From the
We investigated the molecular mechanisms underlying the
transcriptional silencing and the hormone-induced activation of target
genes by thyroid hormone receptor
The nuclear receptors for thyroid hormone belong to the
superfamily of ligand-inducible transcription factors that also include
receptors for steroid hormones, retinoids, and vitamin D
The
biological relevance of transcriptional silencing of cellular target
genes by unliganded TR was suggested by recent analyses of the
transcriptional properties of the avian erythroblastosis virus-encoded
oncoprotein v- erbA, which is a mutated form of TR that has
lost the ability to bind hormonal ligand
(23, 29) . It
is believed that v- erbA induces neoplastic transformation by
arresting normal erythroid differentiation
(30, 31) . In
cell culture experiments, v- erbA acts as a constitutive
silencer of TRE-linked genes and is a dominant negative repressor of
the functions of wild-type TRs
(23, 29, 32) . It
has been reported that v- erbA may also interfere with the
natural functions of other closely related members of the nuclear
receptor superfamily such as the receptors of
all- trans-retinoic acid, which are known regulators of cell
development and differentiation
(33) .
Transcriptional
silencing may also contribute to the physiological perturbations
associated with the naturally occurring mutations in the gene encoding
TR-
The molecular basis of transcriptional
silencing by TR and the hormonal modulation of this activity remains
unclear. Previous studies reported that steroid hormone receptors
stimulate initiation of transcription by facilitating the assembly of a
preinitiation complex (PIC) at the target promoter
(37, 38) . A prediction that follows directly from these
studies is that the mechanism of gene repression by TR, a related
nuclear receptor, may involve inhibition or destabilization of the
initiation complex assembly at the TATA box. The formation of a
functional initiation complex is a multistep process that proceeds
through sequential assembly of RNA polymerase II and several other
general transcription factors at the TATA promoter
(39, 40, 41, 42) . The TATA binding
protein TFIID is the first factor to enter into the complex
(43) . The TFIID
In this report, we investigated the
initiation complex assembly process as a potential target of
transcriptional silencing by unliganded TR-
We employed nickel-affinity chromatography
(50) to
purify human TR-
We
considered the possibility that exogenously added TR may inhibit basal
transcription passively by displacing from the TRE sites an endogenous
transcriptional activator present in HeLa nuclear extracts. We
observed, however, that the basal levels of transcription from
equimolar amounts of test templates containing two copies of TRE or
lacking TREs were essentially equivalent (Fig. 1 C,
compare lanes 1 and 3). If a transcriptional
activator bound to the TRE, one would expect relatively enhanced level
of RNA synthesis from the template containing TREs. This observation
strongly disfavors the idea that a transcriptional activator may bind
to the AGGTCA motifs of a TRE in HeLa nuclear extracts.
In contrast, another
receptor mutant TR
The stepwise assembly of a functional initiation
complex has been studied in vitro by an EMSA
(43, 44, 46) . The various DNA-protein
intermediates that are formed at the TATA promoter due to the
sequential additions of general transcription factors and RNA
polymerase II can be detected and analyzed by this assay. We employed
EMSA to investigate how TR-
As shown in
Fig. 5A, incubation of either TFIIB or TBP alone with an
oligonucleotide containing the AdML gene TATA box sequence did not
result in the formation of any stable DNA-protein complex under our
reaction conditions. However, incubation of a combination of TFIIB and
TBP proteins with the TATA oligonucleotide resulted in the formation of
a retarded DNA-protein complex that we term complex 1
(Fig. 5 A, lane 4). We characterized complex 1
further by employing antibodies directed specifically against TFIIB and
TBP, respectively, in EMSA (Fig. 5 B). Our results showed
that both antibodies could interact with and supershift complex 1,
indicating that complex 1 is an authentic complex of TFIIB and TBP at
the TATA box.
To confirm further the presence of TR-
We also analyzed the
protein-protein interactions between the TFIIB
In EMSA, the mutant TR
We describe here the reconstitution of a cell-free
transcription system to investigate the mechanisms of transcriptional
silencing of a TRE-linked gene by unliganded TR-
Our
studies revealed that the addition of hormone-free human TR-
We identified the TFIIB
The precise
nature of the molecular contacts between TR-
In our experiments TFIIB
We
demonstrate that the specificity of transcriptional silencing by TR is
determined by the presence of specific hormone-response elements in the
target gene promoter. Our studies showed that (i) unliganded TR-
Binding of the hormonal ligand
elicits a conformational change in TR and dramatically alters its
gene-regulatory activity
(55, 56) . We therefore,
reasoned that the interaction of TR-
Recent
studies suggest the possibility that efficient TR-mediated silencing of
basal transcription may require additional cofactors, which may exist
in unfractionated HeLa nuclear extracts. Fondell et al.(48) showed that unliganded TR-
Our in vitro studies provide a plausible mechanism by which
TR, anchored to a nuclear target site, can direct different regulatory
events, namely, repression and hormone-dependent activation of a
cellular gene. To understand precisely how TR controls the assembly of
a functional PIC, further analysis of the composition and structure of
the receptor-bound core promoter complexes and assessment of their
functional activities in cell-free transcriptional assays will be
necessary. Moreover, isolation of potential coactivator or corepressor
molecule(s) may allow us to determine the contribution of these
modulatory factors in TR-mediated gene regulation.
We thank Drs. Bert O'Malley and Ming-Jer Tsai
for providing the plasmids pLovTATA and pAdML200. We are grateful to
Drs. Danny Reinberg and Alexander Hoffmann (Roeder Laboratory) for
generously providing the cDNA clones for human TFIIB and human TBP,
respectively. We also thank Dr. Ronald Evans for the generous gift of
the cDNAs for human TR-
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(TR-
). We developed a
cell-free transcription system containing HeLa cell nuclear extracts in
which unliganded human TR-
represses basal transcription from a
promoter bearing thyroid hormone response elements. Binding of hormonal
ligand to the receptor reverses this transcriptional silencing.
Specific binding of TR-
to the thyroid hormone response element at
the target promoter is crucial for silencing. Studies employing
TR-
mutants indicate that the silencing activity is located within
the C-terminal rather than the N-terminal domain of the receptor. Our
studies reveal further that unliganded TR-
inhibits the assembly
of a functional transcription preinitiation complex (PIC) at the target
promoter. We postulate that interaction with TR-
impairs the
function(s) of one or more assembling transcriptional complexes during
the multistep assembly of a PIC. Consistent with this hypothesis, we
observe that, in the absence of thyroid hormone, TR-
or a
heterodimer of TR-
and retinoid-X-receptor undergoes direct
protein-protein interactions with the transcription factor IIB-TATA
binding protein complex, an early intermediate during PIC assembly.
Binding of hormone to TR-
dramatically reduces the interaction
between the receptor and the transcription factor IIB-TATA binding
protein complex. We propose that the role of ligand is to facilitate
the assembly of functional PICs at the target promoter by reducing
nonproductive interactions between TR-
and the initiation factors.
(1, 2, 3) . The specificity of target gene
recognition by two major types of thyroid hormone receptors
(TRs),
(
)
TR-
and TR-
, is determined by
the interaction of the receptors with short, enhancer-like sequences
termed thyroid hormone response elements (TREs) located near the target
gene promoter
(4, 5) . TREs are composed of repeats of a
consensus hexanucleotide motif, AGGTCA, arranged in a variety of ways:
palindromic, direct repeat, or inverted repeat
(6, 7, 8, 9, 10) . TR can bind
to a TRE in vitro as a monomer, as a TR-TR homodimer, or as a
heterodimer of TR with other nuclear factors termed TR-auxiliary
proteins
(11, 12, 13, 14) . The recently
discovered nuclear receptors of 9- cis-retinoic acid, the
retinoid X receptor (RXR) proteins, behave as TR-auxiliary proteins
(15, 16, 17, 18, 19, 20, 21, 22) .
TR binds to TRE in a ligand-independent manner and functions either as
a transcriptional activator or a repressor of a TRE-linked promoter
depending on the hormonal status of the receptor. In the absence of
hormone, TR functions as a silencer of basal level of transcription
from the target promoter
(23, 24, 25, 26) . Ligand binding to the
receptor releases transcriptional silencing and leads to the activation
of target gene expression
(23, 27, 28) .
in human patients suffering from a genetic disorder termed
generalized resistance to thyroid hormone (GRTH) (for review, see
Refetoff et al.(34) ). All the GRTH mutations
characterized so far result in either failure to bind T
or
reduced binding affinity of the hormone. The heterozygous kindreds
harbor one mutant and three (one TR-
and two TR-
) normal
alleles. It has been suggested that the product of the mutant allele
inhibits normal TR function in a dominant negative manner. In vitro studies indicate that most of these mutants heterodimerize with
RXR and bind to target DNA sites efficiently. Baniahmad et al.(32) using transient transfection assays demonstrated that
two different GRTH mutants displaying drastically reduced or no T
binding activity, respectively, functioned as constitutive
repressors of target genes with strong silencing activity. These
results favor the hypothesis that the dominant negative GRTH mutant is
not simply an intrinsic transcriptionally nonfunctional receptor. It
rather is a receptor that has lost the ability to transactivate but
fully retains an active and constitutive silencing function. This
viewpoint is consistent with the observation that a homozygous GRTH
patient with two mutant (non-hormone-binding but active repressor)
TR-
alleles displayed much more severe clinical symptoms than a
homozygous patient who has a complete deletion of the TR-
gene
(35, 36) .
TATA complex is then recognized by TFIIB
producing the DB
TATA complex
(43) . The presence of TFIIB
is critical for the subsequent recruitment of RNA polymerase II. The
association of RNA polymerase II with the DB complex is mediated by
TFIIF
(44) . The resulting complex, DBpolF, is then recognized
by TFIIE and TFIIH
(45) . This creates DBpolFEH that, in the
presence of ribonucleoside triphosphates, directs a basal level of RNA
synthesis
(46, 47) . It is conceivable that each of
these steps in the complex assembly process can be a potential target
of regulation by a transactivator or a repressor. Consistent with this
scenario, previous studies indicated that (i) unliganded TR efficiently
repressed basal transcription from a TRE-linked minimal promoter
containing only the TATA box
(23) and (ii) both TR-
and
TR-
interact directly with the initiation factor TFIIB
(48, 49) .
. We observed that in a
cell-free reconstituted transcription system, the addition of TR-
during PIC assembly led to the formation of a transcriptionally
inactive complex. Ligand binding to TR reversed this transcriptional
repression. We found that hormone-free TR-
or TR-
-RXR
heterodimer can interact stably with TFIIB
TBP
TATA complex,
an early intermediate during PIC assembly. Binding of TR-
or
TR-
-RXR to the TFIIB
TBP
TATA complex was greatly
reduced in the presence of thyroid hormone. Based on these results, we
propose a hypothesis that transcriptional silencing by TR-
may
involve stable but nonproductive interactions between the unliganded
receptor and an assembling core promoter complex such as
TFIIB
TBP
TATA. Hormone binding to TR reverses silencing by
reducing such abortive interactions and allowing subsequent assembly of
a functional PIC at the promoter of the thyroid hormone-responsive
gene.
Plasmids
The construction of transcriptional
templates pLovTATA and pAdML200 has been described previously
(37, 38) . The test template TRETATA was
constructed by introducing two copies of a palindromic TRE (TREpal)
oligonucleotide (5`-GATCCTCAGGTCATGACCTGA-3`) into the BglII
site of plasmid pLovTATA. The construction of bacterial expression
vectors containing human TBP and human TFIIB cDNAs have been described
before
(50, 51) .
Antibodies
Polyclonal antibodies against human TBP
and human TFIIB were purchased from Upstate Biotechnology, Lake Placid,
NY. Polyclonal antibody against TR- was obtained from Affinity
Bioreagents, Neshanic Station, NJ.
Bacterial Expression and Purification of
Receptors
A full-length cDNA of human TR- (with
5`- NdeI and 3`- BamHI ends) or human RXR
(with
5`- NdeI and 3`- BglII ends) was engineered between
NdeI and BamHI sites of the bacterial expression
vector pET15b (Novagen, Inc., Madison, WI). These constructs were
transformed into the bacterial strain BL21(DE3)plysS. The recombinants
were grown in enriched medium (500 ml) containing Terrific broth and
antibiotics such as ampicillin and chloramphenicol. The bacteria were
grown to an optical density (at 600 nm) of approximately 1.0 and then
induced by 1 mM
isopropyl-1-thio-
-D-galactopyranoside. After 2 h of
induction the bacteria were harvested and stored frozen at -70
°C.
or human RXR
tagged with six histidine
residues at the N-terminal end. The bacterial pellet was thawed and
resuspended in a buffer containing 20 mM Tris-HCl, pH 7.9, 500
mM NaCl, 10 mM mercaptoethanol, 10% glycerol, and a
mixture of protease inhibitors such as phenylmethylsulfonyl fluoride,
leupeptin, aprotinin and pepstatin. The bacteria were lysed by
repeated, mild sonication and the soluble supernatant was collected
following centrifugation at 20,000
g for 15 min. The
supernatant was adjusted to 5 mM imidazole and applied to a
nickel-nitrilotriacetic acid affinity column (Qiagen, CA). The affinity
chromatography was performed according to the procedure of Hoffmann and
Roeder
(50) . The receptor was eluted with a buffer containing
150 mM imidazole and 5 µM zinc chloride. The peak
fractions were pooled, bovine serum albumin was added as a carrier
protein to a final concentration of 0.2 mg/ml and the resulting
fractions were stored at -70 °C. Control protein fractions
were generated from bacteria transformed with pET15b by following the
same protocol. The bacterially expressed TR-
displayed ligand
binding affinity ( K
= 1
nM) similar to that of the native receptor isolated from
tissue sources. The nickel-affinity purified TR-
or RXR
preparations were typically 50-70% pure as estimated by SDS-PAGE.
Human TFIIB and TBP were purified by following the published procedures
(50, 51) .
Cell-free Transcription Assay
The cell-free
transcription conditions and isolation of P-labeled
transcripts have been descibed previously
(38) .
Electrophoretic Mobility Shift Assay (EMSA)
The
EMSAs were performed as described previously
(37, 44) .
Construction of TR-
The TR- Mutants
mutants TR
N, TR
N
D, and TR
C lacking N-terminal 80,
N-terminal 145 and C-terminal 196 amino acids, respectively, were
constructed by polymerase chain reaction-assisted and
oligonucleotide-directed mutagenesis. The resulting DNA fragments
containing NdeI and BamHI ends were subcloned into
pET15b. The mutant proteins were expressed in bacteria and purified by
nickel-nitrilotriacetic acid affinity chromatography as described
above.
Ligand-free TR-
To investigate the mechanism of TR-mediated
transcriptional regulation of target gene promoters, we devised a
cell-free gene expression system using bacterially expressed human
TR- Silences Basal Level of
Transcription from a TRE-linked Promoter in Cell-free Transcription
Extracts
as a source of receptor protein. We employed a synthetic
target gene template TRE
TATA containing two copies of
TREpal linked to a minimal TATA promoter and a G-free cassette as a
reporter sequence. Transcription of this template was carried out in
HeLa cell nuclear extracts which served as a source of RNA polymerase
II and several other general transcription factors that are required
for basal level of RNA synthesis. The nuclear extracts did not contain
any detectable endogenous TR. When the transcription reaction was
performed in the absence of any exogenously added TR, a significant
level of basal transcription was observed from the test promoter.
Addition of increasing amounts of purified TR-
resulted in a
progressive inhibition of basal transcription from the TRE-linked
promoter (Fig. 1 A). This TR-
-induced
transcriptional repression was promoter-specific since transcription
from an internal control adenovirus major-late promoter remained
unaffected by the addition of TR. Quantitation of the transcripts by
densitometric analysis revealed that as much as 80% of basal level of
RNA synthesis was repressed upon addition of 25 nM TR-
to
the transcription reaction (Fig. 1 B). Similar inhibition
of RNA synthesis from a test template containing two copies of a direct
repeat TRE (AGGTCACAGGAGGTCA) was observed also upon addition of
exogenous TR-
(data not shown). Our results, therefore, indicate
that in cell-free nuclear extracts unliganded TR-
exhibits
transcriptional silencing properties similar to that displayed by the
receptor in transient transfection experiments
(23, 24, 25, 26) .
Figure 1:
Silencing of
basal transcription by unliganded TR-. Panel A,
increasing amounts of TR-
(0.2, 0.6, 1.0 pmol) were added to an
in vitro transcription reaction containing HeLa nuclear
extract and a TREpal-linked reporter template, TRE
TATA. The
transcription reactions (volume, 30 µl each) were carried out and
the transcripts were analyzed as described previously (38). The
filled and the hollow arrowheads indicate transcripts
from the TREpal promoter and the internal control AdML promoter,
respectively. Panel B, quantitations of the RNA signals
generated by the test template were performed by densitometry followed
by normalization with respect to the internal control AdML signals. The
plotted results represent an average of three different experiments. In
each of three experiments, the value for basal transcription
(- TR) was set to 100% and the corresponding values for
test (+ TR) measurements were obtained. The error bars represent the standard deviation of the mean. Panel C,
transcription reactions containing either TRE-linked
TRE
TATA ( lanes 1 and 2) or TRE-less
pLovTATA ( lanes 3, 4, and 5) templates were
performed with or without exogenously added TR-
( lane 2,
0.5 pmol; lane 4, 0.5 pmol; and lane 5, 0.75
pmol).
To examine the
functional role of TRE in transcriptional repression by TR-, we
used a template pLovTATA, which exactly resembled the test template
except for a lack of TRE sequences. As shown in Fig. 1 C,
basal transcription from the TRE-less promoter was not affected
significantly by the addition of TR-
(compare lanes 3,
4, and 5), whereas greater than 60% inhibition of
basal transcription from the TRE-linked test promoter was observed upon
addition of the receptor (compare lanes 1 and 2).
These results demonstrate that the TR-mediated silencing is entirely
dependent on the presence of TRE(s) in the target promoter.
Ligand Binding Reverses TR-
Previous studies showed that the gene
regulatory activity of TR is greatly influenced by the binding of
hormonal ligand to the receptor
(23, 24, 25, 26, 27, 28) .
We therefore, examined the effect of Triac, a thyroid hormone analog,
on the transcriptional silencing activity of TR--mediated Silencing of
Basal Transcription
in the cell-free
transcription assay. As shown in Fig. 2, A and
B, while hormone-free TR-
inhibited basal transcription
( lane 2), incubation of TR-
with 1 µM Triac
prior to its addition to the transcription reaction abolished
receptor-mediated transcriptional repression ( lane 3).
Addition of Triac did not affect transcriptional activity of the
control AdML promoter (compare lanes 2 and 3)
indicating that the hormonal effect on RNA synthesis was indeed
mediated through TR-
. These results demonstrate clearly that
TR-
ceases to function as a repressor of target gene transcription
upon hormone binding.
Figure 2:
TR--induced silencing is reversed by
addition of hormone. Panel A, TR-
(1 pmol) was
preincubated with or without 10
M Triac
before addition to the cell-free transcription reaction. Panel
B, the plotted results represent an average of three independent
experiments. The RNA transcripts generated from the test template were
quantitated by densitometry and normalized with respect to the AdML
transcripts. In each experiment the value for basal transcription
(- TR) was adjusted to 100%, and the corresponding values
for test measurements were obtained. The error bars denote
standard deviation of the mean.
Roles of N-terminal and DNA Binding Domains of TR-
Previous studies by Baniahmad et al.(49) indicated that the transcriptional silencing function of
TR-
in Gene Silencing
lies within the C-terminal ligand binding domain between amino
acid residues 168 and 456. The functional role of either the N-terminal
region or the DNA binding domain of TR-
in receptor-mediated
silencing, if any, has not been elucidated. To investigate this, we
constructed mutant TR-
molecules in which various functional
domains were deleted (Fig. 3 A). The mutant receptor
proteins were expressed in bacteria, purified, and assayed for their
gene regulatory activities in cell-free transcription system
(Fig. 3, Panels B and C). These proteins were
also analyzed by EMSA for their DNA binding activities in the presence
or in the absence of RXR.
Figure 3:
Transcriptional activities of TR-
mutants. Panel A, linear structures of human TR-
and its
mutants. Panel B, SDS-PAGE profile of nickel-affinity-purified
TR-
mutants. The proteins were visualized by Coomassie staining.
Lane 1, wild-type TR-
; lane 2, TR
N;
lanes 3 and 4, TR
C; lanes 5 and
6, TR
N
D. Panel C, TR-
and various
deletion mutants (1 pmol of each) were analyzed for silencing
activities in cell-free transcription assays. The protein
concentrations of mutant receptors were determined by comparing the
intensities of stained bands with that of TR-
in SDS-PAGE.
Quantitations of RNA signals were performed by densitometry followed by
normalization with respect to the AdML signals. The value for basal
transcription (- TR) was set to 100% and the
corresponding test (+ TR) values were obtained. The
results of a representative experiment are
shown.
The mutant TRN with intact DNA and
ligand binding domains but harboring a deletion of N-terminal 82 amino
acids, silenced basal transcription as efficiently as the wild-type
receptor (Fig. 3 C, lane 4). TR
N formed
heterodimers with RXR and exhibited high affinity binding to TRE (data
not shown). The addition of the hormonal ligand reversed silencing by
this mutant (Fig. 3 C, lane 5). These results
indicated that the N-terminal 82 amino acids of TR-
are not
critical for receptor-mediated gene silencing.
C containing the N-terminal and the DNA binding
domains but, missing the C-terminal 196 amino acids, failed to repress
basal transcription from the target promoter (Fig. 3 C,
lane 6). TR
C did not display any significant DNA binding
activity either in the presence or in the absence of RXR (data not
shown), presumably due to the loss of the dimerization function
(52) . Recent studies by Au-Fliegner et al.(53) suggest that heterodimerization of TR is necessary for its
silencing activity. A third mutant TR
N
D with a deletion of
the N-terminal 145 amino acids that included the N terminus and most of
the DNA binding domain, also had no effect on basal level of RNA
synthesis from a TRE-linked promoter in the cell-free transcription
assay (Fig. 3 C, lane 8). This mutant, however,
retained the entire ligand binding domain (168-456) that exhibits
silencing activity when fused to a heterologous DNA binding domain
(49) . A deletion of 63 amino acids within the DNA binding
domain of TR
N therefore, converted an active repressor to
TR
N
D that is impaired in both DNA binding and silencing. We
conclude from these studies that specific DNA binding is essential for
transcriptional silencing by TR-
.
TR-
During transcription by RNA polymerase II there are
many steps that can be subjected to inhibition by a gene-specific
repressor. Active repression may occur at the level of assembly or
function of the transcription initiation complex or elongation of the
RNA transcripts. We first examined whether the assembly of the PIC is
the regulatory point at which TR- Inhibits the Assembly of a Functional
Transcription Preinitiation Complex at the Target
Promoter
exerts its gene repression
effects. To test this we added TR-
during or after the formation
of the PIC at the target promoter and monitored RNA synthesis following
the addition of nucleotide triphosphates (Fig. 4). The presence
of low levels of Sarkosyl in the reactions blocked reinitiation of
transcription and ensured that only one round of RNA synthesis occurred
(54) .
Figure 4:
TR- inhibits functional assembly of
the preinitiation complex. In reaction 1, template DNA and HeLa extract
were preincubated at 30 °C for 30 min without ( lane 1) or
with ( lanes 2, 3, and 4) TR-
and then
nucleotide mixture is added to initiate RNA synthesis. In reaction 2, a
mixture of HeLa extract and template DNA is preincubated initially for
15 min at 30 °C. TR-
was then added ( lanes 6,
7, and 8) and the incubation was continued for 15
more min followed by the addition of nucleotide mixture. The
transcription reactions were then carried on for 45 min at 30 °C.
Both reactions were performed in the presence of 0.025% Sarkosyl, a
non-ionic detergent.
When TR- was present during the incubation of the
test template with HeLa nuclear extract in the reaction 1, we observed
strong inhibition of basal RNA synthesis (Fig. 4, compare
lane 1 with lanes 2, 3, or 4). In
contrast, in the reaction 2, when TR-
was added following
preincubation of the test template with the nuclear extract which
generated a PIC at the TATA promoter, no significant repression of
basal transcription was noted (Fig. 4, compare lane 5 with lanes 6, 7, or 8). These results
suggest that TR-
, anchored to a TRE at the target promoter, may
actively inhibit one or more steps during the assembly of a functional
PIC at the TATA box. Once the PIC is fully assembled, it is refractory
to inhibition by TR-
.
TR-
Our
observation that hormone-free TR- Interacts Directly with an Intermediate
TFIIB-TBP-TATA during Preinitiation Complex Assembly
prevents the assembly of a PIC
at a minimal promoter containing only a TATA box suggests that TR-
may function by interacting with components of the basal transcription
machinery either directly or indirectly through a mediator protein(s).
Recent studies indicated that TR-
or TR-
bound directly to
the general transcription initiation factor TFIIB when the two proteins
were combined in vitro(48, 49) . However,
these studies did not reveal either the functional significance of this
interaction or the identity of transcriptional intermediate(s) in the
initiation complex assembly pathway that are targets of inhibition by
TR-
or TR-
. It is also not clear how ligand may modulate the
interactions between the receptor and the target intermediate(s) to
reverse silencing.
may interact with the components of
the basal transcription machinery to regulate the initiation process.
It is known that in the earliest step of initiation complex assembly,
the TBP, a component of the multisubunit basal factor TFIID recognizes
the TATA box. The resulting complex is, however, unstable under
standard EMSA conditions and cannot be easily detected by this assay.
TFIIB, which does not bind to the TATA box by itself, interacts with
TBP bound at the TATA box to generate the TFIIB
TBP complex
(40, 41) . This is the earliest intermediate detectable
under EMSA conditions. We, therefore, analyzed the interaction of
TR-
with the TFIIB
TBP complex.
Figure 5:
Unliganded TR- binds to the
TFIIB
TBP
TATA complex. Panel A, a
P-labeled double-stranded oligonucleotide (40 bp long, 0.1
ng) containing sequences from -30 to +10 of the AdML
promoter including the TATA box was used as a probe in the EMSA which
was performed as described before (44). Bacterially produced human TBP
(10 ng), human TFIIB (7 ng), and human TR-
(1 pmol) were used in
EMSA reactions (10 µl) where indicated. The human TBP and TFIIB
proteins were expressed in E. coli and purified by published
procedures (50, 51). NS1 and NS2 denote nonspecific complexes. NS1 and
NS2 are formed by weak interactions of nonreceptor contaminant proteins
with the
P-labeled DNA probe and complex 1, respectively.
Panel B, preformed complex 1 was incubated with anti-TBP or
anti-TFIIB antibodies as indicated in the figure. Panel C,
preformed complex 1 was incubated with TR-
in the presence of
increasing concentrations (0.2, 0.5, 1.0, and 2.0 µl) of an
anti-TR-
antibody. Panel D, preformed complex 1 was
incubated with increasing concentrations (0.5-1.0 µl) of the
anti-TR-
antibody.
We next examined whether TR- can interact with
the TFIIB
TBP complex. In EMSA, incubation of the TATA
oligonucleotide with purified TR-
alone (Fig. 5 A,
lane 1) or TR-
in combination with either TFIIB or TBP
(data not shown) did not generate any specific DNA-protein complex.
However, when the TATA oligonucleotide was preincubated with both TFIIB
and TBP, and TR-
(100 nM) was subsequently added to this
reaction, a new, stable complex, that is retarded further in the gel,
is formed. We term this higher molecular weight DNA-protein complex as
complex 2 (Fig. 5 A, lanes 5-8). We also
noted the generation of two nonspecific complexes indicated as NS1 and
NS2 (a smeary complex immediately underneath complex 2) in
Fig. 5A ( lanes 5-8) and Fig. 5 C ( lane 1). Unrelated bacterial proteins that were retained
by the nickel-affinity column upon passage of Escherichia coli extract through it, existed as contaminants in our protein
preparations. In control experiments (data not shown), addition of
these irrelevant bacterial proteins to complex 1 generated the
nonspecific complexes NSI and NS2 but not complex 2. These results
indicated strongly that the formation of complex 2 was indeed
TR-
-dependent. Since TR-
itself did not bind to the
TATA-containing probe, complex 2 appeared to be generated by
protein-protein interaction between the receptor and the TFIIB
TBP
complex.
in complex 2,
we studied the effects of adding an antibody specific for TR-
on
the formation of complex 2 in the EMSA. As shown in
Fig. 5C, addition of a specific anti-TR-
antibody
but not a control antibody (data not shown) greatly reduced the
intensity of complex 2 signal indicating that TR-
is indeed a
component of complex 2. The antibody did not affect the formation of
either the NS1 or the NS2 complex, thus demonstrating the specificity
of the antibody-antigen reaction. The anti-TR-
antibody however,
stimulated slightly the formation of complex 1
(Fig. 5 D). Such enhancement of complex 1 was observed
also upon addition of an unrelated, control antibody (data not shown),
which did not inhibit complex 2 formation. It is therefore, clear that
the binding of the anti-TR antibody to the receptor prevented its
physical association with complex 1. These results demonstrated that
under our EMSA conditions, TR-
can interact with the
TFIIB
TBP complex through direct protein-protein interactions. The
presence of a TRE at the target promoter may facilitate stable
protein-protein contacts between the TRE-bound receptor and the
TFIIB
TBP complex at the TATA box under cell-free transcription
conditions. Based on our results, we are tempted to propose that the
mechanism of transcriptional silencing by TR-
may involve the
formation of a stable complex of unliganded receptor, TFIIB and TBP at
a TRE-linked TATA promoter at an early rate-determining step of
initiation complex assembly.
TR-
RXRs exist ubiquitously in all
tissues
(16) . Numerous studies indicate that TR monomers or
homodimers display only weak binding to TRE and heterodimerization with
RXR markedly enhances binding of TR to its response element. It is thus
likely that TR-RXR heterodimers may represent the functional form of TR
in vivo. We therefore investigated whether the TR--RXR Heterodimers Interact with the
TFIIB
TBP
TATA Complex
-RXR
heterodimers can interact with the assembling transcriptional complexes
during the initiation event. As shown in Fig. 6 A,
lane 1, TR-
alone bound weakly to TREpal. In the presence
of an equimolar amount of human RXR
, TR-
-RXR heterodimers
formed readily and were the predominant species that bound to DNA. When
we added increasing concentration of RXR in the presence of excess
TREpal, the amount of the heterodimer-TRE complex increased only
marginally (Fig. 6 A, lanes 2-6). These
results indicated that under our EMSA conditions a 3-fold molar excess
of RXR was sufficient to drive all the TR-
in the reaction into
heterodimeric association with RXR.
Figure 6:
TR--RXR heterodimer binds to
TFIIB
TBP
TATA complex. Panel A, TR-RXR heterodimers
form readily on TREpal. A 20-bp double-stranded oligonucleotide
containing TREpal was end-labeled by
P and used as a probe
in EMSA. TR-
, 0.5 pmol, was combined with increasing amounts of
bacterially produced human RXR
, 0.5, 1.0, 1.5, 2.5, and 5.0 pmol
( lanes 2-6), in EMSA reactions. The notations TR-TR and
TR-RXR point to the positions of migration of the TR homodimer and the
TR-RXR heterodimer, respectively. Panel B, binding of
TR-
-RXR heterodimers to complex 1. Complex 1 was incubated with
none ( lane 1); RXR
, 1 pmol ( lane 2); TR-
,
0.5 pmol + RXR
, 1 pmol ( lane 3); TR-
, 0.5 pmol
+ RXR
, 1.5 pmol ( lane 4); and TR-
, 0.5 pmol
( lane 5). The added proteins were preincubated with unlabeled
TREpal oligonucleotide (1 ng) before addition to preformed complex 1.
Complex 2 and 2` denote TR
TFIIB
TBP
TATA and
TR
RXR
TFIIB
TBP
TATA,
respectively.
We then examined whether these
heterodimers were capable of interacting directly with the
TFIIBTBP complex. For this purpose, TR-
alone or RXR alone
or a combination of TR-
and RXR in a molar ratio of 1:3 was
preincubated in the presence of excess unlabeled TREpal and added to a
reaction containing TFIIB, TBP, and TATA element
(Fig. 6 B). TR-
alone bound to complex 1 and formed
complex 2 as expected (Fig. 6 B, lane 5). The
addition of equivalent amount of RXR alone to complex 1, however, did
not result in the formation of any complex retarded further in the gel.
This result indicated that either RXR does not recognize complex 1 or
the interaction between RXR and complex 1 is rather weak, so that any
RXR
TFIIB
TBP complex that may form is not stable enough to
be detected under the conditions of the EMSA (Fig. 6 B).
An enhancement of TFIIB
TBP complex formation, however, was
reproducibly seen when RXR was added (Fig. 6 B, lanes
1 and 2). The reason for this effect is not clear. The
addition of TR-
-RXR heterodimers to complex 1, on the other hand,
generated a retarded complex 2` (Fig. 5 B, lanes 3 and 4). Complex 2` migrated to the same region of the gel
as complex 2. When gels were run for longer times, however, complex 2`
appeared to migrate slightly slower than complex 2 (data not shown).
This slight difference in migration has been reproducibly observed in
multiple experiments and is consistent with the difference in migration
of homo- and heterodimeric receptor-DNA complexes in mobility shift
assays. These results thus indicated that under conditions that ensure
TR-
-RXR heterodimerization, these heterodimers can undergo direct
protein-protein interaction with the TFIIB
TBP complex at the TATA
box. Our results also demonstrated that TR-
-RXR heterodimers can
interact with the TFIIB
TBP complex even when bound to the hormone
response element (TREpal).
Hormonal Ligand Reduces Interaction of TR-
Transcriptional activity of TR is modulated by thyroid
hormone. Ligand binding is also known to induce an alteration in the
receptor conformation
(55, 56) . We, therefore,
investigated whether binding of the hormonal ligand to TR- and
TR-
-RXR with TFIIB
TBP
TATA
Complex
influences its interaction with the TFIIB
TBP complex. In this
experiment, we preincubated TR-
with or without 1 µM
Triac, added the receptor to complex 1 and monitored the formation of
complex 2 by EMSA. Treatment of TFIIB and TBP with Triac did not
exhibit any effect on the formation of complex 1
(Fig. 7 A, lanes 1 and 2). The addition
of TR-
preincubated with Triac however, generated markedly lesser
amounts of complex 2 compared to that produced by unliganded TR-
(Fig. 7 A, lanes 3 and 4). By our
estimate, complex 2 formation declined by greater than 75% upon ligand
binding to TR-
. If this reduction in complex 2 formation is due to
the inability of ligand-bound TR-
to bind efficiently to complex
1, one may expect to detect unbound complex 1 in these reactions.
Interestingly, as the amount of complex 2 diminished upon ligand
binding to TR-
, there was no concomitant appearance of complex 1
signal in these reactions (compare lanes 3 and 4). We
noted, instead, an increase in the signal of the nonspecific complex
NS2 upon ligand treatment. This may reflect the increased binding of
irrelevant bacterial proteins to complex 1 as the formation of the
specific complex (complex 2) declined. TR-
-RXR heterodimers
treated with Triac also generated significantly lesser amounts of
complex 2` than those incubated without ligand (Fig. 7 B,
lanes 1 and 2). Ligand, therefore, strongly reduced
the interaction between TR-
or TR-
-RXR and the TFIIB
TBP
complex.
Figure 7:
Ligand decreases interaction between
complex 1 and TR- or TR-
-RXR. Panel A, in EMSA
reactions, complex 1 was treated without hormone ( lane 1);
with hormone ( lane 2); hormone-free TR-
( lane
3); and hormone-treated TR-
( lane 4). A
P-labeled TATA oligonucleotide was used as probe. In
lanes 5 and 6, complex 2 was initially formed on the
TATA box and then treated with 1 µM Triac and analyzed by
EMSA. NS2 indicate nonspecific complex. Panel B: TR-
treated with (lane 2) or without ( lane 1) Triac (1
µM) was added to an EMSA reaction containing RXR
, and
complex 1. A
P-labeled TATA oligonucleotide was used as
probe. In lanes 3 and 4, Complex 2` was first formed and then treated
with hormonal ligand as indicated. Panel C, TR-
and
mutants TR
N and TR
C (1 pmol of each) were treated with
( lanes 2, 6, and 9) or without ( lanes
1, 5, and 8) Triac and added to EMSA reactions
containing TFIIB, TBP, and TATA probe as indicated in the
figure.
We next examined the effects of the hormonal ligand on
preformed complex 2. For this purpose, we initially generated complex 2
by incubating unliganded TR- with complex 1 and then treated it
with or without Triac. As shown in Fig. 7 A ( lanes 5 and 6), the amount of preformed complex 2 was
significantly reduced upon incubation with Triac but not with a control
buffer. A similar reduction in complex 2` was observed when ligand-free
TR-
RXR-B
TBP complex was preformed and then incubated
with Triac (Fig. 7 B, lanes 3 and 4).
It is likely that ligand binding induces a conformational change in TR
that perturbs its interaction with TFIIB or TBP or both in complex 2 or
2`. Our results are consistent with the observation of Baniahmad et
al.(49) that ligand binding reduced the interaction of
the C terminus of TR-
with TFIIB.
TBP complex and
each of the three TR-
truncation mutants that we generated. The
N-terminal truncation mutant TR
N, which repressed basal
transcription, displayed direct binding to complex 1
(Fig. 7 C, lane 5). This interaction occurred
presumably through the C-terminal domain of the receptor and generated
a specific complex further retarded in the gel. In contrast,
ligand-bound TR
N, which failed to silence basal transcription, did
not bind to complex 1 efficiently (Fig. 7 C, lane
6). These results are consistent with the idea that the silencing
function resides in the C terminus of TR-
and gene repression is a
consequence of stable interaction between this region of the receptor
and the assembling complex 1. Ligand binding to the C terminus of the
receptor which reduced such interactions, also relieved transcriptional
silencing.
N
D behaved in a manner
essentially similar to that of TR
N. Unliganded TR
N
D
bound to complex 1, while the hormone-bound mutant showed significantly
reduced binding (data not shown). As mentioned earlier, this mutant did
not bind to DNA and failed to inhibit basal RNA synthesis in cell-free
transcription assay. Taken together, these results suggest that
although the C terminus of TR-
can potentially undergo
protein-protein interaction with the basal transcription machinery,
binding of the receptor to its recognition site at the target promoter
is essential for stabilizing such interactions. Interestingly, the
mutant TR
C missing 196 C-terminal amino acids in the ligand
binding domain also displayed binding to complex 1
(Fig. 7 C, lane 8). This interaction was
mediated apparently through the N terminus of the receptor and as
expected, was not affected by the addition of ligand
(Fig. 7 C, lane 9). This result is consistent
with previous reports indicating that the N terminus of TR-
can
also bind to TFIIB
(49) . The functional consequence of such
interaction is, however, not clear. Based on our observation that the
mutant TR
N functions efficiently as a silencer
(Fig. 3 C), we propose that the C terminus of TR-
rather than its N terminus, in concert with the DNA binding domain,
plays a crucial role in gene silencing.
and the
ligand-induced reversal of this repression. A number of thyroid
hormone- and receptor-dependent cell-free transcription systems have
been described recently. Fondell et al.(48) employing
bacterially produced TR-
demonstrated that the hormone-free
receptor repressed basal transcription in vitro, while the
hormone-bound receptor failed to do so. Suen and Chin
(57) have
described an in vitro system in which an endogenous mixture of
the TR isoforms,
and
, in rat GH3 pituitary cell extracts
stimulated (about 4-fold) transcription from a TRE-linked promoter in
the presence of T
. However, the receptor pool being
endogenous in this transcription system, it is not clear whether the
observed hormone-induced activation is indeed over and above the basal
level of transcription or simply represent a ligand-dependent release
of TR-mediated silencing of target gene transcription. Recently, Lee
et al.(58) using baculovirus-expressed rat TR-
have reported a modest (2-fold over minus hormone control)
ligand-induced stimulation of RNA synthesis from a TRE (malic
enzyme)-linked template in B-cell nuclear extracts. It is possible that
the apparent lack of TR-
-dependent RNA synthesis above the basal
level in our transcription reactions is due to a paucity in HeLa
extracts of putative cofactors that may facilitate such activity.
Nevertheless, the HeLa extract-based cell-free transcription system
described here (Figs. 1 and 2) is ideal to work out the mechanism of
TR-mediated gene silencing and its ligand-induced reversal.
during PIC assembly led to the formation of a transcriptionally
inactive complex. In contrast, a fully assembled PIC was refractory to
inhibition by TR-
. Our observations are reminiscent of earlier
studies by Johnson and Krasnow
(59) , who showed that the
Drosophila even-skipped (EVE) homeodomain protein functioned
as a transcriptional repressor by inhibiting an early step in PIC
assembly. The assembling complexes became resistant to repression by
EVE at a subsequent (undefined) step. Similar results have recently
been reported for unliganded TR-
by Fondell et al.(48) indicating an inhibitory role of the receptor during the
formation of a functional PIC. As the function of a fully assembled
complex was not inhibited by TR, we reasoned that the receptor blocked
one or more intermediate steps leading to the formation of the PIC.
TBP complex, an early intermediate in
the PIC assembly pathway, as a potential target of silencing by TR.
Based on the results of our protein-protein interaction experiments, we
postulate that the unliganded receptor represses basal transcription by
interacting with this intermediate transcriptional complex and altering
its function. One can think of at least two possible scenarios by which
this interaction may lead to the inhibition of initiation complex
assembly. Binding of TR-
to the TFIIB
TBP complex may lead to
an impairment in the recruitment of downstream basal factor(s) such as
TFIIF, RNA polymerase II, TFIIE, and TFIIH. Alternatively, TR-
may
bind to TFIIB
TBP, and allow it to recruit some or all of the
downstream factors. The resulting complex(es) may, however, enter
nonproductive pathway due to an improper configuration.
or TR-
-RXR and
the individual basal transcription factors in the context of the
TFIIB
TBP complex remains to be determined. Using
immunoprecipitation technique, Fondell et al.(48) reported that TR-
bound to TFIIB. Baniahmad et al.(49) demonstrated that the N terminus of TR-
interacted with the C terminus of TFIIB, while the C-terminal ligand
binding domain of the receptor recognized the N-terminal region of
TFIIB. Our studies using truncation mutants of TR-
indicated also
that both N and C termini of the unliganded receptor can interact with
the TFIIB
TBP complex. Hisatake et al.(60) reported that the C-terminal domain of TFIIB is involved in
TFIIB
TBP complex formation at the TATA box. Studies by Ha et
al.(61) revealed that the N terminus of TFIIB interacts
with TFIIF. Taken together, these results raise the interesting
possibility that the interaction of the C terminus of TR-
with the
N terminus of TFIIB may affect TFIIF binding and subsequent recruitment
of RNA polymerase II. The role of the N terminus of the receptor in
transcriptional silencing however, remains unclear. Our observation
that a mutant TR-
lacking N-terminal 82 amino acids silenced basal
transcription efficiently indicates that the interaction of the N
terminus of the receptor with the basal transcription complex is not
essential for silencing.
TBP complex
represents a simplified version of the more native TFIIB
TFIID
complex. In the TFIID complex, TBP is associated with a number of
polypeptides which are termed TBP-associated factors
(39) .
TBP-associated factors are essential for activator-induced
transcription and TBP can efficiently replace TFIID for basal level of
RNA synthesis
(39, 62) . It is conceivable that TR-
either in unliganded or in ligand-bound state may contact one or more
TBP-associated factors in the native TFIIB
TFIID complex. One
should also consider the possibility that the receptor may contact
directly additional components of the basal transcription machinery
besides TFIIB, such as RNA polymerase II and regulate the activity of
one or more transcriptional complex(es) which follow the
TFIIB
TFIID complex in the assembly pathway. Future studies
investigating these possibilities will shed further light on how
TR-
, functioning as a hormone-dependent gene regulator, interacts
with the native, assembling initiation complexes at the TATA box.
did not inhibit basal transcription from a control TRE-less promoter
and (ii) a receptor fragment (145-456) containing the putative
repressor domain but lacking the DNA binding function failed to silence
basal transcription from a TRE-linked promoter. These results extend an
earlier observation by Damm et al.(23) that the
silencing effect of TR is TRE-dependent. It is interesting to note
that, although the 145-456 fragment of the receptor exhibited
protein-protein interaction with TFIIB
TBP complex under EMSA
conditions, it did not inhibit basal transcription. These results
indicate that anchoring of the receptor to a TRE at the target promoter
is essential for transcriptional repression. It is likely that in the
intact cell or in the cell-free transcriptional extracts, the receptor
may function as a gene repressor by making stable protein-protein
contacts with complexes such as the TFIIB
TBP at the TATA box,
only when it is promoter-bound.
with the basal transcription
machinery may alter upon ligand binding to the receptor. Consistent
with this line of reasoning, we observed that ligand binding to
TR-
drastically reduced its interaction with the TFIIB
TBP
complex at the TATA box under EMSA conditions. Similar results were
obtained with mutant receptors, TR
N and TR
N
D. The
molecular interactions within the TR-
TFIIB
TBP complex
that are disrupted by ligand binding to the receptor are not entirely
clear at present. Previous reports indicated that ligand binding
weakened the interaction of a C-terminal peptide of TR-
with TFIIB
(49) . Our studies also suggest that ligand binding triggers the
release of the C terminus of the receptor from the TFIIB
TBP
complex. The combined results of the cell-free transcription and in
vitro protein-protein interaction assays presented here, tempt us
to postulate that the mechanism of ligand-dependent reversal of
TR-induced gene silencing may involve the dissolution of an abortive
association between the C terminus of the TRE-bound receptor and the
TFIIB
TBP complex. The ligand-induced release of the inhibitory
function of the receptor may allow the recruitment of downstream
initiation factors and lead to the subsequent assembly of an active
transcription initiation complex at the target promoter.
or TR-
-RXR
heterodimers repressed basal transcription from a TREpal-linked
promoter in a cell-free transcription system reconstituted with
purified basal transcription factors. The overall repression of basal
transcription by TR or TR-RXR in this purified system was, however,
remarkably less efficient compared to that in a nuclear extract. Recent
reports by Casanova et al.(63) and Baniahmad et
al.(64) indicate that coexpression of a ligand binding
domain peptide of either chicken TR-
or human TR-
inhibited
transcriptional repression by a chimeric TR-
or TR-
protein
in the absence of hormone during transient transfection in HeLa or CV1
cells. These results are consistent with the scenario that cofactor(s)
that enhance TR-mediated gene repression may exist in nuclear extracts.
, triiodothyronine; PIC, preinitiation
complex; TREpal, palindromic thyroid hormone response element; EMSA,
electrophoretic mobility shift assay; Triac,
[4-(4-hydroxy-3-iodophenoxy)-3,5-diiodophenyl]acetic acid;
DB, TFIID-TFIIB complex.
and human RXR
. We thank Dr. C. W.
Bardin for his help and support during the entire course of this study.
We acknowledge Dr. Indrani Bagchi for critical reading of the
manuscript and Dr. M. Jeyakumar for preparing transcription factors.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.