From the The retinoid X receptor (RXR) influences gene
activation through heterodimeric and homodimeric association with DNA
and associates with TATA binding protein, TAF110, and cAMP response
element-binding protein-binding protein; yet the molecular mechanisms
responsible for gene activation by RXRs remain incompletely defined.
Since the general transcription factor IIB (TFIIB) is a common target of sequence-specific transcriptional activators, we suspected that RXR
might regulate target genes via an interaction with TFIIB. Co-immunoprecipitation, far Western analysis, and glutathione S-transferase binding studies indicated that murine RXR Nuclear hormone receptors are ligand-dependent
transcription factors that regulate numerous cellular functions,
including growth, development, differentiation, reproduction, and
metabolism (Refs. 1-3 and references therein). Ligand binding to
nuclear hormone receptors leads to assembly of a functional
preinitiation complex (PIC)1 and subsequent RNA
transcription in vitro (4, 5).
Activated transcription of RNA polymerase II (RNAPII) genes requires
binding of transcription factor IID (TFIID) to the TATA element, which is facilitated by TFIIA, and stepwise assembly of the following transcription factors: TFIIB, RNAPII/TFIIF, TFIIE, and TFIIH (reviewed in Refs. 6 and 7). TFIID includes the TATA binding protein (TBP)
associated factors (TAFs) (Ref.7, and references therein). Interaction
of nuclear receptors with the basal transcription complex may occur
directly (8-14) or in association with TAFs (15) or other cofactors
(Refs. 16-25; reviewed in Refs. 26 and 27). Ligand binding to the
receptor results in recruitment of factors that facilitate assembly of
an activated complex (28-30) or, conversely, the release of repressor
molecules, such as SMRT (16) or N-CoR (17, 18) that inhibit
transcription (16-18, 31-32). A significant advance was the
demonstration that TFIIB interacts with the estrogen receptor (10),
progesterone receptor (10), chicken ovalbumin upstream
promoter-transcription factor (10), thyroid hormone receptor (TR) (11,
31), and vitamin D receptor (VDR) (12-14). The interaction of nuclear
hormone receptors with TFIIB is significant since recruitment of TFIIB
to the PIC by transcriptional activators is believed to be a critical,
rate-limiting step for PIC assembly (7, 33, 34) and TFIIB has been
suggested to play a critical role in gene activation by nuclear hormone receptors (12, 31, 32, 35, 36).
The retinoid X receptor (RXR) is a unique member of the
steroid-thyroid-retinoid nuclear receptor family due to its ability to
act as a homodimer or function as a heterodimer partner with other
nuclear receptors (reviewed in Ref. 2). Specifically, RXRs have been
shown to form heterodimers with the TR (37-42), retinoic acid receptor
(37-42), VDR (38, 41), peroxisome proliferator-activated receptor (43,
44), chicken ovalbumin upstream promoter-transcription factor (45, 46),
Arp-1 (47), farnesoidx-activated receptor (48), ubiquitous receptor
(49), liver-X receptor The specific molecular steps governing the temporal and spatial
specificity of ligand-dependent gene activation by RXRs
remain to be precisely defined. RXRs have been shown to interact with transcriptional co-factors including TAFII30 (15), TBP
(58), CBP (29, 30), and TAFII110 (58, 66, 67), as well as Trip1 (24), SUG1 (24), and TIF1 (25). Studies in yeast revealed that
RXRs could associate with either TBP or TAFII110 (58, 67), but these associations could be altered in the presence of a specific RXR agonist (67), suggesting that conformational states of the RXR
ligand-binding domain (LBD) could determine the ability of the receptor
to bind specific transcription factors in vivo. In a
cell-free system, addition of RXR to the PIC reproducibly enhanced TFIIB·TBP complex formation (27); however, a RXR·TFIIB·TBP
complex was not observed in native gel electrophoresis, suggesting that in vitro the complex might be unstable. Since RXRs have been
shown to bind proteins in the transcription complex, it is possible that RXRs might actively augment binding of receptor proteins to the
transcription complex (i.e. not as a silent partner),
perhaps in a holocomplex with CBP (29, 30). In support of this
mechanism, several proteins interact in a ligand-dependent
manner with the carboxyl (AF-2 or In an effort to clarify some of the molecular mechanisms of gene
activation by RXRs, we have tested the interaction of the general
transcription factor TFIIB with mRXR Plasmids--
Murine RXR Unit on the Molecular Mechanisms of
Reproduction,
Laboratory of
Molecular Growth Regulation, and ¶ Laboratory of Molecular
Genetics,
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
(mRXR
) was capable of binding to human TFIIB in vitro.
Functional analysis with a dual-hybrid yeast system and cotransfection
assays revealed the interaction of mRXR
with TFIIB to be
ligand-dependent in vivo. Truncation
experiments mapped the essential binding regions to the carboxyl region
of mRXR
(amino acids (aa) 254-389) and two regions in the carboxyl
region of TFIIB (aa 178-201 and aa 238-271). Furthermore, the
390-410 mRXR
mutant bound to TFIIB in vitro but was
not active in the dual-hybrid yeast system, suggesting that the extreme
carboxyl region of RXR was required for in vivo interaction
with TFIIB. These data indicate that interaction of mRXR
with TFIIB
is specific, direct, and ligand-dependent in vivo and suggest that gene activation by RXR involves TFIIB.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
(50), and NGFI-B/NURR1 (51). RXRs are
activated by the 9-cis-isomer of retinoic acid
(9-cis-RA) (52, 53) and contain specific transcriptional
activation domains in the N terminus and C terminus, termed AF-1 and
AF-2 (which includes the
c or
4 region), respectively (54-58). Some studies have suggested that RXRs principally function as
"silent partners" to augment DNA binding (2, 59, 60), whereas other
reports indicate an active role in gene regulation (48, 50, 51,
61-63). An in vivo role for RXR in gene regulation was
confirmed by disordered development observed in mice that lacked the
receptor (Refs. 1 and 2 and references therein) and by the ability of a
dominant negative mutant of RXR to prevent differentiation of P-19
embryonal cells (64). Given the potential involvement of RXR in the
function of many receptors, ligand-dependent transcription
by the RXR must be tightly regulated to ensure that specific cellular
responses are properly initiated (reviewed in Refs. 26, 62, and
65).
c) region of nuclear hormone
receptors that heterodimerize with RXRs (21-25). Also against an
indirect or silent partner role for RXR is the observation that a
dominant negative RXR mutant (e.g.
AF-2 or
c) could
abrogate the ability of a heterodimer to augment gene activation (64,
66, 69, 70, 71); since RXRs associate weakly, or not at all with SMRT
(16) or N-CoR (17, 18), trans-repression is not easily
explained. Similarly, an indirect mechanism of action does not explain
the ability of RXR to function as a homodimer (68). Furthermore, studies have specifically revealed the ability of liganded RXR to alter
the response of a heterodimer complex associated with DNA (63, 72).
These observations suggest that RXRs may actively participate in
assembly of the transcription complex.
. Using several experimental approaches, we observed the association of mRXR
with TFIIB to be
ligand-dependent, specific, and direct. Furthermore,
interaction required the carboxyl region of mRXR
and two distinct
regions within TFIIB. These data support the conclusion that RXR may
contribute directly to the recruitment of essential transcription
factors involved in ligand-dependent gene activation.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
cDNA was released by
NcoI digestion of pExpress-RXR
(73) and subcloned into
pAS1-CYH2 (74) to produce RXR
-pAS1-CYH2. Unless otherwise stated,
experiments were performed with the mRXR
receptor isoform.
Similarly, NcoI fragments of either wild-type or deletion
mutants of mRXR
(73) were subcloned to pGEX (Pharmacia Biotech Inc.)
to generate GST-RXR plasmids. Expression of appropriately sized GST-RXR
wild-type, or mutant, fusion proteins was confirmed by PAGE. For the
construction of TFIIB-GAL4-AD, pGEX2T-hTFIIB (13) was digested with
BamHI, Klenow filled, digested with EcoRI, and
the fragment subcloned into pACTII, which had been XbaI
cleaved, Klenow filled, and EcoRI digested. For construction
of TFIIB-GAL4-AD (
), the primers 5
-CAATCAACTC CAAGCTTGCG ATGGCGTCTA
CC AGCCGTTT GGAT-3
and 5
-GCGTAGCGAA GCTTAGGTCA GTTATA GCTG TGGTAGTTTG
TCCAC-3
were used to amplify hTFIIB. The polymerase chain reaction
product was then subcloned into the HindIII site of pACTII
(74).
1,
2,
3, a1,
5, and dc3 TFIIB deletion
mutants was performed by polymerase chain reaction using two specific
primers: 5
-CGCCAAGCGC GCAATTAACC CTCACTAAAG GGAAGCCGCC AGCCATGG GG
ATCCCCGCGT CTACCAGCCG TTTGGAT-3
corresponding to the 5
end of hTFIIB,
and 5
-TCTCGAGCTT CGAATTCGTT ATAGCTGTGG TAGTTTGTCC AC-3
corresponding
to the 3
end of TFIIB, except for the dc3 mutant for which the
following 3
primer was used, 5
-CGTCAGCCGA ATTCGGATCC CTTAACG GGC
TATATGTGTA GCTGC-3
. Polymerase chain reaction products were subcloned
into the BamHI/EcoRI site of pACTII. For the
construction of the
116TFIIB-pACTII, the following 5
primer
5
-CGCCAAGCGC GCTAATACGA CTCACTATAG GGCAGCCGCC CGCCATGG GG ATCCCCGCAT
TCAAAGAAAT CACTACC-3
and the same 3
primer was used for the
construction of the
1,
2,
3,
1,
5 TFIIB-pACTII plasmids.
All plasmid constructions were verified by dideoxy sequencing.
Yeast Dual-hybrid System and Immunoblot Analysis--
The yeast
strain Y190 MATa, leu2-3,112, ura3-52,
trp1-901, his3-200, ade2-101,
gal4
gal80
URA3::GAL1-lacZ,
LYS::GAL1-HIS3, cyhr was transformed
using a standard lithium acetate method. Transformants underwent two
rounds of selection on standard dextrose (SD) plates supplemented with
histidine and adenine, were grown to saturation in appropriately
supplemented SD media at 30 °C, re-inoculated into fresh medium, and
grown to mid-log phase overnight. Either 1 µM
9-cis-RA or vehicle (Me2SO) was added to cells
cultured in light-protected tubes for 6 h. Cells were pelleted at
2,000 rpm for 10 min at 4 °C, resuspended in breaking buffer (100 mM Tris-Cl, pH 8, 1 mM dithiothreitol (DTT),
20% glycerol, 0.5 mM phenylmethylsulfonyl fluoride), and
lysed by vortexing at 4 °C with cold, glass beads six times for
15 s at 15-s intervals. Protein extracts were quantified with the
Bio-Rad protein assay (Bio-Rad).
-Galactosidase activity was
detected using 30 µg of protein yeast extracts in the Galactolight chemiluminescence assay (Tropix, Bedford, MA) with a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA). TFIIB
in yeast extracts was detected by Western analysis using antibodies
directed against hTFIIB (sc-4001, Santa Cruz Biotechnology, Santa Cruz,
CA) and an enhanced chemiluminescent detection method (ECL, Amersham
Corp.). Immunoblot analysis of yeast proteins containing mRXR
was
performed with monoclonal antibody (13.17) essentially as described
(40) or with anti-GAL4-DBD (CLONTECH, Palo Alto,
CA) as noted.
Co-immunoprecipitation Assays-- Antibody-protein complexes were precipitated using 20 µl of pre-equilibrated protein A-Sepharose beads as described, with slight modifications (75), and then washed extensively. Bound products were eluted in sample buffer and analyzed by 12% SDS-PAGE and autoradiography.
Far Western Analysis-- Following electrotransfer, nitrocellulose filters were denatured in 6 M guanidine hydrochloride and renatured by stepwise dilutions to 0.187 M guanidine HCl in HBB (25 mM Hepes-KOH, pH 7.4, 25 mM NaCl, 50 mM MgCl2, 1 mM DTT). Filters were blocked 12-14 h in HBB with 5% nonfat milk and 0.05% IPEGAL (Sigma) and rinsed in HYB (20 mM Hepes-KOH, pH 7.4, 75 mM KCl, 0.1 mM EDTA, 2.5 mM MgCl2, 1% nonfat milk, 0.05% IPEGAL, and 1 mM DTT) (21). 500,000 cpm/ml of 32P-labeled GST-RXR or GST fusion proteins2 were added, and incubation was continued at 4 °C for 12 h. Filters were rinsed in HYB three times and subjected to autoradiography.
Glutathione-Sepharose Binding Assays-- 20 µl of glutathione-Sepharose beads coated with bacterially expressed GST fusion proteins were mixed with equal amounts of 35S-labeled in vitro translated RXR or TFIIB and luciferase, added in the presence or absence of 1 µM 9-cis-RA, and rocked at 4 °C for 1 h. Beads were collected, washed extensively in HBB (40 mM Hepes, 75 mM KCl, 0.5 mM EDTA, 5 mM MgCl2, 1 mM DTT, 1 mM 4-2(aminoethyl)benzenesulfonyl fluoride hydrochloride, 0.5 mg/ml bovine serum albumin, and 0.05% Nonidet P-40) containing 1 µM 9-cis-RA or vehicle (Me2SO). Bound proteins were eluted in sample buffer and analyzed by 10% SDS-PAGE and autoradiography. Input lanes show one-tenth of loaded lysate.
For precipitation of TFIIB from nuclear extracts, 25 µg of Namalwa human B lymphocyte nuclear extract was prepared as described (40) and incubated with 10 µl of coated beads at 4 °C for 1.5 h in 20 mM Hepes, 20% glycerol, 0.2 mM EDTA, 50 mM KCl, 0.5% bovine serum albumin, 0.5 mM phenylmethylsulfonyl fluoride. Beads were collected and washed extensively in 20 mM Hepes, 20% glycerol, 0.2 mM EDTA, 100 mM KCl, 0.5 mM phenylmethylsulfonyl fluoride, and 0.05% Triton X. Bound proteins were eluted by boiling and loaded onto 12% SDS-PAGE, blotted to nitrocellulose, and examined using immunoblot analysis.Cell Culture and Transient Transfection--
Murine P19
embryonal carcinoma cells were grown in minimal essential medium
supplemented with 10% heat-inactivated fetal bovine serum, glutamine
(20 mM), and gentamicin (50 mg/ml) and plated at a cell
density of 3 × 106 cells/ml 24 h prior to
transfection by calcium-phosphate precipitation. 0.5 mg of
CRBPII-tk-luciferase (which contains two copies of the rat
CRBPII-RXRE element) or tk-luciferase reporter (64) was added to expression vectors pExpress-RXR (73) and pRSV2-hTFIIB (12),
as indicated. RSV-
-galactosidase (0.5 mg) was used to control for
transfection efficiency (70). Cells were treated with 1 µM 9-cis-RA or vehicle (Me2SO),
harvested 24 h after transfection, and extracts normalized for
-galactosidase activity.
Partial Proteolysis Assays--
35S-Labeled TFIIB
was incubated with 7 µg of Sf9 nuclear proteins prepared from
cells infected with wild-type baculovirus (control) or recombinant
mRXR baculovirus. In other experiments, labeled TFIIB was added to a
20-µl bed volume of Sepharose beads coated with bacterially expressed
GST, or GST-RXR, fusion proteins. After binding for 2 h at
4 °C, trypsin (50 or 500 ng) was added for 5 min at 30 °C and the
reaction stopped with 1 × SDS, and products were resolved by
12-18% SDS-PAGE and subjected to autoradiography.
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RESULTS |
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Interaction of RXR with TFIIB Is Direct-- Since other nuclear hormone receptors have been shown to interact with TFIIB (10-14), and TFIIB is a common target of sequence-specific transcriptional activators (6, 7), we tested whether RXR could precipitate TFIIB from proteins extracted from mammalian cell nuclei. Sepharose beads were coated with either bacterially expressed GST-RXR (RXR) fusion proteins or control proteins (GST), incubated with nuclear proteins, and washed, and immunoblots of bound proteins were performed using antibodies directed against TFIIB. As shown (Fig. 1A), TFIIB could be specifically precipitated from extracts prepared from mammalian cell nuclei using GST-RXR-coated beads. In control experiments, beads that lacked the RXR fusion protein failed to bind TFIIB, thus supporting the specificity of the interaction. TFIIB precipitated from extracts of nuclear proteins co-migrated with endogenous TFIIB detected in immunoblot analysis of nuclear proteins and recombinant TFIIB (Fig. 1A), thus suggesting that RXR could precipitate TFIIB from mammalian cell extracts (see below).
|
TFIIB Augments RXR-dependent Reporter Activity in
Mammalian Cells--
Since mRXR could associate with TFIIB in
vitro, we examined whether overexpression of both TFIIB and RXR
might augment RXR-dependent reporter activity in mammalian
cells. To test this possibility, transient transfections were performed
in undifferentiated P-19 embryonal carcinoma cells (12) using a
luciferase reporter plasmid containing two copies of an RXR response
element coupled to a tk promoter (64). Addition of an
expression plasmid for RXR led to a 7-fold increase in reporter
activity, whereas addition of expression plasmids for both RXR and
TFIIB led to a 19-fold increase; thus addition of TFIIB led to an
additional 2-3-fold ligand-dependent increase in reporter
activity (Fig. 2). There was no increase
in basal reporter activity or ligand-dependent reporter
activity when equivalent amounts of control expression vector were
added, indicating that TFIIB specifically augmented RXR-mediated gene
activation. In addition, the activation depended upon the presence of
the RXR response element and was not observed in experiments performed
using the control tk-luciferase reporter (right
panel). The magnitude of activation in mammalian cells attributable to TFIIB was modest (2-3-fold), largely due to a 7-fold
level of basal activation in the presence of transfected RXR alone. The
increase in basal reporter activation required RXR and was
element-specific, since activity of the tk-luciferase reporter (that lacked an RXRE) was not augmented by RXR alone. At the
quantities of expression plasmids tested (500 ng), our results
resembled levels of activation observed by others using similar
approaches (12). Although Blanco et al. (12) demonstrated a
sizable in vivo effect attributable to TFIIB with VDR, other studies have not indicated a strong in vivo effect in
mammalian transfection systems attributable to basal transcription
factors such as TBP (9) and TFIID (8). Given the modest effect
observed, we concluded that additional experiments in mammalian cells
would not likely elucidate the interaction between RXR and TFIIB,
because endogenous expression of TFIIB, RXR, and available RXR
heterodimerization partners in mammalian cells would confound
interpretation of data. Similarly, we rejected the use of mammalian
two-hybrid approaches, since in that system endogenous RXR
heterodimerization partners have been reported to repress RXR action
(76). For this reason, we turned to an in vivo system devoid
of endogenous RXR expression.
|
Interaction between RXR and TFIIB Is Augmented by Ligand in
Vivo--
We examined the interaction between RXR and TFIIB using the
yeast dual-hybrid system (reviewed in Ref. 77), since yeast do not
contain RXRs (78), and RXRs have been shown to function in yeast (78,
79). A Saccharomyces cerevisiae strain (Y190) containing an
integrated lacZ reporter was transformed with expression plasmids (Fig. 3A) encoding
full-length mRXR fused to the GAL4 DNA binding domain and
full-length hTFIIB fused to the GAL4 activation domain (AD).
Reconstitution of this two-hybrid system would permit quantitative
analysis of reporter activation and not colony survival alone (77).
Furthermore, the incorporation of an integrated lacZ
reporter permitted paired examination of ligand effects from individual
colonies, thus controlling for variability in levels of receptor
expression as determined by immunoblot analysis.
|
The Carboxyl Region of TFIIB Interacts with RXR--
We next
sought to define the specific regions of TFIIB required for interaction
with mRXR via GST binding assays (11). Glutathione-Sepharose beads
were coated with intact, or truncated, GST-TFIIB fusion proteins and
incubated with 35S-labeled mRXR
. As a control for
binding specificity, 35S-labeled luciferase was added to
binding reactions. Labeled RXR did not bind to beads coated with GST
alone nor did labeled luciferase bind to any of the GST-TFIIB-coated
beads (Fig. 4A). Beads coated with the
1,
2,
3,
5, and dN1 GST TFIIB mutant proteins
bound mRXR
as avidly as wild-type TFIIB. In contrast, beads coated with protein corresponding to the
1 (
178-201) and dc3
(
238-316) TFIIB deletion mutants bound less avidly to RXR,
suggesting that regions from amino acids 178-201 and 238-316 of TFIIB
are required for in vitro binding to mRXR
. Under the
conditions tested, addition of 9-cis-RA only slightly
augmented in vitro binding (see also Fig.
5A).
|
|
The C Terminus of RXR Interacts with TFIIB--
To map the regions
of RXR required for binding to TFIIB, we used intact and mutant GST-RXR
fusion proteins immobilized on Sepharose beads (Fig. 5A).
Beads coated with full-length GST-RXR fusion protein specifically bound
labeled TFIIB (lane 2). No binding was observed to beads
coated with GST alone. Also, minimal binding to TFIIB was observed with
a ligand-binding domain RXR mutant (LBD). Furthermore, an N-terminal
deletion mutant (
AF-1) and an AF-2 deletion mutant of RXR
(
390-410) also bound labeled TFIIB, although the
AF-1 mutant
showed reduced binding in the absence of ligand. These data suggest
that amino acids 254-389 of RXR are required for in vitro
binding to TFIIB.
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DISCUSSION |
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Several nuclear hormone receptors have been shown to interact
in vitro with the general transcription factor, TFIIB
(10-14, 31), recruitment of which is thought to be a rate-limiting
step in pre-initiation complex formation (7, 33, 34). The current view
is that nuclear receptors interact with a complex of proteins involving
co-activators, co-repressors, integrators, and other transcription
factors (reviewed in Refs. 26, 27, and 65). In this report, we provide
substantial evidence using both in vivo and in
vitro methods to show that mRXR may interact with hTFIIB in a
direct and ligand-dependent manner to promote gene activation, an observation of significance due to the central role of
RXRs in gene regulation in vivo (1, 2, 48, 50, 51,
61-63).
Specifically, we observed that two regions of TFIIB (aa 178-201 and aa
238-271) interacted with the mRXR LBD (aa 254-389) in
vivo. These results resemble the in vitro results of
Blanco et al. (12) who showed that the C terminus of TFIIB
(aa 118-316), a region encompassing the two direct repeats and the
-helix of TFIIB, interacted with the N-terminal region (aa 132-257)
of the LBD of the VDR. The results of Blanco et al. (12)
contrast with the results of MacDonald et al. (13) who
observed a ligand-independent interaction in vivo between
the N terminus of TFIIB and the ligand-binding domain of VDR, an
observation that was buttressed by site-directed mutagenesis studies
(14). Interestingly, MacDonald et al. (13, 14) also showed
that TFIIB interacts with the extreme C terminus of the VDR, the latter
region lacking in the
390-410 mRXR
mutant. In the case of the TR
(at least in vitro) the N terminus of the hTR
has been
shown to interact with the C terminus of TFIIB, whereas the C terminus
of the LBD of hTR
interacts with the N terminus of TFIIB (11), as
supported by experiments in a cell-free system (28). Baniahmad et
al. (11) observed that two regions in the C terminus of TR (aa
168-259 and 260-465) interacted with TFIIB, and interaction of the
latter region was not hormone-dependent. In comparison,
Fondell et al. (31) showed that the C terminus (aa 213-410)
of hTR
interacted with both TFIIB and TBP, whereas the N terminus
(albeit weakly) and the C terminus of hTR
interacted with the C
terminus of TFIIB (aa 244-316). The findings with different TR
isoforms suggest that different receptors may interact in an isoform-specific manner with TFIIB. Our inability to demonstrate involvement of the N terminus of mRXR
in binding to TFIIB may be
specific for murine RXR
, since mRXR
has not been found to possess
an N-terminal amino acid region homologous to that in TR (36) that
interacted with TFIIB.
Evidence also suggests that TFIIB may present different interfaces for binding to different nuclear receptors, activators, and co-activators. For instance, interaction of RXR, and some other nuclear receptors, does not involve regions of overlap with binding of TFIIB to TFIIF (RAP30) which appears to involve the first 111 aa of TFIIB (33). However, studies have mapped the binding motifs of TBP and RNAPII to regions of TFIIB (33) common to regions utilized in binding of TFIIB to RXR (Fig. 5) and other receptors (11, 13, 14, 31, 32). In addition, it has been shown that other sites in TFIIB are important for co-activator binding (6, 7, 82, 84-87), such as for TAFII40 (aa 195-217) and VP16 (aa 165-217 and aa 269-286) (85). That interfaces of TFIIB involved in binding to RXR differ (in part) from those observed for TR suggests that both RXR and TR might jointly contact TFIIB through distinctly different, but not mutually exclusive, binding interfaces within TFIIB. More definitive proof of this notion will require additional experiments. From the perspective of RXR, different ligands (e.g. LG100268 and LG1007540) have been shown to quantitatively and qualitatively affect interaction of RXR with TBP and TAF110 (67), suggesting that differential interaction of the receptor with basal transcription machinery could be dependent upon the conformation of the LBD (67). These findings support the idea that RXR may augment binding of the heterodimeric complex to the PIC. Thus, our results suggest that RXR may enhance the ability of TR or RAR (for instance) to recruit TFIIB to the PIC, in support of findings in cell-free transcription models (28).
It should be mentioned that MacDonald et al. (13)
demonstrated that VDR interacts with TFIIB using a dual-hybrid yeast
system but did not observe an interaction between hRXR or mRXR
with TFIIB, although RXRs were not the principal focus of the study. The critical difference between the experiments of MacDonald et al. (13) and our experiments is likely that the interaction between RXR and TFIIB in yeast was entirely dependent on
9-cis-RA, a variable not reported in that study (13), and
ligand has been noted to be important for binding to other
transcription factors (29, 58, 67; see also below). Additionally, it is
likely that other significant differences between the two model systems
may account for the differences observed between results of MacDonald et al. (13) and our findings.
Our observations with TFIIB, and those of others with TBP and
TAFII110 (58, 67), suggest that RXR interacts specifically with distinct components of the basal transcription machinery to
augment transcription. In most instances, the in vivo
mapping data agreed with in vitro findings, but the
exceptions serve to elucidate distinct features of receptor function.
First, the association of RXR and TFIIB was not
ligand-dependent in vitro but was
ligand-dependent in vivo. This observation was
supported by results of Baniahmad et al. (11) and Tong
et al. (28) with TR; likewise, we observed only a slight
increase in binding of mRXR to TFIIB in vitro in the
presence of ligand. It is possible that failure of the bacterially expressed, recombinant RXR protein to assume a proper conformation or
damage to (or proteolysis of) the receptor during purification affected
our ability to detect ligand-dependent differences in vitro. In support of the necessity of ligand for association of RXR and TFIIB in vivo, a ligand-dependent
interaction in yeast was also observed between RXR and TBP (58, 67),
CBP (29), and TAF110 (58, 67). Overall, the necessity of ligand for in vivo interaction suggests that specific conformation is
necessary in order for mRXR
to associate with TFIIB.
Second, we observed an interaction between the 390-410 RXR deletion
mutant and TFIIB in vitro but not in vivo,
suggesting that in vivo the extreme carboxyl region of RXR
is required to permit interaction with TFIIB (Fig. 5B), as
was observed for TBP (58). Since the
390-410 RXR deletion mutant
has previously been shown to bind ligand (71), this suggests that
ligand binding alone is not sufficient to promote interaction of RXR
with TFIIB. Our results are consistent with dominant negative activity
reported for the
390-410 mutant (55, 56, 66, 69, 70, 71) and support other studies (66, 69) suggesting that the mechanism involves
binding of the receptor to DNA. In addition, our results suggest the
mutant is unable to interact properly with TFIIB in vivo.
Specifically, use of the dual-hybrid system to characterize this mutant
strongly argues against other mechanisms, since DNA binding and nuclear
localization of the
390-410 mutant to the promoter were ensured by
the inclusion of the GAL-4-DBD in the construct. Others (9, 15) have
drawn similar conclusions based on observations with mutant estrogen
receptor constructs and TFIIB. An alternate explanation for dominant
negative activity of the
390-410 RXR deletion mutant in
vivo could be that an AF-2 binding factor (e.g. CBP,
TIF1, ERAP-160, or RIP 140) is required in vivo for
stabilization of RXR interaction with TFIIB. In support of this
alternate explanation, it has been suggested that yeast proteins, such
as SUG1, interact with basal transcription factors TBP (84) and
hTAFII30 (15) as well as the AF-2 region of nuclear receptors (23, 24), consistent with the notion that binding between
TFIIB and RXR might be influenced by factors present in yeast. Further
experiments are required to definitively distinguish between these two
possibilities.
Since it had been shown that the acidic activator, VP-16, induced a conformational change in TFIIB (82, 83) and bound to a similar region of TFIIB, we performed a partial proteolysis assay to test whether RXR may induce a conformational change within TFIIB. Using in vitro translated TFIIB and baculovirus/or bacterially expressed RXR proteins, we observed that RXR induced an altered proteolytic digestion pattern of TFIIB. These data suggest that one possible action of nuclear receptors may be to induce a conformational change in TFIIB which, as suggested for VP16, then exposes other binding interfaces of TFIIB to other transcription factors, possibly TAFII40 (82, 85), TBP (9, 31, 86, 87), TFIID (8, 34, 85, 88), TFIIF, and RNAPII (89). These results raise the question of whether gene activation by nuclear hormone receptors might result not simply from a passive assembly of transcriptional proteins due to acidic, proline-rich, or glutamine-rich regions, but rather as the active alteration of protein interfaces of TFIIB which in turn present protein interfaces with affinity to other transcription factors, thus influencing assembly of the PIC.
In summary, the demonstration that RXR is capable of interacting with TFIIB in a ligand-dependent manner is consistent with the notion that RXR may contribute to recruitment of TFIIB to heterodimeric DNA-bound receptor complexes as well as homodimeric receptor complexes.
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ACKNOWLEDGEMENTS |
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We sincerely thank Dr. Paul H. Driggers for insightful advice during the study and helpful comments regarding the manuscript; Dr. Charles M. Moehle for expert assistance during the study; Dr. Joseph Grippo for the generous supply of 9-cis-RA (Hoffmann-La Roche, Nutley, NJ); Drs. Ming Jer Tsai and Bert O'Malley for gratefully providing the pGex2T-TFIIB plasmids; and Dr. Stephen Elledge for being so kind to share the Y190 yeast strain and pACTII and pAS-CYH2 plasmids.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant Z01-HD00636-DEB (to J. H. S.).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.
§ Current address: Bone and Mineral Research Division, The Garvan Institute of Medical Research, 384 Victoria St., Darlinghurst, Sydney, New South Wales, 2031, Australia.
** To whom correspondence should be addressed: OSD, NICHD, and Dept. of Obstetrics and Gynecology, Uniformed Services University of the Health Sciences, Bldg. A, Rm. 3078, 4301 Jones Bridge Rd., Bethesda, MD 20814. Tel.: 301-295-3777; Fax: 301-295-6774.
1 The abbreviations used are: PIC, preinitiation complex; TFIID, transcription factor IID; TBP, TATA binding protein; TAF, TATA associated factors; RNAPII, RNA polymerase II; VDR, vitamin D receptor; RSV, Rous sarcoma virus; RXR, retinoid X receptor; aa, amino acid(s); PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; DTT, dithiothreitol; RA, retinoic acid; h, human; m, murine; TR, thyroid hormone receptor; LBD, ligand-binding domain; CBP, cAMP response element-binding protein-binding protein.
2 D. Arbit and J. Segars, unpublished data.
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