University of Queensland (L.J.B., M.D., G.E.O.M.) Centre for
Molecular and Cellular Biology Ritchie Research Laboratories
Brisbane, 4072, Queensland, Australia
Centre Nationale de la
Recherche Scientifique (V.L.) Institut Pasteur Oncologie
Moléculaire Lille-France
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ABSTRACT |
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INTRODUCTION |
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The mRNAs encoding Rev-erbA and RVR are abundantly expressed in most
tissues, although higher levels of expression are seen in skeletal
muscle, brown fat, spleen, and the brain. Evidence for the
physiological/biological role of these receptors has come from cell
culture studies that have demonstrated that Rev-erbA
and RVR
antagonistically regulate mammalian muscle differentiation and affect
the expression of the hierarchical myoD gene family and the
critical cell cycle regulator, p21Cip1/Waf1 (9, 10).
Furthermore, during myogenic differentiation, the expression of
Rev-erbA
and RVR mRNAs is repressed. In contrast, in adipocyte cells
the expression of Rev-erbA
mRNA increases dramatically during
adipogenesis and correlated with the extent of adipocyte
differentiation (11). The basis of these opposing roles in myogenesis
and adipogenesis has not been resolved, but may be dependent on the
respective stimuli that induce differentiation. In situ
hybridization analysis during chicken embryonic development suggested
that RVR plays a role in the complex network of inductive signals
involved in neuronal differentiation (5).
Rev-erbA and RVR bind as monomers to the nuclear receptor half-site
motif, RGGTCA flanked 5' by an AT-rich sequence
[(A/T)6RGGTCA], and as dimers to a novel
direct repeat motif separated by 2 bp [Rev-DR-2,
(A/T)4 AGGTCA CT AGGTCA] (4, 12, 13, 14). The Rev-erb family functions as dominant transcriptional
repressors (4, 6, 7, 9, 10) and encode active transcriptional silencers
in the E region (9, 10). Efficient repression is dependent on a minimal
region (
35 amino acids) in the E domain, that is highly conserved
between Rev-erbA
and RVR (97%). This region spans the
ligand-binding domain (LBD)-specific signature motif,
(F/WAKXXXXFXXLXXXDQXXLL),
helix 3, loop 34, helix 4, and helix 5 (identified in the crystal
structures of the thyroid hormone receptor (TR)/retinoic acid receptor
(RAR) nuclear hormone receptor LBDs).
In the absence of ligand, nuclear receptors silence transcriptional activity. Silencing requires specific cofactors called corepressors. Recently, two closely related but distinct proteins, SMRT (silencing mediator for retinoid and thyroid hormone receptors) (15, 16) and N-CoR (nuclear receptor corepressor) (17) have been identified as candidate corepressor proteins associated with the nuclear receptor superfamily. Multiple isoforms of the N-CoR and SMRT have since been identified and denoted RIP13s [retinoid X receptor (RXR)-interacting proteins (18)] and TRACs (T3 receptor-associating cofactors) (19), respectively. These proteins have been shown to bind to the LBD of thyroid hormone and retinoic acid receptors, in the absence of ligand, and dissociate upon ligand binding.
Repression of transcription by Rev-erbA/RVR is also mediated by the
corepressor N-CoR and its variants RIP13a and RIP13
1 (20, 21).
Detailed analysis of the corepressors has identified a receptor
interaction domain (RID) that consists of two interaction domains, ID-I
and D-II, that efficiently interact with nuclear receptors (18).
Recently it has been demonstrated that RVR and Rev-erbA
interact
very efficiently with the RID from N-CoR and RIP13a, although they
preferentially interact with the RID from the RIP13
1 isoform
(20). Furthermore, it was demonstrated that the E region of RVR and
Rev-erbA
was necessary and sufficient for the interaction with the
RIP13
1-RID (20).
The present study used mammalian two-hybrid and direct in
vitro binding assays to characterize the specific regions in RVR
and Rev-erbA that interact with N-CoR and its variants, RIP13a and
RIP13
1. These experiments identified two corepressor interaction
regions (CIRs) within helices 3 and 11 in the E region of RVR and
Rev-erbA
, denoted CIR-1 and CIR-2, respectively, that mediate
corepressor binding. CIR-1 is a novel domain that is highly conserved
between RVR and Rev-erbA
and is absolutely required for the
interaction with N-CoR, RIP13a, and RIP13
1. Although the E region of
Rev-erbA
was necessary and sufficient for the interaction with
RIP13
1, the D region was also required for N-CoR and RIP13a binding.
This suggested that N-CoR/RIP13a and RIP13
1 differentially interact
with Rev-erbA
. Furthermore, the ability of RVR to function as
dominant transcriptional silencer on a physiological target, the human
Rev-erbA
promoter, was dependent on CIR-1, CIR-2, and corepressor
binding.
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RESULTS |
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Similarly we analyzed the potential of various domains from the
RVR E region to interact with the RID from RIP13a and N-CoR in the
mammalian two- hybrid assay. The chimeric construct consisting of the
GAL4 DBD fused to the N-CoR/RIP13a-RID was expressed in cells with a
set of chimeric constructs containing full-length or various deletions
of the RVR receptor linked to the trans-activation domain of
VP16 (Fig. 1B). Analogously, this demonstrated that the E
region of RVR was necessary and sufficient to support the interaction
with the N-CoR/RIP13a-RID (Fig. 1D
). Deletion of 22 amino acids between
aa 394 and 416 or deletion of 15 aa between aa 561 and 576 also ablated
the receptor-corepressor (N-CoR/RIP13a) interaction. Deletion of the
amino acids between aa 572 and 576, however, only partially reduced
(
1.8-fold) the receptor-corepressor interaction.
In summary, these experiments demonstrated that the E region (but
not the hinge region) of RVR was necessary and sufficient to support
the interaction to three nuclear receptor corepressors, N-CoR and its
variants RIP13a and RIP131. Two domains in RVR denoted CIR-1 and -2
located in helix 3 and 11, respectively, are required to mediate
receptor-corepressor interaction. CIR-1 is a novel conserved
corepressor interaction region. CIR-2 shows homology with the amino
acid residues between aa 597 and 614 in Rev-erbA
(denoted the
Y domain) that support the interaction with N-CoR (21).
Binding of N-CoR, RIP13a, and RIP131 Is Dependent on the
Integrity of CIR-1 in Helix 3 of RVR
CIR-1 is strikingly conserved (90%) in RVR and Rev-erbA
(Fig. 2A
), suggesting a central role of
this region in the interaction with the corepressors and orphan
receptor function. The CIR-1 in RVR contains the amino acid residues
IWEEFSMSFTPAVKEVV between aa 398 and 415 (Fig. 2A
). To identify
specific amino acid residues in CIR-1 of RVR that contact the nuclear
receptor corepressors, we created single- or double-point mutations
that were subsequently examined for their ability to interact with the
RIP13
1 and N-CoR/RIP13a RIDs in the mammalian two-hybrid system
(Fig. 2B
). Two single-point mutations in full-length RVR were
constructed, RVR-E400K and RVR-F402P, and analyzed with respect to
wild-type RVR for their ability to interact with corepressor RIDs.
Consistent with the previous experiment (Fig. 1C
), we saw a very strong
interaction when the VP16-RVR chimera was transfected with the
GAL4-RIP13
1-RID (Fig. 2B
). Neither mutation ablated the
RVR-RIP13
1 interaction; however, the F402P mutation reduced the
strength of the interaction by
3-fold. In contrast, the F402P and
E400K mutations significantly effected the RVR-N-CoR/RIP13a
interaction. The F402P mutation reduced the interaction by
10-fold,
whereas the E400K mutation reduced the interaction by
3-fold.
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Similarly, we examined the potential of the three RVR CIR-1 mutants to
interact with the N-CoR/RIP13a-RID. In contrast to the effect of these
CIR-1 mutations on the RVR-RIP131 interactions, all three mutants
(E400K/E401A, F402A/S403A, and T407A/P408A) ablated the
RVR-N-CoR/RIP13a-RID interaction (Fig. 2D
).
These studies suggested that RVR interacts similarly with the RIP131
and N-CoR/RIP13a-RIDs, except that there may be a larger contact
interface between RVR-N-CoR/RIP13a than the RVR-RIP13
1 interface.
Furthermore, these mutagenesis studies reiterate the core role of CIR-1
in corepressor binding.
RIP131 and N-CoR/RIP13a Differentially Interact with
Rev-erbA
: The Hinge/D Region Has a Role in Corepressor Binding
We had previously demonstrated that full-length Rev-erbA
efficiently interacted with both the N-CoR/RIP13a-RID and
RIP13
1-RIDs (20). Furthermore, we demonstrated that efficient
interaction of Rev-erbA
with RIP13
1 was dependent on an intact E
region. We thus decided to investigate the specific regions and amino
acid residues within Rev-erbA
that were critical to the physical
association with N-CoR and RIP13a-RIDs.
The chimeric construct consisting of the GAL4 DBD fused to the
RIP131-RID (Fig. 3A
) was expressed in
cells with a set of chimeric constructs containing full-length or
various deletions of the Rev-erbA
receptor linked to the
trans-activation domain of VP16 (Fig. 3B
). Consistent with
the previous study, we saw a very strong interaction when VP16-Rev (aa
21614), Rev DE (aa 290614), and Rev E (aa 437614) were
cotransfected with the GAL4-RIP13
1-RID (Fig. 3C
).
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We similarly analyzed the potential of various domains from Rev-erbA
to interact with the N-CoR/RIP13a-RID in the mammalian two-hybrid
assay. The chimeric construct consisting of the GAL4 DBD fused to the
N-CoR/RIP13a-RID (Fig. 3A
) was expressed in cells with a set of
chimeric constructs containing full-length or various deletions of the
Rev-erbA
receptor linked to the trans-activation domain
of VP16 (Fig. 3B
). We saw a very strong interaction when the VP16-Rev
(aa 21614) was cotransfected with the GAL4-N-CoR/RIP13a-RID construct
(Fig. 3D
). Strikingly, further unidirectional deletions of Rev-erbA
(in an N- to C-terminal direction) from aa 290, aa 437, and aa 455
significantly reduced (
3-fold for VP16-Rev-DE, aa 290614) or
ablated the receptor-corepressor interaction (VP16-Rev E, aa 437614;
and VP16-Rev, aa 455614) (Fig. 3D
). Thus the E region of Rev-erbA
,
in contrast to the E region of RVR, was not sufficient to
support/mediate the physical association with N-CoR and RIP13a. Hence,
the D region of Rev-erbA
was required for the interaction with
N-CoR/RIP13a but not to RIP13
1-RIDs.
Interestingly, N-CoR studies to date have suggested that
interaction of N-CoR with TR/RAR is dependent on a highly conserved
domain, denoted the CoR box, that is found in the hinge region of TR,
RAR, and vitamin D receptor (17). The CoR box defined by mutagenesis
contains three invariant amino acids, A, H, and T. Rev-erb contains a
similar region between aa 283 and 300, which displays some homology
(last 10 aa) and retains the A and H residues but not the T residue.
We constructed an identical mutation in full-length Rev-erbA
,
denoted Rev-
CoR, that changed the invariant A, H, and I residues to
G, G, and A (Fig. 3B
) and linked it to the trans-activation
domain of VP16. This chimeric construct, VP16-Rev-
CoR, was
coexpressed in cells containing the GAL4 DBD fused to the RIP13
1-RID
(Fig. 3C
) and the GAL4 DBD fused to the N-CoR/RIP13a-RID (Fig. 3D
).
Consistent with our unidirectional deletion analysis, we observed that
a mutation in the CoR box of full-length Rev-erb did not significantly
affect the strong interaction with the RIP13
1 and N-CoR/RIP13a-RIDs,
in contrast to the significant effect caused by the deletion of CIR-1.
Thus, the CoR box is not involved in the interaction with the
corepressor RIDs.
These data demonstrate that Rev-erbA can form two different
interfaces that are required for the interaction of the receptor with
different corepressor isoforms. This provides the first functional
difference to date between the E regions of Rev-erbA
and RVR.
The CIR-1 of Rev-erbA Is Required for an Efficient Interaction
with the RIP13
1 Splice Variant
The CIR-1 in Rev-erbA is composed of the following amino
acid residues, IWEDFSMSFTPTVREVV between aa 437 and 456 (Fig. 4A
), and is
90% conserved with
respect to RVR. To investigate specific contact points/sites of
interaction, we made alanine substitutions of specific amino acid
residues within CIR-1 in the context of the E region (Fig. 4A
). We
constructed two mutant VP16 Rev E expression plasmids (from aa 437,
Fig. 4B
) that were denoted VP16-Rev E-F441A/S442A and VP16-Rev
E-T446A/P447A (Fig. 4B
). These constructs were then used in the
mammalian two-hybrid assay to investigate whether they could interact
with the RIP13
1-RID linked to GAL4 DBD. Mutation of F441A/S442A in
the CIR-1 ablated the receptor-RIP13
1 interaction, in contrast to
the T446A/P447A, which did not affect the Rev-erbA
-RIP13
1
corepressor interaction (Fig. 4C
).
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The Hinge Region and CIR-1 of Rev-erbA Are Both Required for an
Efficient Interaction with the N-CoR/RIP13a-RID
Our in vivo experiments demonstrated that the D region
and the E region of Rev-erbA are both required for the interaction
with the N-CoR/RIP13a-RID; however, the CoR box of Rev-erb is not
required for corepressor binding. Other studies have suggested that the
X domain of Rev-erbA
(between aa 407 and 418) in the hinge/D region
is involved in N-CoR binding, although the in vitro and
in vivo data are contradictory in this regard (21, 26).
We decided to investigate the role of the D region sequences and the
CIR-1 of Rev-erbA in the interaction with the N-CoR/RIP13a-RID (Fig. 4
, B and D). As can be seen in Fig. 4D
, the ability of the region
between aa 407 and 614 (that includes the X domain, but lacks the
majority of the D region) to interact with the N-CoR/RIP13a-RID is
reduced (
2-fold) with respect to the region between aa 290 and 614.
This suggested that the amino acids between aa 290 and 407 (that
include the CoR Box) affect the efficiency of corepressor binding.
Deletion of an additional 12 amino acids that removed the X domain only
slightly reduced the ability of the region to interact with the
receptor interaction domain (VP16-Rev, aa 419614, Fig. 4D
),
suggesting the X domain was not involved in corepressor binding. This
suggested that Rev-erbA
contained a third corepressor interaction
region (CIR-3) in the N-terminal D region.
We then examined whether CIR-1 in Rev-erbA was also important for
the interaction with the N-CoR/RIP13a-RID. We used the two CIR-1 mutant
VP16 Rev expression plasmids from Rev-aa407 that includes the X domain,
VP16-Rev aa407-F441A/S442A, and VP16-Rev aa 407-T446A/P447A (Fig. 4B
)
and used the mammalian two-hybrid assay to investigate whether they
could interact with the N-CoR/RIP13a-RID (Fig. 4D
). Mutation of
F441A/S442A in the CIR-1 ablated the receptor-corepressor interaction.
However, mutation of T446A/P447A did not significantly affect the
Rev-erbA
-N-CoR/RIP13a interaction. These data demonstrate that CIR-1
and an as yet undefined CIR-3 in Rev-erbA
are required for
N-CoR/RIP13a binding. Furthermore, the data demonstrate that
Rev-erbA
differentially interacts with N-CoR and its variants.
CIR-1 and CIR-2 in the E Region of RVR Mediate Transcriptional
Repression of a Physiological Target, the Human Rev-erbA Promoter:
Overexpression of the Corepressor RIDs Alleviates RVR-Mediated
Repression
RVR has previously been demonstrated to repress the
transcriptional activity of the human Rev-erbA promoter (14). Thus
we decided to investigate the importance of the E region, which
interacts with N-CoR and its variants, in RVR-mediated silencing of a
native physiological target, the human Rev-erbA
promoter. CIR-1
point mutants and CIR-2 deletions were compared with the ability of
native RVR to repress the transcriptional activity of the hRev-erbA
promoter linked to the luciferase reporter (14) in C2C12 cells.
Rev-erbA
and RVR are known to be expressed in mouse C2C12 muscle
cells and to repress the ability of these cells to differentiate.
Deletion of the E region significantly reduced (
6-fold) the ability
of RVR to silence luciferase activity of the hRev-erbA
promoter
(Fig. 5A
). The two-point mutants in
CIR-1, RVR-E400K and RVR-F402P, reduced the ability of RVR by
2.5-
and
5-fold, respectively, to repress luciferase activity of the
hRev-erbA
promoter. Deletion of the C-terminal amino acids in CIR-2
between aa 572 and 576, minimally affected the repression ability of
RVR, whereas deletion of the amino acids between aa 561 and 576 reduced
the silencing ability of RVR by
3-fold. These transfection
experiments demonstrated the importance of CIR-1 and CIR-2 in RVR
function.
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We then investigated the ability of dominant-negative corepressor
expression vectors that contained the corepressor-RIDs but no
repression domains (i.e. pSG5-RIP131-RID and
pSG5-N-CoR/RIP13a-RID) to affect the orphan receptor-mediated
repression of the hRev-erbA
promoter (Fig. 5C
). As seen in Fig. 5C
, overexpression of the corepressor RIDs completely abolished repression
by RVR, thus showing that these RIDs could function as antirepressors.
The native RIP13a and RIP13
1 expression vectors that contained the
functional RIDs and repression domains did not function as
antirepressors (data not shown). These experiments clearly demonstrate
that the corepressor binding is involved in the repression of the
Rev-erbA
promoter by RVR, and that the corepressor RIDs interacted
with the orphan receptors by a different assay.
We then examined the ability of coexpressed full-length N-CoR
(pSG5-N-CoR) to augment repression of the Rev-erbA promoter by
native and mutant RVRs (Fig. 5D
). As seen in Fig. 5D
, overexpression of
N-CoR significantly/synergistically enhances the ability of RVR,
RVR-E400K, and RVR-
572576 to repress promoter expression. In
contrast and comparison, N-CoR has much weaker effects (only additive)
on the ability of RVR-F402P and RVR-
561576 to repress promoter
expression. Interestingly, this correlates with the ability of these
receptors to bind N-CoR in vitro (presented in Fig. 6
); briefly, RVR, RVR-E400K, and
RVR-
572576 efficiently bind the N-CoR/RIP13-RIDs in
vitro, whereas, RVR-F402P and RVR-
561576 have impaired
binding.
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In Vitro Interaction Assays Demonstrate that CIR-1
and CIR-2 Are Required for Corepressor Binding
The demonstration of interaction between RVR and the corepressors
and the characterization of CIR-1 and CIR-2 in the in vivo
mammalian two-hybrid assay strongly suggest these proteins may interact
by a direct mechanism. However, this does not eliminate the possibility
of an indirect mechanism in which additional factor(s) mediate the
interaction. We tested this hypothesis using a biochemical approach,
the in vitro GST pulldown assay, to confirm the direct
interaction between RVR and the RIP131-RID and N-CoR/RIP13a and to
verify the existence/importance of CIR-1/CIR-2 to the RVR-corepressor
interaction.
Glutathione agarose-immobilized GST-RIP131-RID and the
GST-N-CoR/RIP13a-RID were tested for direct interaction with in
vitro 35S-radiolabeled full-length native RVR,
RVR-E400K, and RVR-F402P. GST-RIP13
1- and N-CoR/RIP13a-RIDs showed a
direct interaction with full-length RVR (Fig. 6
, A and B,
respectively). Similarly the RVR-E400K point mutant also interacted
with both corepressor RIDs. In contrast, the RVR-F402P point mutant
failed to interact efficiently with the RIP13
1 and N-CoR/RIP13a-RIDs
(Fig. 6
, A and B, respectively). These direct in vitro
binding data verify that RVR directly interacts with RIP13
1, RIP13a,
and N-CoR. Furthermore, it demonstrates that the F402P mutation in
CIR-1 destroys the interaction with the corepressors, which correlates
with reduced or ablated ability of RVR-F402P to interact in the
mammalian two-hybrid assay, and the inability of RVR-F402P to repress
transcription.
We then examined the ability of in vitro35S-radiolabeled RVR carrying two deletions in the
CIR-2 region, RVR572576 and RVR
561576, to interact with the
immobilized GST-RIP13
1 and N-CoR/RIP13a-RID fusion proteins.
Deletion of the amino acids between 572 and 576 did not affect the
ability of RVR to interact directly with the corepressor-RIDs (Fig. 6C
). However, deletion of the amino acids between 561 and 576 reduced
the specificity and binding of RVR for the RIP13
1- and
N-CoR/RIP13a-RIDs, respectively (Fig. 6
, C and D). These data
demonstrate that the deletion (
561576) in CIR-2 that ablates the
ability to interact in the mammalian two-hybrid assay, and to repress
transcription of the human Rev-erbA
promoter, affects the strength
and specificity of corepressor binding.
We then investigated the ability of in vitro35S-radiolabeled RVR E region and RVR-E-T407A/P408A to
interact with the immobilized GST-RIP131-RID. The E region of RVR
interacts directly with the corepressor RID (Fig. 6E
). However, the
T407A/P408A mutation impaired, but did not ablate, the binding of the
RVR-E region for the RIP13
1-RID, which correlated with the mammalian
two-hybrid data in Fig. 2C
.
We then used the GST-pulldown assay to demonstrate that
mutations/deletion in CIR-1 of Rev-erbA that impaired interactions
in vivo, and the repression of the Rev-erbA
promoter also
affected in vitro binding. In vitro35S-radiolabeled Rev-erb and Rev-F441A/S442A were
tested for their ability to interact with immobilized
GST-N-CoR/RIP13a-RID. Mutation of F441A/S442A in Rev-erb impaired
in vitro binding (Fig. 6F
), which correlated with the two-
hybrid assay and the repression assay. We then compared the ability of
the Rev-erb E region (Rev-E-aa 437614) and a CIR-1-deleted Rev-erb E
region (Rev-E-aa 455614) to bind immobilized GST-RIP13
1-RID.
Deletion of CIR-1 in Rev-erbA between amino acid residues 437 and 455
significantly reduced the ability of Rev-erb to bind the RIP13
1-RID
(Fig. 6G
). Finally, we investigated the ability of Rev-erb carrying a
mutation in the CoR box (Rev-
CoR) to interact with the N-CoR/RIP13a
and RIP13
1 RIDs in vitro. We found that a mutated CoR box
did not affect the ability of Rev-erbA to interact directly with the
corepressor-RIDs (Fig. 6H
). This correlated with the mammalian
two-hybrid assay and the promoter repression assay that demonstrated
the CoR box mutation did not affect in vivo binding to the
corepressor and that the ability of Rev-erbA
to repress its own
promoter was not affected.
Juxtapositioning of CIR-1 and CIR-2 in RVR Leads to Binding of
Corepressors and Transcriptional Repression of the Human
Rev-erbA Promoter
We predicted from the 3D-tertiary structure of the nuclear
receptors RXR, TR, and RAR (22, 23, 24) that CIR-1 and CIR-2 may be
juxtaposed in the tertiary structure of the E region of the Rev-erb
proteins. To examine this, we created an RVR chimera, RVR425556,
that deletes a large portion of the E region to bring CIR-1 and CIR-2
in close proximity (Fig. 7A
). This
construct repressed transcription of the hRev-erbA
promoter linked
to the luciferase reporter as efficiently as native RVR (Fig. 7B
),
indicating the importance of CIR-1 and CIR-2 in repression by RVR. We
also investigated whether in vitro35S-radiolabeled RVR
425556 could bind to the
immobilized GST-RIP13
1-RID and N-CoR/RIP13a-RID fusion proteins. The
chimera, RVR
425556, bound efficiently to both GST-RIP13
1-RID
and N-CoR/RIP13a-RID in vitro. This suggests that CIR-1 and
CIR-2 may be sufficient for corepressor binding to RVR and juxtaposed
in the E region of RVR, forming a single contact interface for the
corepressors.
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DISCUSSION |
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We demonstrated that the E region (that begins with helix 3) of RVR is
necessary and sufficient for the in vivo interaction with
three nuclear receptor corepressors, N-CoR, RIP13a, and RIP131.
Although the E region of Rev-erbA
is necessary and sufficient for
the interaction with RIP13
1, the D region of Rev-erbA
is required
for the physical association with N-CoR and the variant RIP13a. This
suggested that N-CoR/RIP13a and RIP13
1 differentially interact with
Rev-erbA
.
We identified, by in vivo and in vitro
biochemical techniques, two corepressor interaction regions in RVR,
CIR-1 and CIR-2 in helix 3 and helix 11 of the E region, respectively,
that are both required for an efficient interaction with the
N-CoR/RIP13a and RIP131 RIDs. These regions are separated by
150
aa. Furthermore, these regions can mediate corepressor binding and
transcriptional repression when placed in close proximity to one
another. Therefore, we hypothesize that CIR-1 and CIR-2 are juxtaposed
in the putative 3D-tertiary structure of these orphan receptors and
probably form a single-contact interface that interacts with three
nuclear receptor corepressors, N-CoR, RIP13a, and RIP13
1. However,
further experimentation is required to verify this hypothesis.
Interestingly, we had previously shown that helix 3 in RVR (10) and
Rev-erbA (25) was involved in the repression of GAL4VP16-mediated
trans-activation by these orphan receptors. CIR-1, in the
N-terminal part of helix 3, is a novel domain that is strikingly
conserved between RVR and Rev-erbA
. CIR-2 in helix 11 of RVR is
homologous to the previously described Y domain in Rev-erbA
(21)
that is required for the interaction of Rev-erbA
with N-CoR.
Site-specific mutagenesis of CIR-1 in RVR and Rev-erbA
demonstrated
that CIR-1 plays a critical role and is necessary for the in
vivo/vitro interaction with the N-CoR, RIP13a, and
RIP13
1 RIDs. These mutagenesis studies suggested that the orphan
receptor interaction interface with N-CoR/RIP13a is larger than with
RIP13
1. Furthermore, the data suggested that N-CoR/RIP13a and
RIP13
1 differentially interact with Rev-erbA
.
Studies to date have suggested that N-CoR interacts with the CoR box of
TR/RAR and the X domain of Rev-erbA (aa 407419), both located in
the D region of the nuclear receptors. The CoR box was characterized by
functional and biochemical assays (17). Although GST-pulldown assays
initially identified the X domain as an N-CoR-binding site (21),
functional analysis of this domain after complete alanine substitution
strongly suggested it was not required for corepressor binding (26).
Our studies clearly demonstrate that neither RVR nor Rev-erbA
require the D region that includes the CoR box (in helix 1) and the X
domain for binding to the N-CoR variant, RIP13
1. These observations
are in agreement with the hypothesis put forward by Wurtz et
al. (27), who argued it was unlikely that N-CoR interacted with
helix 1 because the triple mutation used to map N-CoR binding would
disrupt the interaction of helix 1 with the LBD core and dislodge helix
1 from its wild-type position. Furthermore, the specified amino acids
are engaged in internal contacts and buried inside the receptor.
In contrast, the E region of Rev-erbA was necessary but not
sufficient for the interaction with the N-CoR and RIP13a RIDs
in vivo. Efficient interaction with N-CoR and RIP13a was
dependent on the D and E regions. Our studies suggest that there exists
a third, as yet undefined, CIR in the D region of Rev-erbA
,
N-terminal of aa 407. Furthermore, deletion/mutation studies in the 1)
mammalian two-hybrid system, 2) transfection system, and 3)
GST-pulldown assay rigorously show that the CoR box in the D region of
Rev-erbA
is not required for corepressor binding and does not encode
the, as yet undefined, CIR-3. Alternatively, a hypothesis analogous to
that proposed by Wurtz et al. (27) could be suggested. The D
region of Rev-erbA
, unlike that of RVR, may contribute to the
positioning of CIR-1/CIR-2 within the E region of Rev-erbA
, allowing
N-CoR/RIP13a binding. However, this study and the work from Zamir
et al. (26) now suggest that if a third CIR exists it does
not involve the CoR box or X domain of Rev-erbA
. Therefore, our
studies have demonstrated a number of important functional differences
between the
- and the ß-isoforms of Rev-erb with respect to
regions and residues required for N-CoR/RIP13a binding.
The ability of RVR to function as a dominant transcriptional silencer
on a physiological target, the human Rev-erbA promoter, is dependent
on the CIR-1 and CIR-2 domains in the E region. This correlates with
the requirement of these regions for corepressor binding. Furthermore,
overexpression of the N-CoR/RIP13a-RID and RIP13
1-RID operated in a
dominant negative manner and blocked RVR-mediated repression of this
promoter. This confirmed that corepressors mediate transcriptional
repression by the Rev-erb family. Furthermore, coexpression of N-CoR
produced synergistic transcriptional repression only with native RVR
and mutants that still bound N-CoR in vitro. This
synergistic repression was not observed in proteins that were impaired
with respect to binding (e.g. RVR-F402P and
RVR-
561576). The functional significance of the
corepressor-binding region has been demonstrated during myogenic
differentiation of C2C12 cells in culture. RVR mRNA is detected in
proliferating myoblasts and is repressed when the cells differentiate
into postmitotic multinucleated cells. This decrease in RVR mRNA
correlates with the appearance of muscle-specific markers (myogenin and
contractile proteins mRNAs) and the induction of the Cdk inhibitor
p21Cip-1/Waf-1 mRNA. Constitutive overexpression of an RVR
construct lacking the E region (i.e. functional silencing
and corepressor interaction domains) in these cells resulted in
precocious morphological and biochemical differentiation of these cells
in culture. Specifically, increased accumulation and precocious
induction of myogenin and p21Cip-1/Waf-1 were observed
(10).
We speculate from the structural studies from Moras, Gronemeyer,
Chambon, and colleagues on the retinoid nuclear receptor LBDs (Ref. 21
and references therein) that the Rev-erb proteins would form an
apo-like orphan receptor LBD [that does not contain helix 12] with a
canonical structure/cavity with a hydrophobic lining. However, the lack
of a holo-LBD type lid structure, normally formed by H11 and H12, would
not carry the N-terminal part of H3 into the ligand-binding cavity.
Therefore, CIR-1 situated at the N-terminal part of H3 and CIR-2 would
not be buried inside the receptor. The differential effects of the
CIR-1 mutants on RIP131 vs. N-CoR/RIP13a binding in
vivo and in vitro support the notion that the CIR-1
interacts directly with the corepressors and forms a true contact
interface. 3D-analysis of the Rev-erb family of orphan receptors will
directly answer these questions.
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MATERIALS AND METHODS |
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5'-GCG AATTCACCATGGTNAAA/GA/TCNAAG/AAAG/ACA-3'
GMUQ297: 5'-GCGAATTCACCNCA/TA/GTCNG/CA/TNAA/ GNGTT/CTCG/ATAT/CTG-3'
GMUQ303: 5'-GCGCGTCGACATATGTTTGCA/CAAG/AA/CG/AGATT/CCCT/CGGC-3'
GMUQ340: 5'-GCGGAATTCACCATGTCAAGTTCAGGTTATCCT-3'
GMUQ342: 5'-GCGGAATTCAGACTTGCTGGAAGAAACATC-3'
GMUQ345: 5'-GCGTCGACTCTGGAAAGCA/CTTC/TTCA/ TATGAGC/TTTC/TACG-3'
GMUQ346: 5'-GCGTCGACTCTGGGAAGAA/CGCC/TTCA/ TATGAGC/TTTC/TAC-3'
GMUQ347: 5'-GCGTCGACTCTGGGAAGAA/CTTC/TTCA/ TATGAGC/TTTC/TGCA/TGTA/GA/CA/GA/GGAG-3'
GMUQ352: 5'-GCGGTCGACTAACCATGGCGCAGACGCAGGGC-3'
GMUQ353: 5'-GCGGTCGACTCAGGCCAACTTGACCTCCTCC-3'
GMUQ354: 5'-GCGTCAGATCTGGGAAG-3'
GMUQ355: 5'-GCGTCAGATCTGGAAAG-3'
GMUQ376: 5'-GCGTCGACTTACCATGCGGCAAGGCAACACCAAG-3'
GMUQ377: 5'-GCGTCGACTTACCATGCCCATGAACATGTATCCC-3'
GMUQ463: 5'-CCCAGGTGGCCAGGGGCGGTCGAGAAG-CCTTCACCTATGCCC-3'
GMUQ464: 5'-GGGCATAGGTGAAGGCTTCTCGACCGC- CCCTGGCCACCTGGG-3'
GMUQ581: 5'-GGACATGAAATCTGGAAAGAATTTTCAAT-GAGTTTTACCC
GMUQ582: 5'-GGGTAAAACTCATTGAAAATTCTTTCCAGATTTCATGTCC
GMUQ583: 5'-GGGAAGAACCTTCAATGAGTTTTACCC
GMUQ584: 5'-GGGTAAAACTCATTGAAGGTTCTTCCC
Plasmids
The expression plasmids pGAL0 (28), pNLVP16 (29), pG5E1bCAT
(30), and pRev-erbAWT (14), which contains the human Rev-erbA
promoter linked to luciferase, have been described previously. pGAL0
contains the yeast GAL4-DBD, and pNLVP16 contains the acidic activation
domain of VP16. All PCR amplifications were performed with
Pfu DNA (Stratagene, La Jolla, CA) or Pwo DNA
polymerase (Boehringer Mannheim, Indianapolis, IN) according to the
manufacturers instructions. End-filling reactions were performed with
Klenow DNA polymerase according to the manufacturers instructions.
All pBluescript and pBS clones and GAL and VP16 chimeras were sequenced
by double-stranded sequencing to verify identity and confirm the
reading frame.
Full-length mouse N-CoR cDNA was amplified from pBluescript SK-NCoR (kindly provided by C. Glass and W. Seol) using the two primers, GMUQ340 and GMUQ297. The product was cleaved with EcoRI, and the resulting fragment was ligated to pSG5/EcoRI to form pSG5-mN-CoR.
To construct pVP16-RVR E aa 394576 and pGAL4-RVR E aa 394576, RVR
aa 394576 was amplified from GV-RVR aa 355576 (10) using the two
primers, GMUQ301 and GMUQ252. The product was ligated to pBluescript
KS/EcoRV to create pBluescript-RVR aa 394576. Antisense
clones were cleaved with SalI, and the resulting fragment
was ligated into pNLVP16/SalI and pGAL0/SalI. To
construct the following pVP16-RVR chimeric expression vectors, primers
were used to amplify regions of RVR from pGAL4-RVR E aa 394576;
pVP16-RVR-E-E400K/E401A (GMUQ345 and GMUQ252), pVP16-RVR
E-F402A/S403A (GMUQ346 and GMUQ252), pVP16-RVR E-T407A/P408A (GMUQ347
and GMUQ252), and pVP16-RVR aa 416576 (GMUQ303 and GMUQ252). These
products were ligated to pBluescript KS/EcoRV to create
pBluescript-RVR aa 398576 mutants and pBluescript-RVR aa 416576.
Antisense clones were cleaved with SalI, and the resulting
fragments were ligated to pNLVP16/SalI. To construct
pVP16-RVR aa 394572 and pVP16-RVR aa 394561, antisense
pBluescript-RVR aa 394576/SalI was partially digested with
DraI, and the required fragments were isolated, end-filled,
and ligated to pNLVP16/NdeI. To construct
pSG5-RVR572576 and pSG5-RVR
561576, pVP16-RVR aa 394572 and
pVP16-RVR aa 394561 were digested with
HincII/XbaI, and the resulting fragments were
ligated to pBS/HincII/XbaI. These pBS clones were
cut with HincII/SmaI, and the resulting fragments
were ligated to pBS-RVR cleaved with HincII to create
pBS-RVR aa 1572 and pBS-RVR aa 1561. These pBS clones were cleaved
with BamHI and ligated to pSG5/BamHI.
pSG5-RVR-E400K and pSG5-RVR-F402P plasmids were constructed with primers that contain the amino acid substitutions E400K (GMUQ581 and GMUQ582) and F402P (GMUQ583 and GMUQ584), respectively, using a Quik-Change site mutagenesis kit (Stratagene) according to the manufacturers instructions.
To construct the following pVP16-Rev chimeric expression vectors,
primers were used to amplify regions of Rev-erbA from pVP16-Rev aa
290614 (25); pVP16-Rev aa 455614 (GMUQ303 and GMUQ132); pVP16-Rev
aa 419416 (GMUQ377 and GMUQ 132); and pVP16-Rev aa 407614 (GMUQ376
and GMUQ132). These products were ligated to pBluescript
KS/EcoRV, antisense clones were cleaved with
SalI, and the resulting fragments were ligated to
pNLVP16/SalI. For construction of the following pVP16-Rev
chimeric expression vectors, primers were used to amplify regions of
Rev-erbA
from pVP16-Rev aa 347614; pVP16-Rev-E-F441A/S442A
(GMUQ346 and GMUQ132); and pVP16-Rev-E-T446A/P447A (GMUQ347 and
GMUQ132). These products were ligated to pBluescript
KS/EcoRV, antisense clones were cleaved with
SalI, and the resulting fragments were ligated to
pNLVP16/SalI. PCR fragments were amplified from the
following chimeras with primers: pVP16-Rev-E-F441A/S442A (GMUQ354 and
GMUQ132); and pVP16-Rev-E-T446A/P447A (GMUQ354 and GMUQ132). These
products contained the BglII site normally found at 1305 bp
in Rev-erbA
and were cut with BglII/BamHI and
ligated to pSG5-Rev
E/BglII to construct full-length
pSG5-Rev-erbA
clones carrying these mutations, denoted
pSG5-Rev-erbA
-F441A/S442A and pSG5-Rev-erbA
T446A/P447A. Mutant
Rev aa 407614 fragments (Rev-407-F441A/S442A and Rev-407-T446A/P447A)
carrying mutations in the CIR-1 were amplified from the full-length
mutated pSG5-Rev-erbA
plasmids with the primers GMUQ376 and GMUQ132
and ligated to pBluescript KS/EcoRV. Antisense clones were
cleaved with SalI, and the resulting fragments were ligated
to pNLVP16/SalI to create pVP16-Rev-407-F441A/S442A and
pVP16-Rev-407-T446A /P447A.
pSG5-Rev-CoR and pVP16-Rev-
CoR plasmids that contain the amino
acid substitutions A295/H296/I299 to G295/G296/A299 were constructed
with the primers GMUQ463 and GMUQ464 using a QuikChange site
mutagenesis kit according to the manufacturers instructions.
All other plasmids and primers have been described previously (10, 20, 25).
Mammalian Two-Hybrid Assay
Each well of a six-well plate of JEG-3 cells (6070%
confluence) was cotransfected with 3 µg pG5E1bCAT reporter, 1 µg
GAL chimeras, and 1 µg VP16 chimeras in 1 ml DMEM containing 5%
charcoal-stripped FCS by the
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium
methylsulfate (DOTAP) (Boehringer Mannheim) mediated procedure as
described previously (31, 32). After 24 h, the medium was replaced
and cells were harvested for the assay of chloramphenicol
acetyltransferase (CAT) activity 3648 h after transfection. Each
transfection was performed at least three times to overcome the
variability inherent in transfections.
C2C12 Transfection
Each well of a 12-well plate of C2C12 cells (6070%
confluence) was cotransfected with 1.0 µg of pRev-erbA WT and
0.331.0 µg of pSG5 constructs in 1 ml phenol red-free DMEM
containing 10% FCS by the DOTAP-mediated procedure as above. The
amount of DNA in each transfection was kept constant by addition of
pSG5. After 24 h, the medium was replaced, and cells were
harvested for the assay of luciferase activity 3648 h after
transfection. Each transfection was performed at least three times to
overcome the variability inherent in transfections.
CAT Assays
Cells were harvested and CAT activity measured as described
previously (33). Aliquots of cell extracts were incubated at 37 C, with
0.10.4 µCi of [14C]chloramphenicol (ICN Nutritional
Biochemicals, Cleveland, OH) in the presence of 5 mM
acetyl-CoA in 0.25 M Tris-HCl, pH 7.8. After a 1- to 4-h
incubation period, 1 ml ethyl acetate was used to extract the
chloramphenicol and its acetylated forms. Extracted materials were
analyzed on Silica gel TLC plates. Quantification of all CAT assays was
performed with an AMBIS ß-scanner (AMBIS, Inc., San Diego, CA).
Luciferase Assays
Luciferase activity was assayed using a Luclite kit (Packard
Instruments, Meriden, CT) according to the manufacturers
instructions. Briefly, cells were washed once in PBS and resuspended in
150 µl phenol red-free DMEM and 150 µl Luclite substrate buffer.
Cell lysates were transferred to a 96-well plate, and relative
luciferase units were measured for 5 sec in a Wallac Trilux 1450
microbeta luminometer (Wallac, Gaithersburg, MD).
In Vitro Binding Assays
GST and GST-fusion proteins were expressed in Escherichia
coli (BL21) and purified using glutathione-agarose affinity
chromatography as described previously (32). The GST-fusion proteins
were analyzed on 10% SDS-PAGE gels for integrity and to normalize the
amount of each protein. The Promega (Madison, WI) TNT-coupled
transcription-translation system was used to produce
[35S]methionine-labeled RVR proteins that were visualized
by SDS-PAGE. In vitro binding assays were performed with
glutathione-agarose beads (Sigma, St. Louis, MO) coated with 500 ng
GST-fusion protein and 230 µl [35S]methionine-labeled
protein in 200 µl of binding buffer containing 100 mM
NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5%
Nonidet P-40, 5 µg ethidium bromide, and 100 µg BSA. The reaction
was allowed to proceed for 12 h at 4 C with rocking. The affinity
beads were then collected by centrifugation and washed five times with
1 ml of binding buffer without ethidium bromide and BSA. The beads were
resuspended in 20 µl SDS-PAGE sample buffer and boiled for 5 min. The
eluted proteins were fractionated by SDS-PAGE, and the gel was treated
with Amersham Amplify fluor (Amersham, Arlington Heights, IL), dried at
70 C, and autoradiographed.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This investigation was supported by the National Health and Medical Research Council (NHMRC) of Australia. The Centre for Molecular and Cellular Biology is the recipient of a Australia Research Council (ARC) special research grant. G. Muscat is a Senior Research Fellow of the NHMRC.
Received for publication August 20, 1997. Revision received October 30, 1997. Accepted for publication November 5, 1997.
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REFERENCES |
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