Requirements for Repression of Retinoid X Receptor by the Oncoprotein P75gag-v-erbA and the Thyroid Hormone Receptors
Gunilla M. Wahlström and
Björn Vennström
Department of Cellular and Molecular Biology Laboratory for
Developmental Biology Karolinska Institute S-171 77 Stockholm,
Sweden
 |
ABSTRACT
|
---|
The oncogenic counterpart of thyroid hormone
receptor-
(TR
), denoted P75gag-v-erbA,
has served as a paradigm for the ability of TRs to repress basal levels
of transcription. We show here that the retinoid X receptor (RXR), when
activated by its specific ligand SR11237, is repressed by both the
normal TR
and the P75gag-v-erbA. The
repression caused by the two proteins is distinct and dependent on both
the cell type and the hormone-response element through which RXR
acts. In HeLa cells only TR repressed efficiently through the
palindromic 2xIR0 element, whereas the proteins were equally efficient
in JEG cells. This demonstrates that proteins distinct in the two cell
types mediate the repression. RXR-dependent induction via the natural
response element of the cellular retinol-binding protein (CRBPII) gene
was likewise (
50%) repressed by TR, whereas
P75gag-v-erbA did not repress during the same
conditions. Furthermore, P75gag-v-erbA and its
variants v-erbAtd359 (lacking repressing
activity on TR) and v-erbAr12 (a highly active
repressor of TR) efficiently repressed induction by a hybrid protein
consisting of the DNA- binding domain of Gal4 and the ligand-binding
region of RXR. The viral proteins did not, however, associate with RXR
unless the two partners were allowed to heterodimerize upon binding to
a specific response element, such as the 2xIR0 element or that of the
CRBPII gene. In conclusion, we suggest that the efficient repression
seen with the the 2xIR0 element is due to heterodimerization of TR or
the viral oncoproteins with RXR and a concomitant inhibition of binding
of the RXR-specific ligand that results in an inability of RXR to
attract a cell type-specific cofactor. In addition, the data suggest
that the interaction between RXR and
P75gag-v-erbA on the CRBPII element is too weak
to inhibit RXR from binding a ligand and therefore also to repress.
 |
INTRODUCTION
|
---|
The retinoid X receptor (RXR) is a heterodimerization
partner for many members of the nuclear hormone receptor superfamily.
The number of nucleotides in the spacer sequence between two half-sites
in a hormone-response element of the direct repeat type (DR),
determines when RXR heterodimerizes with either a retinoic acid
receptor (RAR), a vitamin D3 receptor or a thyroid hormone
receptor (TR) (1). Here, RXR binds to the 5'-half-site (2, 3). RXR also
homodimerizes and activates transcription via synthetic DR elements
spaced by one nucleotide (4, 5) or via a palindromic element (6). Only
one natural promoter gene, the cellular retinol-binding protein type II
(CRBPII) (7), has been shown to be induced by homodimeric RXRs as a
response to ligand. This gene contains four half-sites oriented as DRs
with one nucleotide in each spacer. Chen et al. (8) showed
that the RXR receptor bound to this element as a cooperative high-order
oligomer. Studies by magnetic resonance spectroscopy have demonstrated
that a specific
-helix located immediately after the second zinc
finger in the DNA-binding domain (DBD) of RXR is responsible for
homodimeric protein interaction as well as for protein-DNA interaction
(9). Furthermore, ligand-dependent homodimerization is abolished by
both a 29-amino acid deletion in the C-terminal domain of RXR and by
point mutations within this domain (10).
The RAR activates transcription as a response to both the natural
all-trans- and the 9-cis-retinoic acid ligands,
whereas RXR activates only after binding the latter ligand (11, 12).
Recently, the importance of ligand specificity has been investigated,
and several synthetic ligands have been shown to function in a very
specific manner. Two RXR-specific ligands, SR11217 and SR11237 (13),
have been shown to induce RXR homodimer formation as well as to repress
T3 induction of a reporter construct containing the DR4
element of the myosin heavy chain gene (14).
Not only the ligand is important for activation and repression of
promoter genes. Several cofactors have been found to interact with
different nuclear receptors, thereby exerting a major impact on gene
regulation. The coactivators have been suggested to bind to the
activation domain, AF2, in the C-terminal part of the receptors,
whereas the corepressors, N-CoR (nuclear receptor corepressor) and SMRT
(silencing mediator of retinoic acid and thyroid hormone receptor),
preferentially bind to the hinge region (15, 16, 17, 18).
The oncoprotein P75gag-v-erbA is the viral counterpart to
TR and contains several point mutations, but it still interacts with
corepressors via the hinge region. A transformation-defective mutant of
v-erbA, denoted v-erbAtd359, has no transforming capacity,
whereas its revertant, v-erbAr12, is more potent than the
parental v-erbA gene (19). The loss of oncogenic function in
v-erbAtd359 is due to a single point mutation in the hinge
region (19), a mutation that also causes loss of interaction with the
corepressor SMRT when present in the normal TR (17). The revertant
v-erbAr12 contains a reversal of this mutation. Both the
v-erbAtd359 and v-erbAr12 proteins contain, in
contrast to P75gag-v-erbA, a proline at position 349 in
helix 11 in the C-terminal region, an amino acid suggested to be
essential for efficient heterodimerization with RXR (20, 21).
We have investigated the capacity of different TR isomers,
P75gag-v-erbA and the mutant proteins of
v-erbAtd359 and v-erbAr12, to repress
RXR-induced transactivation. We show that transactivation induced by
the RXR-specific ligand SR11237 via palindromic or natural RXR-specific
response elements is efficiently repressed by both TR
and TRß. Our
data thus extend those of Forman et al. (22). Furthermore, a
dominant negative effect on RXR-specific activation was observed with
P75gag-v-erbA and its variants but only when acting through
the palindromic type of element. Of the three oncoproteins, that of
v-erbAr12 repressed the most efficiently and that of
v-erbAtd359 the least efficiently. This repression was
dependent on DNA binding by the oncoproteins; constructs encoding only
their ligand-binding domains (LBDs) failed to repress RXR, and they all
failed to bind to RXR in the absence of hormone-response elements. Our
studies thus highlight important functional differences between the
P75gag-v-erbA and TR and therefore suggest a new role for
unliganded nuclear receptors in transcriptional repression.
 |
RESULTS
|
---|
TR Interferes with Induction Mediated by RXR Homodimers
Since RXR is important for transactivation by TR due to its role
as a heterodimerization partner, we studied whether TRs could also
modulate the ability of ligand-bound RXR to transactivate its target
genes. The receptors and the response elements used in all the
experiments shown are presented in Fig. 1
.

View larger version (32K):
[in this window]
[in a new window]
|
Figure 1. Schematic View of Nuclear Hormone Receptors and
Response Elements Used
The TR proteins p46 and p40 are distinct only in the
N-terminal A/B domain. Two chicken TRß isomers (ß0 and ß2),
RXR, P75gag-v-erbA, and two transformation-defective
mutants are also shown. The P in circles indicate
important phosphorylation sites, and P, S, and R indicate the amino
acids proline, serine, and arginine located in the LBD of TR and
v-erbA. Two differently oriented response elements with the orientation
of the half-site indicated by arrows and the spacing in
between shown by number, are depicted at the
bottom.
|
|
First, we tested the capacity of the chicken TR
to repress
RXR
-induced transactivation of reporter plasmids containing
different response elements. For this, we transfected JEG cells with
plasmids expressing RXR
and TR
along with relevant reporter
plasmids. Figure 2A
(left
panel) shows that SR11237-induced transactivation via the
synthetic palindromic element (2xIR0) was completely inhibited by an
equal amount of cotransfected TR
plasmid. The observation that
thyroid hormone (T3) could induce a transactivation similar
to that seen when only RXR was active in the cell confirms that the
transfection procedure did not adversely affect the transfected cells.
In a second experiment we tested the RXR-specific response element
found in the CRBPII gene. Figure 2A
(right panel) shows that
SR11237-dependent transactivation was blocked by approximately 50%. To
rule out that the repression was due to squelching, titration of TR in
cotransfection experiments was done as shown in Fig. 2B
. Already a
small proportion of co-transfected TR (20 ng) gave maximal repression
on the 2xIR0 element. The best repression through the element in the
CRBPII gene was achieved when the receptors were transfected at a
1:1 ratio and was at most 50% of the maximal RXR-induced activation.
The 2xIR0 element can be interpreted to contain an everted repeat since
the two single IR0 elements are separated by an eight-nucleotide
linker. We therefore tested repression through response elements of the
ER6, ER8, and also the DR4 types containing typical
T3-responsive sequences. None of these response elements
gave any SR11237-dependent transactivation (data not shown).
To determine whether different forms of TR contain similar
repressing activities on RXR, we co-transfected, into two different
cell lines, equal amounts of plasmids expressing RXR and one of the
four different chicken TRs. The TRs tested were the chicken TR
receptors, p40 and p46, as well as the chicken TRß0 and TRß2.
Figure 2C
(left panel) shows that all TRs tested mediated
repression of RXR-induced transactivation obtained via a 2xIR0
containing reporter in JEG cells to approximately the same degree, and
that addition of T3 activated the TRs. Similar results were
seen when HeLa cells were transfected (Fig. 2C
, right
panel). Our data show that all forms of avian TRs can repress RXR,
thus extending the data of Forman et al. (22).
Repression of RXR by the P75gag-v-erbA
Oncoprotein
P75gag-v-erbA represses ligand-mediated activation of
RAR and TR (21, 23, 24, 25). The mechanisms for this have
been suggested to be an out-titration of the common
heterodimerization partner RXR, resulting in the formation of inactive
heterodimers. To determine whether P75gag-v-erbA also could
repress activation by the RXR-specific ligand SR11237, we cotransfected
equal amounts of plasmids encoding P75gag-v-erbA and RXR
into JEG and HeLa cells along with the 2xIR0 reporter gene. The
experiments show that P75gag-v-erbA abolished activation by
RXR in JEG cells (Fig. 3A
, left
panel). In contrast, only a 50% reduction of SR11237-dependent
transactivation was seen in HeLa cells (Fig. 3A
, right
panel). In addition, the ability of P75gag-v-erbA to
repress RXR via the CRBPII element was tested. Our data demonstrate
that although the viral oncoprotein showed a minor repression of basal
transcription when expressed at high levels (5-fold excess), no
repression was seen in any instances when RXR was activated by the
ligand (Fig. 3B
). The oncoprotein formed heterodimers with RXR on the
CRBPII element, as shown by a gel retardation assay (Fig. 3C
).
Moreover, the complexes formed were supershifted with anti-v-erbA or
anti-RXR antibodies, thus verifying that the receptors formed a
heterodimer.

View larger version (41K):
[in this window]
[in a new window]
|
Figure 3. Repression of RXR by the v-erbA,
v-erbAtd359, and v-erbAr12 Genes
Panel A shows transfections with vectors expressing RXR along
with plasmids encoding v-erbA or the variants
v-erbAtd359 or v-erbAr12. The
reporter contained the 2xIR0 element, and the SR11237 ligand was added
as indicated (+). The transfections were done with either JEG or HeLa
cells as indicated. Two representative and independent experiments are
shown. Panel B shows transfections in JEG cells done with the CRBPII
element as reporter and a plasmid expressing RXR. One- or 5-fold excess
of a P75gag-v-erbA expressing plasmid was added (1x, 5x)
in two instances. Panel C shows a gel retardation assay, demonstrating
binding of the Vaccinia virus-expressed TR, RXR, or
P75gag-v-erbA receptors to the CRBPII element. The
complexes were supershifted with antibodies directed against either RXR
or erbA as indicated. The arrowhead indicates unbound
oligonucleotides.
|
|
The two mutants of v-erbA, v-erbAtd359 and
v-erbAr12, have properties distinct from those of
the wild-type oncogene. V-erbAtd359 yields a protein that
has lost the ability to repress TR and RAR, whereas the
v-erbAr12 protein is more effective in this than
P75gag-v-erbA. We therefore tested whether these variant
proteins retained their respective properties when acting on RXR
through the 2xIR0 element. Figure 3A
(left panel) shows that
in JEG cells the v-erbAr12 protein was, as expected, more
potent than P75gag-v-erbA (96% and 92% repression,
respectively). Surprisingly, the v-erbAtd359 protein also
gave a good repression, although it was less efficient than with the
other two proteins (80%). To exclude that the transfected mutants
expressed different amount of proteins, nuclear extract was made from
the transfected cells and compared by Western blotting (data not
shown). In HeLa cells the repressing activity of the mutant proteins
was similar to that seen with P75gag-v-erbA (Fig. 3A
, right panel).
Dependence of the DNA-Binding Region for Repression
Previous experiments have shown that repression of TR-mediated
activation by P75gag-v-erbA is dependent on elements that
allow receptor dimerization to occur (24, 26). Furthermore,
heterodimerization of P75gag-v-erbA with RXR was found to
be dependent on binding of the receptor proteins to specific response
elements (26). We therfore determined whether the repression of RXR is
dependent on the DNA-binding region of P75gag-v-erbA. For
this, we compared the repressional activity of full-length
P75gag-v-erbA with that of a construct that lacks the DBD.
JEG cells were transfected with increasing amounts of plasmid
expressing the respective viral proteins, a reporter containing the
2xIR0 response element, and an RXR-encoding vector. Figure 4B
shows that the construct containing
only the hinge region and the LBD of P75gag-v-erbA reduced
the induction to about 50%, regardless of the fold excess of the viral
protein. In contrast, transfection of as little as 20 ng of
the P75gag-v-erbA-encoding plasmid reduced the
SR11237-induced transactivation by 75%, and a 6-fold (640 ng) excess
of P75gag-v-erbA expressing construct abolished all
induction (Fig. 4A
). The data show that the DBD of
P75gag-v-erbA is required for efficient repression and,
furthermore, indicate that the LBD/hinge region of
P75gag-v-erbA weakly interferes with a cofactor for RXR or
with the basal transcription machinery.

View larger version (30K):
[in this window]
[in a new window]
|
Figure 4. Fig. 4. Repression by the Full-Length or the LBD of
P75gag-v-erbA
Plasmids (100 ng) expressing full-length (panels A and B) or Gal4-fused
RXR protein (panel C) was cotransfected along with increasing
concentrations (20640 ng) of either the full-length (panels A and C)
or the LBD of the P75gag-v-erbA protein (panel B). As
reporters the 2xIR0 (panels A and B) or Gal4 (panel C) elements were
used. Fold repression is shown as percentage of RXR activation.
|
|
To substantiate that the efficient repression is dependent on response
elements that allow heterodimerization, we investigated the capacity of
the full-length P75gag-v-erbA to repress the
SR11237-induced activation of a hybrid protein consisting of the DBD of
Gal4 and the LBD of RXR (denoted
Gal4DBD-RXRLBD). JEG cells were transfected as
described above, except that a reporter containing five binding sites
for the chimeric Gal4 protein was used. Figure 4C
shows that
transfection of equal amounts (100 ng) of plasmids encoding the
receptor protein reduced transactivation by only about 65%, and that a
6-fold excess of P75gag-v-erbA led only to a 85%
reduction. We conclude that DNA binding by the
P75gag-v-erbA is required for efficient repression of the
SR11237-dependent induction by RXR.
The poor repression seen could be due to an inability of
P75gag-v-erbA to associate with
Gal4DBD-RXRLBD in the absence of specific DNA
binding (26). Such a deficiency of P75gag-v-erbA has been
suggested to be due to the point mutation (P
S) at position 349 in
helix 11 of TR (20, 21, 26, 27). The v-erbAtd359 and
v-erbAr12 proteins do not contain this mutation. We
therefore compared the ability of these viral proteins and of TR to
repress the Gal4DBD-RXRLBD protein. Figure 5A
shows that an equal amount of
transfected v-erbAtd359 construct was as inefficient as
P75gag-v-erbA and TR in repression, and that the
cotransfected v-erbAr12 construct was only slightly more
effective.

View larger version (26K):
[in this window]
[in a new window]
|
Figure 5. Inability of the v-erbA and the Variant
v-erbAtd359 and v-erbAr12 Proteins to Associate
with RXR in the Absence of Specific DNA Response Elements
Panel A shows SR11237-dependent transactivation from
cotransfections with a 5xGal4 reporter and expression constructs
containing the Gal4DBD-RXRLBD. In addition,
plasmids expressing the TR, v-erbA, or the variant
v-erbAtd359 or v-erbAr12 proteins were
cotransfected with the Gal4DBD-RXRLBD construct
in equal amounts. The experiments was done in the presence or absence
of the SR 11237 ligand. Panel B shows the interaction between RXR and
P75gag-v-erbA and its variants -/+ T3 as
tested by the receptor-dependent two-hybrid method. Again, the RXR LBD
was fused to the DBD of Gal4 but then cotransfected with plasmids
expressing a full-length P75gag-v-erbA containing an
N-terminal VP16 domain, or with similar plasmids expressing
VP16-v-erbAr12 or VP16-v-erbAtd359 proteins.
VP16- denotes a construct to which no receptor was fused and which
therefore shows only background activation. In panel C the same
VP16-receptor constructs were allowed to bind to the DR4 element in an
in vivo assay. The binding is shown as VP16-mediated CAT
activity. The figures show both the results of one representative
experiment of a total of four done.
|
|
The modest repression seen with the proteins could be due to a
general repression of transcriptional activity in the cells or to a
weak association with the Gal4DBD-RXRLBD that
only marginally reduces the ability of the LBD of RXR to bind the
ligand SR11237. We therefore tested whether oncoprotein constructs
fused to an N-terminal VP16 transactivation domain associate with the
Gal4DBD-RXRLBD construct in vivo.
Figure 5B
shows that in contrast to a VP16-TR control, none of the
VP16-oncoprotein chimeras gave transactivation, indicating a lack of
association with Gal4DBD-RXRLBD. This was
independent of the presence of thyroid hormone. Control experiments
have shown that a VP16-gag-TR construct is as efficient as VP16-TR in
associating with Gal4DBD-RXRLBD (26). Moreover,
the VP16 constructs used above expressed comparable amounts of proteins
in the transfected cells, as demonstrated by gel retardation assay
using cell extracts from the transfected cells (data not shown). In
addition, they bind efficiently to DNA in vivo, since a
reporter containing the DR4 element gave high transactivation levels
when transfected into JEG cells along with the respective expression
plasmids (Fig. 5C
). Taken together, our data suggest that the viral
oncoproteins do not associate with RXR unless they are allowed to
heterodimerize on a specific response element.
 |
DISCUSSION
|
---|
Repression via the 2xIR0 Element
The dependence on RXR by other nuclear hormone receptors for
ligand-induced activation or repression of target gene expression has
been well studied. Some studies have also addressed how these receptors
affect RXR and its specific ligands in transcriptional regulation. For
instance, a heterodimerization partner prevents RXR from binding ligand
when the dimer complex has bound to certain response elements (4).
Moreover, such a heterodimer forms a complex with corepressors
(15, 16, 17). In this paper, we show that different TRs repress
SR11237-induced homodimeric RXR activation from a synthetic palindrome
(2xIR0) and from the natural CRBPII response element. Repression via
2xIR0 was very efficient, and the data suggest that it is due to a
heterodimerization between RXR and TR that inhibits RXR from binding
its ligand. This would prevent RXR from expressing its transactivating
properties e.g. through attraction of coactivators.
Also the oncoprotein P75gag-v-erbA repressed RXR-mediated
transactivation efficiently in JEG cells via the 2xIR0 element. The
mechanism is likely to be the same as that proposed for TR, since
P75gag-v-erbA heterodimerizes efficiently with RXR when
binding to this type of element (26). The reason for the much less
efficient repression in HeLa cells is unclear but may be due to
interaction with other cofactors that have distinct affinities for TR
and P75gag-v-erbA, respectively, in the two types of cells.
Alternatively, the cells contain different coactivators for RXR that
exhibit distinct activating functions.
The JEG cells are known to contain detectable amounts of RXR
but
presumably also other endogenous factors. Recently, Teboul and
collaborators (28) showed that RXR can form heterodimers with the
orphan receptor OR1, and that RXR in such a heterodimer can be
activated by ligand through elements of the DR4 type. We have tested
whether TR or P75gag-v-erbA can repress activation of such
heterodimers: our results obtained by transfection of an OR1-expressing
plasmid showed that OR1 did not contribute to SR11237-dependent
transactivation of RXR from the 2xIR0 element, and that OR1 did not
affect the ability of P75gag-v-erbA to repress RXR (G.
Wahlström, unpublished data).
Repression through the CRBPII Element
TR exhibited a modest repression via the the natural RXR element
in the CRBPII gene. Our gel retardation data showed that TR forms a
heterodimer with RXR on such an element. However, the RXR/TR
heterodimer has a relatively low affinity to elements of the DR1 type
as compared with an IR0 element [a dissociation constant
(kd) of 5.8 nM vs. 1.4
nM (26, 29)]. It is thus possible that the modest
repression is due to an inability of TR to form a stable heterodimer
with RXR on the CRBPII element.
P75gag-v-erbA completely failed to represss RXR through the
CRBPII element, even when present in vast excess over RXR within the
cell (Fig. 3B
). This could be due to distinct abilities of TR
and P75gag-v-erbA to bind corepressors, or to a receptor
function deficiency in P75gag-v-erbA. Our data suggest that
the latter is the case. Gel mobility shift assays showed that
RXR/P75gag-v-erbA heterodimers bound only weakly to the
CRBPII element (Fig. 3C
), showing that RXR and
P75gag-v-erbA interact poorly. Previously published data
suggested that a P
S mutation found in the C-terminal region of
P75gag-v-erbA is responsible for the low heterodimerizing
ability of the oncoprotein (20, 21, 27). We explored this further by
testing the v-erbAtd359 and v-erbAr12 variant
oncoproteins that have a proline in the C-terminal end like TR and
should theoretically be able to interact with RXR. Other mutations in
the LBDs of these VP16 oncoprotein constructs also contribute
efficiently to the lack of interaction with RXR as suggested by our
observation that none of them associated with the
Gal4DBD-RXRLBD protein but yet transactivated
in vivo. It is therefore likely that a
RXR/P75gag-v-erbA heterodimer bound to a CRBPII element is
disrupted by the SR11237 ligand, thus allowing RXR to bind ligand,
homodimerize, and transactivate.
The oncoproteins gave a modest (7550%) but consistent repression
of Gal4DBD-RXRLBD-mediated transactivation
despite the fact that they do not associate with the LBD of RXR. This
type of repression was also seen with the hinge-LBD portion of
P75gag-v-erbA (Fig. 5A
) when RXR-dependent transactivation
was measured with the 2xIR0 element. We suggest that this is due to an
interference with transcriptional cofactors needed by RXR for maximal
activation.
Role of Repression by TR and
P75gag-v-erbA in Vivo?
The recently described corepressors SMRT and N-CoR (15, 16, 17) appear
to play little role in P75gag-v-erbA-mediated repression of
RXR on a 2xIR0 element, since transfections of plasmids expressing
these proteins did not increase the repressing capacity of TR or any of
the oncoproteins (G. Wahlström, unpublished data). Alternatively,
they are already expressed at saturating levels in the cell types
tested.
That TR represses transactivation by RXR both via synthetic and natural
response elements in transfection experiments suggests that it could
have similar properties in vivo. It is well known that TRs
are expressed in several organisms before the development of the
thyroid gland (30, 31, 32). Several investigators have suggested that a
role for the unliganded receptor is to repress basal level
transcription of target genes for TR (33, 34, 35, 36). A special example of
this has been described for Xenopus development (Refs. 37
and 38 and references therein). TR
is already expressed in the
oocyte and in the developing embryo, and treatment of such embryos with
T3 induces the TRß gene (39), resulting in a precocious
metamorphosis of the tadpole. In contrast, microinjection of a
plasmid expressing P75gag-v-erbA into newly fertilized frog
oocytes results in defective brain and facial development, which are
abnormalitites usually associated with retinoid deficiency (40, 41).
The published data on the action of TR and P75gag-v-erbA
also show that they can oppose the teratogenic action of retinoids
during Xenopus development (38, 42). Our data provide
molecular information on how P75gag-v-erbA and unliganded
TRs may interfere with the functions of the retinoid pathway mediated
through RXR.
The v-erbA oncogene elicits its most prominent effects in
erythroblasts. Here, the oncoprotein blocks the differentiation of
immature cells to erythrocytes (for review, see Refs. 43 and 44).
However, treatment of v-erbA- expressing cells with
all-trans-retinoic acid causes the cells to overcome the
differentiation block, and they terminally differentiate if provided
with appropriate growth factors (45, 46). All-trans-retinoic
acid is quickly metabolized to derivatives that bind to both RXR and
RAR, and erythroblasts express high levels of both RXR and RAR. It is
therefore possible that the differentiation-inducing effect of
all-trans-retinoic acid alluded to above is mediated by
RXR.
 |
MATERIALS AND METHODS
|
---|
Plasmid Constructs
One copy of double-stranded oligonucleotides representing the
natural CRBPII and rGH promoters was inserted into the
HindIII site of the pBLCAT2 vector (47). Single
elements of the IR0 and DR4 type (rGH A and B) from the rat GH were
cloned individually into the pBLCAT2 vector (48). In
addition, the synthetic response elements DR4, ER6, ER8, IR0 and 2xIR0
(26) were cloned inte the same reporter vector. The chicken
TR
1 receptors (p40 and p46), mouse RXR
, and v-erbA were cloned
into the pSG5 expression vector (49). cDNAs for v-erbA,
v-erbAtd359, and v-erbAr12 were cloned into an
Rous sarcoma virus (RSV) expression vector (23). Full length gag-erbA
(V3) (50) or gag-v-erbA, v-erbAtd359 and
v-erbAr12 were fused to the transactivating domain of VP16
by cloning into the KpnI/BamHI site of the
pCMX-VP16 vector (51), placing the VP16 transactivation domain in the N
terminus of the chimeric nuclear receptors. The v-erbA mutant lacking
the DNA binding domain (DBD) was constructed by Casanova et
al. (27). The vectors pCMX-Gal4-RXR and pCMX-VP16-TRß have
previously been described by Perlmann and Jansson (51).
Gel Retardation Experiments
The gel retardation experiments were done with
Vaccinia virus-expressed receptor proteins as previously
described in Wahlström et al. (52). The receptor/DNA
complexes were supershifted with polyclonal antibodies raised against
either RXR or v-erbA.
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 later the medium was replaced with
DMEM containing 8% calf serum depleted of retinoic acid and/or
T3 and T4 by ion exchange resin (53).
Approximately 2 h later the cells were cotransfected with
expression vectors encoding 100 ng mouse RXR
, 100500 ng chicken
TR
1, or v-erbA mutants plus 500 ng of different reporter constructs,
unless indicated otherwise. A plasmid containing the cytomegalovirus
(CMV) promoter driving the ß-galactosidase (ß-gal) gene was
cotransfected to provide an internal control. The cells were maintained
in the presence or absence of 0.51 mM SR 11237 and
12100 nM T3, harvested 24 h after
hormone treatment, and assayed for chloramphenicol acetyltransferase
activity. The ß-galactosidase activity was assayed using
o-nitrophenyl-ß-D-galactopyranoside as
substrate. Quantifications were done with a Molecular Dynamics
(Sunnyvale, CA) PhosphorImager (54). All transfections of JEG and HeLa
cells were repeated at least three times with similar results.
Duplicate sample points were used in each experiment and varied by less
than 15%.
 |
ACKNOWLEDGMENTS
|
---|
We are greatful to Dr. H. Samuels and Dr. T. Perlmann for
receptor constructs and Dr. Louise Foley (Roche Diagnostics, Nutley,
NJ) for the SR11237 ligand. The SMRT constuct was kindly provided by
Dr. R. Evans, and we are greatful to Dr. J. Rosenfeldt for the N-CoR
construct. We also thank Mrs. Gunnel Jönsson for secreterial
help.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Bjorn Vennstrom, Department of Cellular and Molecular Biology, Medical Nobel Institute, Karolinska Institute, Stockholm, Sweden S-171 77.
This project was funded by grants from the Swedish Cancer Society
(RMC), The Beijer Foundation, and funds at the Karolinska Institute. In
addition, G. M. Wahlström was supported by the Swedish
Society for Medical Research.
Received for publication May 7, 1997.
Revision received December 3, 1997.
Accepted for publication January 13, 1998.
 |
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]
-
Perlmann T, Rangarajan PN, Umesono K, Evans R 1993 Determinants for selective RAR and TR recognition of direct repeat
HREs. Genes Dev 7:14111422[Abstract]
-
Kurokawa R, Yu VC, Näär A, Kyakumoto S, Han Z,
Silverman S, Rosenfeld MG, Glass CK 1993 Differential orientations of
the DNA-binding domain and carboxy-terminal dimerization interface
regulate binding site selection by nuclear receptor heterodimers. Genes
Dev 7:14231435[Abstract]
-
Kurakowa R, DiRenzo J, Boehm M, Sugarman J, Gloss B,
Rosenfeld MG, Heyman RA, Glass CK 1994 Regulation of retinoid
signalling by receptor polarity and allosteric control of ligand
binding. Nature 371:528531[CrossRef][Medline]
-
Yang YZ, Subauste S, Koenig RJ 1995 Retinoid X receptor alpha
binds with the highest affinity to an imperfect direct repeat response
element. Endocrinology 136:28962903[Abstract]
-
Zhang X-K, Lehmann J, Hoffmann B, Dawson MI, Cameron J,
Graupner G, Hermann T, Pfahl M 1992 Homodimer formation of retinoid X
receptor induced by 9-cis retinoic acid. Nature 358:587591[CrossRef][Medline]
-
Mangelsdorf DJ, Umesono K, Kliewer SS, Borgmeyer U, Ong ES,
Evans RM 1991 A direct repeat in the cellular retinol binding protein
type II gene confers differential regulation by RXR and RAR. Cell 66:555561[Medline]
-
Chen H, Privalsky M 1995a Cooperative formation of high-order
oligomers by retinoid X receptors: an unexpected mode of DNA
recognition. Proc Natl Acad Sci USA 92:422426
-
Lee MS, Kliewer SA, Provencal J, Wright PE, Evans RM 1993 Structure of the retinoid X receptor a DNA binding domain: a helix
required for homodimeric DNA binding. Science 260:11171121[Medline]
-
Zhang X-K, Salbert G, Lee M-O, Pfahl M 1994 Mutations that
alter ligand-induced switches and dimerization activities in the
retinoid X receptor. Mol Cell Biol 14:43114323[Abstract]
-
Heyman RA, Mangelsdorf DJ, Dyck JA, Stein RB, Eichele G, Evans
RM, Thaller C 1992 9-cis retinoic acid is a high affinity ligand for
the retinoid X receptor. Cell 68:397406[Medline]
-
Levin AA, Sturzenbecker LJ, Kazmer S, Bosakowski T, Huselton
C, Allenby G, Speck J, Kratzeisen C, Rosenberger M, Lovely A, Grippo JF 1992 9-cis retinoic acid stereoisomer binds and activates the nuclear
receptor RXR alpha. Nature 355:359361[CrossRef][Medline]
-
Lehmann JM, Jong L, Fanjul A, Cameron JF, Lu XP, Haefner P,
Dawson MI, Pfahl M 1992 Retinoids selective for retinoid X receptor
response pathway. Science 258:19441946[Medline]
-
Lehmann JM, Zhang X-K, Graupner G, Lee M-O, Hermann T,
Hoffmann B, Pfahl M 1993 Formation of retinoid X receptor homodimers
leads to repression of T3 response: hormonal cross talk by
ligand-induced squelching. Mol Cell Biol 13:76987707[Abstract]
-
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 1995b A transcriptional co-repressor that
interacts with nuclear hormone receptor. Nature 377:454459
-
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]
-
Damm K, Beug H, Graf T, Vennström B 1987 A single point
mutation in erbA restores the erythroid transforming potential of a
mutant avian erythroblastosis virus (AEV) defective in both erbA and
erbB oncogenes. EMBO J 6:375382[Abstract]
-
Selmi S, Samuels HH 1991 Thyroid hormone receptor/and v-erbA.
A single amino acid difference in the C-terminal region influences
dominant negative activity and receptor dimer formation. J Biol
Chem 266:1158911593[Abstract/Free Full Text]
-
Barettino D, Bugge TH, Bartunek P, Vivanco-Ruiz MdM,
Sonntag-Buck V, Beug H, Zenke M, Stunnenberg H 1993 Unliganded T3R, but
not its oncogenic variant, v-erbA suppresses RAR-dependent
transactivation by titrating out RXR. EMBO J 12:13431354[Abstract]
-
Forman BM, Umesono K, Chen J, Evans RM 1995 Unique response
pathways are established by allosteric interactions among nuclear
hormone receptors. Cell 81:541550[Medline]
-
Damm K, Heyman RA, Umesono, K, Evans, R 1993 Functional
inhibition of retinoic acid response by dominant negative retinoic acid
mutants. Proc Natl Acad Sci USA 90:29892993[Abstract]
-
Hermann T, Hoffmann B, Piedrafita FJ, Zhang X-K, Pfahl M 1993 V-erbA requires auxiliary proteins for dominant negative activity.
Oncogene 8:5565[Medline]
-
Chen H-W, Privalsky M 1993 The erbA oncogene represses the
action of both retinoid X and retinoid A receptors but does so by
distinct mechanisms. Mol Cell Biol 13:59705980[Abstract]
-
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]
-
Casanova J, Helmer E, Selmi-Ruby S, Qi J-S, Au-Fliegner M,
Desai-Yajnik V, Koudinova N, Yarm F, Raaka BM, Samuels HH 1994 Functional evidence for ligand-dependent dissociation of thyroid
hormone and retinoic acid receptors from an inhibitory cellular factor.
Mol Cell Biol 14:57565765[Abstract]
-
Teboul M, Enmark E, Li Q, Pelto-Huikko M, Gustafsson J-Å 1995 OR-1, a member of the nuclear receptor superfamily that interacts with
the 9-cis-retinoic acid receptor. Proc Natl Acad Sci USA 92:20962100[Abstract]
-
Wahlström GM 1996 Transcriptional Regulation by the
Thyroid Hormone Receptor and Its Viral Counterpartthe
P75gag-v-erbA Oncoprotein. Thesis
-
Baker BS, Tata JR 1990 Accumulation of proto-oncogene c-erbA
related transcript during Xenopus development: association
with early acquisition of response to thyroid hormone and estrogen.
EMBO J 9:879885[Abstract]
-
Forrest D, Hallböök F, Persson H, Vennström
B 1991 Distinct functions for thyroid hormone receptors a and b in
brain development indicated by differential expression of receptor
genes. EMBO J 10:269275[Abstract]
-
Prati M, Calvo R, Morreale G, Morreale de Escobar G 1992 L-Thyroxine and 3,5,3'-triiodothyronine concentrations in
the chicken egg and in the embryo before and after the onset of thyroid
function. Endocrinology 130:26512659[Abstract]
-
Baniahmad A Kohne AC, Renkawitz, R 1992 A transferable
silencing domain is present in the thyroid hormone receptor, in the
v-erbA oncogene product and in the retinoic acid receptor. EMBO J 11:10151023[Abstract]
-
Piedrafita FJ, Bendik I, Ortiz MA, Pfahl M 1995 Thyroid
hormone receptor homodimers can function as ligand-sensitive
repressors. Mol Endocrinol 9:563578[Abstract]
-
Fondell JD, Roy AL, Roeder RG 1993 Unliganded thyroid hormone
receptor inhibits formation of a functional preinitiation complex:
implications for active repression. Genes Dev 7:14001410[Abstract]
-
Fondell JD, Brunel F, Histake K, Roeder RG 1996 Unliganded
thyroid hormone receptor a can target TATA-binding protein for
transcriptional repression. Mol Cell Biol 16:281287[Abstract]
-
Kawahara A, Baker BS, Tata JR 1991 Developmental and regional
expression of thyroid hormone receptor genes during Xenopus
metamorphosis. Development 112:933943[Abstract]
-
Shi YB, Wong J, Puzianowska-Kuznicka M, Stolow MA 1996 Tadpole
competence and tissue-specific temporal regulation of amphibian
metamorphosis: roles of thyroid hormone and its receptors. Bioessays 18:3919[Medline]
-
Machuca I, Esslemont G, Fairclough L, Tata JR 1995 Analysis of
structure and expression of the Xenopus thyroid hormone
receptor-beta gene to explain its autoinduction. Mol Endocrinol 9:96107[Abstract]
-
Schuh TJ, Hall BL, Kraft JC, Privalsky ML, Kimelman D 1993 v-erbA and citral reduce the teratogenic effects of all-trans retinoic
acid and retinol, respectively, in Xenopus embryogenesis.
Development 119:785798[Abstract/Free Full Text]
-
Nagl SB, Nelson CC, Romaniuk PJ, Allison LA 1995 Constitutive
transactivation by the thyroid hormone receptor and a novel pattern of
activity of its oncogenic homolog v-ErbA in Xenopus oocytes.
Mol Endocrinol 9:152232[Abstract]
-
Banker DE, Eisenman RN 1993 Thyroid hormone receptor can
modulate retinoic acid-mediated axis formation in frog embryogenesis.
Mol Cell Biol 13:754052[Abstract]
-
Beug H, Vennström B 1991 Nuclear Hormone Receptors.
Academic Press, New York, pp 355375
-
Beug H, Mullner EW, Hayman MJ 1994 Insights into erythroid
differentiation obtained from studies on avian erythroblastosis virus.
Curr Opin Cell Biol 6:816824[Medline]
-
Schroeder C, Gibson L, Zenke M, Beug H 1992a Modulation of
normal erythroid differentiation by the endogenous thyroid hormone and
retinoic acid receptors: a possible target for v-erbA oncogene action.
Oncogene 7:217227
-
Schroeder C, Gibson L, Beug H 1992b The v-erbA oncogene
requires cooperation with tyrosine kinase to arrest erythroid
differentiation induced by ligand-activated endogenous c-erbA and
retinoic acid receptor. Oncogene 7:203216
-
Luckow B, Schutz G 1987 Nucleic Acid Res 15:5490
-
Sjöberg M, Vennström B 1995 Ligand-dependent and
-independent transactivation by thyroid hormone receptor b2 is
determined by the structure of the hormone response element. Mol Cell
Biol 15:47184726[Abstract]
-
Green S, Issemann I, Sheer E 1988 A versatile in
vivo and in vitro eukaryotic expression vector for
protein engineering. Nucleic Acid Res 16:369[Medline]
-
Munoz A, Zenke M, Gehring U, Sap J, Beug H, Vennström B 1988 Characterization of the hormone-binding domain of the chicken
c-erbA/thyroid hormone receptor protein. EMBO J 7:155159[Abstract]
-
Perlmann T, Jansson L 1995 A novel pathway for vitamin A
signaling mediated by RXR heterodimerization with NGFI-B and NURR1. Gen
Dev 9:76982[Abstract]
-
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]
-
Samuels HH, Stanley F, Casanova J 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.
Endocrinolgy 105:8085[Abstract]
-
Johnston RF, Pickett SC, Barker DL 1990 Autoradiography using
storage phosphor technology. Electrophoresis 11:355360[Medline]