The Adenovirus E1A Protein Is a Potent Coactivator for Thyroid Hormone Receptors
Gunilla M. Wahlström1,
Björn Vennström and
Maria Bondesson Bolin1
Department of Cell and Molecular Biology Medical Nobel
Institute Karolinska Institutet S-171 77 Stockholm, Sweden
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ABSTRACT
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The thyroid hormone receptors interact with
several different cofactors when activating transciption. In this
study, we show that the adenovirus E1A oncoprotein functions as a
strong coactivator for the thyroid hormone receptor (TR), and that TR
and E1A synergistically activate transcription via direct (DR4) or
palindromic (IR0) hormone-responsive sites. Cotransfection experiments
using different isoforms of the chicken TR and E1A show synergistic,
ligand-enhanced transactivation. This transactivation is accomplished
through a direct, ligand-independent interaction between TR and E1A.
The interaction domains in TR are localized to the DNA-binding domain
and to the carboxy-terminal part of the ligand-binding domain. In E1A,
the regions of interactions are localized to the conserved regions 1
and 3. Both of these domains in E1A are required for a 40-fold
enhancement of TR-mediated activation in transfection experiments.
Taken together, we show that E1A strongly enhances transcriptional
activation, which suggests that it serves as a bridging factor between
the receptor and other components of the transcription machinery.
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INTRODUCTION
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Thyroid hormones play a fundamental role in the regulation of
normal cell function and differentiation by interacting with
intracellular thyroid hormone receptors (TRs). These receptors belong
to a family of nuclear hormone receptors that includes receptors for
steroids as well as those for retinoic acid (RAR),
9-cis-retinoic acid (RXR), and vitamin D3. TRs
interact with specific thyroid response elements (TREs) within
the promoters of target genes and thereby activate or repress
transcription. TREs consist of hexameric motifs (half-sites) arranged
as inverted, everted, or direct repeats separated by a specific number
of nucleotides (1, 2, 3). TR preferentially binds as a heterodimer with
RXR to a direct repeat spaced by four nucleotides (DR4). In the same
manner, heterodimers of RXR/RAR activate transcription via elements of
the DR5 and DR2 type (4, 5, 6, 7). Corepressors, such as N-CoR and SMRT, have
been shown to interact with TR in its unliganded state. These
corepressors bind to the hinge region in between the DNA- and
ligand-binding domain (DBD and LBD) in the absence of T3
(8, 9, 10, 11). Upon binding of T3, the corepressors are released
and coactivators are recruited. The coactivators, such as SRC-1 and
CBP/p300, have been suggested to preferentially bind to an activation
domain (AF2) in the C-terminal part of TR (12).
The adenovirus E1A gene products have been shown to be
multifunctional, playing central roles in the control of viral and
cellular gene expression and transformation. Two structurally
homologous proteins, of 243 and 289 amino acids (243R and 289R), are
the major products encoded by E1A. E1A-289R contains three, among
adenoviruses, highly conserved regions, designated conserved region
(CR) 1, 2, and 3. These regions are important for the multiple
activities ascribed to E1A. The major E1A transcription activation
domain is contained within CR3 and is unique to E1A-289R. This domain
participates in transcriptional activation by physically interacting
with both basal and upstream binding transcription factors (13).
E1A has been shown to directly interact with retinoic acid receptor ß
(RARß) and thereby function as a cofactor for activation of the
RARß2 promoter (14). In cotransfection experiments, efficient
activation of RARß2 by E1A and RARß also requires the addition of
the TATA binding protein (TBP) (15). Furthermore, E1A has been shown to
directly bind to TBP (16, 17, 18). These results suggest that E1A-289R
mediates transcriptional activation by providing a physical bridge
between TBP and RARß.
Retinoids play an important role in differentiation of embryonic
carcinoma (EC) cells. Undifferentiated murine P19 EC cells
differentiate into neurons, astrocytes, and fibroblast-like cells after
addition of retinoic acid (RA) (19). An early and essential step in the
differentiation process is the activation of the RARß2-promoter.
However, activation of the RARß2-promoter in P19 cells does not
require the viral E1A protein; instead, an endogenous E1A-like activity
(E1A-LA) is used as a bridging factor between RARß and TBP (15). The
existence of E1A-LA was first suggested when nonviable adenovirus E1A
mutants were able to grow in undifferentiated EC cells (20).
Since RAR uses E1A/E1A-LA as a cofactor, we investigated whether
TR and E1A/E1A-LA also cooperate in transcriptional activation. In this
paper, we show that E1A and TR synergistically activate transcription
of promoters containing TR recognition sites. This activation is seen
for three different TR recognition elements [DR4>IR0>
-myosin
heavy chain (
MHC)] and is mediated by all the isoforms of chicken
TRs tested.
The synergistic activation is accomplished through a direct interaction
between TR and E1A. We have mapped the interacting domains of E1A to
CR1 and CR3, both of which are also required for efficient enhancement
of TR-mediated transcription. E1A interacts with the LBD and DBD of TR.
The results presented here indicate that a complex containing TR and
E1A is formed on a promoter containing a DR4 type of
cis-acting element and that this complex is of
sufficient importance to gain efficient transcriptional
activation.
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RESULTS
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TR and E1A Synergistically Activate Transcription of Promoters
Containing TREs
Since E1A interacts with several DNA-bound transcription
factors that are localized to promoter regions, we tested whether E1A
had the potential to activate TR-mediated transcription. For this,
plasmids expressing either E1A or chicken TR
-p46 were cotransfected
along with a chloramphenicol acetyltransferase (CAT)-reporter construct
(pBLCAT-TRE) containing a TR-responsive element into human chorion
carcinoma cells (JEG). These cells contain low endogenous TR (Fig. 1A
, right) (21) and no
endogenous E1A-like activity. We first studied the effects of E1A on
thyroid-mediated transcription via a direct repeat separated by four
nucleotides, since this is the the most commonly found TRE. Figure 1A
shows that E1A increased the level of TR-mediated transcription at
least 40-fold. As shown previously, TR alone activated transcription
3-fold via the single DR4 element in the presence of T3
(21). It is noteworthy that the basal activity in the absence of
T3 was also several fold increased by E1A, although not to
the same extent as when the ligand was added, 11- and 40-fold,
respectively (Fig. 1
). When E1A was transfected without exogenous TR, a
small amount of T3-induced transcription was seen on all
elements tested (Fig. 1
). This is most likely explained by interaction
of E1A with endogenous expressed TR in the JEG cells (21).

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Figure 1. E1A Potentiates TR-Mediated Transactivation
Three different response elements, cloned into the pBLCAT2 reporter
vector, were tested for transcriptional activation in JEG cells by the
TR and E1A proteins. Panel A, left, shows the relative
TR-mediated activation on the thyroid DNA element DR4 in the presence
or absence of RXR and E1A, as indicated. To the right,
the endogenous levels of TR in JEG and HeLa cells are shown by Western
blot anlysis. The numbers refer to the protein sizes.
Panel B shows the relative TR-mediated activation on the natural
response element rat MHC and the palindromic element IR0 in the
presence or absence of E1A, as indicated. pBLCAT2 represents an
reporter vector containing no binding sites for TR. Addition of
T3 to the samples is presented by striped
bars. The organization of the half-sites in the response
element are indicated at the top of the graphs.
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To investigate the activation of TR and E1A on a DR4 type of element
located in a natural gene, we analyzed the level of activation of a
reporter construct that contained an element from the promoter region
of the
MHC gene, which is normally expressed in heart muscle cells.
This element consists of two half-sites oriented as direct (DR4) and
palindromic (IR2) repeats separated by four and two nucleotides,
respectively. E1A was shown to enhance the TR-mediated activation
approximately 40-fold both via the heart-specific element and via a
palindromic (IR0) TR-responsive element (Fig. 1B
).
Neither TR nor E1A could activate transcription of the corresponding
control reporter plasmid containing only the RSV (Rous sarcoma virus)
TATA box in front of the CAT-gene (pBLCAT), thus verifying that the
activation obtained on the various TREs was TR and DNA specific.
The ubiquitously expressed RXR is known to heterodimerize with TR and
thereby increase transactivation. However, exogenous RXR had no
additional effect on the E1A-enhanced TR-mediated activation in our
cotransfection studies (Fig. 1A
). Endogenous RXRs are present in low
amounts in JEG cells as shown by Wahlström et al.
(21).
During an adenovirus infection E1A efficiently activates transcription
of the adenoviral E4 and E1A promoters. Therefore, the influence
of TR on E1A- regulated transcription was assayed using these
promoters as reporter constructs in cotransfections. Addition of
TR had no effect on the activation by E1A (data not shown).
Different Isoforms of TR Cooperate with E1A in Transactivation
Several variants of the chicken thyroid hormone receptor
that are expressed in different tissues and at different developmental
times have been described (22, 23, 24, 25). The receptors differ in their N
termini, regions that have been shown to contain putative
phosphorylation sites and nuclear localization signals (26). We
therefore investigated whether the synergism between E1A and cTR
was
TR isoform specific. Cotransfection experiments were done in the
presence of either TR
-p46, TR
-p40, TRß0, or TRß2 in the
absence or presence of E1A and T3 hormone (Fig. 2
). Figure 2B
shows that the synergistic
effect was obtained with all the chicken thyroid hormone receptors,
tested on a DR4 type of DNA element. These different receptors have
previously been shown to bind equally well to DR4 half-sites in
vitro (Ref. 3 and data not shown). Interestingly, the N-terminal
shorter variants (TR
-p40 and TRß0) gave a slightly lower level of
activation.

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Figure 2. Synergistic Effect with Different Isoforms of
Chicken TR
Panel A shows the schematic representation of the various chicken TRs
tested in transactivation studies. TR -p40 is an N-terminal shorter
variant lacking the two phosphorylation sites present in the longer
variant p-46. In addition to the -receptors, two TRß receptors and
the E1A early protein with three conserved regions are shown. Panel B
shows the relative transcriptional activation obtained with the various
TRs in the presence or absence of E1A and T3. Addition of
T3 to the samples is indicated by stripes.
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Efficient Activation Requires CR1 and CR3 in E1A
The conserved regions in E1A have been shown to exert
different functions in transcription. CR1 is of importance in the
activation of the proliferating cell nuclear antigen (PCNA) promoter,
while CR3 activates for example the adenoviral E4 promoter (27). Both
CR3 and CR1 separately activate transcription as Gal4 fusion proteins
on promoters with Gal4-binding sites (28).
To investigate how the CR1, CR2, and CR3 domains of the E1A protein
exert their effects on TR-mediated activation, we transfected plasmids
encoding E1A deletion mutants along with TR into JEG cells. Figure 3
shows that the
CR3 mutant activated
transcription to 30% of the wild-type (wt) E1A protein, indicating
that the E1A-enhanced activation is mainly dependent on this domain. A
mutant lacking CR1 activated TR-regulated transcription to 50% of
E1Awt, and the mutant lacking CR2 enhanced the transactivation to the
same extent as E1Awt, whereas a double mutant of CR1 and CR3 gave, as
expected, the lowest transactivation. Taken together, preferentially
CR3, but also CR1, contributes to E1A enhancement of TR-mediated
transactivation in JEG cells.

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Figure 3. Transcriptional Activation of E1A Deletion Mutants
The transcriptional activation by TR and the full-length E1A protein
(E1Awt) was compared with various E1A deletion mutants in
cotransfection experiments in JEG cells. The relative CAT activity
obtained by constructs deleted in one of the three conserved regions is
shown. Addition of T3 to the samples is as indicated.
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TR and E1A Interact Directly; Domains of Interaction in E1A
E1A has been shown to directly interact with several upstream
binding transcription factors, such as RARß and ATF-2 (14, 29, 30).
To study a potential direct interaction between E1A and TR,
35S-labeled in vitro translated E1A proteins
were added to column-bound glutathione-S-transferase
(GST)-TR fusion proteins. The retained E1A proteins were analyzed using
SDS-PAGE. Figure 4B
shows that the wt E1A
protein strongly bound to GST-TR, both in the presence and absence of
T3. As expected, in vitro translated RXR was
also bound by GST-TR. The E1A carboxy-terminal binding protein, CtBP
(31), here used as a negative control, did not bind efficently to
GST-TR compared with input levels. The binding of the E1A 289-amino
acid protein to GST-TR was strong since E1A still bound efficiently in
high-salt buffer (500 mM NaCl, data not shown).

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Figure 4. Direct Interaction between E1A and GST-TR
Panel A shows a schematic representation of the E1A-truncated or
-deleted proteins tested in GST-TR binding experiments. Panel B shows
retention of 35S-labeled E1Awt, RXR, RAR, and CtBP proteins
on columns with GST-TR, GST, or Sepharose beads alone. In panels C and
D, E1A proteins with truncations or internal deletions are retained by
GST-TR in the presence or absence of T3, as indicated.
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To map the TR-interacting domains in E1A, the E1A protein was truncated
at amino acids 221, 130, and 89. All three truncated proteins were
retained by GST-TR, although the proteins truncated at amino acids 130
and 89 bound to a lesser extent than E1A 1221 and E1A wt (Fig. 4C
).
As the E1A 1130 and E1A 189 proteins both lack CR3, the result
suggests that CR3 is important for efficient binding to TR.
An E1A mutant containing both CR1 and CR3, but lacking the
carboxy-terminal amino acids 193245, was retained by GST-TR at the
same efficiency as the wt E1A protein (Fig. 4C
). This deletion spans
the AR1 region that is required for activation of the E4F transcription
factor (32).
The E1A mutant with an internal deletion of CR1 bound to GST-TR,
although to a lesser extent than the wt protein (Fig. 4D
). The mutant
with an internal deletion of CR3 also weakly bound to GST-TR (Fig. 4D
).
The double mutant, lacking both CR1 and CR3, was totally deficient in
binding GST-TR. These results demonstrate that the CR1 and CR3 domains
of E1A cooperate to form a stable complex with TR.
The bacterially expressed GST-TR bound to the DR4 recognition sequence,
as determined by gel-shift analysis. The GST-TR protein was also
recognized by an
-TR antibody in Western blots (data not shown).
This indicates that expression of TR in bacteria does not destroy its
conformation and function.
Domains of Interaction in TR
In a set of reciprocal experiments, different GST-E1A fusion
proteins were used to assay binding of in vitro translated
TR proteins. Both the TR
variants, p46 and p40, bound to GST-E1A
containing either CR1 or CR3 (GST-E1A
CR3 and GST-E1A76289) (Fig. 5B
). None of the TR proteins bound to the
GST-E1ACt mutant, which contains amino acids 200243 of the
carboxy-terminal domain of E1A (Fig. 5B
). This is in agreement with the
results presented above in Fig. 4
. RAR
bound to GST-E1A proteins
containing CR1 or CR3, whereas RXR did not associate with any of the
GST-E1A proteins. CtBP, now used as a positive control, was retained by
all E1A proteins, as they all have the carboxy-terminal domain
present.

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Figure 5. Direct Interaction between TR and GST-E1A
Panel A shows a schematic illustration of GST-E1A proteins and TR
isoforms and mutant proteins. Panel B shows binding of
35S-labeled in vitro translated p46 and p40
TR isoforms, as well as RAR, RXR, and CtBP to GST-E1A 76289
(containing CR3), GSTE1A CR3 (containing CR1), and GST-E1ACt
(containing amino acids 200243 of E1A). Panels C and D show retention
of PCR-amplified, in vitro translated TR mutants on the
various GST-E1A fusion protein columns.
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To further map the domains of interaction in TR, a number of TR mutants
were synthesized and in vitro translated using PCR primers
(Fig. 5A
). The TR mutant encoding amino acids 49408 bound efficiently
to GST-E1A 76289 and GST-E1A
CR3. This TR mutant contains both the
DBD and LBD. The p46 122408 mutant, containing the LBD, also bound to
GST-E1A 76289 and GST-E1A
CR3, although not as strongly as p46
49408 (Fig. 5C
). The mutants p46 1256, p46 49256, and p46
49118, all containing the DBD, bound weakly to GST-E1A 76289, but
not to GST-E1A
CR3 (Fig. 5
, C and D). This suggests that there is a
weak interaction between TR DBD and E1A CR3.
A clear interaction was found between p46 257408 and GSTE1A 76289
(Fig. 5D
). This result indicates that the main interactions are made
between the carboxy-terminal part of the LBD in TR and CR3 in E1A. The
p46 1118 and p46 122256 mutants were not retained by any of the
GST-E1A proteins.
The shorter amino terminus TR variant, p40, bound more efficiently to
GST-E1A than the full-length variant, p46 (Fig. 5B
). Similarly, the
mutants p46 49256 and p46 49118 bound GST-E1A better than the
corresponding amino termini containing mutants p46 1256 and p46
1118, respectively (Fig. 5
, C and D). In all these cases, addition of
the amino terminus to the TR mutant weakened its ability to bind to
E1A. This indicates that the amino terminus has a negative influence on
the interaction between TR and E1A.
In summary, the CR3 in E1A interacts with both the carboxy terminus and
the DBD of TR, while CR1 in E1A makes contact with TR somewhere in the
LBD. The strongest binding is seen when both the DBD and LBD are
present in TR. In contrast, the amino terminus of TR has a negative
effect on binding to E1A.
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DISCUSSION
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On the basis of transfection experiments and binding studies, we
here show that E1A directly interacts with TR and that E1A enhances
TR-dependent activation of promoters driven via TREs. E1A has
previously been shown to directly bind to the basal transcription
factor TBP (16, 17, 18). Based on these and our results, we propose that
E1A may simultaneously interact with TR and TBP, thereby functioning as
a supplementary bridge between TR and the basal transcription
machinery.
E1A has been shown, in an analogous way, to interact with RARß, and
thereby activate transcription of the RARß2 gene. The direct
interaction between E1A and RARß was shown to depend on CR3 in E1A
and the LBD including AF2 in RARß (14). In our study, two regions in
E1A, CR1 and CR3, bound to TR, with stronger affinity for CR3. These
two regions appear to cooperate to obtain an efficient binding. Our
results from the binding experiments are in agreement with our
cotransfection experiments, which demonstrated that a mutant containing
CR3 gave 50% and a mutant containing CR1 gave 30% of the activation
normally observed with E1Awt. In the study of Folkers and van der Saag
(14), RARß-mediated transactivation was dependent on CR3. The
observed differences between their study and the results presented in
this paper may be explained by different requirements of E1A domains
for TR- and RARß-induced transactivation and by the use of different
cell lines.
The region in TR required for interaction with E1A is primarly the
carboxy-terminal portion of the LBD, including AF-2. This is in
agreement with the results of Folkers and van der Saag, as they have
suggested that the region required for E1A binding in RARß is in the
LBD. In addition, we also see a minor binding between E1A and the DBD
of TR. Thus, we suggest that two separate regions in E1A interact with
two separate regions in TR.
Several TR-interacting cofactors have been identified recently. Two of
these cofactors, p300/CBP and SRC-1, have been found to possess
intrinsic histone acetyltransferase activities; consequently, it was
suggested that these cofactors may remodel transcriptionally
repressed chromatin to make the promoter accessible for general
transcription factors (33, 34, 35, 36). Other cofactors are thought to enhance
and stabilize the assembly of the preinitiation complex. The latter is
likely one function of E1A, since it interacts with both upstream and
basal transcription factors. On the other hand, recent reports have
suggested that E1A may inhibit p300/CBP histone acetyltransferase
activity and also the transcription mediated by nuclear hormone
receptors. (37, 38). However, the histone acetyltransferase activity of
p300/CBP is necessary for transcriptional activation by the
transcription factor Stat1
, but not for the nuclear retinoid
receptor RAR, (39, 40). Instead, E1A inhibits RAR transactivation by
preventing the association of CBP with nuclear receptor coactivators
(40). Taken together, the recent reports suggest that E1A activates
transcription of nuclear receptors when E1A interacts with the receptor
and thereby localizes to the promoter. Not juxtaposed to the promoter,
E1A would instead repress nuclear receptor-mediated transcription
through disassembling receptor-coactivator complexes. The ability of
E1A to function as a coactivator or a corepressor may be influenced by
the promoter context and by the composition of nuclear receptor dimers
as well as by cell type-specific cofactors.
What is the biological significance of an interaction between a nuclear
receptor and the adenovirus E1A oncoprotein? As these two proteins can
only coexist in adenovirus-infected cells, one function could be
that the virus uses TR for modifying E1A-regulated gene transcription.
However, since TR did not affect E1A-induced activation of the E1A- and
E4-promoters, this is not likely to be the case. Instead, we
hypothesize that the adenovirus E1A oncoprotein mimics the function of
E1A-LA of undifferentiated EC cells, and that the interaction between
TR and E1A-LA would be confined to early, pluripotent cells. E1A-LA
would, in such a model, enhance the activating potential of TR on
differentiation-specific genes. Our preliminary results suggest that
activation of TR-driven transcription through DR4 sites is not further
enhanced by the addition of viral E1A in undifferentiated P19 cells
(data not shown). This indicates that E1A-LA could cooperate with TR to
induce efficient transactivation in P19 cells. Furthermore,
T3 treatment of P19 cells induces the cells to
differentiate into cardiac myocytes (41). The authors suggest that this
differentiation is accompanied by preferential binding of as yet
undefined factors to DR4-sites (42). Whether or not these factors are
TR and E1A-LA remains to be elucidated.
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MATERIALS AND METHODS
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Plasmid Constructs
One copy of the double-stranded DR4 oligonucleotide with the
four- nucleotide spacer sequence CTTC was inserted into the
HindIII site of the pBLCAT2 vector. The MHC and
IR0 elements were cloned in the same way (21). The chicken TR
1 (p40
and p46) and TRß0 and ß2 receptors were cloned into the pSG5
expression vector (3, 43). The plasmid pML005, used for expressing
E1Awt in transfections, contains nucleotides 11773 of genomic
adenovirus type 2. The CR1, CR2, and CR3 mutants of pML005 has been
described previously (44). GSTE1A 76289 and GSTE1A
CR3 (GSTE1A12S)
have been described previously (45). GST-TR was cloned by inserting the
EcoRI fragment of pSG65-p46 into the EcoRI site
of the pGEX1lambdaT vector (Pharmacia Biotech, Piscataway,
NJ). Gst-E1ACt (aa 200243) and pcDNA3-CtBP have been described
previously (31, 46). pML00512S, pML00513S (47), and pSG5p46 were used
as templates for making in vitro translated E1A and TR
proteins, respectively.
Transfections
Human chorion carcinoma cells (JEG) were plated at a density of
2 x 105 per 3-cm dish in DMEM (Biological
Industries) supplemented with 8% FCS. One day after plating,
the medium was replaced with DMEM containing 8% calf serum depleted of
T3 and T4 by ion exchange resin (48).
Approximately 2 h later the cells were cotransfected with
expression vectors encoding 100200 ng of different chicken TRs, E1A,
or E1A mutants plus 500 ng of reporter constructs containing a TRE. The
cells were maintained in the presence or absence of 1230
nM T3, harvested 24 h after hormone
treatment, and assayed for chloramphenicol acetyltransferase activity.
Quantifications were done with a Molecular Dynamics, Inc.
(Sunnyvale, CA) phosphorimager (49). All transfections were repeated at
least three times with similar results. Duplicate sample points were
used in each experiment and varied by less than 15%.
Protein Binding Analysis
In vitro GST-fusion proteins were produced in
Escherichia coli and purified with gluthathione agarose
beads (Current Protocols in Molecular Biology). Protein
concentrations were estimated on a Coomassie-stained SDS-polyacrylamide
gel. Approximately equal amounts of GST fusion proteins were mixed with
510 µl of [35S]methionine-labeled in vitro
synthesized proteins (TNT-coupled reticulocyte lysate systems,
Promega Corp., Madison, WI). The proteins were incubated
rotating at 4 C for 3 h in binding buffer (250500 mM
NaCl, 50 mM HEPES, pH 7.9, 0.5 mM EDTA, 0.1%
NP40, 1 mM dithiothreitol, and 0.2 mM
phenylmethylsulfonylfluoride). Beads were washed four times in
binding buffer, and bound proteins were separated on a polyacrylamide
gel and visualized by autoradiography. In Figs. 4D
and 5
, C and D, the
35S-labeled proteins were synthesized using PCR-amplified
DNA templates. The 5'-PCR primers contained the sequence for a T7 RNA
polymerase start site.
Western Blot
Equal amounts of crude extracts made from human chorion
carcinoma (JEG) and human cervical carcinoma (HeLa) cells were run on a
10% denatured gel. Separated proteins were transferred overnight to
enhanced chemiluminescence (ECL) nitrocellular membranes, which were
then hybridized to a primary rabbit polyclonal antibody directed
against TR diluted 1:1000 (FL-408, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After incubation with a secondary
peroxidase-conjugated goat antirabbit antibody diluted 1:1000
(DAKO Corp., Carpinteria, CA), the membranes were washed
and the migrated proteins were detected with the ECL system
(Amersham, Arlington Heights, IL).
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ACKNOWLEDGMENTS
|
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We are grateful to Dr. K. Sollerbrandt for the gift of
plasmids.
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FOOTNOTES
|
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Address requests for reprints to: Maria Bondesson Bolin, Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, S-171 77 Stockholm, Sweden.
This project was funded by grants from Cancerfonden, the Lars Hierta
Foundation, the Magnus Bergvalls Foundation, Human Fronties, Karolinska
Institutet, and the Swedish Society for Medical Research. M.B-B. was
also supported by the Swedish Natural Science Research Council.
1 These authors have contributed equally to the work. 
Received for publication December 17, 1998.
Revision received April 12, 1999.
Accepted for publication April 16, 1999.
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REFERENCES
|
---|
-
Umesono K, Murakami KK, Thompson CC, Evans RM 1991 Direct
repeats as selective response elements for the thyroid hormone,
retinoic acid, and vitamin D3 receptors. Cell 65:12551266[Medline]
-
Näär AM, Boutin JM, Lipkin SM, Yu VC, Holloway
JM, Glass CK, Rosenfeld MG 1991 The orientation and spacing of core
DNA-binding motifs dictate selective transcriptional response to three
nuclear receptors. Cell 65:12671279[Medline]
-
Wahlström GM, Sjöberg M, Andersson M,
Nordström K, Vennström B 1992 Binding characteristics of
the thyroid hormone receptor homo- and heterodimers to consensus AGGTCA
repeat motifs. Mol Endocrinol 6:10131022[Abstract]
-
Kliewer SA, Umesono K, Mangelsdorf DJ, Evans RM 1992 Retinoid
X receptor interacts with nuclear receptors in retinoic acid, thyroid
hormone and vitamin D3 signalling. Nature 355:446449[CrossRef][Medline]
-
Yu VC, Delsert C, Andersen B, Holloway JM, Devary OV,
Näär AM, Kim SY, Boutin J-M, Glass CK, Rosenfeld MG 1991 RXRb: a coregulator that enhances binding of retinoic acid, thyroid
hormone, and vitamin D receptors to their cognate response element.
Cell 67:12511266[Medline]
-
Zhang X-k, Hoffmann B, Tran PB, Graupner G, Pfahl M 1992 Retinoid X receptor is an auxiliary protein for thyroid hormone and
retinoic acid receptors. Nature 355:441446[CrossRef][Medline]
-
Leid M, Kastner P, Lyons R, Nakshatri H, Saunders M,
Zacharezski T, Chen J-Y, Staub A, Garnier J-M, Mader S, Chambon P 1992 Purification, cloning, and RXR identity of the HeLa cell factor with
which RAR or TR heterodimerizes to bind target sequences efficiently.
Cell 66:377395
-
Hörlein AJ, Näär AM, Heinzel T, Torchia J,
Gloss B, Kurakowa R, Ryan A, Kamei Y, Söderström M, Glass
CK, Rosenfeld MG 1995 Ligand-independent repression by the thyroid
hormone receptor mediated by a nuclear receptor co-repressor. Nature 377:397404[CrossRef][Medline]
-
Kurakowa R, Söderström M, Hörlein A,
Halachmi S, Brown M, Rosenfeld MG, Glass CK 1995 Polarity-specific
activities of retinoic acid receptors determined by a co-repressor.
Nature 377:451454[CrossRef][Medline]
-
Chen JD, Evans RM 1995 A transcriptional co-repressor that
interacts with nuclear hormone receptor. Nature 377:454459[CrossRef][Medline]
-
Baniahmad A, Leng X, Burris TP, Tsai SY, Tsai M-J, OMalley
BW 1995 The T4 actvation domain of the thyroid hormone receptor is
required for release of a putative corepressor(s) necessary for
transcriptional silencing. Mol Cell Biol 15:7686[Abstract]
-
Koenig RJ 1998 Thyroid hormone receptor coactivators and
corepressors. Thyroid 8:703713[Medline]
-
Akusjärvi G 1993 Proteins with transcription regulatory
properties encoded by human adenoviruses. Trends Microbiol 1:163170[CrossRef][Medline]
-
Folkers GE, van der Saag PT 1995 Adenovirus E1A functions as a
cofactor for retinoic acid receptor ß (RARß) through direct
interaction with RARß. Mol Cell Biol 15:58685878[Abstract]
-
Berkenstam A, Ruiz MM, Barettino D, Horikoshi M, Stunnenberg
HG 1992 Cooperativity in transactivation between retinoic acid receptor
and TFIID requires an activity analogous to E1A. Cell 69:401412[Medline]
-
Meyer M, Sonntag-Buck V, Keaveney M, Stunnenberg HG 1996 Retinoid-dependent transcription: the RAR/RXR-TBP-EIA/EIA-LA
connection. Biochem Soc Symp 62:97109[Medline]
-
Lee WS, Kao CC, Bryant GO, Liu X, Berk AJ 1991 Adenovirus E1A
activation domain binds the basic repeat in the TATA box transcription
factor. Cell 67:365376[Medline]
-
Horikoshi N, Maguire K, Kralli A, Maldonado E, Reinberg D,
Weinmann R 1991 Direct interaction between adenovirus E1A protein and
the TATA box binding transcription factor IID. Proc Natl Acad Sci USA 88:51245128[Abstract]
-
Rudnicki MA, McBurney MW 1987 Cell culture methods and
induction of differentiation of embryonal carcinoma cell lines. In:
Robertson EJ (ed) Teratocarcinomas and Embryonic Stem Cells: A
Practical Approach. IRL Press, Oxford, Washington DC, pp 1949
-
Imperiale MJ, Kao HT, Feldman LT, Nevins JR, Strickland S 1984 Common control of the heat shock gene and early adenovirus genes:
evidence for a cellular E1A-like activity. Mol Cell Biol 4:867874[Medline]
-
Wahlström GM, Harbers M, Vennström B 1996 The
oncoprotein P75gag-v-erbA represses thyroid hormone induced
transcription only via response elements containing palindromic
half-sites. Oncogene 13:843852[Medline]
-
Sap J, Munoz A, Damm K, Goldberg Y, Ghysdael J, Leutz A, Beug
H, Vennstrom B 1986 The c-erb-A protein is a high-affinity receptor for
thyroid hormone. Nature 324:635640[Medline]
-
Bigler J, Eisenman RN 1988 c-erbA encodes multiple proteins in
chicken erythroid cells. Mol Cell Biol 8:41554161[Medline]
-
Forrest D, Sjöberg M, Vennström B 1990 Contrasting
developmental and tissue-specific expression of
and ß thyroid
hormone receptor genes. EMBO J 9:15191528[Abstract]
-
Showers MO, Darling DS, Kieffer GD, Chin WW 1991 Isolation and
characterization of a cDNA encoding a chicken ß thyroid hormone
receptor. DNA Cell Biol 10:211221[Medline]
-
Lee Y, Mahdavi V 1993 The D domain of the thyroid hormone
receptor a1 specifies positive and negative transcriptional regulation
functions. J Biol Chem 268:20212028[Abstract/Free Full Text]
-
Kannabiran C, Morris GF, Labrie C, Mathews MB 1993 The
adenovirus E1A12S product displays functional redundancy in activating
the human proliferating cell nuclear antigen promoter. J Virol 67:507515[Abstract]
-
Bondesson M, Mannervik M, Akusjärvi G, Svensson C 1994 An adenovirus E1A transcriptional repressor domain functions as an
activator when tethered to a promoter. Nucleic Acids Res 22:30533060[Abstract]
-
Liu F, Green MR 1990 A specific member of the ATF
transcription factor family can mediate transcription activation
by the adenovirus E1a protein. Cell 61:12171224[Medline]
-
Liu F, Green MR 1994 Promoter targeting by adenovirus E1A
through interaction with different cellular DNA-binding domains. Nature 368:520525[CrossRef][Medline]
-
Sollerbrant K, Chinnadurai G, Svensson C 1996 The CtBP binding
domain in the adenovirus E1A protein controls CR1-dependent
transactivation. Nucleic Acids Res 24:25782584[Abstract/Free Full Text]
-
Bondesson M, Svensson C, Linder S, Akusjärvi G 1992 The
carboxy-terminal exon of the adenovirus E1A protein is required for
E4F-dependent transcription activation. EMBO J 11:33473354[Abstract]
-
Spencer TE, Jenster G, Burcin MM, Allis CD, Zhou J, Mizzen CA,
McKenna NJ, Onate SA, Tsai SY, Tsai MJ, OMalley BW 1997 Steroid
receptor coactivator-1 is a histone acetyltransferase. Nature 389:194198[CrossRef][Medline]
-
Bannister AJ, Kouzarides T 1996 The CBP co-activator is a
histone acetyltransferase. Nature 384:641643[CrossRef][Medline]
-
Ogryzko VV, Schiltz RL, Russanova V, Howard BH, Nakatani Y 1996 The transcriptional coactivators p300 and CBP are histone
acetyltransferases. Cell 87:953959[Medline]
-
Jenster G, Spencer TE, Burcin MM, Tsai SY, Tsai MJ, OMalley
BW 1997 Steroid receptor induction of gene transcription: a two-step
model. Proc Natl Acad Sci USA 94:78797884[Abstract/Free Full Text]
-
Chakravarti D, Ogryzko V, Kao H-Y, Nash A, Chen H, Nakatani Y,
Evans R 1999 A viral mechanism for inhibition of p300 and PCAF
acetyltransferase activity. Cell 96:393403[Medline]
-
Hamamori Y, Sartorelli V, Ogryzko V, Puri PL, Wu H-Y, Wang
JYJ, Nakatani Y, Kedes L 1999 Regulation of histone
acetyltransferases p300 and PCAF by the bHLH protein twist and
adenoviral oncoprotein E1A. Cell 96:405413[Medline]
-
Korzus E, Torchia J, Rose DW, Xu L, Kurakowa R, McInerney EM,
Mullen T-M, Glass CK, Rosenfeld MG 1998 Transcription
factor-specificity requirements for coactivators and their
acetyltransferase functions. Science 279:703707[Abstract/Free Full Text]
-
Kurakowa R, Kalafus D, Ogliastro M-H, Kioussi C, Xu L, Torchia
J, Rosenfeld MG, Glass CK 1998 Differential use of CREB binding
protein-coactivator complexes. Science 279:700703[Abstract/Free Full Text]
-
Rodriguez ER, Tan CD, Onwuta US, Yu ZX, Ferrans VJ, Parrillo
JE 1994 3,5,3'-Triiodo-L-thyronine induces cardiac myocyte
differentiation but not neuronal differentiation in P19 teratocarcinoma
cells in a dose dependent manner. Biochem Biophys Res Commun 205:652658[CrossRef][Medline]
-
Rodriguez ER, Tan CD, Onwuta US, Parrillo JE 1994 Cardiac
myocyte differentiation induced by 3,5,3'-triiodo-L-thyronine (T3) in
P19 teratocarcinoma cells is accompanied by preferential binding of
RGG(T/A)CA direct repeats spaced by 4 base pairs in the DNA. Biochem
Biophys Res Commun 05:18991906[CrossRef]
-
Sjöberg M, Vennström B, Forrest D 1992 Thyroid
hormone receptors in chick retinal development: differential expression
of mRNAs for
and N-terminal variant ß receptors. Development 114:3947[Abstract]
-
Svensson C, Bondesson M, Nyberg E, Linder S, Jones N,
Akusjärvi G 1991 Independent transformation activity by
adenovirus-5 E1A-conserved regions 1 or 2 mutants. Virology 182:553561[Medline]
-
Fattaey AR, Harlow E, Helin K 1993 Independent regions of
adenovirus E1A are required for binding to and dissociation of
E2F-protein complexes. Mol Cell Biol 13:72677277[Abstract]
-
Schaeper U, Boyd JM, Verma S, Uhlmann E, Subramanian T,
Chinnadurai G 1995 Molecular cloning and characterization of a cellular
phosphoprotein that interacts with a conserved C-terminal domain of
adenovirus E1A involved in negative modulation of oncogenic
transformation. Proc Natl Acad Sci USA 92:1046710471[Abstract]
-
Sollerbrant K, Akusjärvi G, Svensson C 1993 Repression
of RNA polymerase III transcription by adenovirus E1A. J Virol 67:41954204[Abstract]
-
Samuels HH 1979 Depletion of
L-3,5,3'-triiodothyronine and L-thyroxine in
euthyroid calf serum for use in cell culture studies of the action of
thyroid hormone. Endocrinology 105:8085[Abstract]
-
Johnston RF, Pickett SC, Barker DL 1990 Autoradiography using
storage phosphor technology. Electrophoresis 11:355360[Medline]