Silencing Subdomains of v-ErbA Interact Cooperatively with Corepressors: Involvement of Helices 5/6
Kerstin Busch,
Bernd Martin1,
Aria Baniahmad,
Joseph A. Martial,
Rainer Renkawitz and
Marc Muller
Laboratoire de Biologie Moléculaire et de Génie
Génétique (K.B., B.M., J.A.M., M.M.) Institut de
Chimie-B6 Université de Liège B-4000 Liège,
Belgium
Genetisches Institut (K.B., A.B., R.R.)
Justus-Liebig-Universität D-35392 Giessen, Germany
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ABSTRACT
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Members of the thyroid hormone receptor (TR)
family act on vertebrate development and homeostasis by activating or
repressing transcription of specific target genes in a ligand-dependent
way. Repression by TR in the absence of ligand is mediated by an active
silencing mechanism. The oncogene v-ErbA is a variant form of TR unable
to bind hormone and thus acts as a constitutive repressor. Functional
studies and mutation analysis revealed that the TR/v-ErbA silencing
domain is composed of three silencing subdomains (SSD13) which,
although nonfunctional individually, synergize such that silencing
activity is restored when they are combined in a heteromeric complex.
Here we demonstrate, using protein interaction assays in
vitro and in vivo, that the inactive v-ErbA point
mutant L489R within helix 5/6 in SSD2 fails to interact with the two
corepressors N-CoR (nuclear receptor corepressor) or SMRT
(silencing mediator of retinoic acid and thyroid hormone
receptor). Furthermore, mutants in SSD1 and SSD3 exhibit a
reduced corepressor recruitment corresponding to their weak residual
silencing activity. In mammalian two-hybrid assays, only the
combination of all three silencing subdomains, SSD13, leads to a
cooperative binding to the corepressors N-CoR or SMRT comparable to
that of the full-length v-ErbA repression domain. In conclusion, full
silencing activity requires corepressor interaction with all three
silencing subdomains, SSD13. Among these, SSD2 is a new target for
N-CoR and SMRT and is essential for corepressor binding and function.
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INTRODUCTION
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Transcriptional regulation by steroid/thyroid hormones and
retinoids is a critical component in controlling many aspects of animal
development, reproduction, and metabolism (1, 2, 3, 4). The functions of
these hormones are mediated by intracellular receptors, which comprise
a large superfamily of ligand-dependent transcription factors (2, 4).
The thyroid hormone receptor (TR) is bound to its cognate DNA sequence
upstream of specific target genes (5) and activates transcription in
the presence of its ligand, but represses transcription in the absence
of hormone (6, 7). Repression by TR is mediated by its C-terminal
ligand binding domain (LBD) (8, 9), which is known to harbor additional
functions including ligand binding, transcriptional activation, nuclear
localization, and dimerization (1, 10). The LBD represents a separable
silencing domain that confers a true silencing activity (11, 12) when
fused to the heterologous DNA-binding domain (DBD) of the yeast
transcription factor Gal4 (8). A similar silencing function is found in
the oncogene v-ErbA, a viral derivative of the chicken TR
, which is
unable to bind hormone and functions as a constitutive repressor of
target genes (6, 13). V-ErbA lacks the activation domain
AF-2-AD/
C/
4 at the C-terminal end (14, 15, 16). Deletion or mutation
of this domain converts TR or retinoic acid receptor (RAR) into potent
constitutive transcriptional repressors (9, 17, 18, 19). V-ErbA and
constitutively repressing TR or RAR mutants were shown to cause defects
in cell differentiation and development (20, 21, 22, 23, 24, 25, 26, 27, 28). Thus, transcriptional
repression by unliganded receptors appears to play an important role in
regulating cell growth and differentiation.
The molecular mechanisms of the repression by nuclear hormone receptors
have been the object of intense research. Interactions of the silencing
domain of TR with factors of the basal transcription machinery, such as
transcription factor (TF)IIB, TATA-binding protein, and
TBP-associated factor (TAF) 110, have been shown in
vitro (29, 30, 31, 32, 33, 34). Also, transcriptional regulation was shown to
involve additional cofactors (reviewed in Refs. 35, 36, 37). In the absence
of hormone, interaction of the TR with the corepressors N-CoR (nuclear
receptor corepressor) and SMRT (silencing mediator of retinoic
acid and thyroid hormone receptor) (38, 39, 40, 41, 42, 43) leads to subsequent
recruitment of a Sin3/histone deacetylase complex able to change
nucleosomes into a repressive state. In the presence of hormone, a
conformational change involving helix 12 in the AF-2-AD/
C/
4
activation domain of TR or RAR (9, 44, 45, 46) induces the release
of corepressors and the binding of coactivators. Protein interaction
studies identified the N-CoR-box within the hinge region (38, 39) and
the ninth heptad repeat in helix 11 (47, 48) of TR as direct
interaction regions with these corepressors.
We previously described that the repression domain of TR and v-ErbA
consists of three different subdomains (SSD1, SSD2, and SSD3; see Fig. 1
), which show no silencing activity
alone but which synergize in repression when they are combined (49, 50). The molecular mechanism for this functional reconstitution of the
three subdomains is still unknown. The structural organization of the
v-ErbA silencing domain in three subdomains is further illustrated by
the positions of different point mutations that affect the repression
function (50). Mutants located in SSD1 (P396R and P398R) and in SSD3
(L544R) present a severely weakened activity and mutants in SSD2 (P481R
and L489R) were completely inactive. In a first attempt to investigate
the involvement of limiting cofactors in the silencing activity, we
performed in vivo titration (squelching) experiments (50).
Expression of the chimeric effector protein Gal-ErbA and of large
amounts of the v-ErbA silencing domain as competitor resulted in relief
of silencing on the pUAS-tk-CAT reporter plasmid. Overexpression of
mutants mediating wild-type repression activity also alleviates the
repression. In sharp contrast, none of the inactive mutants was able to
compete for silencing cofactors.

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Figure 1. Point Mutants within the Three Silencing Subdomains
SSD13 of the V-ErbA Repression Domain
The v-ErbA C-terminal repression domain is shown and the three
silencing subdomains SSD1 (aa 362434), SSD2 (aa 434508), and SSD3
(508639) (50 ) are indicated. The position of several functionally
relevant regions are shown: the hinge region including the N-CoR-box
(38 ) in SSD1, helix 5/6 in SSD2, and helix 8 and the ninth heptad
repeat/helix 11 (47 48 ) in SSD3. Three types of point mutants have
been used in this work: P475R in SSD2 and I537R and L540R in SSD3
display wild-type repression activity (+); P396R and P398R in SSD1 and
L544R in SSD3 exhibit a weakened silencing function (+/-); and mutant
L489R in SSD2 is fully inactive (-).
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Here, we directly investigate the protein-protein interaction of the
wild-type and mutant v-ErbA silencing domain or the three silencing
subdomains with the two corepressors N-CoR or SMRT in vitro
and in vivo. We show that the silencing activity of several
mutants of v-ErbA correlates with their capacity to recruit the
corepressors N-CoR and SMRT. We show that the combination of the three
subdomains results in a cooperative association with the corepressors,
presumably leading to the reconstitution of the full repression.
Furthermore, our results indicate that helices 5/6 (44) in SSD2 play a
crucial role in these interactions and thus constitute a new direct
interaction interface for corepressors.
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RESULTS
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Mutants in SSD2 Cannot Be Complemented in Silencing by Mutants in
Other Subdomains
Our previous results showed that none of the silencing-deficient
v-ErbA mutants (Fig. 1
) were able to compete with wild-type v-ErbA for
silencing cofactors in vivo (50). A simple model for
corepressor interaction with v-ErbA silencing subdomains would predict
that inactive mutants in different subdomains should be able to
complement each other. Therefore, we performed complementation
experiments by combining repression-defective v-ErbA mutants in each of
the subdomains. For this purpose, we used the well established
Gal4-DBD-fusion system, in which amino acids 1147 of Gal4 are
sufficient to mediate nuclear translocation, dimerization, and specific
binding to the upstream activator sequence (UAS), whereas the
transactivation functions are deleted. In particular, we previously
showed that two different Gal4 fusion proteins are able to
heterodimerize via this domain (49). We cotransfected combinations of
expression plasmids for different Gal-ErbA mutants, defective in
silencing, with the reporter plasmid p(UAS)x4-tk-CAT in
L-tk- cells. The wild-type Gal-ErbA and the indicated
mutants are expressed at similar levels in mammalian cells (Ref. 50 ;
see also Fig. 3
). Three different point mutants within each of the
previously defined silencing subdomains SSD13 were used.

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Figure 3. V-ErbA/SMRT Interaction in Vitro
Requires SSD2 and Is Weakened by Mutations in Other Subdomains
EMSA analysis was performed to analyze SMRT interactions with wild-type
and mutant v-ErbA. Extracts from COS-1 cells transfected with the
indicated expression plasmids were incubated with bacterially expressed
and purified nuclear receptor interaction domain of SMRT (C.SMRT aa
982-1495) fused to GST (600 ng) and tested with the UAS sequence as a
probe. Extracts from cells transfected with pGal4-DBD or the empty
expression vector gal (C) with or without GST.C.SMRT are shown as
controls. The complex obtained with the Gal-ErbA fusions (lower
arrow) and the supershift with GST.C.SMRT are indicated
(upper arrow).
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Expression of the Gal4 DBD alone did not affect the activity of the
p(UAS)x4-tk-CAT reporter gene, while expression of the wild-type
Gal-ErbA resulted in 18-fold repression (Fig. 2B
). As expected, the two mutant Gal-ErbA
in SSD1 and SSD3 (P398R and L544R) displayed reduced silencing activity
(2-fold) while mutant L489R in SSD2 was completely inactive.
Coexpression of the mutant P398R in SSD1 and L544R in SSD3 restored
10-fold repression activity. In contrast, the completely
silencing-deficient mutant within SSD2 (L489R) could not be
complemented by coexpression of either mutant L544R or mutant P398R.
Similar results were obtained when other mutants in each of the
silencing subdomains were used (not shown). The synergistic effect
observed when two point mutants, respectively, in SSD1 and SSD3 are
coexpressed indicates that the inactivating mutation of only one
silencing subdomain can be complemented by a mutant in the other
subdomain. However, the mutation in the central subdomain SSD2 cannot
be rescued by coexpression of any of the mutants in other
subdomains.

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Figure 2. The SSD2 Mutant Cannot Be Complemented by Mutants
in SSD1 or SSD3
A, Schematic representation of wild-type Gal-erbA and indication of the
position of the mutations in the different silencing subdomains. B,
Cotransfection of the reporter plasmid p(UAS)x4-tk-CAT with expression
vectors for the wild-type or mutant Gal-ErbA fusion proteins was
performed into L-tk- cells, as indicated. In control
experiments, a vector coding for the Gal4-DBD was added to keep the
amount of expression plasmid constant. The graph represents fold
repression obtained relative to Gal4-DBD alone. C, Cotransfection of
p(UAS)x4-tk-CAT and wild-type Gal-erbA with a 10-fold excess of
expression vectors for the Gal4-DBD or mutant Gal-ErbA fusions was
performed into L-tk- cells, as indicated. Fold repression
relative to Gal4-DBD alone is shown.
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To check the functional integrity of the different mutants, we combined
the wild-type Gal-ErbA with a 10-fold excess of Gal4-DBD or the
Gal-ErbA mutants. Cotransfection of Gal-ErbA with the Gal4-DBD
expression vector resulted in a significant reduction of its silencing
(from 20- to 4-fold repression) probably by formation of inactive
heterodimers, while coexpression of the P398R or L489R mutants hardly
affected the silencing function (Fig. 2C
). Thus, wild-type/mutant
Gal-erbA heterodimers appear to be functional, suggesting that the
overall structure of the mutant silencing domains is not destroyed.
Mutation in SSD2 of the V-Erba Silencing Domain Completely
Abolishes in Vitro Interaction with the SMRT
Corepressor
To understand the differential behavior of defective v-ErbA
mutants in the complementation experiments, we decided to investigate
directly the interaction between the wild-type or mutant v-ErbA
repression domain and corepressors. We used a bacterially expressed
fusion protein between glutathione-S-transferase (GST) and
the C-terminal receptor interaction domain of SMRT [amino acids (aa)
980-1495] which was affinity purified (GST-C.SMRT). The wild type and
mutant (see Fig. 1
) Gal-ErbA fusion proteins were expressed in COS-1
cells, whole cell extracts were prepared and used in electrophoretic
mobility shift assay (EMSA) experiments (Fig. 3
).
Incubation of extracts containing Gal-ErbA fusion proteins in the
presence of a Gal4 binding site (UAS) probe resulted in the formation
of a specific complex, which was not obtained with extracts from
mock-transfected cells (Fig. 3
, lane 20). Addition of GST-C.SMRT to the
wild-type Gal-ErbA led to the appearance of a slower migrating
SMRT-ErbA complex (Fig. 3
, lane 16), supershifting the original
DNA-protein complex (lane 15) and thus indicating an interaction
between these two factors. No such complex was obtained in the negative
controls, testing either the Gal4-DBD alone with GST-C.SMRT (lane 19)
or wild-type Gal-ErbA with only GST (lane 17). Point mutants P475R,
I537R, and L540R, displaying wild-type silencing activity, are not
affected in their interaction with GST-C.SMRT (Fig. 3
, lanes 6, 10, and
12). Interestingly, the point mutants P396R and P398R within SSD1 and
L544R within SSD3 showed a weaker supershift with GST-C.SMRT (lanes 2,
4, and 14) as compared with the one obtained with the wild-type
Gal-ErbA, whereas the completely inactive point mutant L489R, located
in SSD2, was unable to form a SMRT-receptor complex (Fig. 3
, lane
8).
In conclusion, in vitro interaction studies of v-ErbA
mutants with the corepressor SMRT reveal a correlation between
repression function and corepressor interaction.
Correlation between the Silencing Activity and Corepressor Binding
of Several Point Mutants of V-ErbA in Vivo
To extend the results concerning the in vitro
interaction between v-ErbA mutants and corepressors, we performed
in vivo mammalian two-hybrid experiments. We coexpressed the
Gal-ErbA silencing domain fusion proteins together with the receptor
interaction domain of N-CoR (aa 15852453) or SMRT (aa 10731495) as
VP16 fusions in CV-1 cells. Using the reporter plasmid
p(UAS)x5-SV40-LUC, a specific interaction between the Gal-ErbA fusions
and the VP16-corepressor protein (VP16-N-CoR-C' or VP16-C.SMRT)
results in a high luciferase expression via the transactivation domain
of VP16.
Expression of the VP16-corepressor fusion proteins only marginally
affected the transcription activation by Gal4-DBD (not shown). We
tested three defective point mutants, P398R, L489R, and L544R (Fig. 1
),
each localized in one of the silencing subdomains SSD13. The point
mutants P398R in SSD1 and L544R in SSD3 exhibit a reduced silencing
activity, while mutant P489R within SSD2 was completely inactive (see
Fig. 2
). The wild-type Gal-ErbA displayed a strongly enhanced
transcription in the presence of both VP16-N-CoR-C' or VP16-C.SMRT, the
interaction with N-CoR being significantly stronger (Fig. 4
). This observation is consistent with
the observed weaker interaction of TR with SMRT as compared with N-CoR
(51). The two weakly active mutants, P398R and L544R, showed a reduced
interaction with both corepressors, and the extent of the decrease was
similar for both N-CoR and SMRT. In contrast, the repression-defective
mutant L489R within SSD2 was unable to recruit either of the two
corepressors.

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Figure 4. In Vivo Interaction between V-ErbA
Mutants and N-CoR or SMRT Depends on SSD2
Mammalian two-hybrid experiments of v-ErbA mutants with the two
corepressors N-CoR and SMRT were performed. The indicated wild-type or
mutant Gal-ErbA fusions were transiently transfected into CV-1 cells
together with the VP16-fusions of the C-terminal receptor interaction
domains of N-CoR (aa 15852453) or SMRT (aa 10731495) and the
reporter plasmid p(UAS)x5-SV40-LUC. Protein interactions between the
v-ErbA mutants and the corepressors will result in high luciferase
activity via the transactivation domain of VP16. The resulting data are
presented as fold activation compared with the corresponding Gal-ErbA
fusion coexpressed with the transactivation domain VP16 alone.
Coexpression of the VP16 activation domain has no effect on the
repression levels of the Gal-ErbA fusions (not shown).
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In conclusion, we can separate the v-ErbA point mutants into different
classes according to their ability to interact with the corepressors
N-CoR and SMRT: mutants with wild-type activity show wild-type
interaction, mutants with a residual silencing activity exhibit a
reduced corepressor recruitment, and the completely inactive mutant is
unable to interact with any of the corepressors.
Silencing Subdomains Are Not Able to Fully Recruit the Corepressor
N-CoR
To analyze the role of the isolated v-ErbA silencing subdomains in
corepressor interaction, we first performed GST pull-down experiments.
Fusion proteins of the full-length silencing domain of v-ErbA or the
three subdomains with the GST were bacterially expressed, coupled to
glutathione-Sepharose beads, and used to precipitate in
vitro translated 35S-labeled nuclear
receptor interaction domain of N-CoR (N-CoR-C', aa 15852453).
Addition of 35S-labeled luciferase as an internal
control showed no nonspecific interaction with any silencing
subdomain (not shown). The three subdomain fusions, GST-SSD1 (aa
360434), GST-SSD2 (aa 434508), and GST-SSD3 (aa 508639), exhibit
a very weak interaction (Fig. 5
) compared
with that observed with the wild-type full-length silencing domain of
v-ErbA (aa 346639). Interestingly, GST-SSD2 as well as GST-SSD1/2 (aa
362508), containing both SSD1 and SSD2, showed a slightly higher
N-CoR interaction in this very sensitive protein-protein interaction
assay. No interaction was observed with GST alone (Fig. 5
). These
results indicate that the isolated subdomains interact marginally with
the corepressors in vitro and that SSD2 is able to interact
weakly with N-CoR.

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Figure 5. The Isolated Subdomains SSD13 Only Weakly
Interact with N-CoR in Vitro
GST-pull down experiments between N-CoR and the three silencing
subdomains (SSD13) of v-ErbA were performed. Bacterially expressed
GST or GST fusions with the full-length v-ErbA repression domain or the
silencing subdomains SSD1, SSD2, SSD3, and SSD1/2 were tested for
interaction with in vitro translated nuclear receptor
interaction domain of N-CoR, as indicated. The input lane contains 10%
of the 35S-radiolabeled protein used for the interaction
studies.
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Cooperative Binding of the Corepressors N-Cor and SMRT by V-ErbA
Silencing Subdomains in Vivo
To analyze the binding of corepressors to the v-ErbA silencing
subdomains in vivo, we used the mammalian two-hybrid assay
in CV-1 cells. Each of the three silencing subdomains was expressed as
a fusion protein with the Gal4-DBD together with VP16-C.SMRT or
VP16-N-CoR-C'. The transcriptional activities and the interactions were
analyzed by cotransfection with the reporter plasmid p(UAS)x5-SV40-LUC.
As expected, none of the subdomains SSD13 alone was able to
substantially repress transcription, and all three failed to interact
with the corepressors N-CoR and SMRT (Fig. 6
). Combination of two of the three
silencing subdomains was not sufficient to restore corepressor binding,
consistent with the lack of silencing activity. In contrast,
cotransfection of all three silencing subdomain fusions (Gal-SSD1,
-SSD2, and -SSD3) resulted in a nearly 5-fold repression and restored
the recruitment of both VP16-N-CoR-C' and VP16-C.SMRT.
Additionally, in squelching experiments none of the silencing
subdomains was able to titrate out corepressors for silencing activity
via the full-length v-ErbA repression domain (not shown).

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Figure 6. Silencing Subdomains Bind Corepressors
Cooperatively in Vivo
Interaction between the three silencing subdomains (SSD13) and the
two corepressors, N-CoR and SMRT, was tested in mammalian two-hybrid
assays, performed as described in Fig. 4 . The Gal fusions of the v-ErbA
full-length repression domain or the silencing subdomains were
cotransfected with the nuclear receptor interaction domains of N-CoR
(aa 15852453) or SMRT (aa 10731495) fused to VP16 and the reporter
plasmid p(UAS)x5-tk-LUC into CV-1 cells. For the combinations of the
silencing subdomains SSD13, expression vectors were cotransfected in
equal amounts (0.1 pmol). Amounts of expression vector were kept
constant by addition of Gal4-DBD expression vector. Fold repression is
shown relative to Gal4-DBD and is measured in the presence of
pCMX-VP16. Fold induction is defined as the luciferase activity in the
presence of the VP16-corepressor fusion relative to that obtained with
VP16 alone. The silencing function of v-ErbA or its subdomains was not
affected by expression of VP16, and the VP16-corepressor fusions only
marginally affected the basal level of Gal4-DBD (not shown).
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Our results clearly show that the combination of the three silencing
subdomains, which each interact marginally with corepressors, restores
the in vivo interaction between the heteromeric
transcription factor and the corepressors N-CoR or SMRT, probably
involving a cooperative recruitment by the combination of SSD1, SSD2,
and SSD3.
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DISCUSSION
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We previously showed that the silencing subdomain of v-ErbA
consists of three inactive subdomains that restore activity when
brought together on the same promoter. We also described different
mutations located in each of these subdomains leading to the loss of
the silencing function, and we showed that these inactive mutants are
unable to compete for silencing cofactors in squelching experiments
(50). In a first attempt to understand the function of each of the
subdomains in repression, we tested whether an inactive mutant in one
of the subdomains could be rescued by coexpression with a mutant in a
different subdomain (Fig. 2B
). Mutant P398R in SSD1 could indeed be
complemented by coexpression of mutant L544R in SSD3, while the
silencing function of mutant L489R in SSD2 could not be restored by
coexpression of any of the other mutants. Coexpression in excess of
this mutant does not lead to a dominant impairment of the silencing
function of the wild-type Gal-ErbA, in contrast to the Gal4-DBD alone
(Fig. 2C
). This observation, together with the fact that homodimers are
readily formed in gel retardation experiments and that their migration
is similar to that of the wild-type Gal-ErbA or other mutants (Ref. 50
and Fig. 3
), strongly suggests that the overall structure of mutant
L489R is intact.
We then tested several v-ErbA mutants for their ability to interact
with the corepressors N-CoR and SMRT in vitro and in
vivo. Supershift experiments and mammalian two-hybrid studies
clearly revealed that mutant L489R in SSD2 is unable to recruit
corepressors, while mutant P398R in SSD1 or mutant L544R in SSD3 shows
a reduced interaction with N-CoR and SMRT (Figs. 3
and 4
). These
results correlate with the silencing function of the different mutants;
mutations in SSD2 completely abolish repression, while mutations in
SSD1 or SSD3 only weaken this function. We suggest that the residual
repression activity of mutant P398R and L544R, involving their weak
interaction with corepressors, is sufficient to restore wild-type
interaction and silencing when they are combined. Such an effect would
be impossible for the completely inactive mutant L489R in SSD2.
The functional differences between point mutants in SSD1, SSD2, or SSD3
raises the question concerning the precise role of each of these
silencing subdomains, which are inactive on their own but synergize in
transcriptional repression (50). Previous mutation studies of TR showed
that deletion of the hinge region (N-CoR-box in SSD1) (38) or the ninth
heptad repeat/helix 11 in SSD3 (47, 48) abolishes its interaction with
N-CoR or SMRT. However, these regions were not tested individually for
corepressor binding. In GST pull-down experiments we show a weak N-CoR
interaction with SSD2 and a very weak N-CoR recruitment by SSD1 or
SSD3, as compared with the complete silencing domain of v-ErbA (Fig. 5
). This result is further supported by the fact that none of the
silencing subdomains, when coexpressed as fusion with a nuclear
localization signal, was able to titrate corepressors for repression
activity via the full-length repression domain of v-ErbA (not
shown).
Furthermore, no interaction between the isolated subdomains and N-CoR
or SMRT was observed in mammalian two-hybrid experiments (Fig. 6
). Even
the combination of the two previously defined corepressor interaction
regions, the N-CoR-box (38) in SSD1 and the ninth heptad repeat/helix
11 in SSD3 (47, 48), is not sufficient for corepressor binding. We show
that N-CoR and SMRT are only recruited when all three silencing
subdomains (SSD13) are combined, regaining repression activity
similar to the full-length repression domain. Our studies suggest a
cooperative binding of corepressors by the three silencing subdomains,
resulting in the formation of a functional repression complex. Isolated
SSD13 appear to be able to refold in a native-like structure able to
present a correct protein surface for corepressor binding.
Recently, the human protein Alien was described as a new corepressor
for TR that is structurally unrelated to N-CoR and SMRT (52). Alien is
able to interact constitutively with v-erbA and with TR in the absence
of hormone. No Alien interaction was observed with repression-defective
TR or v-erbA mutants or with the TR SSD1 or SSD2/3 in a yeast
two-hybrid experiment. These observations correlate well with our
results concerning v-erbA mutants and subdomains, suggesting that the
molecular interactions between TR or v-erbA and the corepressors N-CoR,
SMRT, and Alien are similar, with only subtle differences.
Analysis of the silencing-deficient mutants allows a closer inspection
of the different subdomains. Weakly active mutants are altered in SSD1
and SSD3, both of which contain regions previously shown to interact
with corepressors. Mutations in SSD1 (P396R and P398R) are localized in
the so-called hinge region, close to the previously described
AHT-mutation in TR (38). SSD3 contains the ninth heptad repeat/helix 11
(44), previously shown to be involved in corepressor binding (47, 48).
The mutation described here, L544R, is localized in helix 8 (44),
suggesting that this region may contribute to corepressor interaction
as well. However, these mutants retain some ability to interact with
corepressors, consistent with their residual silencing activity. Our
results identified SSD2, consisting of helices 36 in the TR
structure (44, 46), as a new important corepressor targeting domain.
Mutations in this subdomain most strongly affect silencing by v-ErbA
and preclude rescue by mutants in other subdomains. A slightly stronger
interaction of SSD2 with N-CoR, as compared with SSD1 and SSD3, was
observed in the GST pull-down experiments, which increase the
sensitivity for weak protein interactions. In the crystal structure of
the apoform of hRXR
(46) or the holoform of rTR
(44), the
inactivating point mutation of L489R is located within helices 5/6
(50). Interestingly, this location corresponds to the end of the
LBD-signature motif [(F/W)AKxxxxFxxxDQxxLL], which was shown to be
involved in corepressor binding by Rev-erbA
and Rev-erbAß (RVR)
(53). However, Rev-erbA
and RVR interaction with N-CoR was
mapped to the N-terminal end of this motif, in helix 3 (54), and
requires both interaction domains of N-CoR (RID-I and -II), in contrast
to TR (54). In addition, corepressor interaction with RVR does not
involve an N-CoR-box in region D. These observations suggest that
important differences exist in the way Rev-erbAa/RVR interacts with
corepressors compared with v-erbA/TR; whether helix 3 in Rev-erbA
or
helices 5/6 in v-erbA are responsible for this divergent behavior
remains to be established. Our results present the first evidence for
the involvement of helices 5/6 in TR or v-erbA corepressor interaction.
These helices are positioned close to helix 1, identified as the
corepressor interaction domain N-CoR-box in TR (38). Whether the
precise mechanism is the presentation of a larger interaction surface
to the corepressor or a mutual structural stabilization of the
different subdomains is at present unclear.
Our results indicate a good correlation between the strength of
corepressor recruitment and the extent of the silencing activity.
However, a mechanism proposing merely an accumulation of corepressor
interaction surfaces would be too simplistic. We clearly show that all
three subdomains must be present for functional interaction, suggesting
that each subdomain has a vital role to play, which cannot be filled by
the others. The complexity of the multiprotein complexes involved in
the repression process offers numerous additional possibilities for
modulation. Each subdomain might make optimal contacts with a different
part of the corepressors. A precise sequence of events might be
required for high-affinity interaction. The corepressor interaction
might be nonproductive, resulting in an impaired interaction with
additional cofactors involved in the repression mechanism, such as the
Sin3a/HDAC histone deacetylase complex shown to be recruited by the
corepressors N-CoR or SMRT (55, 56, 57). Recent studies concluded that the
phosphorylation status via the epidermal growth factor signaling
pathway is important for binding of the corepressor SMRT to nuclear
receptors (58). SMRT, N-CoR, and Sin3a were shown to interact with
TFIIB (59, 60), which was previously also described to bind TR (29).
Further investigations will be required to understand the precise
arrangement of all these factors and their role in transcriptional
repression.
In conclusion, the silencing subdomain SSD2 is a new targeting domain
for corepressor interaction of v-erbA, which acts only in the entire
repression domain of v-ErbA. In our model we assume that the identified
corepressor interaction regions, like the N-CoR-box in SSD1, helix 5/6
in SSD2, and the ninth heptad repeat/helix 11 in SSD3, represent
different cooperative interfaces for the previously shown nuclear
receptor interaction domains RID-I and -II of N-CoR (38, 42, 51) and
SMRT (39, 41, 61).
 |
MATERIALS AND METHODS
|
---|
Plasmids
The expression plasmids pGST-SSD1/2 (aa 362508) and pGST-SSD3
(aa 508639) were described previously (49). Expression vectors coding
for GST-ErbA wt (aa 346639), GST-SSD1 (aa 360439), and GST-SSD2 (aa
434508) were cloned by inserting the
SmaI/HindIII inserts from the corresponding
pAB-gal plasmids (8, 50) into pGEX-KG-GST. The reporter plasmids used
in this study contain four (UASx4-tk-CAT) or five tandem copies
(UASx5-SV40-LUC) of a Gal4 binding site (62) inserted in front of the
tk-CAT gene (8) or the SV40-LUC gene (63) (kindly provided by R.
Thiesen, Rostock, Germany).
The expression plasmids coding for the Gal4-DBD (aa 1147), wild-type
Gal-ErbA (aa 346639), and the control vector
gal (C) have been
described previously (8). Expression plasmids coding for the Gal-ErbA
point mutants P396R, P398R, P475R, L489R, I537R, L540R, and L544R, as
well as for the three silencing subdomains SSD13, Gal-SSD1 (hTRß aa
173265), Gal-SSD2 (aa 434508), and Gal-SSD3 (aa 508639), were
described previously (9, 49, 50). The plasmids pGEX-GST-C.SMRT (39),
for bacterial expression, and the eukaryotic expression vector for the
VP16 activation domain pCMX-VP16 (64) were kindly provided by R.
M. Evans (San Diego, CA). The plasmid
pSK+(T7)-N-CoR-C' (aa 15852453) used for
in vitro transcription/translation was cloned as a
BamHI fragment from pBKS-N-CoR (38) (kind gift from M.
G. Rosenfeld, San Diego, CA) into pSK+. For the
mammalian two-hybrid assays, the HindIII/EcoRI
fragment containing the nuclear receptor interaction region C.SMRT (aa
10731495) was cloned into pCMX-VP16, whereas the homologous region of
N-CoR-C' (aa 15852453) was cloned as a BamHI-fragment from
pSK+(T7)-N-CoR-C' into pCMX-VP16.
Cell Culture and Transfections
L-tk-, CV-1, and COS-1 cells were grown
in DMEM (Life Technologies, Inc., Gaithersburg, MD)
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 (65). For each 10-cm dish with
1 x 106 cells, we used 1.5 pmol reporter
plasmid cotransfected with 0.5 pmol expression plasmids. For the
mammalian two-hybrid experiments, equal amounts of Gal-fusion plasmid
(0.25 pmol) and VP16-fusion plasmid (0.25 pmol) were transfected
together with 1.5 pmol of reporter plasmid. Cells were harvested after
72 h, and the luciferase activity in the supernatant was
determined as described (66). For the complementation experiments,
1 x 106 L-tk- cells
were transfected with 1 pmol reporter and 0.25 pmol of each expression
plasmid using the diethylaminoethyl-Dextran method, as described
previously (50), and seeded on a 6-cm dish containing 7 ml medium and
grown 48 h before harvesting. CAT assays were performed 48 h
after transfection as described (67). Transfections were done in
duplicate and performed in at least three independent experiments.
Transfections into COS-1 cells and the preparation of whole-cell
extracts from these transfected cells were described previously (49, 68).
EMSA
EMSA 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
polydeoxyinosinic-deoxycytidylic [p(dIdC)], 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%
polyacryamide gel in 25 mM Tris, 192 mM Glycin.
For the supershift experiments, GST-C.SMRT was expressed in
Escherichia coli BL21 cells (69). Protein extracts were
prepared as described (49), bound to glutathione-Sepharose 4B beads
(Pharmacia Biotech , Piscataway, NJ) in 50
mM Tris, pH 8.0, and eluted in the presence of 10
mM reduced glutathione. GST-C.SMRT protein
extracts (600 ng) were incubated 20 min on ice with Gal-receptor fusion
proteins before the 32P-labeled UAS-DNA probe was
added.
Protein-Protein Interactions in Vitro
Protein-protein interactions were assayed essentially as
described previously (49). GST-fusion proteins were expressed in
E. coli BL21 cells. Cells were harvested by centrifugation
and resuspended in NENT buffer [100 mM NaCl, 1
mM EDTA, 20 mM Tris (pH
8.0), 0.5% NP-40]. Cells were lysed by freeze-thaw cycles, and the
cellular debris was removed by centrifugation. Glutathione-Sepharose 4B
beads (Pharmacia Biotech) were washed with NENT and 10
µl of beads were incubated with 100 µl lysate containing the
GST-fusion protein for 30 min at room temperature. Subsequently, the
supernatant was removed and the beads were incubated with 15% milk
powder in NENT for 10 min at room temperature. The beads were washed
twice with 1 ml NENT and once with 1 ml transcription washing buffer
[20 mM HEPES (pH 7.9), 60
mM NaCl, 1 mM
dithiothreitol, 6 mM MgCl2,
8% glycerine, 0.1 mM EDTA]. The volumes of the
different lysates used were adjusted to obtain similar amounts
of the fusion proteins, as analyzed by Coomassie-stained SDS-PAGE.
In vitro translated and
35S-radiolabeled proteins were obtained using a
TNT-kit (Promega Corp., Madison, WI) following the
manufacturers instructions. Crude lysate (5 µl) was incubated with
the beads in 100 µl transcription washing buffer for 1 h at room
temperature. In vitro translated luciferase was used as a
nonspecific negative control. Finally, the beads were washed (5 x
1 ml NENT buffer), and the proteins were solubilized in SDS-loading
buffer and analyzed on SDS-PAGE. Gels were treated with fluorigraphic
amplifier reagent (Amersham Pharmacia Biotech, Arlington
Heights, IL) and the bands were visualized by autoradiography.
 |
ACKNOWLEDGMENTS
|
---|
We would like to thank M. G. Rosenfeld for the pBKS-N-CoR
plasmid and R.M. Evans for the pGEX-GST-C.SMRT and pCMX-VP16
constructs. We are grateful to K. Krueger for excellent technical
assistance.
 |
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, Liege, Belgium.
This work was supported by grants from the Région Wallone (ULg
1815); the Services Fédéraux des Affaires Scientifiques,
Techniques et Culturelles (PAI P3042, P3044, and P4/30) and Actions
de Recherche Concertees (95/00193); and the Fonds National de la
Recherche Scientifique (FNRS) (-3.4537.93 and -9.4569.95). M. M.
is a Chercheur qualifié at the FNRS. This work contains part of
the Ph.D. thesis of K. Busch.
1 Present adress: Humanbiologie, Universität Frankfurt,
Siesmayerstraße 70, D-60323 Frankfurt, Germany. 
Received for publication April 9, 1999.
Revision received September 21, 1999.
Accepted for publication October 21, 1999.
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