At Least Three Subdomains of v-erbA Are Involved in Its Silencing Function
Kerstin Busch,
Bernd Martin,
Aria Baniahmad,
Rainer Renkawitz and
Marc Muller
Laboratoire de Biologie Moléculaire et de Génie
Génétique Institut de Chimie-B6 Université de
Liège B-4000 Sart-Tilman, Belgium
Genetisches
Institut (A.B. R.R.) Justus-Liebig-Universität D-35392
Giessen, Germany
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ABSTRACT
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Several members of the thyroid hormone receptor
(TR) family are able to switch from a transcriptional repressor to a
transcriptional activator upon binding of their ligand. The oncogene
v-erbA is a variant form of the TR unable to bind hormone and thus acts
as a constitutive repressor. We demonstrate, using fusion proteins
between the DNA-binding domain of the yeast factor GAL4 and the
silencing domains of v-erbA and TRß, that point mutations in three
different regions severely affect their repression function.
Furthermore, the three regions, each as an inactive fusion protein with
the GAL4 DNA-binding domain, restore silencing activity when assembled
on the same promoter. These observations define at least three
silencing subdomains, SSD1SSD3, which are involved in the silencing
function of v-erbA. We propose a model in which full silencing activity
is brought about by the combined interaction of each silencing
subdomain with corepressors and/or basal transcription factors.
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INTRODUCTION
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Thyroid hormone receptors (TRs) belong to a large family of
regulatory proteins that include receptors for steroid hormones,
vitamin D, and vitamin A derivatives. These receptors function as
ligand-activated transcription factors that bind to their cognate DNA
sequences located in the vicinity of specific genes (1). In general, TR
activates transcription in the presence of hormone (T3),
but represses transcription of a target gene in the absence of ligand
(2). This repression represents a true silencing activity (3), as it is
independent of the target promoter and even functions on a minimal
promoter containing only a TATA-box (4). Furthermore, a separable
silencing domain of the TR could be defined that is active when fused
to the heterologous DNA-binding domain (DBD) of the yeast transcription
factor GAL4 (5). A similar silencing function is found in the oncogene
v-erbA, a viral derivative of the chicken TR that is unable to bind
hormone and thus functions as a constitutive repressor on the same
target genes (2).
The TR (and v-erbA) silencing domain is localized in the C-terminal
hormone binding domain (HBD), which also contains activation functions
in the presence of T3 (5). Using deletion analysis on GAL4
DBD fusion proteins, the repression function was assigned to a minimal
silencing domain encompassing amino acids (aa) 389639 of v-erbA (5, 6). Cotransfection experiments of different inactive deletion mutants
defined two subdomains that restore silencing when combined in a
heterodimeric complex (6, 7). These complementary subdomains consist of
aa 173265 and 265461 or 362508 and 508639, respectively, in
human (h)TRß or v-erbA. The C-terminal subdomain of TRß was shown
to interact in vitro with the basal transcription factor
TFIIB only in the absence of T3 (7). Similarly, interaction
with the recently described corepressors N-CoR and SMRT is relieved in
the presence of hormone (8, 9, 10). On the other hand, repression was
obtained in a reconstituted in vitro transcription system
using bacterially expressed TR (11, 12, 13). Little is known about the
precise structural requirements for the silencing function in TR or
v-erbA.
Here we describe mutations in three distinct regions of the silencing
domain of v-erbA and TRß that severely affect the repression
function. Furthermore, three subdomains, each as an inactive fusion
protein with the GAL4 DBD, restore silencing activity when assembled on
the same promoter. These observations define at least three silencing
subdomains, SSD1SSD3, which are involved in the silencing function of
v-erbA.
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RESULTS
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Two Prolines in the Hinge Region Are Required for Full Silencing
Activity
The region (hinge region) between the v-erbA DBD and the domain
homologous to the TR hormone binding domain was shown to contain the
transactivation domain
2 (aa 207217) (14) and to interact directly
with the corepressors SMRT and N-CoR (8, 9). In a GAL-v-erbA fusion
protein, the deletion of aa 389 to 409 resulted in a complete loss of
the silencing function (6). A natural mutant of v-erbA was described
previously with a change of Pro398 to Arg abolishing the silencing
function (15).
We generated the P398R and other point mutations in this region (Fig. 1A
) in a fusion protein of the GAL4 DBD with the
silencing domain of v-erbA and tested their effect on a UAS-tkCAT
reporter gene in L-tk- and CV-1 cells. Fold repression was
determined relative to the promoter activity obtained after
coexpressing only the GAL4 DBD protein.
Mutation of Pro 398 to Arg resulted in a 3- to 4-fold decrease of the
repression function in both cell types (Fig. 1B
). A similar mutation at
Pro 396, very close to the first one (see Fig. 1A
), leads to a similar
decrease in the silencing capacity, suggesting that the overall
structure flanking Pro 396 and 398 is required for silencing function
in v-erbA and TR. In contrast, changing the hydrophobic Iso 389 to Arg
did not affect the repression function of v-erbA (Fig. 1B
).
Point Mutations in the Ti-Region Abolish Silencing Function
The previously identified Ti-region is highly conserved among the
members of the TR family; an internal deletion of this region was shown
to severely reduce silencing of v-erbA (5, 6). It covers the structures
defined as helices H3, H4, and H5/H6 of rat (r)TR
(16) (Fig. 2A
), which present a clear amphipathic character and
would therefore be good candidates for a protein-protein interaction
interface. Mutations in this region affected the repression function in
different ways (Fig. 2B
). While the change of Pro 475 to Arg had no (in
L-tk- cells) or a marginal (in CV-1 cells) effect,
mutation of Pro 481 to Arg drastically reduced the silencing ability.
The exchange of Leu 489 to Arg similarly abolished repression, whereas
mutation of the Cys 493 to Leu resulted in close to wild type activity,
in both cell types tested.
Our results indicate that amino acids corresponding to the interhelical
region H4 to H5 and to the N-terminal part of H5 of the TR
are of
great importance for the silencing function of v-erbA.
Helix 8 Is Involved in the Silencing Function of v-erbA
An interesting feature in the C-terminal half of the v-erbA
silencing domain is the region corresponding to helices H8 and H9 of
rTR
(16) (Fig. 3A
), as it corresponds to the
activation domain
3 in hTRß (aa 339368) (14) and is also highly
conserved among nuclear receptors. On the other hand, H8 and H9 could
be involved in the dimerization function of TR, as was shown for the
homologous region in human retinoid X receptor-
(hRXR
) (17).
Therefore, we concentrated on H8 and particularly on the amphipathic
structure formed by helix H8. Leucines and one isoleucine, all located
on the same side of the
-helix, were changed to the basic arginine,
and the repression function of the mutant GAL4-fusion proteins was
tested (Fig. 3B
). While mutants L530R, I537R, and L540R displayed wild
type activity, replacement of Leu 544 by Arg resulted in a 3-fold
decrease of the silencing activity. Similar results were obtained using
L-tk- or CV-1 cells. Thus, the C-terminal part of helix H8
contributes to the repression function of v-erbA.
The Mutant Phenotype Is Seen in the Context of the Full Length
v-erbA
The v-erbA mutant P398R was previously described to be completely
deficient in silencing activity in cotransfection experiments (15). In
our system, the GAL-v-erbA fusion protein mutant P398R still retained
some activity (6-fold repression). To understand the reasons for this
discrepancy, we decided to test some of the mutants in the context of
the full length v-erbA protein using as a reporter gene the tkCAT
fusion controlled by three copies of the everted palindromic binding
site F2 (4). Expression of the wild type v-erbA results in a 4-fold
repression (Fig. 4
), as did the functional mutant P475R
(compare with the 20-fold repression obtained using GAL4 fusions). In
this assay, mutant P398R displayed only a marginal activity, while
mutants L544R, P481R, and P396R had no effect on transcription. We
conclude that the use of GAL4 fusion proteins represents a much more
sensitive method to study transcriptional effects, allowing in
particular a finer classification of the mutants.

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Figure 4. The Mutant Phenotype Is Conserved in the Full
Length v-erbA
CV-1 cells were cotransfected with the reporter plasmid (F2)x3-tkCAT
and wild type (WT) or mutant full-length v-erbA expression vectors. The
graph represents fold repression relative to
cotransfection with an empty expression vector (C).
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Mutations in the Ti-Region and
3 Affect the Silencing Function
of rTRß
To detect possible relationships between the silencing function
without ligand and the activation function of TR in the presence of
ligand, we decided to analyze rTRß mutants for their repression and
activation capacity in our system. Some of the mutants we used were
shown to superactivate a reporter gene in the absence of hormone, as
compared with wild type, when tested in yeast (18), possibly reflecting
a lack of silencing activity in higher eukaryotes. To test this
possibility, we constructed fusion genes coding for the GAL4 DBD and
the HBD of wild type (WT) and mutant TRß (see Fig. 5A
). After transfection into L-tk- or CV-1
cells, the ability of the chimeric proteins to repress or activate
transcription of a UAS-tkCAT gene was assessed. The wild type
GAL-hTRß led to a 7- or 5-fold repression in the absence of ligand,
and a 15- or 40-fold activation in the presence of T3, in
L-tk- or CV-1 cells, respectively (Fig. 5B
). All of the
mutants displayed close to wild type transactivation function in the
presence of hormone, but mutants K419E, K415E, and V279E/K283R/K301Q
seem to mediate a slightly reduced ligand-dependent activation in CV-1
cells. Interestingly, the superactivation described in yeast is not
detected in higher eukaryotic cells. In contrast, three of the mutants
(K419E, K415E, and V279E/K283R/K301Q) displayed a 3- to 4-fold lower
silencing activity as compared with wild type. Mutant N359S showed
close to wild type repression in CV-1 cells. These results support the
involvement of the Ti region and of helix H8 in the silencing function,
and they further point to helix H11 (see Fig. 5A
) as an important
component of the silencing domain. On the other hand, the fact that
none of the TRß mutants displayed an important difference in its
activation capacity clearly shows that both functions, transactivation
and silencing, represent two independent activities of the same
protein.

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Figure 5. Several Mutations in the TRß HBD Affect
Silencing, but Not Transactivation
A, Amino acid sequences of the concerned regions (Ti, 3, and helix
11) in TRß. The point mutations in the repression domain of TRß are
indicated. L-tk- (black boxes) and CV-1
cells (stippled boxes) are cotransfected with the
reporter pUAS-tkCAT and the expression plasmids coding for the GAL4 DBD
(GAL), the fusion proteins GAL94-hTRß (aa 173461) (WT),
or the GAL147-rTRß point mutants (aa 172456). B, The
graph represents fold repression of the WT or the
different mutants of GAL-TRß relative to GAL. C, Fold activation
mediated by the WT or the mutants of GAL-TRß relative to the GAL4 DBD
(GAL).
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Expression and DNA Binding of the Fusion Proteins
To ensure that the lack of activity of the defective mutants was
not due to a lower expression or a defect in DNA binding of the mutant,
we expressed the fusion proteins in COS-1 cells and prepared whole cell
extracts of the transfected cells. Electrophoretic mobility shift
assays were performed using an oligonucleotide containing a GAL4
binding site as a probe. Each of the extracts gave rise to a specific
DNA-protein complex (Fig. 6
) not present in extracts
from mock-transfected cells (lane 19). The complexes can be competed by
the unlabeled specific oligonucleotide, but not with an unrelated
sequence (data not shown). A faster migrating complex was formed using
each extract, presumably originating from a degraded GAL4 DBD (lane
13), as it was specific and similar for every extract. Expression of
GAL-v-erbA (lanes 112) or GAL-TRß (lanes 1418) fusion proteins
gave rise to complexes of lower mobility, as expected. The results show
that expression and DNA binding are similar for the wild type and each
of the mutants.

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Figure 6. Expression and DNA Binding of the Mutants of v-erbA
and TRß Fusion Proteins
Gel retardation analysis using whole cell extracts from transfected
COS-1 cells on a polynucleotide kinase-labeled UAS DNA probe
(GAL4-binding site). COS-1 cells are transfected with the expression
plasmids of GAL4 DBD (aa 1147) (lane 13) or the different fusion
proteins, GAL-erb 346 wild type (lane 12), point mutants thereof (lane
111), the GAL-hTRß wild type (lane 18), and the GAL-rTRß-mutants
(lanes 1417). Note that the GAL-hTRß wild type fusion consists of
the GAL4 DBD (aa 194) and the repression domain of hTRß (aa
173461). Lane 19 shows the whole cell extract of COS-1 cells
transfected with an empty expression vector (C).
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Three Subdomains Cooperate for Silencing Function when Placed on
Different Molecules
Our mutational analysis defined three regions that seemed to be
important for silencing function. Therefore we decided to test whether
the silencing domain of v-erbA could be split into three regions (Fig. 7A
), which would restore repression when combined. We
showed previously that different GAL-v-erbA chimeras are able to
heterodimerize via the GAL4 region aa 1147 (6). These heterodimers,
appropriately matched, were able to mediate silencing on a
reporter plasmid (6) controlled by a single palindromic GAL4-binding
site (UAS). To assemble simultaneously three different fusion proteins
on a regulatory region, we decided to use the tkCAT transcription unit
placed downstream of a hexamerized UAS. This construct can bind up to
six fusion protein dimers; thus each of the three tested chimeras
should be present.

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Figure 7. Three Subdomains Cooperate for Silencing Function
When Placed on Different Molecules
A, Diagram of the linear structure of hTRß, v-erbA, and the GAL4 DBD
(GAL) fusions with the different C-terminal subdomains of hTRß and
v-erbA. Note that SSD1 was derived from hTRß, as it is functional in
this type of experiment, whereas the other silencing subdomains (SSD
1/2, 2/3, 2 and 3) are parts of the repression domain of v-erbA. B,
L-tk- cells are cotransfected with the reporter plasmid
(UAS)x6-tkCAT and several combinations of the indicated C-terminal
subdomains of hTRß and v-erbA fused to the GAL4 DBD. Fold repression
of the GAL-erb 346 wild type (WT) and the different combinations is
relative to GAL.
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The experiments were carried out in L-tk- cells by
cotransfecting the reporter plasmid (UAS)x6-tkCAT together with three
expression vectors coding for different GAL-v-erbA or GAL-TRß fusion
proteins. We decided to use the TRß fusion protein for GAL-SSD1 (aa
173265), as this domain was shown before to form a silencing
subdomain (7), whereas the other silencing subdomains are taken from
v-erbA. In the controls, when no, one, or two chimeras were expressed,
the total amount of transfected DNA was kept constant. The results are
presented in Fig. 7B
. The repression is indicated relative to
transfection of pGAL4 DBD alone. Transfection of 0.6 pmol of
pGAL-v-erbA, coding for a fusion protein containing the complete
v-erbA-silencing domain, leads to 18-fold repression of the basal
level, as expected. Expression of each of the chimeric proteins,
GAL-SSD1, GAL-SSD2, GAL-SSD3, GAL-SSD1/2, or GAL-SSD2/3 alone, does not
result in any silencing effect. Coexpression of GAL-SSD1 + GAL-SSD2/3
or GAL-SSD1/2 + GAL-SSD3 resulted in a synergism between the expressed
subdomains leading to 11-fold or 14-fold repression, respectively.
Synergism in this system is strong enough to yield silencing effects
similar to the one obtained with the complete silencing domain. Neither
of the other dual combinations led to a significant repression. In
contrast, coexpression of the three fusion proteins, GAL-SSD1,
GAL-SSD2, and GAL-SSD3, results in 6-fold silencing.
These results suggest that the simultaneous presence of the three
v-erbA subdomains, SSD1, SSD2, and SSD3, restores silencing activity,
whereas each one alone or any combination of two is nonfunctional.
The silencing domain of v-erbA is thus composed of three defined
subdomains, all of which represent separable structural entities and
which cooperate to result in the repression function.
Competition for Silencing Cofactors Requires the Intact,
Full-Length Silencing Domain of v-erbA
To test for the possible involvement of (a) corepressor(s) in
silencing activity, we performed cotransfection experiments in which we
expressed GAL-erb 346 as a silencer protein and large amounts of the
silencing domain of v-erbA (WT) or functionally characterized point
mutants as competitors in L-tk--cells. Relief of silencing
was tested on the p(UAS)-tkCAT reporter plasmid (Fig. 8
).

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Figure 8. Relief of Silencing Activity by in
Vivo Competition Experiments
Cotransfection experiments in L-tk- cells with the
reporter plasmid UAS-tkCAT, the expression plasmid pGAL-erb 346, and
wild type (WT) or mutant ligand binding domain of v-erbA as competitor.
Repression activity is mediated by cotransfection of pGAL-erb 346 (280
ng) with the empty expression plasmid (C) (10 µg) in comparison to
GAL. Silencing activity obtained after additional cotransfection of an
excess (10 µg) of expression vector coding for the v-erbA-silencing
domains is shown for wild type (WT) and each mutant.
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Transfection of pGAL-erb 346 (280 ng) together with 10 µg empty
pAB-
gal (C) led to an 11-fold repression of the CAT activity as
compared with transfection of pGAL4-DBD. This repression was relieved
more than 4-fold by coexpression of the wild type v-erbA silencing
domain (WT), showing the requirement of one or several titratable
corepressors for silencing activity. In contrast, mutants P396R and
P398R, located in SSD1, were clearly unable to titrate out the
cofactor(s), in correlation with their weak silencing activity (see
Fig. 1
). The next point mutation cluster is located in the Ti-region
(Fig. 2
). The active mutant P475R clearly displays an ability to relief
silencing, while the mutants P481R and L489R, which show no silencing
activity (Fig. 2
), do not compete for (a) corepressor(s). Point mutants
L530R and I537R, located within the
3-region and presenting wild
type repression function, also compete the silencing activity. Mutant
L540R still shows silencing activity and displays a weak competition
function, but the nearly inactive mutant L544R does not relieve the
repression activity of v-erbA.
In conclusion, our results show that the full length silencing domain
of v-erbA is able to relieve the silencing activity of GAL-erb 346.
Mutants presenting a wild type repression function are also able to
titrate out (a) corepressor(s), although at different levels. Most
importantly, none of the inactive mutants is able to compete for (a)
silencing corepressor(s), again supporting the view that the complete,
intact silencing domain is required for repression function.
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DISCUSSION
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Our study suggests that the silencing domain of v-erbA is composed
of at least three functional subdomains, each of which is inactive on
its own, but which synergize in transcriptional repression. Two lines
of evidence support this conclusion. First, we describe inactivating
point mutations in each of the proposed subdomains. Second, the
combination of the three subdomains, on different molecules bound to
the same promoter, restores the silencing function.
In our experiments, we used fusion proteins consisting of the
C-terminal v-erbA silencing domain joined to the DBD of the yeast GAL4
transcription factor. The GAL4 DBD ensures correct nuclear
translocation (19) and DNA binding to the specific UAS sequence (20).
In addition, we previously showed that homo- and heterodimerization of
these fusion proteins is mediated by the dimerization activity of GAL4
(6). Moreover, we clearly show, using gel retardation experiments with
transfected cell extracts, that the mutations in the silencing domain
do not affect the synthesis or DNA binding of the fusion proteins.
A further advantage in using GAL4 fusion proteins is the increased
sensitivity with respect to silencing capacity. Mutants P398R, P396R,
and L544R are completely inactive when tested in the full-length
protein, but still present a 10- to 15-fold repression ability in the
GAL4 fusions. In contrast, mutants P481R and L489R retain only a
severely reduced (in L-tk- cells) or no (in CV-1 cells)
silencing function as GAL4 fusion proteins, while they are
indistinguishable from the other inactive mutants when tested in the
natural context. This approach thus allows a more precise evaluation of
the effects of individual mutations on the repression function.
Trans-acting complementation analysis of individual protein domains has
been successfully performed in several cases (6, 7, 21, 22). Here we
use a similar strategy to test three different, nonoverlapping
subdomains of v-erbA and TRß. To achieve the simultaneous presence of
the three different fusion proteins on the same promoter, we used a
reporter plasmid controlled by multimerized UAS sequences. Based on the
different heterodimer combinations, each subdomain is expected to be
bound to the promoter. Indeed, we could observe a recovery of silencing
function only upon coexpression of the three subdomain fusion proteins.
Our result suggests that the proposed subdomains are able to adopt a
functional conformation when isolated from the rest of the protein.
This approach might prove useful in the future study of other
multifunctional proteins.
We tested the effects on silencing activity of single amino acid
substitutions in three regions of the v-erbA-silencing domain. The most
N-terminal one (SSD1, aa 173265) corresponds to the TR hinge region
and transactivation domain
2. This region was recently shown to
interact with the corepressors N-CoR (8) and SMRT (9). In a GAL-v-erbA
fusion protein, the deletion of aa 389 to 409 resulted in a complete
loss of the silencing function (6). A natural mutant of v-erbA was
described previously with a change of Pro 398 to Arg abolishing the
silencing function (15). A homologous mutation in the TR
similarly
abolished its repression function in the absence of ligand, without
affecting its ability to activate transcription in the presence of
hormone. The authors proposed Pro 398 to be required for the precise
positioning of the structures flanking it. Our data confirm this
finding; in addition, we show that Pro 396 is required for silencing
function as well, supporting the view that these amino acids form a
backbone to precisely arrange the
-helical structures flanking them.
It is unclear whether the prolines are directly involved in
protein-protein interactions in addition to this putative structural
role, but it was shown that a mutation corresponding to the P398R does
abolish the TR interaction with the corepressor SMRT (9). In addition,
mutation of amino acids AHxxT at the end of helix 1 in hTRß (see Fig. 1
) abolished both repression and interaction with N-CoR (8).
Surprisingly, mutation of only HxxT at the same site to AxxA results in
a silencing domain able to compete for a corepressor (23). The same
authors describe a mutant V174A/D177A (corresponding to positions 363
and 365 in v-erbA) that is unable both to repress transcription and to
interact with the corepressor. We show that isoleucine 389, located in
the same region, is not involved in silencing.
The second domain is identified by the inactive v-erbA mutants P481R
and L489R and the triple TRß mutant V279E/K283R/K301Q. It covers the
highly conserved, so-called Ti region-spanning helices H4, H5, and H6
in TR
(16). These mutants appear to be most strongly affected in
their silencing activity, pointing to a crucial role of this region in
silencing function. Helices H5 and H6 form a highly hydrophobic surface
(see the arrangement of L and I in Fig. 2A
) which would be disrupted by
the mutations. Insertion of an additional L in mutant C493L has no
effect on repression function.
The
3 region was previously defined as a transactivation domain in
hTRß (14) and was shown to be involved in T3 binding and
heterodimerization with RXR (24). A mutation in this region of v-erbA
(mutant L544R) clearly affected the silencing function as well, again
altering the amphipathic character of an
-helix (H8).
An other functional region is suggested by the rTRß mutants K415E and
K419E located in helix H11. This region corresponds to the previously
described ninth heptad repeat and was shown to be involved in
homodimerization and heterodimerization with RXR (25). Furthermore,
mutation L365R of the cTR
(position 605 in v-erbA) results in loss
of repression function (25, 26). As a slight effect of these mutations
is also observed on induction in the presence of T3, at
least in CV-1 cells, we cannot rule out the possibility that a lack of
interaction between the silencing domains in the homodimers of the
GAL-TRß chimeric proteins is the basis for their reduced activities.
These and other mutants have been described to act as superactivators
in yeast (18). Our results, showing a similar hormone induction of the
mutants compared with wild type TRß in mammalian cells, are
consistent with those obtained by Uppaluri et al., 1995
(18). The authors propose that their selection for highly activating
receptors in yeast resulted in the identification of TRs adapted to the
yeast transcriptional machinery. As a result of this adaptation, these
receptors are less well suited for activation in mammalian cells. Here
we show that these mutants, in addition, lose their silencing function.
Recently, it was shown that coexpression of hormone-binding deficient
TR mutants or of v-erbA is able to enhance the hormone-dependent
activation by GAL-TR fusion protein (27) in HeLa cells, suggesting that
an inhibitory factor interacts with the TR even in the presence of
T3. Similarly, a mutation abolishing the interaction of the
TR with the putative inhibitor would result in a superactivation. Such
a superactivation in mammalian cells, due to the loss of a residual
silencing activity in the presence of T3, is not observed.
It is unclear whether the effect could be masked by the concomitant
loss of activation capacity of the mutants.
Our cotransfection experiments clearly show that the assembly of the
three defined subdomains in heteromeric complexes on the promoter of a
reporter gene restores silencing function. In particular, the
combination of three GAL4 DBD fusion proteins, each containing a
different subdomain, restores repression, suggesting that each
subdomain is able to adopt an active conformation on its own.
To visualize a potential protein-protein interaction region in the
v-erbA/TR silencing domain, we wanted to localize the mutations
abolishing the repression function in the three-dimensional structure
of the receptor. The structure has been determined for three nuclear
receptors, hRXR
(17), hRAR-
(28), and rTR
1 (16), only one of
which (hRXR
) was generated in the absence of ligand. As the
structure of different receptors is quite similar, in contrast to the
conformational change induced in each receptor upon ligand binding, we
used the structure determined for RXR in the absence of ligand.
Figure 9
shows the RXR-ligand-binding domain
structure with the highlighted (red) positions of the amino
acids involved in silencing. Strikingly, these amino acids are all
located in one region of the HBD, opposite to the C-terminal activation
domain
4/AF-2AD (14, 29, 30). Moreover, they all face to the outside
of the molecule, supporting the notion that these regions represent
interaction interfaces with other factors. Helices H1 and H2, located
in subdomain SSD1, are shown in yellow. Unfortunately, the
arrangement of the hinge region is so far unknown, and no information
on the structure of the complete SSD1 is available. Subdomains SSD2 and
SSD3 are represented in violet and green,
respectively. The position of the amino acids required for silencing in
the subdomains is consistent with a model where each subdomain, when
isolated in a GAL4 DBD fusion protein, is able to adopt a conformation
leading to the correct spatial arrangement of the crucial amino
acids.
Our results led us to propose a model for the interactions
responsible for the silencing mechanism by TR/v-erbA. SSD1 was shown to
interact with the corepressors N-CoR and SMRT only in the absence of
hormone. On the other hand, it was shown that SSD1 alone, fused to the
GAL4 DBD, is unable to repress a UAS-containing reporter gene. The
observation that the other subdomains, SSD2 and SSD3, are necessary to
restore silencing activity suggests that other interactions are
absolutely required for function. In additional experiments, we found
that none of the silencing-deficient mutants (Fig. 8
) and none of the
silencing subdomains (data not shown) is able to titrate out (a)
corepressing factor(s), further supporting the view that all three
subdomains are required for functional corepressor interaction(s). A
repression model in which each subdomain interacts with a different
corepressor would imply that each mutant deficient in only one
subdomain should be able to titrate out the other cofactors, thus
resulting in a relief of silencing. This does not appear to be the
case, although we cannot completely rule out the possibility that more
than one cofactors are involved in silencing. The fact that some of the
still active mutants display a reduced capacity to relief silencing
indicates, however, that small changes in corepressor interaction might
occur without affecting silencing function.
Previously, Hörlein et al. (8) performed
interaction studies between TRß deletion mutants and N-CoR, using GST
pull down and yeast two-hybrid experiments (8). They showed that
deletion of aa 203230 (part of SSD1) abolishes interaction with
N-CoR, while deletion of either aa 260335 (SSD2) or aa 335456
(SSD3) clearly weakened the interaction as compared with the
full-length ligand-binding domain. These results are consistent with a
model where interactions of intermediary factors with one single
subdomain are too weak for repression activity, but are stabilized in
the presence of the other subdomains to result in a functional
silencing complex.
The C-terminal region of TRß, corresponding to SSD2/3, was previously
shown to interact with the general transcription factor TFIIB in the
absence of ligand (7), and this interaction was shown to be involved in
repression by TR in in vitro transcription (11, 12, 13). Thus, a
complex picture of transcriptional silencing emerges involving
interaction of SSD1 with N-CoR and/or SMRT, strengthening of this
interaction by other subdomains, and interaction of SSD2 and/or SSD3
with TFIIB or with a still unknown factor. Further experiments will be
required to ultimately understand the precise molecular interactions
leading to transcriptional silencing.
 |
MATERIALS AND METHODS
|
---|
Plasmid Constructions
The reporter plasmids used in this study contain a single
(UAS-tkCAT) or six tandem copies (UASx6-tkCAT) of a GAL4 binding site
(20) inserted in front of the tkCAT gene (5). The reporter F2x3-tkCAT
was described previously (4).
The expression plasmids coding for the GAL4 DBD (aa 1147), GAL-erb
346, GAL-erb 362508 (SSD1/2), GAL-erb 508639 (SSD3), and GAL-erb
434 (SSD2/3), as well as the control vector
gal (C), have been
previously described (4). The expression plasmid pGAL-erb 434508
(SSD2) was obtained by cutting pGAL-erb 362508 with PvuII
and EcoRV and religating, thereby destroying these
restriction sites. The pABGAL94,
pABGAL94-hTRß (aa 173461), and
pABGAL94-hTRß 173265 (SSD1) vectors were described
previously (14).
For the competition experiments, the PvuII/BamHI
fragments from wild type and mutant pABGAL147-erb 346 were
cloned into the expression vector pAB-
gal, resulting in expression
vectors for wild type and mutant silencing domains.
The rat TRß mutants were kindly provided by H. C. Towle (18):
pG2M/GAL-TRß (aa 172456)-S4 (K419E), -S10 (K415E), -S20 (V279E,
K283R, K301Q), and -112 (N359S) are cloned into the
pABGAL147 vector by digestion with
HpaI/SalI to obtain the corresponding GAL-TRß
mutant fusion proteins.
Expression vectors for the full-length v-erbA mutants were constructed
by cloning the gag-coding KpnI/PvuII fragment
from pRSV-v-erbA (2) in the KpnI/PvuII-digested
expression plasmid pGAL-erb 346 mutant, thus replacing the GAL4 DBD
with the original v-erbA N-terminus.
Site-Directed Mutagenesis
The v-erbA-mutants were generated by site-directed mutagenesis
as described (31). Briefly, a single-stranded uracil-containing
template containing sequences coding for v-erbA aa 346639 was
prepared and annealed with a specific primer carrying the mutation. The
annealed primer was elongated using T4 DNA-Polymerase (in the presence
of the single-strand binding protein gene 32 from phage T4). After
ligation, the product was transformed into Escherichia
coli.
The resulting point mutants were confirmed by sequencing. The primers
used to obtain the 11 mutants are shown below; the modified bases are
underlined. I 389 R:
5'-G-GAG-GAG-ATG-AGG-AAA-TCC-CTG-C-3' P 396 R:
5'-G-CAC-CGG-CGC-AGC-CCC-3' P 398 R:
5'-GG-CCC-AGC-CGC-AGC-GCA-GAG-G-3' P 475 R:
5'-GCC-AAA-AAC-CTG-CGC-ATG-TTC-TCG-G-3' P 481 R:
5'-C-TCG-GAG-CTG-CGG-TGC-GAG-GAT-CAG-3' L 489 R:
5'-CAG-ATC-ATC-CTG-CGG-AAG-GGC-TGC-3' C 493 L:
5'-G-AAG-GGC-TGC-TTG-ATG-GAG-ATC-ATG-3' L 530 R:
5'-C-GGA-GGG-CGG-GGG-GTC-G-3' I 537 R:
5'-C-GTG-TCT-GAT-GCC-AGG-TTC-GAC-CTC-G-3' I 540
R: 5'-GCC-ATC-TTC-GAC-CGC-GGC-AAG-TCG-C-3' I 544 R:
5'-C-GGC-AAG-TCG-CGG-TCT-GCC-TTC-AAC-3'
Cell Culture and Transfections
L-tk- cells, CV-1, and COS-1 cells were grown in
DMEM (GIBCO, Grand Island, NY) supplemented with 10% FCS, 100 U/ml
penicillin, and 100 µg/ml streptomycin.
DNA transfer into CV-1 cells was performed using the calcium phosphate
precipitation method (32). L-tk- cells were transfected
using the following protocol (33): 1 x 106 cells were
suspended in DNA-diethylaminoethyl-dextran solution (1 pmol reporter
and 0.5 pmol expression plasmids) and incubated for 60 min at room
temperature. Cells were seeded on a 6-cm dish containing 7 ml medium
and grown for 48 h before harvesting. For hormonal induction
experiments, the serum was depleted of thyroid hormone by extensive
charcoal stripping (34). The cells were kept for at least 24 h in
depleted medium before transfection; after transfection
10-6 M T3 was added when
indicated. CAT assays were performed as described (35).
Transfections were done in duplicate and performed in at least three
independent experiments. Transfections into COS-1 cells were done using
a similar diethylaminoethyl-dextran suspension method using 25 µg of
DNA on 2 x 106 cells. After 1 h incubation in
the DNA solution, a dimethylsulfoxide shock was performed for 3 min,
the cells were taken up in 30 ml TBS and 10 ml DMEM, spun down, seeded
on a 15-cm dish, and grown for 48 h before harvesting (6).
DNA-Protein-Binding Assays
Whole cell extracts were prepared from COS-1 cells transfected
with various expression vectors as described (36). Gel retardation
experiments were performed using 20,000 cpm of polynucleotide
kinase-labeled UAS DNA probe, 5 µg whole cell extract in an
incubation mix containing 1 µg of poly-deoxyinosinic-deoxycytidylic
acid, 6 mM HEPES. pH 7.8, 133 mM KCl, 6%
glycerol, 0.6 mM dithiothreitol. The DNA-protein complexes
formed were analyzed on a 5% polyacrylamide gel in 25 mM
Tris, 192 mM Glycin.
 |
ACKNOWLEDGMENTS
|
---|
We would like to thank K. Krueger for excellent technical
assistance. We are grateful to D. Moras for providing the coordinates
of the three-dimensional structure of the RXR and to W. Wende, G.
Schlauderer, and A. Jeltsch for the computer visualization of the
mutants in the RXR structure. Furthermore, we thank H. C. Towle for
sending us the TRß mutants.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Marc Muller, Laboratory of Molecular Biology and Genetic Engineering, Institut de Chimie-B6, University of Liege, B-4000 Sart-Tilman, Belgium.
This work was supported by the Fonds der Chemischen Industrie, the
Deutsche Forschungsgemeinschaft (SFB 249), the Services
Fédéraux des Affaires Scientifiques, Techniques, et
Culturelles PAI P3042 and PAI P3044, Fonds National de la Recherche
Scientifique (FNRS)-3.4537.93 and 9.4569.95, and the Actions de
Recherche concertée-95/00193. K. Busch was supported by a
fellowship from the DAAD (Doktorandenstipendium aus Mitteln des zweiten
Hochschulsonder-programmes") and B. Martin by a fellowship from the
Boehringer Ingelheim Fonds.
This work contains part of the Ph.D. theses of K. Busch and B. Martin
(University of Giessen, Germany).
Received for publication July 26, 1996.
Revision received December 20, 1996.
Accepted for publication December 30, 1996.
 |
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