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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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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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) 389–639 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 173–265 and 265–461 or 362–508 and 508–639, 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, SSD1–SSD3, which are involved in the silencing function of v-erbA.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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2 (aa 207–217) (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.
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). 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.
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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 339–368) (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.
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), 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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3 Affect the Silencing Function
of rTRß). 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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) 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 1–12) or GAL-TRß (lanes 14–18) 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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), which would restore repression when combined. We
showed previously that different GAL-v-erbA chimeras are able to
heterodimerize via the GAL4 region aa 1–147 (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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. 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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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 173–265) 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 rTR1 (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.
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) 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 203–230 (part of SSD1) abolishes interaction with N-CoR, while deletion of either aa 260–335 (SSD2) or aa 335–456 (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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The expression plasmids coding for the GAL4 DBD (aa 1–147), GAL-erb
346, GAL-erb 362–508 (SSD1/2), GAL-erb 508–639 (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 434–508
(SSD2) was obtained by cutting pGAL-erb 362–508 with PvuII
and EcoRV and religating, thereby destroying these
restriction sites. The pABGAL94,
pABGAL94-hTRß (aa 173–461), and
pABGAL94-hTRß 173–265 (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 172–456)-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 346–639 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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 P3–042 and PAI P3–044, Fonds National de la Recherche Scientifique (FNRS)-3.4537.93 and 9.4569.95, and the Actions de Recherche concertée-95/00–193. 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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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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can target TATA-binding protein for
transcriptional repression. Mol Cell Biol 16:281–287[Abstract]
4 activation domain of the thyroid hormone receptor is
required for release of a putative corepressor(s) necessary for
transcriptional silencing. Mol Cell Biol 15:76–86[Abstract]
. Proc Natl Acad Sci USA 90:10668–10672. Nature 375:377–382[CrossRef][Medline]
ligand-binding domain
bound to all-trans retinoic acid. Nature 378:681–689[CrossRef][Medline]
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B. Altincicek, S. P. Tenbaum, U. Dressel, D. Thormeyer, R. Renkawitz, and A. Baniahmad Interaction of the Corepressor Alien with DAX-1 Is Abrogated by Mutations of DAX-1 Involved in Adrenal Hypoplasia Congenita J. Biol. Chem., March 10, 2000; 275(11): 7662 - 7667. [Abstract] [Full Text] [PDF]
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K. Busch, B. Martin, A. Baniahmad, J. A. Martial, R. Renkawitz, and M. Muller Silencing Subdomains of v-ErbA Interact Cooperatively with Corepressors: Involvement of Helices 5/6 Mol. Endocrinol., February 1, 2000; 14(2): 201 - 211. [Abstract] [Full Text]
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T. Cook, B. Gebelein, M. Belal, K. Mesa, and R. Urrutia Three Conserved Transcriptional Repressor Domains Are a Defining Feature of the TIEG Subfamily of Sp1-like Zinc Finger Proteins J. Biol. Chem., October 8, 1999; 274(41): 29500 - 29504. [Abstract] [Full Text] [PDF]
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U. Dressel, D. Thormeyer, B. Altincicek, A. Paululat, M. Eggert, S. Schneider, S. P. Tenbaum, R. Renkawitz, and A. Baniahmad Alien, a Highly Conserved Protein with Characteristics of a Corepressor for Members of the Nuclear Hormone Receptor Superfamily Mol. Cell. Biol., May 1, 1999; 19(5): 3383 - 3394. [Abstract] [Full Text] [PDF]
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 R. Nicol, G. Zheng, P. Sutrave, D. N. Foster, and E. Stavnezer Association of Specific DNA Binding and Transcriptional Repression with the Transforming and Myogenic Activities of c-Ski Cell Growth Differ., April 1, 1999; 10(4): 243 - 254. [Abstract] [Full Text]
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P. Bartunek and M. Zenke Retinoid X Receptor and c-erbA/Thyroid Hormone Receptor Regulate Erythroid Cell Growth and Differentiation Mol. Endocrinol., September 1, 1998; 12(9): 1269 - 1279. [Abstract] [Full Text]
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 T. Tagami, P. Kopp, W. Johnson, O. K. Arseven, and J. L. Jameson The Thyroid Hormone Receptor Variant {alpha}2 Is a Weak Antagonist because It Is Deficient in Interactions with Nuclear Receptor Corepressors Endocrinology, May 1, 1998; 139(5): 2535 - 2544. [Abstract] [Full Text] [PDF]
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G. Elberg, J. M. Gimble, and S. Y. Tsai Modulation of the Murine Peroxisome Proliferator-activated Receptor gamma 2 Promoter Activity by CCAAT/Enhancer-binding Proteins J. Biol. Chem., September 1, 2000; 275(36): 27815 - 27822. [Abstract] [Full Text] [PDF]
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