From the Section of Microbiology, Division of Biological Sciences, University of California, Davis, California 95616
Received for publication, November 3, 2000, and in revised form, January 17, 2001
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ABSTRACT |
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Nuclear hormone receptors, such as the thyroid
hormone receptors (T3Rs) and retinoid X receptors (RXRs), are
ligand-regulated transcription factors that control key aspects of
metazoan gene expression. T3Rs can bind to DNA either as receptor
homodimers or as heterodimers with RXRs. Once bound to DNA, nuclear
hormone receptors regulate target gene expression by recruiting
auxiliary proteins, denoted corepressors and coactivators. We report
here that T3R homodimers assembled on DNA exhibit particularly strong interactions with the SMRT corepressor, whereas T3R·RXR heterodimers are inefficient at binding to SMRT. Mutants of T3R that exhibit enhanced repression properties, such as the v-Erb A oncoprotein or the
T3R Small, hydrophobic hormones, such as steroids, retinoids, and the
thyroid hormones T3 and T4 thyronine, regulate many important aspects
of metazoan differentiation, reproduction, and homeostasis. These
hydrophobic hormones function by binding to specific nuclear receptors
that operate as hormone-regulated transcription factors (1-6).
Different nuclear receptors bind to different DNA sequences, denoted
hormone response elements
(HREs),1 and modulate the
expression of adjacent target genes. In this fashion, different sets of
target genes are regulated in response to different hormone ligands,
leading to physiologically appropriate changes in target cell phenotype
(1-3, 5, 6).
Many nuclear hormone receptors possess bipolar transcriptional
properties and can either repress or activate expression of target
promoters (7-9). These bimodal regulatory properties are manifested
through the ability of nuclear receptors to interact with auxiliary
proteins, denoted corepressors and coactivators (reviewed in Refs.
10-14). Thyroid hormone receptors (T3Rs) and retinoic acid receptors
(RARs), for example, typically interact with a corepressor complex in
the absence of hormone and thereby confer transcriptional repression
(15-22). The corepressor complex includes SMRT or its paralog N-CoR,
which contacts the nuclear receptors directly, as well as additional
proteins that are tethered indirectly through interactions with SMRT,
such as mSin3, SAPs, Ski, and an assortment of histone deacetylases
(23-32). The addition of cognate hormone induces a conformational
change in the T3Rs and RARs, leading to dissociation of the SMRT
corepressor complex from the receptor and recruitment of a novel set of
coactivator proteins that confer transcriptional activation (33-36).
Coactivators include the p160 family, such as SRC-1 and GRIP-1,
CBP/p300, and the DRIP-TRAP-SMCC complex (10-14, 37, 38). Once
recruited, coactivators and corepressors regulate transcription through
modification of the chromatin template and by interactions with the
general transcriptional machinery (12-14, 23, 25, 39-42).
Nuclear receptors bind to DNA with each receptor molecule recognizing a
conserved, 6-8-base DNA sequence referred to as a "half-site" (3).
Given that most nuclear receptors are able to bind to DNA as protein
dimers, a prototypic HRE is composed of two half-sites (43, 44). The
sequence of the individual half-sites, their spacing, and their
orientation contribute to the specificity of DNA recognition by the
different nuclear receptors (45-47). Notably, however, a surprisingly
diverse array of DNA sequences can serve as response elements for a
given nuclear receptor. T3Rs, for example, are able to bind to
HREs comprised of two half-sites oriented as a direct repeat with a
4-base spacer (DR-4), an inverted repeat with no spacer (INV-0), or a
divergent repeat with a 6-base spacer (DIV-6) (46-52). Imposed on this
diversity of HREs is the ability of many nuclear receptors to bind to
DNA not only as homodimers but also as heterodimers with other members
of the nuclear receptor family. Retinoid X receptors (RXRs) are
particularly important heterodimer partners for many nuclear receptors,
and T3R·RXR and RAR·RXR heterodimers can form with higher affinity
and exhibit stronger transcriptional activation properties than do the
corresponding homodimers (3, 4, 45).
We wished to determine if the ability of nuclear receptors to recruit
SMRT corepressor might differ for receptor homodimers versus
heterodimers and if the nature of the DNA response element could
influence, directly or indirectly, this receptor/corepressor interaction. We report here that T3R homodimers assembled on DNA exhibit particularly strong interactions with the SMRT corepressor, whereas T3R·RXR heterodimers are inefficient at binding to
corepressors. Mutants of T3R that exhibit enhanced repression
properties, such as the v-Erb A oncoprotein or the T3R Molecular Clones, Oligonucleotides, and Antisera Electrophoretic Mobility Shift Assays--
T3R
EMSAs were also performed using nuclear receptors synthesized from pSG5
vectors by use of a coupled in vitro
transcription/translation system (TnT system; Promega). A total of 6 µl of each in vitro synthesized receptor preparation was
incubated with the radiolabeled oligonucleotide probe (100,000 cpm,
representing 100 ng of DNA) for 20 min at 25 °C in 40 mM
HEPES, pH 7.8, 50 mM KCl, 5 mM
MgCl2, 1 µM ZnCl2, 6% glycerol,
0.2 µg/µl bovine serum albumin, 0.066 µg/µl poly(dI-dC). The
resulting protein/DNA complexes were then resolved by electrophoresis
on a 5% polyacrylamide gel containing 1% glycerol and were visualized
and quantified as above.
Avidin-Biotin-DNA Binding Assay--
Approximately 0.05 µg of
each biotin-tagged oligonucleotide were immobilized by incubation with
20 µl (packed volume) of streptavidin-agarose. The
oligonucleotide-agarose complexes were then incubated for 30 min at
4 °C with 250 ng of T3R Transient Transfections--
CV1 cells were transfected by use
of a Lipofectin protocol (Life Technologies, Inc.). Typically 50 ng of
the pSG5-T3R SMRT Corepressor Preferentially Interacts with Homodimers,
Not Heterodimers, of T3R on DR-4 DNA Response Elements--
We
and others have reported that SMRT interacts more avidly with nuclear
receptor dimers than with receptor monomers (16, 58-60). However, most
of these prior studies employed two-hybrid and GST pull-down assays and
did not address the corepressor interaction properties of defined
receptor complexes assembled on authentic DNA response elements. We
therefore employed an EMSA to determine the ability of T3R alone or of
RXR and T3R together to interact with SMRT when these receptors were
bound to a suitable DNA response element. We first examined the ability
of T3R or T3R·RXR to bind to a DR-4 DNA element and the ability of
SMRT to "supershift" the mobility of the resulting receptor-DNA
complexes. T3R
A GST-SMRT derivative, purified from E. coli, did not
detectably bind to the DR-4 probe when tested alone (Fig.
1B, lane 10). However, the addition of
the GST-SMRT derivative to the T3R protein preparation resulted in a
further reduction in the electrophoretic mobility of the T3R homodimer
complex (i.e. a "supershift") indicative of an SMRT/T3R
interaction (Fig. 1B, lane 3; labeled
T3R/T3R/SMRT). No supershift was observed with a
nonrecombinant GST preparation (Fig. 1B, lane
1). As anticipated for an authentic SMRT·T3R complex, antibodies to T3R, but not to RXR, further shifted the migration of the
T3R·T3R·SMRT complex to a still slower mobility, and the supershifted T3R·T3R·SMRT species could be dissociated by the addition of T3 hormone (Figs. 1, B and C)
(20).
In contrast to the robust supershift observed for T3R·T3R homodimers,
the addition of GST-SMRT to T3R·RXR heterodimers assembled on the
same DR-4 DNA element resulted in a very weak supershift, with the bulk
of the heterodimer failing to detectably interact with the corepressor
(Fig. 1B, lanes 5-8; quantified in
Fig. 2A). The relatively poor
supershift of the T3R·RXR heterodimer compared with the T3R·T3R
homodimer was observed under a variety of conditions, using a range of
GST-SMRT concentrations, and was not enhanced by the addition of an
RXR-specific ligand; the DNA probe was in excess in all experiments
(data not shown). Of note, the modest amount of SMRT-supershifted
complex that did form in the presence of T3R·RXR heterodimers reacted
primarily with anti-T3R but not with anti-RXR antibodies (see below);
these results suggest that even in the presence of a T3R·RXR
heterodimer, the corepressor may be able to recruit T3R homodimers,
perhaps by shifting the receptor equilibrium away from the heterodimer
toward the homodimeric species.
Our results suggested that T3R homodimers, when bound to a DR-4
element, interacted more strongly with corepressor than did T3R·RXR
heterodimers. To more rigorously test this hypothesis, we employed the
DR-4 DNA element again and adjusted our EMSA conditions to generate a
mixed receptor population, so that both T3R·T3R homodimers and
T3R·RXR heterodimers were present in the same reaction (Fig.
2B, lane 7). We then challenged this
mixed receptor population by the addition of the GST-SMRT construct.
Under these conditions, virtually all of the T3R·T3R homodimer was
supershifted into the T3R·T3R·SMRT complex, whereas very little of
the T3R·RXR heterodimer, present in the same reaction, was
supershifted by the addition of GST-SMRT (Fig. 2B,
lane 8). Close inspection did reveal that introduction of SMRT under these conditions resulted in a modest reduction in the amount of T3R·RXR heterodimer (compare
lane 8 with lane 7);
nonetheless, the predominant SMRT-containing supershifted complex even
under these conditions was the T3R·T3R·SMRT species (compare
lane 8 with lane 3), and
this supershifted complex did not contain significant amounts of RXR
when probed with RXR-directed antibodies (compare lane
10 with lanes 8 and 9).
These results support the hypothesis that SMRT preferentially binds to
T3R·T3R homodimers, not to T3R·RXR heterodimers, and indicate that
this preferred binding of the homodimer by SMRT may be sufficient to partially shift the receptor equilibrium away from the heterodimer species.
The Preferential Interaction of SMRT Corepressor with T3R
Our results, obtained by EMSA on receptors bound to DNA, appeared to
contradict studies, using GST pull-down or two-hybrid approaches, that
suggested T3R interacts with SMRT more strongly in the presence of RXR
than in its absence (58, 60). We therefore confirmed our results by use
of an alternative methodology. We incubated T3R The Preferential Association of SMRT with T3R Homodimers Was Also
Observed for a Variety of Natural and Synthetic DNA Response
Elements--
We next sought to determine if the preferential binding
of SMRT to homodimers of T3R, observed on DR-4s, extended to other classes of DNA response elements. We first compared DR-4, DIV-6, and
INV-0 response elements (49). T3R T3Rs Bound to Different Response Elements Exhibited a Similar
Release of Corepressor in Response to Hormone--
The experiments
detailed above tested the interaction of T3R and T3R·RXR with
corepressor in either the absence of hormone or the presence of a large
excess (1 µM) of T3 ligand. We wished to determine if
binding of these receptor complexes to different response elements
might subtly influence the ability of hormone to displace corepressor
at intermediate hormone concentrations. We therefore repeated our
previous experiments using T3R Overexpression of RXR in Transfected Cells Counteracts T3R-mediated
Repression--
Our results suggested that homodimers, and not
heterodimers, of T3R may serve to confer target gene repression
in vivo. To test a prediction of this hypothesis, we
examined the ability of T3Rs to repress reporter gene expression in
transfected cells in the presence of increasing amounts of RXR. CV-1
cells lack endogenous T3Rs, and a DR-4-luciferase reporter displayed a
basal level of expression when introduced into these cells (Fig.
5). Co-introduction of a T3R A T3R
The v-Erb A, an Oncogenic Form of T3R
To resolve this apparent conflict in the literature, we determined if
v-Erb A preferentially forms homodimers, rather than heterodimers, only
on specific DNA elements and, if so, what the consequences might be for
corepressor recruitment. We first examined the ability of v-Erb A to
bind to the DR-4 and the cLYS (DIV-6) elements. In the absence of RXR,
the v-Erb A protein bound to both DNA elements with a mobility
characteristic of v-Erb A homodimers; no equivalent complex was
observed in control experiments using unprimed reticulocyte lysates
(Fig. 7B). Intriguingly, v-Erb
A formed homodimers on these DNA probes more readily than did T3R
Are these different dimerization properties of v-Erb A manifested as
different interactions with corepressor? In the absence of RXR, v-Erb A
homodimers formed on either the DR-4 or the cLYS elements were
supershifted by SMRT, with the enhanced formation of v-Erb A homodimers
on the cLYS element reflected as an enhanced formation of SMRT complex
(Fig. 8, lanes 5 and 6 and
lanes 14 and 15). This difference
between the ability of SMRT to bind to v-Erb A on the two different
response elements was further magnified in the presence of RXR. The
addition of RXR to v-Erb A on the DR-4 element resulted in a partial
conversion of the v-Erb A homodimer to a v-Erb A·RXR heterodimer
complex; the addition of SMRT to this mixed population preferentially
supershifted the v-Erb A homodimeric complex but left unaltered the
majority of the v-Erb A·RXR heterodimeric complex (Fig. 8, compare
lanes 8 and 7). In contrast to the
DR-4, the cLYS element, which selected virtually exclusively for v-Erb
A homodimers, generated an extremely strong interaction of v-Erb A with
SMRT corepressor (Fig. 8, compare lanes 19 and
18). We conclude that v-Erb A displays an elevated ability to form homodimers and a decreased ability to form heterodimers relative to the T3R RARs, Unlike T3Rs, Interact with SMRT Corepressor both as Receptor
Homodimers and as Heterodimers with RXRs--
We examined if the
preferential recruitment of SMRT by T3R homodimers extended to other
members of the nuclear receptor family. Unliganded RARs, like
unliganded T3Rs, interact with SMRT and can form either homodimers or
heterodimers with RXR (3, 4, 15, 17, 20). We therefore tested RARs in
our EMSA procedure. RAR The Efficacy of Corepressor Recruitment by T3Rs Is Contingent on
the Nature of the Receptor Dimer--
Our studies demonstrate that the
SMRT corepressor is recruited at high affinity to homodimers of T3R
assembled on DNA but not to heterodimers composed of T3R and RXR. The
strong preference of SMRT for T3R homodimers over heterodimers extended
to an assortment of synthetic and natural DNA response elements, was
observed for a variety of T3R and RXR isoforms, and was detected by
both an EMSA supershift protocol and by an avidin-biotin DNA binding
procedure. The selectivity of the SMRT/receptor interaction was most
clearly demonstrated in experiments employing a mixed population of T3R homodimers and T3R·RXR heterodimers. The addition of SMRT to this mixed receptor population resulted in a selective interaction of the
corepressor with the T3R homodimers; in contrast, little or no direct
interaction of SMRT was observed with the T3R·RXR heterodimers
simultaneously present in the same receptor population. Intriguingly,
the addition of SMRT was able to reduce the abundance of T3R·RXR
heterodimers in these experiments without inducing formation of a
corresponding T3R·RXR·SMRT complex. Experimental dissection of this
phenomenon revealed that the loss of T3R·RXR heterodimers was
probably a mass action effect. Presumably, by selectively interacting
with T3R homodimers, SMRT reduces the availability of T3R for
heterodimerization with RXR, thereby altering the dynamic equilibrium
between receptor homodimers and receptor heterodimers. This ability of
SMRT to recruit T3R homodimers at the expense of T3R·RXR heterodimers
may also play a role in stabilizing the formation of T3R homodimers
in vivo (see below).
At first glance, our results might appear to contradict prior studies
reporting RXR as stimulating, rather than inhibiting, SMRT recruitment
by T3Rs (58, 60). However, most of these previous studies employed GST
pull-down and two-hybrid methods to assay the corepressor/receptor
interaction, leaving the precise nature of the resulting receptor
"dimers" undefined. It is likely that authentic T3R homodimers do
not form in GST-pull-down and two-hybrid assays; the effects of RXR in
these cases may reflect a preference of SMRT for T3R·RXR heterodimers
(which do form in solution) relative to T3R monomers, not relative to
homodimers. In support of our own findings with SMRT, N-CoR, a protein
that displays 50% sequence relatedness to SMRT, displays a similar preference for T3R homodimers in EMSA experiments (75, 76). In this
regard, we (and others) have found that SMRT and N-CoR interact
comparably with T3Rs (20, 58, 60); a suggestion to the contrary,
that N-CoR interacts with T3Rs more strongly than SMRT, may be
the result of differences in the C terminus of the SMRT and N-CoR
constructs employed in these prior experiments (76).
Do T3R homodimers mediate transcriptional repression in
vivo? In this regard, a number of studies have implicated RXR
heterodimers in transcriptional activation, but much less is known as
to the nature of the receptor entity involved in transcriptional
repression (77, 78). Indeed, several features of T3R molecular biology support a role for T3R homodimers in transcriptional repression. (a) It is difficult to reconcile the poor ability of
T3R·RXR heterodimers to interact with SMRT in vitro with
the proposal that heterodimers function in repression in
vivo. (b) Although T3R homodimers are generally less
stable than are T3R·RXR heterodimers, the presence of SMRT can change
this equilibrium by stabilizing homodimers and destablizing
heterodimers. The levels of SMRT protein in vivo may be
sufficient to allow the formation of T3R homodimers on appropriate DNA
elements in preference to T3R·RXR heterodimers. (c)
Conversely, the addition of T3 hormone can destabilize T3R homodimers
but not T3R·RXR heterodimers (43, 52, 61, 79, 80). Thus, binding of
hormone may induce release of SMRT by two means: a direct allosteric
change in the receptor and a shift in the nature of the receptor-DNA
complex from a corepressor-interactive T3R homodimer to a
corepressor-noninteractive T3R·RXR heterodimer. (d)
Ectopic expression of high levels of RXR in transfected cells counteracts the ability of T3Rs to repress. This RXR-mediated inhibition of repression is opposite to the stimulatory effects of RXR
on T3R-mediated activation and suggests that altering the T3R
equilibrium from homodimers to heterodimers is paralleled by a shift
from repression to activation. (e) DNA response elements and
mutants of T3R that favor formation of T3R homodimers also favor
receptor-mediated transcriptional repression; this last point is
discussed at greater length below.
What Is the Molecular Basis of the Specificity Exhibited by SMRT
for T3R Homodimers?--
The ability of receptor dimers to recruit
SMRT or N-CoR more efficiently than receptor monomers is well
established and presumably relates to the stoichiometry of the
receptor-corepressor complex (60). SMRT contains two
(L/I)XXII motifs, each of which can interact with a
hydrophobic groove on the surface of the nuclear receptor; the
corepressor molecule therefore contains two potential receptor
interaction sites, whereas only one corepressor interaction site has
been mapped on the corresponding surface of the nuclear receptor (33,
35, 36). Receptor dimer formation may, therefore, stabilize the
corepressor interaction by permitting occupancy of both of the two
receptor interaction sites within a single corepressor molecule.
Alternatively the enhanced ability of SMRT to be recruited by receptor
dimers may reflect the ability of the corepressor itself to dimerize,
thereby allowing a dimeric corepressor/dimeric receptor interaction to occur.
Stoichiometric considerations may account for the preference of SMRT
for receptor dimers, but they do not explain why T3R homodimers recruit
corepressor better than do T3R·RXR heterodimers. RXR alone, when
assayed by GST pull-down or two-hybrid procedures, has a much lower
affinity for SMRT than does T3R (9, 15, 18, 20, 21, 58). The presence
of two strong SMRT interaction sites in the T3R·T3R homodimer might
therefore tether corepressor more strongly than the one strong and one
weak interaction sites available in the T3R·RXR heterodimer. However,
this hypothesis does not account for the strong SMRT interaction we
observe for the RAR·RXR heterodimer, which would be expected to
display an analogous mix of one strong and one weak interaction site
(RARs, when tested alone, possess a similar affinity for SMRT as do
T3Rs). Of note, the low affinity of RXR for corepressor has been
attributed to a steric inhibition by the RXR C-terminal helix 12 domain
(81). Formation of a RAR·RXR heterodimer might reposition the RXR C terminus so as to alleviate this steric inhibition, resulting in a
strong RXR/SMRT interaction, whereas the geometry of the T3R·RXR
heterodimer may be unable to alleviate the RXR helix 12-mediated inhibition. Indeed, deletion of the RXR helix 12 modestly enhances the
ability of the T3R·RXR heterodimer to recruit SMRT although not to
the level observed for the T3R
homodimer.2 Other receptor
and corepressor determinants may also be involved in this phenomenon;
for example, T3Rs interact with both (L/I)XXII interaction domains within SMRT, whereas RARs and RXRs display preferences for individual interaction domains (18, 20). We are
currently pursuing experiments to define the contributions of these
different interaction surfaces to the homodimer/heterodimer phenomenon.
The v-Erb A Protein Exhibits an Enhanced Ability to Form Homodimers
and to Recruit Corepressor, Compared with the T3R
v-Erb A has been reported to differ from T3R
Notably, the ability of v-Erb A to bind to the cLYS element very
strongly as a homodimer resulted in a very efficient recruitment of
SMRT corepressor, and this ability to recruit SMRT was not inhibited by
the presence of high levels of RXR. In contrast, v-Erb A homodimers
formed on the DR-4 element could be driven into heterodimers by the
addition of large amounts of RXR, and these v-Erb A·RXR heterodimers
were significantly less able to recruit SMRT corepressor than were the
v-Erb A homodimers. We therefore propose that the enhanced
homodimerization and impaired heterodimerization properties of the
v-Erb A protein are likely to contribute to the ability of the viral
protein to recruit corepressor and to act as a transcriptional
repressor in cells where RXR is present. The enhanced homodimerization
properties of v-Erb A may therefore operate together with the defects
in hormone binding and altered DNA recognition properties, noted
previously, to enhance the leukemogenic proclivities of the v-Erb A
oncoprotein by making it a stronger antimorph. The extent to which this
enhanced homodimerization phenomenon is manifested is dependent on the
nature of the DNA binding site and therefore is likely to differ for
different promoters. Of note, the T3R The Nature of the DNA Response Element and the Nature of the
Receptor Dimer Determine Corepressor Recruitment--
The T3Rs are
unique among the nuclear hormone receptors in their ability to bind to
response elements exhibiting an extraordinary range of different
half-site spacing and orientations. Different thyroid hormone response
elements can mediate different transcriptional regulatory properties.
For example, DIV-6 sequences such as the cLYS element mediate strong
transcriptional repression and relatively weak transcriptional
activation by T3Rs, whereas DR-4 elements tend to exhibit the
reciprocal properties (48, 52, 82). We therefore examined if the
different topological organizations of these different elements could
influence the ability of the tethered T3Rs to bind or release from
corepressor. When using pure T3R homodimers, the orientations of the
half-sites did not appear to directly influence corepressor
recruitment; all three DNA elements tested (DR-4, INV-0, and DIV-6)
exhibited comparable corepressor association and hormone-mediated
dissociation. Notably, however, these elements do differ in the ability
to recruit T3Rs as homodimers or as heterodimers with RXR, and, as a
consequence, corepressor recruitment by the different elements differed
in the presence of a mix of T3Rs and RXRs. Most obvious was the
enhanced homodimerization and impaired heterodimerization properties of v-Erb A on the cLYS element, which represents a DIV-6 half-site orientation. Interestingly, the cLYS element was initially isolated from avian erythroblastosis virus-transformed erythroid cells by virtue
of its ability to confer v-Erb A-mediated repression of lysozyme
expression (48). Native T3R
Presenting an interesting contrast to this phenomenon for T3Rs, RAR-
432 mutant found in human resistance to thyroid hormone syndrome, display enhanced homodimerization properties and exhibit unusually strong interactions with the SMRT corepressor. Significantly, the topology of a DNA binding site can determine whether that site
recruits primarily homodimers or heterodimers and therefore whether
corepressor is efficiently or inefficiently recruited to the resulting
receptor-DNA complex. We suggest that T3R homodimers, and not
heterodimers, may be important mediators of transcriptional repression
and that the nature of the DNA binding site, by selecting for receptor
homodimers or heterodimers, can influence the ability of the receptor
to recruit corepressor.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-
432 mutant
found in human resistance to thyroid hormone (RTH) syndrome (8, 53),
display enhanced homodimerization properties and exhibit unusually
strong interactions with corepressor. Significantly, the orientation and spacing of the half-sites in a response element can influence whether a given response element recruits v-Erb A primarily as a
homodimer or as a v-Erb A/RXR heterodimer and therefore whether corepressor is efficiently or inefficiently recruited to the v-Erb A-DNA complex. Transfection experiments further support the suggestion that transcriptional repression may be mediated primarily through the
actions of receptor homodimers rather than heterodimers with RXR. In
contrast to T3Rs, however, RARs efficiently recruit corepressor both as
homodimers and as heterodimers with RXR, indicating that the nature of
the receptor dimer may play a less decisive role in corepressor
recruitment by RARs.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
The pSG5
mammalian expression vectors have been described previously and include
the wild-type and
432 mutant T3R
clones (53), the wild-type avian
T3R
and v-ErbA clones (54), and the human RAR
and RXR
clones
(34). The baculovirus expression vectors for T3R
, T3R
, RAR
,
and RXR
were also detailed in a previous publication (55). For
expression as a glutathione S-transferase (GST) fusion
protein in bacteria, a suitable restriction fragment representing amino
acids 1056-1495 of SMRT was inserted into the pGEX-KG vector and
transfected into Escherichia coli strain DH5
(53, 56).
RXR-directed antiserum was a generous gift from Pierre Chambon (INSERM,
Strasborg, France). The oligonucleotides used in the DNA binding
assays were as follows: DR-4, 5'-TCGAATAAGGTCAAATAAGGTCAGAG-3'; DIV-6, 5'-TCGATACGATCGTGACCTATTAGGAGGTCAACAGACGGG-3'; INV-0,
5'-TCGAGTTCTCAGGTCATGACCTGAGAAC-3'; rGH,
5'-TCGAGGAAAGGTAAGATCAGGGACGTGACCGCAGGAG-3'; cLYS,
5'-TCGAATTATTGACCCCAGCTGAGGTCAAGTTACG-3'; DR-5,
5'-TCGACTCTGACCTCTCGTTGACCTGCT-3'; biotin-DR-4,
5'-XTCGAATAAGGTCAAATAAGGTCAGAGTCTGA-3'; biotin-DR-4mut,
5'-XTCGAATAAGATCAAATAAGATCAGAGTCGA-3'.
, T3R
,
RAR
, and RXR
were prepared by expression in a recombinant
baculovirus/Sf-9 cell system (55). GST and GST-SMRT proteins were
expressed in E. coli and were purified by
glutathione-agarose affinity chromatography (53); the resulting
proteins were reconstituted at a concentration of 2-10 mg/ml in 50 mM Tris, pH 7.4, 20% glycerol, 200 µg/ml bovine serum
albumin. Electrophoretic mobility shift assays (EMSAs) were performed
by mixing 1-2 ng of each nuclear receptor preparation with a
32P-radiolabeled oligonucleotide probe (40,000-60,000 cpm,
representing 20-60 ng of DNA) at 25 °C for 25 min in 15 µl of
binding buffer (10 mM Tris-Cl, pH 7.5, 3% glycerol, 66.7 mM KCl, 2 mM MgCl2, 13.3 µg/µl
bovine serum albumin, 0.113 µg/µl poly(dI-dC). The resulting
DNA-protein complexes were resolved by nondenaturing gel
electrophoresis through a 5% polyacrylamide gel (30:1
acrylamide/bisacrylamide); the free and bound DNA probe was visualized
by PhosphorImager analysis (STORM system; Molecular Dynamics, Inc.,
Sunnyvale, CA) and was quantified using ImageQuant software (Molecular
Dynamics). For supershift experiments using corepressor, the nuclear
receptor preparations were preincubated with either GST or GST-SMRT for 10 min on ice prior to the addition of the oligonucleotide probe. For
antibody supershifts, the protein/DNA complexes were allowed to form
for 15 min at 25 °C, and then suitable antisera were added, and the
incubation continued for an additional 15 min at 25 °C prior to gel electrophoresis.
, or of T3R
and RXR
, in 500 µl of
PBST buffer (150 mM NaCl, 16 mM
Na2HPO4, 4 NaH2PO4, 2 mM EDTA, 1 mM dithiothreitol, and 1% Triton
X-100) supplemented with 10 mg/ml bovine serum albumin and 1× Complete
protease inhibitor (Roche Molecular Biochemicals). The
agarose-DNA-receptor complexes were then washed three times with 1 ml
of PBST buffer and once with 1 ml of HEMG buffer (53) prior to
incubation with 5 µl of 35S-labeled, in vitro
transcribed/translated SMRT protein. The agarose DNA-protein complexes
were finally washed four times with 1 ml of HEMG buffer, and the
proteins were eluted in SDS sample buffer. The eluted proteins were
resolved by SDS-polyacrylamide gel electrophoresis and were
characterized by Western and PhosphorImager analyses (57).
vector was used per well of a 12-well plate (~5 × 104 cells), together with 200 ng of a thymidine kinase
promoter-luciferase reporter containing a DR-4 element, 200 ng of
pCH110-lacZ (employed as an internal control), and various
amounts of a pSG5-RXR
expression vector (53).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
formed a single predominant complex when incubated
with the radiolabeled DR-4 probe (Fig.
1A, denoted T3R·T3R). This
protein-DNA complex displayed all of the characteristics previously
noted for a T3R homodimer: the protein-DNA complex migrated at an
appropriate electrophoretic mobility for a homodimer, it was
supershifted with anti-T3R antibodies but not with RXR-directed
antibodies, the presumptive homodimer complex was destabilized by the
addition of T3 hormone (61), and no protein-DNA complex was observed using otherwise identical control lysates of nonrecombinant
baculovirus/SF9 cells (Fig. 1A). The addition of RXR to the
T3R preparation resulted in the formation of a novel complex exhibiting
the properties of a T3R·RXR heterodimer (Fig. 1A, denoted
T3R/RXR). This complex migrated at a slower mobility than
that of the T3R·T3R complex, was stable to T3 hormone (61), and could
be supershifted with either anti-T3R or anti-RXR antibodies. RXR alone
failed to bind at significant levels to the DR-4 DNA probe under these
conditions (Fig. 1A).
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Fig. 1.
Preferential interaction of SMRT corepressor
with T3R homodimers on a DR-4 DNA response
element. A, T3R
binds to a DR-4 DNA element either
as T3R·T3R homodimers or as T3R·RXR heterodimers. Either 2 ng of
T3R
alone, 2 ng of RXR
alone, or 1 ng each of T3R
and human
RXR
together were incubated with a 32P-labeled DR-4 DNA
probe at 25 °C for 15 min in either the presence or absence of T3,
as indicated below. Antiserum directed against RXR
(X), T3R (T), or normal rabbit serum
(N) was also introduced to several of the receptor/DNA
mixtures as indicated below. The resulting protein-DNA
complexes were resolved by gel electrophoresis and were visualized by
PhosphorImager analysis. A nonrecombinant baculovirus preparation was
employed as a negative control (lane 1).
Complexes corresponding to the T3R·T3R homodimer and to the T3R·RXR
heterodimer are indicated to the right; the corresponding
protein-DNA complexes, supershifted by antisera, are indicated
with asterisks. The portion of the electrophoretogram
representing free probe has been omitted to permit a clearer display of
the protein-DNA complexes; the DNA probe was in excess in all cases.
B, SMRT interacts efficiently with T3R
·T3R
homodimers but not with T3R
·RXR
heterodimers, bound to a
DR-4 DNA element. A similar analysis was performed as in A,
except GST (
) or GST-SMRT (+) proteins were also included in the
DNA binding reaction, as indicated below. The
positions of the T3R·T3R, T3R·RXR, and T3R·T3R·SMRT complexes
are indicated to the right. Nonrecombinant baculovirus/Sf-9
lysates were employed as a negative control in lanes
9 and 10 (No). C, the
T3R·T3R·SMRT complex contains T3R but not RXR. The
receptor-SMRT-DNA complex analyzed in B was incubated with
either normal serum, T3R-specific antiserum, or RXR-specific antiserum,
denoted as in A. Protein-DNA complexes supershifted
by antisera are indicated with asterisks.
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Fig. 2.
Selectivity of SMRT for homodimers, not
heterodimers, of T3R. A, quantification. The results in
Fig. 1B were quantified by PhosphorImager analysis. The
amount of receptor-DNA-SMRT complex formed (black
bars) is displayed relative to the total amount of
receptor-DNA complex (stippled bars). The amount
of receptor-DNA complex formed in the absence of SMRT and T3 was
defined as 100%. B, a similar analysis was performed as in
Fig. 1, B and C, except the amount of RXR was
titrated so as to yield a mixed population of T3R
·T3R
homodimers and T3R
·RXR
heterodimers (lane
7). This mixed receptor population was challenged by the
addition of GST-SMRT (lanes 8 and 10),
GST alone (lanes 7 and 9), and/or an
antibody against RXR (lanes 9 and 10).
Lanes 1-6 represent a comparison experiment
performed in an identical fashion on T3R
·T3R
homodimers in the
absence of RXR.
Extends to Other Receptor Isoforms and Was Also Detected by an
Avidin-Biotin Protocol--
In addition to the T3R
tested above, a
second, T3R
, locus is present in all vertebrates (1, 2). T3R
exhibits a distinct expression pattern from that of T3R
and plays a
distinct, if partially overlapping, role in organismal development and
physiology. In common with our results with T3R
, T3R
homodimers
bound to a DR-4 DNA element and interacted readily with SMRT to
generate a T3R·T3R·SMRT complex, whereas T3R
·RXR heterodimers,
assembled on the same DR-4 element, interacted with SMRT with much
lower efficiency (data not shown). Similarly, RXRs are encoded by three different loci: RXR
, -
, and -
(4). Use of the RXR
or RXR
isoforms instead of RXR
did not alter the relative inability of the
T3R·RXR heterodimer to interact with SMRT (data not shown). Therefore, the preferential interaction of SMRT with T3R homodimers, rather than with T3R·RXR heterodimers, extends to both of the major
T3R isoforms and to all three of the major RXR isoforms.
-1 with a
biotinylated, but otherwise unlabeled, DR-4 DNA element. The proteins
binding to the DNA probe were subsequently isolated by adsorption of
the biotinylated DNA to a streptavidin matrix. The
DNA-protein-streptavidin complexes were washed, and the proteins
were eluted and were resolved by SDS-polyacrylamide gel
electrophoresis. The proteins that bound to the DNA element were then
visualized by using either a radiolabeling or immunoblotting procedure.
As expected, little or no T3R bound to the streptavidin matrix in the
absence of DNA or if a biotinylated DR-4 probe bearing a dysfunctional,
mutated response element sequence was employed (Fig.
3A, lanes
1, 4, and 5). In contrast, significant
amounts of T3R bound to a biotinylated DNA probe representing a DR-4
HRE (Fig. 3A, lanes 2 and
3; the T3R homodimer was not detectably destabilized by T3
hormone when assayed in this fashion, probably reflecting minor
technical differences between the avidin-biotin and the EMSA
approaches). The addition of RXR increased the amount of T3R bound by
the biotinylated probe, and the RXR protein itself was now also found
in the biotinylated DNA complex, consistent with formation of a
T3R·RXR heterodimer (Fig. 3B; quantified in Fig.
3C). Consistent with our EMSA experiments, the T3R complexes formed on the biotinylated DR-4 element in the absence of RXR were able
to interact with the corepressor, resulting in copurification of SMRT
on streptavidin-agarose (Fig. 3, B and C).
Conversely, although the addition of RXR greatly increased the amount
of nuclear receptors bound to the biotinylated DR-4 probe, the
resulting T3R·RXR complexes displayed a significantly reduced ability
to bind to SMRT compared with the T3R homodimer complexes (Figs. 3,
B and C). Of note, several SMRT variants have
been described (e.g. Refs. 15, 19, 20). The 1495-codon SMRT
version was used for Fig. 3; comparable results, however, were obtained
with a longer, 2517-codon form of SMRT (data not shown). We conclude that, both by EMSA and by avidin/biotin protocols, the T3R homodimer recruits SMRT more efficiently than does the T3R·RXR heterodimer.
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Fig. 3.
Preferential interaction of SMRT corepressor
with T3R homodimers detected by an
avidin-biotin assay. A, T3R
complexes assemble in a
sequence-specific fashion on biotinylated DNA response elements.
Biotin-conjugation DR-4 oligonucleotides representing either the
wild-type DR-4 sequence (WT) or a mutated DR-4 sequence
(mut) were immobilized on streptavidin-agarose and were
incubated with 35S-labeled T3R
in the absence or
presence of T3 hormone, as indicated below. The
receptor-DNA-agarose complexes were then washed, the proteins remaining
bound to the DNA-agarose matrix were eluted, and the eluted proteins
were analyzed by SDS-polyacrylamide gel electrophoresis and
PhosphorImager analysis. As an additional negative control, the ability
of the radiolabeled T3R
to bind to streptavidin-agarose in the
absence of any oligonucleotides was also tested (lane
1). B and C, homodimers of
T3R
recruit corepressor SMRT more efficiently than do heterodimers
of T3R
/RXR
. Either T3R
alone or T3R
and RXR
together, as
indicated, were incubated with the immobilized wild-type DR-4
oligonucleotide as in A. The resulting receptor-DNA-agarose
complexes were further incubated with 35S-labeled SMRT. The
protein-DNA complexes were then washed, eluted from the DNA-agarose
matrix, and resolved by SDS-polyacrylamide gel electrophoresis. The
electrophoretograms were visualized by immunoblotting and/or
PhosphorImager analysis (B), and the relative amounts of T3R
(light stippled bars), RXR
(dark stippled bars), and SMRT
(black bars) bound to the DR-4 DNA under the
different conditions were quantified (C). Although the
experiment was quantified in both the absence and presence of T3
hormone, electrophoretograms are provided only for the experiment
performed in the absence of T3.
was able to form homodimers on all
of the DNA elements tested, and in all three cases these T3R
homodimer complexes were efficiently supershifted by the addition of
GST-SMRT in the absence but not in the presence of 1 µM
T3 hormone (Fig. 4A). For all
three elements, the addition of RXR led to the formation of a novel
complex with properties characteristic of a T3R·RXR heterodimer, and
in all cases this T3R·RXR heterodimer was less susceptible to
supershift by GST-SMRT than was the T3R·T3R homodimer (Fig.
4A). The DR-4, DIV-6, and INV-0 elements are synthetically
derived, artificially optimized response elements; we therefore also
extended our studies to two naturally occurring HREs that play a role
in T3R function in vivo: the rat growth hormone promoter
element and the chicken lysozyme silencer element (cLYS) (48, 62). T3R
homodimers formed on these naturally occurring elements were
efficiently supershifted by GST-SMRT, whereas the corresponding
T3R·RXR heterodimers were not (data not shown). We conclude that for
all DNA elements tested, the precise sequence and topology of the
response element does not directly affect the ability of the tethered
T3R to interact with SMRT corepressor and that the T3R·T3R homodimer
displayed a significantly stronger interaction with SMRT than did the
T3R·RXR heterodimer.
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Fig. 4.
Preferential interaction of SMRT with
T3R homodimers as detected on different DNA
response elements. A, the ability of SMRT to
preferentially interact with T3R
·T3R
homodimers, relative to
T3R
·RXR
heterodimers, was examined by EMSA for a DR-4 element,
a DIV-6 element, or an INV-0 element. The EMSAs were performed and
quantified as in Fig. 2A. For each DNA element, at least
three independent experiments were performed, with the averages and the
S.D. values shown. B, T3R
homodimers bound to different
response elements exhibit a similar release of corepressor in response
to hormone. The EMSA analysis was performed as described in
A, except that a range of hormone concentrations from 0 to
1000 nM T3 was used. At each hormone
concentration, the amount of T3R·T3R·SMRT complex was determined
relative to the amount of total T3R·T3R complex. The amount of
T3R·T3R·SMRT complex formed in the absence of T3 is
defined as 100% for comparison purposes.
, three different response elements,
and a range of hormone concentrations from 0.1 to 1000 nM.
The results are quantified in Fig. 4B. No significant
difference could be detected among the different response elements in
the ability of hormone to release SMRT from the T3R homodimer complex.
Approximately 12 nM T3 was sufficient to displace 50% of
the SMRT complexes from any of the elements tested: the DIV-6, INV-0,
and DR-4. We conclude that at the level of resolution of this in
vitro experiment, different DNA response elements do not alter the
ability of hormone to release corepressor.
expression plasmid in the absence of hormone resulted in a reduction in
luciferase activity to below basal levels (Fig. 5); this repression
reflects the ability of T3R to recruit the SMRT corepressor complex
(e.g. Refs. 15 and 20). If the same levels of T3R
were
maintained but increasing amounts of an RXR expression construct were
introduced into the CV-1 cells, the T3R-mediated repression was
counteracted, resulting in partial restoration of basal reporter gene
expression (Fig. 5). These results were most clearly observed with the
DR-4 and INV-0 elements but were also seen more weakly with a DIV-6
element. These effects of RXR on luciferase activity were not observed in the absence of T3R, were not observed with an otherwise identical reporter construct lacking a hormone response element, and were not due
to a reduction in the levels of T3R expressed in the transfected cells
(Fig. 5 and data not shown). Therefore, these results, together with
our in vitro data, suggest that formation of T3R·RXR
heterodimers from T3R·T3R homodimers inhibits the ability of
corepressor to interact with the receptor and to mediate
transcriptional repression.
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Fig. 5.
Effect of increasing RXR
on T3R-mediated repression. The ability of T3R
to repress
reporter gene expression in transfected cells was analyzed in the
presence of increasing amounts of an RXR
expression vector. CV-1
cells were transfected with 50 ng of either an empty pSG5 vector
(None) or a pSG5-T3R
vector, together with 200 ng of a
pCH110 lacZ vector and 200 ng of a thymidine
kinase-luciferase reporter containing a DR-4, DIV-6, or INV-0 response
element, as indicated below. 0 ng (light
stippled bars), 10 ng (dark
stippled bars), or 50 ng (black
bars) of a pSG5-RXR
vector were included in each
transfection; appropriate amounts of an empty pSG5 vector were used to
keep the total amount of vector equal in all cases. The cells were
subsequently incubated and harvested, and the luciferase activity was
determined and normalized relative to the
-galactosidase activity.
The averages and S.D. values of at least two experiments are
presented.
Mutant Associated with Human Resistance to Thyroid Hormone
Syndrome,
432-T3R
, Exhibits Increased Homodimer Formation,
Increased Interaction of the Homodimer with SMRT, and Enhanced Dominant
Negative Repression Properties in Transfections--
Mutations in the
T3R
locus occur naturally in the human population and can manifest
as an endocrine disorder, RTH syndrome. In most cases characterized,
the mutant T3Rs associated with RTH syndrome are unable to release
properly from corepressor in response to T3 hormone (56, 63-66).
Apparently as a consequence, these RTH-T3Rs act as repressors and can
interfere in a dominant negative fashion with the functions of the
wild-type T3Rs. Intriguingly, several RTH-T3R mutants not only fail to
release from corepressor upon the addition of T3 hormone but also
display an enhanced corepressor interaction in the absence of hormone
(53). We wished to determine if the elevated interaction of these
RTH-T3R mutants with SMRT, previously characterized in solution, was
also observed when the receptor was bound to DNA and if this enhanced
corepressor interaction was specific to the homodimeric form of the receptor.
432-T3R
mutant, representing an in frame deletion within
helix 11 of the hormone binding domain, displays a 4-8-fold enhanced
interaction with SMRT compared with wild-type receptor in a GST
pull-down protocol (53). Intriguingly, the
432-T3R
mutant formed
homodimers on the DR-4 DNA element somewhat more readily than did the
wild-type T3R
(Fig. 6A,
compare lane 7 with lane
3), and virtually all of the
432-T3R
homodimer complex was supershifted by SMRT under conditions where less than half of the
wild-type T3R
homodimer was supershifted (Fig. 6A,
compare lane 9 with lane
5). We conclude that the enhanced ability of the
432-T3R
mutant to interact with SMRT, previously seen in solution, extends to receptor homodimers formed on HREs. We next asked
if the enhanced SMRT interaction observed with the
432 mutant
applied only to T3R homodimers or was also manifested for heterodimers.
Paralleling our results with the wild-type receptor, heterodimers
formed by the
432-T3R
mutant in the presence of excess RXR were
much less susceptible to supershift by SMRT than were the corresponding
432-T3R
homodimers (Fig. 6A, compare lanes
13 and 14 with lanes 11 and
12). Consistent with these enhanced homodimerization and
SMRT association properties in vitro, repression by the
432-T3R
mutant was more resistant to the inhibitory effects of
ectopic RXR than was repression by wild-type T3Rs (Fig. 6B), and the
432-T3R
mutant exhibits enhanced dominant negative
properties in transfected cells (53). We conclude that the elevated
ability of the
432-T3R
mutant to recruit SMRT is largely
restricted to homodimers of this receptor and that this enhanced
corepressor/homodimer interaction in vitro is paralleled by
an augmented ability of the
432 mutant to repress transcription
in vivo.
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Fig. 6.
Enhanced homodimer formation and enhanced
corepressor binding by the 432 mutant
T3R
. A, SMRT corepressor
strongly interacts with
432-T3R
homodimers, but not with
RXR·
432-T3R
heterodimers. Wild-type or
432 mutant T3R
receptors, bound to a DR-4 DNA element, were examined for the
ability to interact with GST-SMRT. EMSAs were performed as described in
the legend to Fig. 1B, except that in vitro
transcription/translations were employed as a source of receptor (see
"Experimental Procedures"). Two faint, background complexes
(denoted by an asterisk) were observed in many
lanes, including DNA binding reactions performed with an
unprogrammed transcription/translation lysate (lanes
1 and 2). Wild-type T3R
alone, the
432-T3R
mutant alone, or the
432-T3R
mutant together with
RXR were tested in the presence or absence of SMRT, as indicated
below. B, repression by the
432-T3R
mutant
is more resistant to the effects of RXR than is repression by wild-type
T3R. The same experimental strategy as in Fig. 5 was employed but
testing the
432-T3R
mutant in addition to the wild-type T3R
and -
alleles, as indicated below.
, Displays an Enhanced
Ability to Homodimerize on Certain Response Elements and, as a
Consequence, an Enhanced Interaction with SMRT
Corepressor--
The v-erb A
oncogene was first identified as a locus involved in leukemogenesis by
avian erythroblastosis virus and was subsequently recognized to be a
virally transduced version of the T3R
gene (7, 67). Compared with
the wild-type receptor, v-Erb A bears a C-terminal deletion that
prevents hormone binding; as a consequence, v-Erb A interacts with
corepressor in both the absence and the presence of hormone and is
thought to function in oncogenesis as a dominant negative inhibitor of
T3Rs and retinoid receptors (8, 55, 68-70). Notably, v-Erb A has
sustained multiple mutations compared with the wild-type T3R
,
including 13 amino acid substitutions (71); many of these mutations
enhance the oncogenic abilities of the v-Erb A protein and appear to
have been selected for during viral propagation (e.g. Refs.
72 and 73). Among other differences between v-Erb A and its T3R
progenitor, it has been reported that v-Erb A is unable to form
heterodimers with RXR (74); however, contrary results have also been
obtained that suggest v-Erb A does heterodimerize with RXRs, although
at reduced efficiency compared with T3R
(55). No role for the
altered dimerization properties of v-Erb A has previously been ascertained.
, with this effect particularly evident for the cLYS element (Fig. 7B, compare lanes 8 with
lanes 3). The addition of RXR to the v-Erb A
preparation enhanced the ability of the oncoprotein to bind to the DR-4
element but had little or no effect on the already strong ability of
v-Erb A to bind to the cLYS element (Fig. 7B). Due to their
comparable charge/mass ratios, v-Erb A·RXR heterodimers migrate at
virtually the same position in these electrophoretograms as do v-Erb A
homodimers (Fig. 7B). Use of receptor-specific antibodies, however, confirmed that the enhanced binding of v-Erb A to the DR-4
element upon the addition of RXR corresponds to the formation of v-Erb
A·RXR heterodimers, whereas the bulk of v-Erb A on the cLYS element
remains a homodimer and does not associate with RXR under the same
conditions (Fig. 8 and data not shown).
In contrast, the addition of increasing amounts of RXR to the T3R
preparation resulted in the formation of receptor heterodimers and
increased DNA binding on both the DR-4 and cLYS elements (Fig.
7B, lanes 4-7), although heterodimer
formation was again somewhat more efficient on the DR-4 (Fig.
7B, compare lanes 4-7 in the
upper and lower panels). We conclude
that v-Erb A forms homodimers more efficiently than does T3R
,
particularly on the cLYS element, and that v-Erb A can associate and
form heterodimers with RXR on a DR-4, whereas v-Erb A remains a
homodimer and exhibits little or no ability to form heterodimers with
RXR on the cLYS element.
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Fig. 7.
More efficient formation of homodimers by
v-Erb A compared with T3R . A,
schematic representations of the protein structure of avian T3R
(top) and v-Erb A (bottom) are shown. Deletions
in v-Erb A compared with chicken T3R
are indicated, as are amino
acid substitutions (vertical bars);
gag refers to retroviral structural sequences present on the
N terminus of the viral protein. B, the ability of v-Erb A
to form heterodimers with RXR
is compared with that of T3R
. EMSAs
were performed using either 6 µl of unprogrammed translation product
(lanes 1 and 2), 4 µl of T3R
with
increasing amounts of RXR
translation product (0, 0.5, 1, or 2 µl;
lanes 3-7), or 4 µl of v-erb
A with increasing amounts of RXR
(0, 0.5, 1, or 2 µl;
lanes 8-11). Analyses were performed on both the
DR-4 (top panel) and the cLYS (bottom
panel) elements in the presence or absence of T3 as
indicated. The positions of the v-Erb A homodimer and v-Erb A·RXR
heterodimer complexes are indicated to the right (denoted
vA/vA and vA/RXR, respectively).
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Fig. 8.
Enhanced corepressor recruitment by v-Erb A
homodimers. EMSAs were performed as described in the legend to
Fig. 7 using either the DR-4 or the cLYS elements and including either
100 ng of GST or GST-SMRT in the binding reactions, as indicated.
RXR-directed (X) or normal sera (N) were also
included in the reactions as indicated. The locations of protein-DNA
complexes corresponding to v-Erb A·v-Erb A homodimers, v-Erb
A·v-Erb A homodimers supershifted by SMRT, and v-Erb A·RXR
heterodimers supershifted by RXR antibody are indicated to the
right (vA/vA, vA/vA/SMRT, and
vA/RXR*, respectively).
progenitor, that this preferential formation of
homodimers by v-Erb A is dependent on the nature of the DNA response
element, and that enhanced homodimerization properties of v-Erb A are
reflected as an enhanced ability of v-Erb A to recruit SMRT corepressor.
formed receptor homodimers when incubated
with a suitable DNA response element (i.e. a DR-5 element)
(Fig. 9A). The RAR
homodimers were efficiently supershifted to a slower electrophoretic mobility by the addition of GST-SMRT, whereas the addition of a hormone
agonist (all-trans-retinoic acid; ATRA)
dissociated this presumptive RAR·RAR·SMRT complex (Fig.
9A, compare lanes 3 and 4 with lanes 1 and 2). The addition of
RXR
to the RAR
preparation resulted in the formation of receptor
heterodimers that bound to the DR-5 element with higher affinity than
did the RAR homodimers (Fig. 9A, lanes
5-8; only one-eighth of the RAR
preparation employed in
lanes 1-4 was utilized in lanes
5-8). In common with the RAR
homodimers, but in marked
contrast to T3R·RXR heterodimers, the RAR
·RXR
heterodimers
were efficiently supershifted by the addition of GST-SMRT (Fig.
9A, compare lanes 7 and
5). This presumptive RAR· RXR·SMRT complex
appeared to be authentic; it was not observed if RXR or SMRT was
employed in the EMSA in the absence of RAR
, the mobility of the
RAR·RXR·SMRT complex was further supershifted by RXR-directed
antibodies, and the complex was dissociated by the addition of
all-trans-retinoic acid (Fig. 9B and data not
shown). We conclude that the preference of SMRT for T3R homodimers does
not extend to all members of the nuclear receptor family and that the
nature of the RAR
dimer does not significantly alter the ability of
this receptor to interact with corepressor under the conditions tested
here.
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Fig. 9.
Interaction of SMRT both with RAR homodimers
and with RAR·RXR heterodimers. A, the ability of SMRT
to interact with both RAR homodimers and RAR·RXR heterodimers was
analyzed by EMSA. Either RAR alone (lanes
1-4) or RAR
and RXR
together (lanes
5-8) were incubated with a 32P-labeled DR-5 DNA
element in the presence or absence of all-trans-retinoic
acid (ATRA), as indicated below. GST (
) or
GST-SMRT (+) was included in the reactions as indicated. The positions
of the protein-DNA complexes corresponding to RAR·RAR homodimers,
RAR·RXR heterodimers, and the corresponding complexes supershifted
with SMRT are indicated to the right. B,
RAR·RXR heterodimers interact with SMRT and can be supershifted by
RXR antibody. A similar experiment was performed as in A,
except that in vitro transcription/translation reactions
were used as a source of RAR. RXR-directed antiserum (X) was
included in the binding reactions as indicated. The positions of the
protein-DNA complexes supershifted by the RXR-directed antiserum are
indicated by asterisks.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(c-Erb A)
Progenitor--
The v-erb A gene was first
identified as an oncogenic locus within the avian erythroblastosis
virus and was later recognized to be a virally transduced version of
the normal avian T3R
transcript (7, 67). v-Erb A has sustained 13 internal amino acid substitutions, as well as N- and C-terminal
deletions, compared with the T3R
progenitor, and as a consequence
v-Erb A acts as a constitutive repressor of genes normally regulated by
T3Rs and by RARs (7, 8, 55, 68). When examined, many of these
differences between v-Erb A and the T3R
progenitor contribute to the
oncogenic properties of the former, and these v-Erb A mutations may be
the result of a selection for increased oncogenic virulence during
viral propagation. For example, mutations in the C terminus of v-Erb A
result in an inability of the viral protein to release from corepressor in response to hormone, thereby generating the constitutive repressor phenotype (70). Notably, four different mutations, within the v-Erb A
DNA binding domain, have changed the DNA recognition properties of
v-Erb A, and this alteration in DNA recognition also appears necessary
for efficient oncogenesis (55, 72, 73).
in at least one
additional aspect: the ability to interact with RXR. It has been
reported that v-Erb A forms homodimers but does not heterodimerize with
RXR (74). However, alternative reports have been published that
indicate v-Erb A can form heterodimers with RXR although at a reduced
efficiency relative to T3R
(55). The results obtained in the current
study help reconcile these apparently opposing views; v-Erb A is
impaired in its ability to form heterodimers with RXR, but the extent
of this impairment is highly dependent on the nature of the DNA
response element. On a DR-4 element, v-Erb A can form both homodimers
and heterodimers with RXR, although the latter occurs with reduced
efficacy compared with T3R
. On a DIV-6 element (such as the cLYS
promoter sequence) v-Erb A binds very strongly as a homodimer and
exhibits little or no ability to form heterodimers with RXR. In this
regard, v-Erb A displays, in a highly magnified manner, an otherwise
similar preference observed for T3R; the cLYS element recruits T3R
homodimers better than does the DR-4 element, and more RXR must be
added to generate T3R·RXR heterodimers on the cLYS element than on
the DR-4 element. Nonetheless, on all DNA elements we tested here,
v-Erb A was significantly more resistant to RXR heterodimer formation
than was T3R
, with v-Erb A binding to the cLYS element as a receptor
homodimer even at very high RXR concentrations.
432 mutation implicated in
human RTH syndrome also displays an increased ability to form
homodimers, an enhanced interaction with SMRT, and an enhanced ability
to function as a dominant negative repressor. Therefore, mutations that
increase the ability of T3Rs to homodimerize may have the general
effect of increasing the ability of the receptor to tether SMRT and to function as a transcriptional repressor.
also formed homodimers more readily (and
heterodimers less readily) on the cLYS element than on a DR-4 element,
although this phenomenon was more muted than for v-Erb A. We suggest
that the ability of different hormone response elements to exhibit
different transcriptional properties may reflect, in part, differences
in the ability of these DNA sequences to recruit receptor homodimers
versus heterodimers, which in turn can influence the
interactions of these receptor complexes with corepressors.
interacts strongly with SMRT corepressor whether tested as a receptor
homodimer or as a heterodimer with RXR. Presumably, the nature of the
receptor dimer does not play as critical a role in determining
RAR-mediated transcriptional regulation as it does for the T3Rs;
however, an explanation of the physiological or evolutionary basis
behind this phenomenon remains elusive. It may be relevant in this
regard that retinoic acid does not destabilize the binding of RAR
homodimers to DNA, and thus, unlike T3Rs, hormone would not be expected
to potentiate an exchange of RAR·RXR heterodimers for RAR homodimers.
Nonetheless, it should be noted that an oncogenic fusion protein,
PML-RAR
, derived by chromosomal translocation of the normal RAR
locus, exhibits an enhanced ability to form homodimers (and
homo-oligomers) compared with the unmodified RAR
, and this enhanced
homodimer formation appears to manifest as an enhanced ability of
PML-RAR
to bind to corepressor (83, 84). Although we have not
observed differences in the corepressor interaction properties of homo-
and heterodimers of native RAR, our experiments do not exclude the
possibility that aberrant RAR homodimers, formed by chromosomal
rearrangements or by artificial means, may exhibit the enhanced
corepressor interactions that we report here for T3R homodimers.
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ACKNOWLEDGEMENT |
---|
We thank Valentina Taryanik for dedicated technical assistance.
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FOOTNOTES |
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* This work was supported by Public Health Services/National Institutes of Health Grants R37 CA-53394, R01 DK-53528, and RO1 DK-54064.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.: 530-752-3013;
Fax: 530-752-9014; E-mail: mlprivalsky{at}ucdavis.edu.
Published, JBC Papers in Press, February 21, 2001, DOI 10.1074/jbc.M010022200
2 S. M. Yoh and M. L. Privalsky, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: HRE, hormone response element; T3R, thyroid hormone receptor; RAR, retinoic acid receptor; RXR, retinoid X receptor; RTH, resistance to thyroid hormone; EMSA, electrophoretic mobility shift assay; cLYS, chicken lysozyme silencer element.
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