Identification and Characterization of a Novel Corepressor Interaction Region in RVR and Rev-erbA{alpha}

Les J. Burke1, Michael Downes1, Vincent Laudet and George E. O. Muscat

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Rev-erbA{alpha} and RVR are orphan nuclear receptors that function as dominant transcriptional silencers. Ligand-independent repression of transcription by Rev-erbA{alpha} and RVR is mediated by the nuclear receptor corepressors, N-CoR and its variants RIP (RXR interacting protein) 13a and RIP13{Delta}1. The physical association between the corepressors and Rev-erbA{alpha} and RVR is dependent on the presence of a receptor interaction domain (RID) in the N-CoR family. Our previous study demonstrated that the E region of RVR and Rev-erbA{alpha} is necessary and sufficient for the in vivo interaction with the nuclear receptor corepressor, RIP13{Delta}1. The present investigation demonstrates that two corepressor interaction regions, CIR-1 and CIR-2, separated by ~150 amino acids in the E region of RVR, are required for the interaction with N-CoR, RIP13a, and RIP13{Delta}1. The D region is not required for the physical interaction. In contrast, the D and E regions of Rev-erbA{alpha} were necessary for the interaction with the N-CoR and RIP13a-RIDs in vivo, suggesting that RIP13{Delta}1 and N-CoR/RIP13a differentially interact with Rev-erbA{alpha}. Mutagenesis of CIR-1, a novel domain that is highly conserved between RVR and Rev-erbA{alpha}, demonstrated that the N-terminal portion of helix 3 plays a key role and is absolutely necessary for the interaction with RIP13{Delta}1, RIP13a, and N-CoR. The phenylalanine residues, F402 and F441, in RVR and Rev-erbA{alpha}, respectively, were critical residues in supporting corepressor interaction. Cotransfection studies demonstrated that repression of a physiological target, the human Rev-erbA{alpha} promoter, by RVR was significantly impaired by mutation of CIR-1 or deletion of CIR-2. Furthermore, overexpression of either the N-CoR/RIP13a or RIP13{Delta}1-RIDs alleviated RVR-mediated repression of the Rev-erbA{alpha} promoter, demonstrating that corepressor binding mediates the repression of a native target gene by RVR. A minimal region containing juxtapostioned CIR-1 and CIR-2 was sufficient for corepressor binding and transcriptional repression. In conclusion, our study has identified a new corepressor interaction region, CIR-1, in the N terminus of helix 3 in the E region of RVR and Rev-erbA{alpha}, that is required for transcriptional silencing. Furthermore, we provide evidence that CIR-1 and CIR-2 may form a single corepressor interaction interface.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Members of the nuclear receptor superfamily bind specific DNA elements and can function as ligand-activated transcription factors (1, 2). This group includes the orphan receptors, which have no known ligands in the classical sense and appear to be the ancient progenitors of this receptor superfamily (3). The Rev-erb family of proteins, Rev-erbA{alpha}/ear-1 and RVR/Rev-erbß/BD73, are orphan members of this superfamily. These two proteins are highly conserved in the DNA-binding domain (DBD) (95%) and the E region (75%); however, major differences between the two isoforms occur within the hypervariable A/B and D regions of the proteins. These orphan receptors are closely related to the ROR/RZR gene family (retinoic acid receptor-related orphan receptor) and the Drosophila orphan receptor, E75A, particularly in the DBD and E region (4, 5, 6, 7, 8).

The mRNAs encoding Rev-erbA{alpha} 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{alpha} 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{alpha} and RVR mRNAs is repressed. In contrast, in adipocyte cells the expression of Rev-erbA{alpha} 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{alpha} 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{alpha} and RVR (97%). This region spans the ligand-binding domain (LBD)-specific signature motif, (F/WAKXXXXFXXLXXXDQXXLL), helix 3, loop 3–4, 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{alpha}/RVR is also mediated by the corepressor N-CoR and its variants RIP13a and RIP13{Delta}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{alpha} interact very efficiently with the RID from N-CoR and RIP13a, although they preferentially interact with the RID from the RIP13{Delta}1 isoform (20). Furthermore, it was demonstrated that the E region of RVR and Rev-erbA{alpha} was necessary and sufficient for the interaction with the RIP13{Delta}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{alpha} that interact with N-CoR and its variants, RIP13a and RIP13{Delta}1. These experiments identified two corepressor interaction regions (CIRs) within helices 3 and 11 in the E region of RVR and Rev-erbA{alpha}, 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{alpha} and is absolutely required for the interaction with N-CoR, RIP13a, and RIP13{Delta}1. Although the E region of Rev-erbA{alpha} was necessary and sufficient for the interaction with RIP13{Delta}1, the D region was also required for N-CoR and RIP13a binding. This suggested that N-CoR/RIP13a and RIP13{Delta}1 differentially interact with Rev-erbA{alpha}. Furthermore, the ability of RVR to function as dominant transcriptional silencer on a physiological target, the human Rev-erbA{alpha} promoter, was dependent on CIR-1, CIR-2, and corepressor binding.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Two Domains in the E Region of RVR Are Required for the Interaction with the Nuclear Receptor Corepressors, RIP13{Delta}1, RIP13a, and N-CoR
We had previously demonstrated that full-length RVR and Rev-erbA{alpha} efficiently interacted with the RIDs (see Fig. 1AGo) from N-CoR, RIP13a, and RIP13{Delta}1. The RIDs from N-CoR and RIP13a are identical; however, the RID from RIP13{Delta}1 has an internal deletion of 120 amino acids (18). Furthermore, we demonstrated that the E region of both these orphans was necessary and sufficient for the interaction with RIP13{Delta}1 (20). We embarked on further mammalian two-hybrid analysis, site-specific mutagenesis, and direct in vitro binding assays to rigorously identify the specific domains and amino acid residues within the E region that were critical to the physical association with the N-CoR family.



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Figure 1. Identification of the Domains of RVR Necessary for Interaction with the RIDs of RIP13{Delta}1 and N-CoR-RIP13a

A, Schematic representation of the various corepressor(s) and RIDs used in this study. B, Schematic representation of the VP16-RVR chimeras used in the mammalian two-hybrid assay. The boundaries of the AB, C, D, and E regions are denoted. CIR-1 and CIR-2 are shown and are located between aa 394 and 416, and between aa 561 and 576, respectively. C, JEG-3 cells were cotransfected with the indicated plasmids (+) in the presence of pG5E1bCAT reporter plasmid and assayed for CAT activity. Results shown are mean ± SD and were derived from three independent transfections. Fold activation is expressed relative to CAT activity obtained after transfection of GAL4-RIP13{Delta}1-RID and the VP16 vector alone arbitrarily set to 1.0. D, JEG-3 cells were cotransfected with the indicated plasmids (+) in the presence of pG5E1bCAT reporter plasmid and assayed for CAT activity. Results shown are mean ± SD and were derived from three independent transfections. Fold activation is expressed relative to CAT activity obtained after transfection of GAL4-N-CoR/RIP13a-RID and the VP16 vector alone arbitrarily set to 1.0.

 
Hence, we investigated the potential of various deletions from the RVR E region to interact with the RIP13{Delta}1-RID in the mammalian two-hybrid assay. The chimeric construct consisting of the yeast GAL4 DBD fused to the RIP13{Delta}1-RID was expressed in cells with a set of chimeric constructs containing full-length or various deletions of RVR linked to the trans-activation domain of VP16. We examined the impact of fine N- and C-terminal deletions of the E region (Fig. 1BGo) because 1) they encode the amino acid residues that would putatively encode helix 3 and helix 11; 2) the three-dimensional (3D)-tertiary structure of the nuclear receptors RXR, TR, and RAR (22, 23, 24) would suggest that these domains are in close proximity; and 3) helix 3 is crucial to transcriptional repression (10, 20, 25). Consistent with the previous study, the RVR E region [amino acids (aa) 394–576] was necessary and sufficient for the interaction with RIP13{Delta}1-RID (Fig. 1CGo). Deletion of ~20 amino acids between aa 394 and 416 in the N-terminal E region (in helix 3) ablated the receptor-corepressor interaction. Similarly, deletion of 15 amino acids between aa 561 and 576 in the C-terminal E region (in helix 11) ablated the receptor-corepressor (RIP13{Delta}1) interaction, whereas deletion of the four amino acids between aa 572 and 576 did not affect the receptor-corepressor interaction.

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. 1BGo). Analogously, this demonstrated that the E region of RVR was necessary and sufficient to support the interaction with the N-CoR/RIP13a-RID (Fig. 1DGo). 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 RIP13{Delta}1. 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{alpha} (denoted the Y domain) that support the interaction with N-CoR (21).

Binding of N-CoR, RIP13a, and RIP13{Delta}1 Is Dependent on the Integrity of CIR-1 in Helix 3 of RVR
CIR-1 is strikingly conserved (~90%) in RVR and Rev-erbA{alpha} (Fig. 2AGo), 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. 2AGo). 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{Delta}1 and N-CoR/RIP13a RIDs in the mammalian two-hybrid system (Fig. 2BGo). 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. 1CGo), we saw a very strong interaction when the VP16-RVR chimera was transfected with the GAL4-RIP13{Delta}1-RID (Fig. 2BGo). Neither mutation ablated the RVR-RIP13{Delta}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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Figure 2. Characterization of the CIR-1 of RVR Necessary for Interaction with the RIP13{Delta}1 and the N-CoR/RIP13a-RIDs

A, The sequence and alignment of the CIR-1 and CIR-2 of RVR and the corresponding region of Rev-erbA{alpha}. Identical amino acids are indicated by dots. Mutations introduced into the CIR-1 of RVR are shown. B, Point mutation of the CIR-1 of full-length RVR disrupts binding to corepressor-RIDs. JEG-3 cells were cotransfected with the indicated plasmids (+) in the presence of pG5E1bCAT reporter plasmid and assayed for CAT activity. Results shown are mean ± SD and were derived from three independent transfections. Fold activation is expressed relative to CAT activity obtained after transfection of the GAL4-corepressor RID and the VP16 vector alone arbitrarily set to 1.0. C, Mutation of the CIR-1 of RVR E disrupts binding of RIP13{Delta}1-RID. JEG-3 cells were cotransfected with the indicated plasmids (+) in the presence of pG5E1bCAT reporter plasmid and assayed for CAT activity. Results shown are mean ± SD and were derived from three independent transfections. Fold activation is expressed relative to CAT activity obtained after transfection of GAL4-RIP13{Delta}1-RID and the VP16 vector alone arbitrarily set to 1.0. D, Mutation of the CIR-1 of RVR E disrupts binding of N-CoR/RIP13a-RID. The mutations in the CIR-1 of RVR shown in panel A were tested for the ability to bind to N-CoR/RIP13a-RID in the mammalian two-hybrid assay.

 
We then constructed a number of double-point mutations in CIR-1 and examined their effect on the ability of the E region to interact with the corepressor RIDs. The three mutants were denoted VP16-RVR E-E400K/E401A, VP16-RVR E-F402A/S403A, and VP16-RVR E-T407A/P408A. Consistent with the previous experiment (Fig. 1CGo), we saw a very strong interaction when the VP16-RVR E chimera was transfected with the GAL4-RIP13{Delta}1-RID construct (Fig. 2CGo). The E400K/E401A mutation ablated the receptor-corepressor interaction whereas the two mutations, F402A/S403A and T407A/P408A, did not abrogate the receptor-corepressor interaction, but significantly reduced it 5- and 3-fold, respectively (Fig. 2CGo).

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-RIP13{Delta}1 interactions, all three mutants (E400K/E401A, F402A/S403A, and T407A/P408A) ablated the RVR-N-CoR/RIP13a-RID interaction (Fig. 2DGo).

These studies suggested that RVR interacts similarly with the RIP13{Delta}1 and N-CoR/RIP13a-RIDs, except that there may be a larger contact interface between RVR-N-CoR/RIP13a than the RVR-RIP13{Delta}1 interface. Furthermore, these mutagenesis studies reiterate the core role of CIR-1 in corepressor binding.

RIP13{Delta}1 and N-CoR/RIP13a Differentially Interact with Rev-erbA{alpha}: The Hinge/D Region Has a Role in Corepressor Binding
We had previously demonstrated that full-length Rev-erbA{alpha} efficiently interacted with both the N-CoR/RIP13a-RID and RIP13{Delta}1-RIDs (20). Furthermore, we demonstrated that efficient interaction of Rev-erbA{alpha} with RIP13{Delta}1 was dependent on an intact E region. We thus decided to investigate the specific regions and amino acid residues within Rev-erbA{alpha} that were critical to the physical association with N-CoR and RIP13a-RIDs.

The chimeric construct consisting of the GAL4 DBD fused to the RIP13{Delta}1-RID (Fig. 3AGo) was expressed in cells with a set of chimeric constructs containing full-length or various deletions of the Rev-erbA{alpha} receptor linked to the trans-activation domain of VP16 (Fig. 3BGo). Consistent with the previous study, we saw a very strong interaction when VP16-Rev (aa 21–614), Rev DE (aa 290–614), and Rev E (aa 437–614) were cotransfected with the GAL4-RIP13{Delta}1-RID (Fig. 3CGo).



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Figure 3. Identification of the Domains of Rev-erbA{alpha} Necessary for Interaction with the RIP13{Delta}1 and the N-CoR/RIP13a-RIDs

A, Schematic representation of the various corepressor(s) and RIDs used in this study. B, Schematic representation of the VP16-Rev-erbA{alpha} chimeras used in the mammalian two-hybrid assay. The boundaries of the AB, C, D, and E regions are denoted. CIR-1 and CIR-2 are shown and are located between aa 437 and 455 and aa 602 and 614, respectively. CIR-3 is an undefined region in the N-terminal part of the hinge. The region in Rev-erbA{alpha} corresponding to the previously identified CoR box in TR and RAR is indicated. C, JEG-3 cells were cotransfected with the indicated plasmids (+) in the presence of pG5E1bCAT reporter plasmid and assayed for CAT activity. Results shown are mean ± SD and were derived from three independent transfections. Fold activation is expressed relative to CAT activity obtained after cotransfection of GAL4-RIP13{Delta}1-RID and the VP16 vector alone arbitrarily set to 1.0. D, JEG-3 cells were cotransfected with the indicated plasmids (+) in the presence of pG5E1bCAT reporter plasmid and assayed for CAT activity. Results shown are mean ± SD and were derived from three independent transfections. Fold activation is expressed relative to CAT activity obtained after transfection of GAL4-N-CoR/RIP13a-RID and the VP16 vector alone arbitrarily set to 1.0.

 
Deletion of 23 amino acids between aa 432 and 455 (N-terminal part of helix 3) in the E region of Rev-erbA{alpha}, which spanned the CIR-1 residues, ablated the receptor-corepressor interaction in the mammalian two-hybrid assay (Fig. 3CGo). These experiments demonstrated that the interaction of RIP13{Delta}1 with Rev-erbA{alpha} (like RVR) was also dependent on the CIR-1 and correlated with the highly conserved nature of these residues. The CIR-2 (or Y domain) of Rev-erbA{alpha} was demonstrated previously to be required for receptor-corepressor interactions; therefore, we did not reexamine this issue (21). These experiments indicated that the E region from Rev-erbA{alpha}, like RVR, is necessary and sufficient for the interaction with the corepressor, RIP13{Delta}1. Furthermore, the D region of Rev-erbA{alpha}, like RVR, was not required for the interaction with RIP13{Delta}1.

We similarly analyzed the potential of various domains from Rev-erbA{alpha} 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. 3AGo) was expressed in cells with a set of chimeric constructs containing full-length or various deletions of the Rev-erbA{alpha} receptor linked to the trans-activation domain of VP16 (Fig. 3BGo). We saw a very strong interaction when the VP16-Rev (aa 21–614) was cotransfected with the GAL4-N-CoR/RIP13a-RID construct (Fig. 3DGo). Strikingly, further unidirectional deletions of Rev-erbA{alpha} (in an N- to C-terminal direction) from aa 290, aa 437, and aa 455 significantly reduced (~3-fold for VP16-Rev-DE, aa 290–614) or ablated the receptor-corepressor interaction (VP16-Rev E, aa 437–614; and VP16-Rev, aa 455–614) (Fig. 3DGo). Thus the E region of Rev-erbA{alpha}, 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{alpha} was required for the interaction with N-CoR/RIP13a but not to RIP13{Delta}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{alpha}, denoted Rev-{Delta}CoR, that changed the invariant A, H, and I residues to G, G, and A (Fig. 3BGo) and linked it to the trans-activation domain of VP16. This chimeric construct, VP16-Rev-{Delta}CoR, was coexpressed in cells containing the GAL4 DBD fused to the RIP13{Delta}1-RID (Fig. 3CGo) and the GAL4 DBD fused to the N-CoR/RIP13a-RID (Fig. 3DGo). 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{Delta}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{alpha} 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{alpha} and RVR.

The CIR-1 of Rev-erbA{alpha} Is Required for an Efficient Interaction with the RIP13{Delta}1 Splice Variant
The CIR-1 in Rev-erbA{alpha} is composed of the following amino acid residues, IWEDFSMSFTPTVREVV between aa 437 and 456 (Fig. 4AGo), 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. 4AGo). We constructed two mutant VP16 Rev E expression plasmids (from aa 437, Fig. 4BGo) that were denoted VP16-Rev E-F441A/S442A and VP16-Rev E-T446A/P447A (Fig. 4BGo). These constructs were then used in the mammalian two-hybrid assay to investigate whether they could interact with the RIP13{Delta}1-RID linked to GAL4 DBD. Mutation of F441A/S442A in the CIR-1 ablated the receptor-RIP13{Delta}1 interaction, in contrast to the T446A/P447A, which did not affect the Rev-erbA{alpha}-RIP13{Delta}1 corepressor interaction (Fig. 4CGo).



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Figure 4. Characterization of the CIR-1 of Rev-erbA{alpha}, Which Is Necessary for Interaction with the RIP13{Delta}1 and the N-CoR/RIP13a-RIDs

A, The sequence of the Rev-erbA{alpha} CIR-1, mutations introduced in the CIR-1 of Rev-erbA{alpha}, and alignment of this region to corresponding regions of RVR, RAR, and TR. B, Schematic representation of the VP16-Rev-erbA{alpha} chimeras used in the mammalian two-hybrid assay. The boundaries of the AB, C, D, and E regions are denoted. The CIRs are shown. C, Analysis of the effects of Rev-erbA{alpha} CIR-1 mutations on RIP13{Delta}1-RID binding. JEG-3 cells were cotransfected with the indicated plasmids (+) in the presence of pG5E1bCAT reporter plasmid and assayed for CAT activity. Results shown are mean ± SD and were derived from three independent transfections. Fold activation is expressed relative to CAT activity obtained after transfection with GAL4-RIP13{Delta}1-RID and the VP16 vector alone arbitrarily set to 1.0. D, Analysis of the contribution of CIR-1 in Rev-erbA{alpha} to N-CoR/RIP13a-RID binding. JEG-3 cells were cotransfected with the indicated plasmids (+) in the presence of pG5E1bCAT reporter plasmid and assayed for CAT activity. Results shown are mean ± SD and were derived from three independent transfections. Fold activation is expressed relative to CAT activity obtained after cotransfection with GAL4-N-CoR/RIP13a-RID and the VP16 vector alone arbitrarily set to 1.0.

 
The X domain of Rev-erbA{alpha} (between aa 407 and 418) in the hinge/D region has been implicated in N-CoR binding, based on in vitro glutathione-S-transferase (GST) pulldown assays, however, alanine mutagenesis and functional analysis indicated this domain was not required for N-CoR binding in vivo (21, 26). Hence, we also examined the ability of the amino acids between aa 407 and 614 vs. the E region alone (aa 437–614) to interact with the RIP13{Delta}1-RID to resolve these discrepancies (Fig. 4Go, B and C). Interestingly, there was no significant difference in the ability of either region to support the Rev-erbA{alpha}-RIP13{Delta}1 interaction. Identical mutations in CIR-1 as above, in constructs including the X domain (VP16-Rev aa 407-F441A/S442A), reinforced the critical role of CIR-1 in the receptor-RIP13{Delta}1 interaction (Fig. 4CGo). However, we note that the F441A/S442A mutation in the context of the aa 437 start point ablated Rev-erbA{alpha}-RIP13{Delta}1 interaction, whereas the F441A/S442A mutation in the context of the aa 407 start point only reduced the interaction by 3-fold.

The Hinge Region and CIR-1 of Rev-erbA{alpha} 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{alpha} 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{alpha} (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{alpha} in the interaction with the N-CoR/RIP13a-RID (Fig. 4Go, B and D). As can be seen in Fig. 4DGo, 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 419–614, Fig. 4DGo), suggesting the X domain was not involved in corepressor binding. This suggested that Rev-erbA{alpha} contained a third corepressor interaction region (CIR-3) in the N-terminal D region.

We then examined whether CIR-1 in Rev-erbA{alpha} 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. 4BGo) and used the mammalian two-hybrid assay to investigate whether they could interact with the N-CoR/RIP13a-RID (Fig. 4DGo). 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{alpha}-N-CoR/RIP13a interaction. These data demonstrate that CIR-1 and an as yet undefined CIR-3 in Rev-erbA{alpha} are required for N-CoR/RIP13a binding. Furthermore, the data demonstrate that Rev-erbA{alpha} 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{alpha} 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{alpha} 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{alpha} 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{alpha} promoter linked to the luciferase reporter (14) in C2C12 cells. Rev-erbA{alpha} 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{alpha} promoter (Fig. 5AGo). 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{alpha} 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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Figure 5. Analysis of the Repression of the Human Rev-erbA{alpha} Promoter by RVR and the N-CoR Family of Corepressors

C2C12 cells were cotransfected with the indicated plasmids (+) in the presence of pRev-erb{alpha}WT reporter plasmid in 12-well plates and assayed for luciferase activity. Results shown are mean ± SD and were derived from at least three independent transfections. Fold repression is expressed relative to repression obtained after cotransfection of pRev-erb{alpha}WT and pSG5 alone arbitrarily set to 1.0. A, Luciferase activity was determined after cotransfection of pRev-erb{alpha}WT (1 µg) and pSG5-RVR plasmids (0.3 µg). B, Luciferase activity was determined after cotransfection of pRev-erb{alpha}WT (1 µg) and pSG5-Rev plasmids (0.3 µg). C, Luciferase activity was determined after cotransfection with pRev-erb{alpha}WT (1 µg) and pSG5-RVR (0.33 µg) and RIDs of RIP13{Delta}1 or N-CoR/RIP13a (0.66 µg). D, Luciferase activity was determined after cotransfection with pRev-erb{alpha}WT (1 µg), pSG5-RVR (0.33 µg), and pSG5 or pSG5-N-CoR (0.5 µg). RLUs, Relative luciferase units.

 
Similarly, we decided to investigate the importance of the E region on Rev-erbA{alpha}-mediated silencing of the human Rev-erbA{alpha} promoter and the impact of a number of point mutants. As previously observed, Rev-erbA{alpha} represses the transcription of its own promoter by 2- to 3-fold (14). Deletion of the E region ablated the ability of Rev-erbA{alpha} to silence the human Rev-erbA{alpha} promoter (Fig. 5BGo). The two-point mutants in CIR-1, Rev-F441A/S442A and Rev-T446A/P447A, abrogated and did not affect the ability of Rev-erb, respectively, to repress the expression of the Rev-erbA{alpha} promoter. This correlates with the mammalian two-hybrid analysis (Fig. 4Go, C and D), which demonstrated that the Rev-F441A/S442A mutation very significantly reduced N-CoR/RIP13a and RIP13{Delta}1-RID binding, whereas the Rev-T446A/P447A mutation did not affect corepressor binding in vivo. Furthermore, mutation of the CoR box did not affect the ability of Rev-erbA{alpha} to repress the expression of the Rev-erbA{alpha} promoter (Fig. 5BGo) and similarly correlated with the mammmalian two-hybrid analysis (Fig. 3Go, C and D). These transfection experiments demonstrated the importance of the E region and CIR-1 in Rev-erbA{alpha} function.

We then investigated the ability of dominant-negative corepressor expression vectors that contained the corepressor-RIDs but no repression domains (i.e. pSG5-RIP13{Delta}1-RID and pSG5-N-CoR/RIP13a-RID) to affect the orphan receptor-mediated repression of the hRev-erbA{alpha} promoter (Fig. 5CGo). As seen in Fig. 5CGo, overexpression of the corepressor RIDs completely abolished repression by RVR, thus showing that these RIDs could function as antirepressors. The native RIP13a and RIP13{Delta}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{alpha} 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{alpha} promoter by native and mutant RVRs (Fig. 5DGo). As seen in Fig. 5DGo, overexpression of N-CoR significantly/synergistically enhances the ability of RVR, RVR-E400K, and RVR-{Delta}572–576 to repress promoter expression. In contrast and comparison, N-CoR has much weaker effects (only additive) on the ability of RVR-F402P and RVR-{Delta}561–576 to repress promoter expression. Interestingly, this correlates with the ability of these receptors to bind N-CoR in vitro (presented in Fig. 6Go); briefly, RVR, RVR-E400K, and RVR-{Delta}572–576 efficiently bind the N-CoR/RIP13-RIDs in vitro, whereas, RVR-F402P and RVR-{Delta}561–576 have impaired binding.



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Figure 6. Interaction of the Reverbs and Corepressors in Vitro

Panels A to G, Point mutations and deletions in CIR-1 and CIR-2 of RVR and Rev-erbA{alpha} were radiolabeled with [35S]methionine by in vitro transcription/translation and tested for interaction with GST-alone, GST-RIP13{Delta}1, and GST-N-CoR/RIP13a. Inputs of the radiolabeled [35S]methionine protein are also shown. Panels A and B, Point mutations in CIR-1 of RVR affect binding to the RIP13{Delta}1 and N-CoR/RIP13a corepressor RIDs. Panels C and D, Deletions in CIR-2 of RVR affect binding to the RIP13{Delta}1 and N-CoR/RIP13a corepressor RIDs. E, The E region of RVR is sufficient for corepressor binding; the T407A/P408A mutation in RVR reduces binding to the RIP13{Delta}1-RID in vitro. F, The F441A/S442A mutation in CIR-1 of Rev-erbA{alpha} impairs the ability of Rev-erbA to interact with the N-CoR/RIP13a RID in vitro. G, Deletion of CIR-1 reduces the ability of the Rev-erbA E -region to bind RIP13{Delta}1 in vitro. H, Mutation of the Rev-erbA{alpha} CoR box does not alter the interaction of Rev-erbA with the N-CoR/RIP13a and RIP13{Delta}1-RIDs in vitro. The input lanes in all gels contain between 5% and 10% input.

 
These transfection studies correlated with the mammalian two-hybrid experiments that demonstrated CIR-1 and -2 in RVR were required for efficient corepressor binding.

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 RIP13{Delta}1-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-RIP13{Delta}1-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{Delta}1- and N-CoR/RIP13a-RIDs showed a direct interaction with full-length RVR (Fig. 6Go, 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{Delta}1 and N-CoR/RIP13a-RIDs (Fig. 6Go, A and B, respectively). These direct in vitro binding data verify that RVR directly interacts with RIP13{Delta}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, RVR{Delta}572–576 and RVR{Delta}561–576, to interact with the immobilized GST-RIP13{Delta}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. 6CGo). However, deletion of the amino acids between 561 and 576 reduced the specificity and binding of RVR for the RIP13{Delta}1- and N-CoR/RIP13a-RIDs, respectively (Fig. 6Go, C and D). These data demonstrate that the deletion ({Delta}561–576) in CIR-2 that ablates the ability to interact in the mammalian two-hybrid assay, and to repress transcription of the human Rev-erbA{alpha} 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-RIP13{Delta}1-RID. The E region of RVR interacts directly with the corepressor RID (Fig. 6EGo). However, the T407A/P408A mutation impaired, but did not ablate, the binding of the RVR-E region for the RIP13{Delta}1-RID, which correlated with the mammalian two-hybrid data in Fig. 2CGo.

We then used the GST-pulldown assay to demonstrate that mutations/deletion in CIR-1 of Rev-erbA{alpha} that impaired interactions in vivo, and the repression of the Rev-erbA{alpha} 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. 6FGo), 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 437–614) and a CIR-1-deleted Rev-erb E region (Rev-E-aa 455–614) to bind immobilized GST-RIP13{Delta}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{Delta}1-RID (Fig. 6GGo). Finally, we investigated the ability of Rev-erb carrying a mutation in the CoR box (Rev-{Delta}CoR) to interact with the N-CoR/RIP13a and RIP13{Delta}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. 6HGo). 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{alpha} 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{alpha} 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, RVR{Delta}425–556, that deletes a large portion of the E region to bring CIR-1 and CIR-2 in close proximity (Fig. 7AGo). This construct repressed transcription of the hRev-erbA{alpha} promoter linked to the luciferase reporter as efficiently as native RVR (Fig. 7BGo), indicating the importance of CIR-1 and CIR-2 in repression by RVR. We also investigated whether in vitro35S-radiolabeled RVR{Delta}425–556 could bind to the immobilized GST-RIP13{Delta}1-RID and N-CoR/RIP13a-RID fusion proteins. The chimera, RVR{Delta}425–556, bound efficiently to both GST-RIP13{Delta}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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Figure 7. Juxtaposed CIR-1 and CIR-2 Mediate Transcriptional Repression and in Vitro Corepressor Binding

A, Schematic representation of plasmids used in the transfection analysis and in vitro binding assays. B, Analysis of the repression of the human Rev-erbA{alpha} promoter by RVR and RVR{Delta}425–556. C2C12 cells were cotransfected with the indicated plasmids (+) in the presence of pRev-erb{alpha}WT reporter plasmid in 12-well plates and assayed for luciferase activity. Results shown are mean ± SD and were derived from at least three independent transfections. Fold repression is expressed relative to repression obtained after cotransfection of pRev-erb{alpha}WT and pSG5 alone arbitrarily set to 1.0. Luciferase activity was determined after cotransfection of pRev-erb{alpha}WT (1 µg) and pSG5-RVR plasmids (0.3 µg) RVR. C, RVR and RVR{Delta}425–556 were radiolabeled with [35S]methionine by in vitro transcription/translation and tested for interaction with GST alone, GST-RIP13{Delta}1, and GST-N-CoR/RIP13a.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
This study used in vivo mammalian two-hybrid assays, transfection analysis, and biochemical techniques to characterize in detail the interaction between RVR and Rev-erbA{alpha} and three nuclear receptor corepressors, N-CoR and the variants RIP13a and RIP13{Delta}1.

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 RIP13{Delta}1. Although the E region of Rev-erbA{alpha} is necessary and sufficient for the interaction with RIP13{Delta}1, the D region of Rev-erbA{alpha} is required for the physical association with N-CoR and the variant RIP13a. This suggested that N-CoR/RIP13a and RIP13{Delta}1 differentially interact with Rev-erbA{alpha}.

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 RIP13{Delta}1 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{Delta}1. However, further experimentation is required to verify this hypothesis.

Interestingly, we had previously shown that helix 3 in RVR (10) and Rev-erbA{alpha} (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{alpha}. CIR-2 in helix 11 of RVR is homologous to the previously described Y domain in Rev-erbA{alpha} (21) that is required for the interaction of Rev-erbA{alpha} with N-CoR. Site-specific mutagenesis of CIR-1 in RVR and Rev-erbA{alpha} demonstrated that CIR-1 plays a critical role and is necessary for the in vivo/vitro interaction with the N-CoR, RIP13a, and RIP13{Delta}1 RIDs. These mutagenesis studies suggested that the orphan receptor interaction interface with N-CoR/RIP13a is larger than with RIP13{Delta}1. Furthermore, the data suggested that N-CoR/RIP13a and RIP13{Delta}1 differentially interact with Rev-erbA{alpha}.

Studies to date have suggested that N-CoR interacts with the CoR box of TR/RAR and the X domain of Rev-erbA{alpha} (aa 407–419), 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{alpha} require the D region that includes the CoR box (in helix 1) and the X domain for binding to the N-CoR variant, RIP13{Delta}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{alpha} 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{alpha}, 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{alpha} 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{alpha}, unlike that of RVR, may contribute to the positioning of CIR-1/CIR-2 within the E region of Rev-erbA{alpha}, 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{alpha}. Therefore, our studies have demonstrated a number of important functional differences between the {alpha}- 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{alpha} 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{Delta}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-{Delta}561–576). 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 RIP13{Delta}1 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Primer Sequences
GMUQ296:

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-erbA{alpha}WT (14), which contains the human Rev-erbA{alpha} 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 manufacturer’s instructions. End-filling reactions were performed with Klenow DNA polymerase according to the manufacturer’s 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 394–576 and pGAL4-RVR E aa 394–576, RVR aa 394–576 was amplified from GV-RVR aa 355–576 (10) using the two primers, GMUQ301 and GMUQ252. The product was ligated to pBluescript KS/EcoRV to create pBluescript-RVR aa 394–576. 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 394–576; 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 416–576 (GMUQ303 and GMUQ252). These products were ligated to pBluescript KS/EcoRV to create pBluescript-RVR aa 398–576 mutants and pBluescript-RVR aa 416–576. Antisense clones were cleaved with SalI, and the resulting fragments were ligated to pNLVP16/SalI. To construct pVP16-RVR aa 394–572 and pVP16-RVR aa 394–561, antisense pBluescript-RVR aa 394–576/SalI was partially digested with DraI, and the required fragments were isolated, end-filled, and ligated to pNLVP16/NdeI. To construct pSG5-RVR{Delta}572–576 and pSG5-RVR{Delta}561–576, pVP16-RVR aa 394–572 and pVP16-RVR aa 394–561 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 1–572 and pBS-RVR aa 1–561. 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 manufacturer’s instructions.

To construct the following pVP16-Rev chimeric expression vectors, primers were used to amplify regions of Rev-erbA{alpha} from pVP16-Rev aa 290–614 (25); pVP16-Rev aa 455–614 (GMUQ303 and GMUQ132); pVP16-Rev aa 419–416 (GMUQ377 and GMUQ 132); and pVP16-Rev aa 407–614 (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{alpha} from pVP16-Rev aa 347–614; 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{alpha} and were cut with BglII/BamHI and ligated to pSG5-Rev{Delta}E/BglII to construct full-length pSG5-Rev-erbA{alpha} clones carrying these mutations, denoted pSG5-Rev-erbA{alpha}-F441A/S442A and pSG5-Rev-erbA{alpha}T446A/P447A. Mutant Rev aa 407–614 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{alpha} 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-{Delta}CoR and pVP16-Rev-{Delta}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 manufacturer’s 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 (60–70% 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 36–48 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 (60–70% confluence) was cotransfected with 1.0 µg of pRev-erbA{alpha} WT and 0.33–1.0 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 36–48 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 0.1–0.4 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 manufacturer’s 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 2–30 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 1–2 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.


    ACKNOWLEDGMENTS
 
We sincerely thank Udani Abeypala and Katrina Franke for excellent technical assistance with plasmid preparation and tissue culture. We also thank Dr. W. Chin and Dr, V. Giguère for the kind gifts of Rev-erbA{alpha} and RVR cDNA clones. Special thanks to Drs. David Moore and Wongi Seol for the RIP13a and RIP13{Delta}1 cDNA clones.


    FOOTNOTES
 
Address requests for reprints to: Dr. George E. O. Muscat, Centre for Molecular and Cellular Biology, University of Queensland, Ritchie Research Laboratories, Brisbane, 4072 Queensland, Australia.

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.

1 Joint first authors Back

Received for publication August 20, 1997. Revision received October 30, 1997. Accepted for publication November 5, 1997.


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