCUSSION |
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Similar Stoichiometrical Principles Dictate Interactions of TRAP220
and TIF2 with NRs--
Recent studies by Roeder and co-workers (29)
indicate that TRAP220 is responsible for anchoring other TRAPs to the
TR AF-2 domain and, in addition to TR, interacts with many NRs, which is in agreement with previous studies (30, 31, 39). As in case of
p160s, the interactions of TRAP220 depend on the integrity of the NR
box motifs (17, 25, 26, 29). Previous work has characterized some
mechanistic principles in NR box mediated interactions of p160
coactivators including TIF2 with NR dimers (Ref. 36 and references
therein). Although certain AF-2-binding proteins (for example, TIF1
(44)) contain only one NR box and would be expected to bind in a
two-to-two stoichiometry to activated NR dimers, all p160 coactivators,
as well as TRAP220, contain at least two NR box motifs within the
central NR interaction domain, separated by approximately 40-50
residues. This may allow them to bind in an also energetically
favorable one-to-two ratio, i.e. binding of one coactivator
molecule per NR dimer, as suggested recently in structural studies
analyzing binding of the SRC-1 NR box domain to the dimeric PPAR
LBD/AF-2 domain (10). Both alternatives are compatible with the
envisaged binding of one NR box motif to the conserved
coactivator-binding surface within the AF-2 domain of each monomeric
receptor subunit (10, 14).
Irrespective of the binding mode, our study suggests that TRAP220 and TIF2 bind with comparable affinities to the TR AF-2 domain, as well as to TR/RXR heterodimers, allowing them efficiently to compete for binding. Competition most likely explains why in previously described biochemical far-Western-based or pull-down-based interaction assays, p160s and associated cofactors are excluded from the TRAP (and DRIP) complex, and vice versa (20, 29, 39). Although p160 and TRAP subunits appear to be ubiquitously expressed in most tissues and cell lines (see, for example, Refs. 29 and 45), competition, together with expression differences at the protein level, may contribute to cell type-specific interaction patterns. In light of recent reports describing affinity differences between NRs with regard to p160 coactivators and CBP/p300 (46), future quantitative binding studies on TRAP220, as well as other coactivators, will be required to determine carefully their affinity to different NR dimers in the context of different ligand scenarios and bound to different DNA response elements. Further, it also needs to be examined whether protein-protein interactions within the TRAP complex influence the binding affinity of TRAP220 to the AF-2 domain. Such differences have been observed, for example, in case of ADA2 as part of the yeast GCN5 complex (Ref. 47 and references therein). To our knowledge the interaction characteristics of TR with isolated TRAP220 correspond well to those observed with the entire TRAP complex (29, 39).
Implications for NR Activation Models--
The recent discovery
that all components of the putative p160 complex exhibit HAT activity
strongly suggests functional connections to chromatin by modification
of histones (for references, see Refs. 27 and 28). Intriguingly, it is
now known that even non-histone proteins, such as the general
transcription factors THIIE and TFIIF (48) or the tumor suppressor
protein p53 (49), can become acetylated. Thus, it is tempting to
speculate that transcriptional activation mediated by p160 and
associated coactivators is more than just a matter of chromatin
modification. In case of the TRAP complex, its coactivator function in
in vitro transcription systems strongly suggests direct
links to the basal transcription machinery (20, 29). In support of a
direct bridging function, we demonstrated here that TRAP220 exhibits
intrinsic transcriptional activity and may contact putative targets,
such as TBP, ADA2, or CBP. Intriguingly, each of these proteins is
considered a putative target factor for "classical" transcription
activation domains (Ref. 35 and references therein), and TRAP220 may
function in conjunction with other TRAPs as a true bridging factor
between the unique AF-2 activation domain of NRs and the RNA polymerase II complex. Although no evidence has been provided yet for functional connections of the TRAP complex to chromatin, it is interesting to note
that the TRAP-related DRIP complex apparently exhibited HAT activity
(39) and that we have observed in vitro interactions of
TRAP220 to CBP and ADA2, which exhibit intrinsic HAT activity and
function within HAT complexes, respectively (33, 47, 50, 51).
Under consideration of envisaged functional differences between p160 (HAT) complexes and the TRAP complex, Roeder and co-workers (29) recently proposed a sequential multistep activation model for NRs. Briefly, in the absence of activating ligands, NRs remain transcriptionally inactive or repress due to the binding of corepressors, which may connect them to histone deacetylase complexes and chromatin repression mechanisms. Upon ligand binding or other activating signals, structural changes within the LBD cause dissociation of corepressors and promote the association of chromatin-modifying HAT complexes containing p160 coactivators, CBP/p300, and/or PCAF/GCN5 to NRs. Subsequently, HAT complexes are suggested to work either in concert with or (consistent with our competition model) to be replaced by the TRAP complex, which now directly connects NRs to the RNA polymerase II complex. Because our results indicate competitive interactions of TRAP220 and TIF2 with NRs, the proposed sequential activation model would require regulated changes in either the relative affinity or the relative availability of TIF2 and TRAP220 to their receptor targets.
Although a sequential NR activation model is very attractive, we would not exclude the possibility that NRs may use p160 or TRAP complexes as alternative coactivator complexes. In such a model, both complexes would independently fulfill all functions required for transcriptional activation. In view of the coexpression of p160 and TRAP proteins in many tissues, it could be relevant for receptors that display different affinities to different coactivators. Furthermore, in light of the envisaged complex mode of interactions of NR box domains with NR dimers (see above), dimer-specific affinity differences and additional influences of ligands, binding sites, or competitive coregulatory proteins have to be considered. Additional support for the alternative model comes from the possibility that TRAP220 contacts CBP/p300 and ADA2. As suggested earlier, p160 coactivators might be required for the recruitment of CBP/p300 to NRs (52). Because we demonstrated that TRAP220 interacts with CBP in vitro, it may be able not only to displace p160s from NR heterodimers, but it may also substitute for these coactivators in fulfilling a bridging function between NRs and CBP/p300. Moreover, because ADA2 is part of the PCAF/GCN5 complex (47, 53), and because we observed interactions of TRAP220 to ADA2 via its putative activation domain(s), it is possible that TRAP220 may function even without the TRAP complex. This is consistent with the functionality of TRAP220 in yeast.
Finally, it is interesting to note that the TRAP220 interaction domain
of CBP (aa 1678-1868) contains docking sites for other cofactors
implicated to be directly or indirectly involved in certain aspects of
NR signaling: PCAF (54, 55), RNA helicase A, which may serve as direct
bridging molecule to the RNA polymerase II complex (56), and the
adenovirus E1A protein, which competes with PCAF (54) for binding to
CBP. It was recently suggested that E1A might inhibit NR activation in
part through competition with p160/SRC-1 coactivators for CBP binding
(18). Our new findings imply that E1A may even compete for binding of
TRAP220 to CBP. However, because CBP was not found within the TRAP
complex (20), future studies have to investigate the relevance of our
in vitro data for the function of TRAP220 in
vivo, including the consequences of E1A expression on
TRAP220-dependent coactivator function. Additionally, the
functional interconnection or independence of both TRAP and p160
coactivator complexes has to be addressed experimentally by directly
comparing their functionality in, for example, in vitro
transcription systems utilizing chromatin and non-chromatin templates,
or by performing comparable in vivo inhibition studies, for
example by microinjection of antibodies and by generation of knockout
animals for individual complex subunits.
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ACKNOWLEDGEMENTS |
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We thank Drs. B. W. O'Malley, R. M. Evans, J. K. Reddy, T. Kouzarides, F. Saatciogliu, P. Kushner, and A. Berkenstam for providing plasmids. We acknowledge Dr. R. G. Roeder for stimulating discussions and comments during the progress of this work. We are extremely grateful to Dr. S. Nilsson (KaroBio) for providing purified TR protein and plasmids. We thank members of the Nuclear Receptor Unit at Novum for providing materials and helpful suggestions. We acknowledge U. Jukarainen for expert technical assistance.
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FOOTNOTES |
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* This work was supported by a postdoctoral fellowship of the Deutsche Forschungsgemeinschaft (to E. T.) and by grants from the Medical Research Fund of the Tampere University Hospital (to M. P.-H.), from the Wenner-Gren Foundation (to M. S.), and from the Swedish Cancer Society (to J.-Å. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 46-8-608-9160;
Fax: 46-6-774-5538; E-mail: eckardt.treuter{at}csb.ki.se.
§ These authors contributed equally to this work.
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ABBREVIATIONS |
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The abbreviations used are: NR, nuclear receptor; TR, thyroid hormone receptor; TRAP, TR-associated protein; RXR, retinoid X receptor; PPAR, peroxisome-proliferator activated receptor; PBP, PPAR-binding protein; LBD, ligand-binding domain; AF, activation function; TIF, transcriptional intermediary factor; CBP, CREB-binding protein; SRC, steroid receptor coactivator; HAT, histone acetyltransferase; DRIP, vitamin D receptor-interacting protein; RIP, receptor-interacting protein; TBP, TATA box-binding protein; GST, glutathione S-transferase; EMSA, electrophoretic mobility shift assay; aa, amino acid(s); PAGE, polyacrylamide gel electrophoresis; RU, resonance unit(s); SPR, surface plasmon resonance.
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REFERENCES |
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