Genetisches Institut der Justus-Liebig Universität D-35392 Giessen, Germany
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ABSTRACT |
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INTRODUCTION |
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Nuclear hormone receptors share common structural features. They are
composed of a well conserved DNA-binding domain (C region), a variable
amino (N) terminus (A/B-region), and a conserved C terminus (D, E, and
F region) (for reviews, see Refs. 2, 4, 11, and 12). The receptor C
terminus harbors various functions such as silencing, hormone binding,
dimerization, and transactivation (4). It was shown that RAR and other
nuclear hormone receptors contain at least two activation functions:
one is localized in the receptor N terminus and the other in the C
terminus. The latter is dependent on the binding of hormone (2, 4, 11, 12). A small stretch of conserved amino acids have been identified to
be responsible for ligand-dependent transactivation (3, 4, 9, 13, 14)
and is referred to as AF2, AF2-AD, 4, or
c (2, 4, 9, 14, 15).
Both activation domains retain their functions when transferred on
heterologous DNA-binding proteins (9, 14, 15, 16, 17, 18).
Interestingly, similar to other nuclear hormone receptors, both activation domains of RAR function in a cell-specific manner (19, 20, 21, 22, 23, 24, 25). The detailed mechanisms for these cell-specific hormonal responses are unknown. One explanation may be that receptor-interacting cofactors are distinct from cell to cell. Therefore, the levels or the nature of cofactors may differ, which could lead to cell-specific hormonal responses. Thus, the analysis of cell specificity of retinoic acid responses will shed light into their role in gene regulation and development.
Here, we analyzed the RAR, which displays cell-specific
transcriptional properties (5, 20, 21, 22). In the absence of hormone, in
contrast to the TR, the RAR is a transcriptional activator in CV1
cells. However, RAR acts as a silencer in L cells. Addition of hormone
leads to a further activation in CV1 cells, while in L cells
transcriptional silencing is relieved and the receptor is converted to
a gene activator. In general, the RAR-mediated hormonal response is
much weaker in CV1 cells compared with that of L cells. It was shown
that the RAR C terminus lacks silencing in CV1 cells and is responsible
for the cell type specificity (5).
We show, that the cell specificity is due to the AF2/c activation
domain of RAR and is not due to a general lack of silencing in CV1
cells. Interestingly, silencing in CV1 cells is achieved by replacing
RAR-AF2/
c with TR-AF2/
c. This suggests a cell-specific role of
AF2/
c for regulation of transcriptional silencing in the context of
its receptor. Furthermore, we show that coexpression of SMRT (silencing
mediator of retinoic acid and thyroid hormone receptor), but not N-CoR
(nuclear receptor corepressor), in CV1 cells enhances strongly the
hormonal response. Therefore, the transactivation by RAR is reduced in
the absence of hormone, and silencing is regained. This indicates that
SMRT is one of the limiting factors for RAR-mediated silencing in CV1
cells. Thus, the level of certain cellular corepressors is an important
factor in regulation of target genes and the hormonal response of the
cell.
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RESULTS |
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It was previously shown that the CV1-specific lack of RAR-mediated
silencing is localized in its C terminus (Fig. 2 and Ref.5). Here, we
have focused on the RAR C terminus and used the established fusion of
the C-terminal part of RAR to the DNA-binding domain of Gal4 (Gal-RAR,
Fig. 1
). This expression plasmid was
cotransfected together with a reporter containing the Gal4-binding site
[upstream activating sequence (UAS) or 17 mer] in front of the tkCAT
fusion. Cotransfection showed that the receptor fusion acted as a
transcriptional silencer in the absence of ligand and as a retinoic
acid-dependent transactivator in L cells (Fig. 2
). In contrast, the same fusion acted as
a weak activator in CV1 cells even in the absence of ligand (Fig. 2
).
Addition of hormone increased the transcriptional activity. This
indicates that the cell-specific effect is localized in the receptor D,
E, or F region. Thus, in CV1 cells, retinoic acid induces only a
marginal hormonal response by RAR
(
3-fold), quite different from
L cells (
200-fold).
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AF2/c of RAR Is the Cause for the Lack of RAR Silencing in CV1
Cells
To test whether the F region or AF2/c of RAR is inhibiting
silencing in CV1 cells, we generated a receptor missing only the F
region (RAR
C43; Fig. 1
). As seen in Fig. 2
, this receptor deletion
exhibited similar transcriptional activity as the origin receptor: lack
of silencing in CV1 cells in ligand-free conditions and weak hormone
induction. Therefore, we conclude that the receptor F region is not
required, and that the conserved 17 amino acids (aa 403419) of RAR
AF2/
c are sufficient to inhibit silencing in CV1 cells.
Furthermore, we fused the corresponding AF2/c of the thyroid hormone
receptor (TR-
4 (Ref.9); TR-AF2/
c) to the C terminus of
RAR
C60, creating Gal-RAR-TR-AF2/
c. This receptor chimera acted as
a retinoic acid-dependent transcriptional activator in both L- and CV1
cells (Fig. 2
). Figure 2
shows that AF2/
c of TR can substitute the
activation function of RAR and promote hormone-dependent activation.
Interestingly, in the absence of hormone, RAR-TR-AF2/
c acts as a
transcriptional silencer in both cell types (Fig. 2
). Therefore,
hormone induction in CV1 cells is enhanced dramatically to about
150-fold, compared with an about 4-fold induction of the original
receptor (Fig. 2
). This suggests that the CV1-specific effect (lack of
silencing) is conferred specifically by the RAR AF2/
c domain and not
by the homologous domain of TR.
To determine whether the endogenous/intrinsic transactivation function
of AF2/c is responsible for the difference in retinoic acid response
between RAR
C43 and RAR-TR-AF2/
c, we tested for their ability to
transactivate in both cell types. As seen in Fig. 3
, both Gal-RAR402462 (harboring both
AF2/
c and the F region) and Gal-RAR402419 (RAR-AF2/
c)
transactivate only very weakly, if at all, when tested on one UAS
element of the reporter 17 mer tkCAT in both cell types. As also seen
in Fig. 3
, AF2/
c derived from TR (TR-AF2/
c) harbors even a
stronger transactivation function, at least in CV1 cells, compared with
RAR-AF2/
c. Nevertheless, the receptor fusion RAR-TR-AF2/
c
exhibits silencing function in CV1 cells. This indicates that the
almost constitutive transactivation of RAR in CV1 cells is not caused
by a cell-specific activity of AF2/
c.
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Taken together, the lack of silencing in the absence of hormone in CV1
cells is due to the specific sequence of RAR-AF2/c. Our data suggest
that the CV1-specific effect is uncoupled from the strength of AF2/
c
intrinsic transactivation function, since replacing the conserved
sequence AF2/
c of RAR with that of TR rendered the receptor to a
transcriptional silencer. Thus, the small AF2/
c sequence of RAR
harbors other requirements to inhibit silencing in CV1 cells and
exhibit cell type specificity in the context of its own receptor.
The Lack of Silencing by RAR in CV1 Cells Can Be Overcome by the
Corepressor SMRT, But Not by N-CoR
Another possibility for the failure of RAR to silence in CV1 cells
in the absence of hormone is that one or few corepressors are
present in a limiting amount in CV1 cells. These corepressors may be
required to overcome the effect of AF2/c of RAR
. To test this
possibility, we cotransfected the known corepressors SMRT or N-CoR in
CV1 cells and tested for hormonal response. In the absence of
cotransfected corepressors, RAR exhibited about a 2- to 4-fold
ligand-mediated induction (Figs. 2
and 4A
). Coexpression of SMRT resulted in a
profound hormonal induction of about 22-fold. This was achieved by
reducing the activation function by Gal-RAR in the absence of hormone
(Fig. 4A
). As a control, SMRT expression affected the promoter activity
only weakly, while it changed the RAR-specific transcriptional activity
to a level slightly below that of the promoter activity (Fig. 4A
).
Interestingly, coexpression of N-CoR did not enhance hormonal
induction; rather the promoter activity was enhanced in the presence or
absence of RAR. Similar results were obtained using Gal-RAR
C43 in
CV1 cells. RAR
C43 with only the F region deleted lacked silencing
function (Fig. 2
). Coexpression of N-CoR did not affect RAR
C43
transcriptional properties significantly. In contrast, SMRT
coexpression abolished the RAR
C43-mediated activation and led to
transcriptional silencing of about 6-fold in the absence of hormone,
while the induced level was unchanged. Thus, a strong hormonal response
of above 60-fold was achieved when SMRT was coexpressed in CV1 cells
(Fig. 4A
).
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This suggests that SMRT is at least one of the cofactors that are limited in CV1 cells for RAR being a transcriptional silencer in the absence of hormone. In addition, we show that the hormone response is strongly enhanced after coexpression of SMRT, but not N-CoR, which indicates that SMRT and N-CoR have differential effects on nuclear hormone receptors. Furthermore, our results indicate that receptor target genes can be regulated by magnitudes with hormone, dependent on the cell type and on the presence of specific cofactors.
Replacing AF2/c of RAR by that of TR Enhances SMRT Binding to
the RAR-Silencing Domain
Replacing the RAR activation domain AF2/c by that of TR
(RAR-TR-AF2/
c) resulted in a receptor fusion that exhibited
silencing function in CV1 cells (Fig. 2
). One explanation for this
effect may be that the interaction of SMRT with RAR is affected by
exchanging the activation domain AF2/
c. Therefore, we performed band
shift experiments using extracts from retinoic acid-free transfected
COS-cells. As seen in Fig. 5A
, addition
of bacterially expressed glutathione-S-transferase (GST)-SMRT
(30) to COS extracts that expressed Gal-RAR led to a supershift, which
represents the receptor-SMRT complex. Addition of the cognate ligand,
retinoic acid, abolished the supershift with SMRT, as expected.
Previously, we have shown that neither GST nor GST-SMRT alone can bind
to the UAS-probe (31). To test the possibility that AF2/
c affects
the receptor-SMRT complex formation, increasing amounts of bacterially
expressed and purified GST-SMRT (30) were incubated with Gal-RAR or
Gal-RAR-TR-AF2/
c containing extracts and with the UAS probe (5). As
seen in Fig. 5B
, both the Gal-RAR or the Gal-RAR-TR-AF2/
c bands were
supershifted when SMRT was added, resulting in a GAL-RAR/SMRT complex.
Increasing amounts of added GST-SMRT resulted in an increase of each of
the receptor-SMRT complexes. However, addition of the same amount of
SMRT enabled more of Gal-RAR-TR-AF2/
c than of Gal-RAR to supershift.
This suggests that SMRT interacts more strongly with the
Gal-RAR-TR-AF2/
c fusion. This is also in accordance with yeast 2
hybrid results (31). Thus, a greater affinity may be the cause for the
RAR-TR-AF2/
c fusion to act as a transcriptional silencer in CV1
cells. For this fusion we can speculate that the corepressor SMRT is
not limiting in obtaining transcriptional silencing by
RAR-TR-AF2/
c.
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Cellular SMRT Levels May Account for Cell Type Specificity
To analyze the expression levels of SMRT, we performed
Western experiments comparing extracts from equal cell numbers of L-
and CV1 cells. Using an anti-SMRT antibody directed against the C
terminus of SMRT (Santa Cruz Biotechnology, Santa Cruz, CA) we show
that the amount of SMRT is distinct between both cell types (Fig. 6). The SMRT antibody recognizes in CV1
cells a slightly slower migrating band compared with that of L cells
(Fig. 6
). This indicates that, in addition to endogenous SMRT levels,
there may be also cell type-specific variants of SMRT. Both different
levels and perhaps variants of SMRT may explain the cell type-specific
lack of RAR- mediated silencing in CV1 cells.
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DISCUSSION |
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Cell-Specific Silencing in the Absence of Hormone
The RAR silencer core (RARC60, aa 143403) exhibited strong
silencing function in the presence or absence of hormone. Although it
was shown that this RAR deletion is able to bind
all-trans-retinoic acid with similar affinity as that of the
wild-type receptor (8, 37). This confirms data that ligand binding is
not sufficient to relieve silencing (9, 38). Interestingly, the RAR
deletion RAR
C43, harboring an additional 17 aa, which correspond to
its activation function AF2/
c sequence, does not mediate silencing
in CV1 cells.
We tested, therefore, whether the strength of the endogenous/intrinsic
activation function may be a major cause for the cell specificity. We
compared AF2/c from TR and RAR as well as heterologous activation
domains such as that of p65 and Oct2 (26). When we replaced
RAR-AF2/
c with TR-AF2/
c, we saw a hormone-dependent receptor
transactivation, with a similar level of activity as Gal-RAR or
Gal-RAR
C43. Nevertheless, the intrinsic transactivation of the
TR-AF2/
c activation domain, shown in Fig. 3
, is stronger as that of
RAR-AF2/
c. Replacing AF2/
c sequences with the activation domain
p65, which had similar intrinsic transactivation properties compared
with TR-AF2/
c in CV1 cells, was only able to relieve silencing only
by addition of hormone. Similarily, AF2/
c and the F region of RAR
was significantly weaker in transactivation, but in the context of the
receptor it enabled strong hormone-induced activation. This suggests
that the strength of activation function is not the critical event in
ligand-induced activation and indicates that strong coactivator binding
to AF2/
c, which may displace corepressors in the absence of hormone,
can be excluded.
We show, rather, that the levels or type of cellular corepressor SMRT
is the cause of the lack of silencing mediated by RAR in CV1 cells.
Coexpression of SMRT (6) was able to overcome the lack of silencing of
RAR and RARC43 and rendered the receptor into a transcriptional
silencer in the absence of hormone, while the level of ligand-induced
transactivation of RAR remained unchanged. Thus, our data suggest a
direct involvement of SMRT in the magnitude of hormonal response of a
cell.
Interestingly, N-CoR was unable to yield similar effects as seen with SMRT, although both proteins are related to each other and share sequence homologies (36). We have shown previously that both SMRT and N-CoR are able to bind to the DNA-bound Gal-RAR fusions (31). While RAR is able to bind to both corepressors, only coexpression of SMRT enhanced silencing. This may indicate that N-CoR, despite the homologies to SMRT, harbors different functions in mediating silencing. Also a difference between the homologous corepressors SMRT and N-CoR was observed for the orphan receptor RevErb (39). SMRT and N-CoR interacted differentially with this orphan receptor: N-CoR, but not SMRT, potentiated the RevErb repression, when bound to DNA.
Another possibility for the cell-specific silencing of RAR may be due to lower levels or to specific variants of the corepressor SMRT in CV1 cells. Based on our Western experiments we have indeed seen a weaker SMRT band in CV1 cells compared with that of L cells. However, there may exist variants of SMRT in CV1 cells. SMRT variants can be generated by differential splicing events or be caused by posttranslational modifications (e.g. phosphorylation). Variants of a corepressor have been described for the SMRT homologous factor N-CoR/RIP13 (40). In any case, we show that the coexpression of SMRT can overcome the endogenous SMRT function. Thus, different SMRT levels and/or variants may be the basis for cell type specificity of receptor-mediated gene repression.
Multiple Functions of Nuclear Receptor AF2/c Sequence
Our data show that silencing is inhibited by the AF2/c
(17 aa) of RAR specifically in CV1 cells. Fusion of AF2/
c from TR
(9) encompassing 17 aa rendered the RAR silencer core into a
hormone-dependent transactivator, indicating that the conserved
AF2/
c sequences can be exchanged without loss of ligand-induced
transactivation, which is in accordance with the findings of Durand
et al. (15). Interestingly, this receptor-AF2/
c fusion
(RAR-TR-AF2/
c) exhibited silencing function in the absence of ligand
in CV1 cells. This may be due to a stronger interaction of corepressors
with this receptor fusion. Our electrophoretic mobility shift assay
(EMSA) experiments indeed show an enhanced interaction of SMRT with the
RAR-TR-AF2/
c fusion. Similarily, mutations of AF2/
c of RXR
increased SMRT binding to the RAR/RXR heterodimer in EMSA experiments
(30). Also, naturally occurring mutations of TRß, derived from
patients with resistance to thyroid hormone with mutations outside of
the characterized SMRT interaction region, also exhibit an unusually
strong interaction with the corepressor SMRT (38).
Our data indicate that specifically the RAR-AF2/c sequence prevents
silencing in the context of its receptor. This may indicate that
despite sequence conservation, TR-derived AF2/
c and RAR-derived
AF2/
c differ in their functionality in that AF2/
c can have an
influence on corepressor binding to the silencing domain in the absence
of hormone.
Thus, amino acids of the conserved C-terminal activation domain
AF2/c are required not only for corepressor release in the presence
of hormone, but also have an influence on corepressor binding in the
absence of hormone and consequently can influence silencing function of
nuclear receptors.
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MATERIALS AND METHODS |
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Cell Culture
CV1 and L cells were grown in DMEM with 10% FCS at 37°
C/5% CO2. Cotransfections were carried out using the
CaPO4-method (27, 28) for CV1 cells and
diethylaminoethyl-Dextran method (5) for L cells. Expression vector
(0.5 pmol) was cotransfected with the 1.5 pmol of indicated reporter
plasmid. Coexpression of SMRT was performed by addition of 1.75 pmol
pCMX-SMRT (6) into the DNA transfer mix. For hormone treatment, cells
were seeded out in charcoal-treated 10% FCS.
EMSA and Protein Source
Extracts from transfected COS cells (29, 41) were modified
as follows. Transfected COS1 cells were harvested in PBS, pelleted, and
resuspended in 5x packed cell volume binding buffer containing 20
mM HEPES, pH 7.8, 400 mM NaCl, 20% glycerol,
and 2 mM dithiothreitol. Cells were lysed by freezing (-80
C) and thawing on ice. Cell debris were removed by centrifugation. EMSA
was performed by preincubation of the protein extract with 1 µg
deoxyinosinic-deoxycytidylic acid, 0.25 µg denatured calf thymus DNA
in 0.5times] binding buffer. Bacterially expressed GST-SMRT (30) was
purified and used (25500 ng) according to Chen and Evans (6). After
10 min incubation on ice the 17 mer probe (5) was added and incubated
for an additional 15 min on ice before loading a polyacrylamide gel,
which was run with Tris/Glycine (25 mM/192
mM).
Western Analysis
Equal numbers of L or CV1 cells were lysed directly in SDS
loading buffer and sonified for Western analysis. Goat anti-SMRT
antibody directed against a peptide corresponding to aa 14771495 of
SMRT C terminus (Santa Cruz Biotechnology) and anti-goat horseradish
peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) was
used for detection with the enhanced chemiluminescence analysis system
(Amersham) according to the manufacturers protocol.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by grants from the Sonderforschungsbereich SFB 249 of the Deutsche Forschungsgemeinschaft and from the Fonds der Chemischen Industrie.
This work contains parts of the Ph.D. thesis of U. Dressel.
Received for publication October 20, 1997. Revision received January 8, 1998. Accepted for publication January 12, 1998.
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REFERENCES |
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