Transcriptional Activation and Repression by ROR{alpha}, an Orphan Nuclear Receptor Required for Cerebellar Development

Heather P. Harding1, G. Brandon Atkins1, Aron B. Jaffe, William J. Seo and Mitchell A. Lazar

Division of Endocrinology, Diabetes, and Metabolism, Departments of Medicine and Genetics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mutation of the orphan nuclear receptor ROR{alpha} results in a severe impairment of cerebellar development by unknown mechanisms. We have found that ROR{alpha} activates transcription from only a subset of sites to which it binds strongly as a monomer. ROR{alpha} also selectively binds as a homodimer to a direct repeat of this monomer site with a 2-bp spacing between the AGGTCA sequences (Rev-DR2 site) and is a much more potent transcriptional activator on this site than on monomer sites or other direct repeats. To better understand the transcriptional regulatory functions of ROR{alpha}, we fused its C terminus to a heterologous DNA-binding domain. Mutational analysis revealed that ROR{alpha} contains both transcriptional activation and transcriptional repression domains, with the repression domain being more active in some cell types. The abilities of ROR{alpha} polypeptides to repress transcription correlate with their abilities to interact with the nuclear receptor corepressors N-CoR and SMRT in vitro. However, the AF2 region of ROR{alpha} inhibits corepressor interaction on DNA, consistent with the lack of repression by the full-length receptor. Thus, transcriptional regulation by ROR{alpha} is complex and likely to be regulated in a cell type- and target gene-specific manner.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ROR{alpha} is a member of the nuclear hormone receptor superfamily that was originally isolated and named on the basis of its similarity to retinoic acid receptor (RAR) (1). There are two other ROR subtypes, known as RORß or RZRß (2) and ROR{gamma} (3), which are encoded on separate genes. ROR{alpha} is referred to as an orphan nuclear receptor because it is not known whether it requires a ligand to activate gene transcription (4). ROR{alpha} is particularly interesting because it has been shown that mutation of the ROR{alpha} gene results in the failure of normal cerebellar development in the Staggerer mouse (5). However, the mechanism by which ROR{alpha} mutation impairs cerebellar development is not known.

ROR{alpha} is a constitutive transcriptional activator in the absence of exogenous ligand (1). This differs from ligand-activated nuclear receptors such as thyroid hormone receptor (TR) and RAR that interact with corepressor proteins such as nuclear receptor corepressor (N-CoR) and silencing mediator of retinoid and thyroid receptors (SMRT) and function as repressors of transcription in the absence of lipophilic ligand (6, 7). Ligand binding causes a conformational change in these receptors that leads to the dissociation of the corepressor and recruitment of a putative transcriptional coactivator, candidates for which include thyroid receptor-interacting protein 1 (8), steroid receptor coactivator 1 (9), receptor-interacting protein 140 (10), transactivation domain-interacting factor 1 (TIF1) (11), glucocorticoid receptor-interacting protein (GRIP1, also called TIF2) (12, 13), and cAMP response element-binding protein (14, 15, 16). The main effect of ligand binding is to reorient the C-terminal activation function 2 (AF2) amphipathic helix toward the hydrophobic core of the receptor where ligand is bound (17, 18, 19). In the case of ROR, it is uncertain whether the protein is a true orphan that is constitutively active because it can achieve an activated conformation in the absence of ligand or whether it is activated by interaction with a ligand that is endogenous in the cell systems used to study its function.

ROR{alpha} is a prototypical member of the subfamily of nuclear receptors that bind DNA strongly as monomers. This subfamily includes TR (20, 21, 22, 23) as well as the orphan receptors NGFI-B (24), SF-1 (25), and RevErb (26). All of these monomer-binding orphan receptors recognize the nuclear receptor half-site AGGTCA flanked 5' by nucleotides that increase the affinity and determine the receptor specificity of the interaction. Although ROR is named after RAR, it is more highly related to RevErb (27, 28). Unbiased determination of the optimal binding sites for RevErb and ROR{alpha} independently uncovered remarkably similar monomer binding sites. In each case, the half-site hexamer was flanked 5' by an extended A/T-rich sequence, the consensus of which is (A/T)(A/T)(A/T)NT (1, 26). However, whereas ROR{alpha} activates transcription constitutively (i.e. in the absence of exogenous ligand) from this site (1), RevErb is inactive (29, 30, 31). Rather, RevErb function requires binding to a direct repeat of this site spaced by 2 bp (RevDR2) (32). On this site RevErb interacts with the nuclear hormone receptor corepressor N-CoR (6) and potently represses transcription (33, 34).

Because of the similarities between RevErb and ROR{alpha}, we studied the transcriptional activity of ROR{alpha} on a variety of binding sites. Remarkably, although we confirmed that ROR{alpha} can function as a monomer, it is a much more potent transcriptional activator on a RevDR2, on which it forms a dimer. The transcriptional regulatory properties of ROR{alpha} were further delineated by fusion to a heterologous DNA binding domain. This analysis revealed a cell type-specific transactivation function, as well as a previously unidentified repression function that was revealed by mutation. The repression domain was found to interact specifically with putative transcriptional corepressor proteins. These data indicate that the transcriptional regulatory properties of ROR{alpha} are complex and are likely to vary in a manner that is dependent upon DNA-binding site and cell type.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ROR{alpha} Activates Transcription on a Subset of Monomer-Binding Sites
Although ROR{alpha} and RevErb have similar monomeric binding sites, one difference between the proteins is that ROR{alpha} is apparently able to regulate transcription from its monomer site whereas RevErb functions only from a related site which it binds as a dimer (RevDR2) (34). To further study the relationship between DNA binding and transcriptional function of ROR{alpha}, we examined its DNA binding and activation on multiple sites to which it binds as a monomer. Figure 1BGo shows ROR{alpha} bound avidly as a monomer to four different sites referred to as A through D in Fig. 1AGo. However, the function of the binding sites was qualitatively different. As shown in Fig. 1CGo, ROR{alpha} transfected into JEG-3 cells activated transcription from a reporter containing a single copy of monomer site A but had little effect upon transcription of a reporter gene containing a single copy of binding site D. Monomer binding site B was also unable to support transactivation by ROR{alpha} (data not shown).



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Figure 1. ROR{alpha} Activates Transcription from Only a Subset of Monomer-Binding Sites

A, Monomer binding sites. The half-sites are marked with arrows. B, ROR{alpha} binds as a monomer to multiple sites. EMSA analysis of ROR{alpha} binding to the sites shown in panel A. C, ROR{alpha} activates transcription from only a subset of monomer sites. JEG-3 cells were transiently transfected with ROR{alpha} expression vector and TK-luciferase reporter containing no ROR{alpha}-binding site or the monomer sites indicated. RORE refers to the ROR binding site used.

 
ROR{alpha} Preferentially Activates Transcription on a Direct Repeat to Which it Binds with a Stoichiometry of 2:1
The lack of transcriptional activity of ROR{alpha} from the monomer site D in spite of in vitro binding was similar to what we have observed for RevErb, which functions specifically as a homodimer on a direct repeat of the monomer site with 2-bp spacing between the AGGTCA motifs (RevDR2, Fig. 2AGo) (32). Remarkably, Fig. 2BGo shows that ROR{alpha}, like RevErb, functioned most potently on the RevDR2, and not on the other direct repeats shown in Fig. 2AGo. In the case of ROR{alpha}, the function was as an activator of transcription. ROR{alpha} potently activated transcription from a reporter gene containing one copy of the RevDR2 sequence (Fig. 2BGo). In multiple experiments this activity was 2- to 3-fold greater than that obtained from Monomer A in Fig. 1Go (data not shown). Furthermore, ROR{alpha} had minimal effects on the other direct repeats, as well as on a DR2 site in which the flanking sequences were replaced by C’s.



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Figure 2. ROR{alpha} Selectively Activates Transcription from the RevDR2 Site

A, RevDR series of binding sites. The 5'-flank and spacer sequences are indicated in boldface, and the half-sites are marked with arrows. B, ROR{alpha} selectively activates transcription from the RevDR2. JEG-3 cells were transfected with the indicated reporters containing no ROR{alpha}-binding site or the sites indicated. The DR2-C flank is a DR2 with C’s flanking the upstream half-site and as the spacer between the half-sites. C, ROR{alpha} binds as a dimer only to the RevDR2. EMSA analysis of ROR{alpha} binding to the sites shown in panel A. Monomer (M) and Dimer (D) complexes are indicated. D, ROR{alpha} activates transcripton from a reporter containing two widely separated monomer sites. Transient transfection study, using reporter containing monomer D, RevDR2, or the monomer D x 2 site (with a spacing of 14 bp between the two AGGTCA half-sites).

 
We next investigated whether DNA binding by ROR{alpha} correlated with its function on these binding sites. Figure 2CGo shows that ROR{alpha} bound strongly as a monomer to all of the RevDR-binding sites. In contrast, two ROR{alpha} molecules bound only to the RevDR2 site. Unlike RevErb binding to the RevDR2, ROR{alpha} binding was not cooperative, in that monomer binding was preferred at low concentrations of ROR{alpha}. This result suggested that the failure of two ROR{alpha} molecules to bind to the other sites may be related to steric hindrance preventing two molecules from binding simultaneously. To test whether the 2-bp spacer of the RevDR2 had a more significant functional role then allowing two molecules to bind to the reporter, we examined ROR{alpha} activation on two sets of reporters containing either a single monomer site D, two copies of monomer site D separated by 14 bp, or the RevDR2 (which is a direct repeat of monomer site D with a spacing of 2 bp between the AGGTCA sequences). As shown in Fig. 2DGo, the presence in cis of two monomer sites that were ineffective on their own allowed ROR{alpha} to activate transcription even though the sites were widely spaced. Thus the ability of two ROR{alpha} molecules to interact with an enhancer region directly correlated with the ability of ROR{alpha} to activate transcription, with the RevDR2 spacing being modestly more favorable. ROR{alpha} activated transcription somewhat more potently on the RevDR2-containing reporter, but the degree to which the RevDR2 was more active varied somewhat between experiments. Similar results were obtained using other sequences containing two widely separated monomer sites in cis (data not shown). Thus, two monomer sites that were nearly inactive on their own functioned as potent ROR-response elements both in the DR2 and DR14 configurations, suggesting a qualitative significance of having the two ROR-binding sites present in cis on the reporter gene.

Comparison of Monomeric and Dimeric DNA Binding by ROR{alpha}
Because of the striking increase in ROR{alpha} activity on the RevDR2 despite reduced binding of the dimer relative to the monomer, we compared the characteristics of ROR{alpha} monomer and dimer binding to the RevDR2. Figure 3AGo shows the results of a methylation interference study. As predicted, the ROR{alpha} monomer preferentially contacted the upstream half-site since this half-site is in the context of a more favorable monomer-binding site than is the downstream half-site. In contrast, the ROR{alpha} dimer contacted both half-sites, including the two G residues in the downstream half-site (marked with arrowheads in Fig. 3AGo), as well as the A/T-rich flank characteristic of its monomer-binding site. The results of methylation and diethyl pyrocarbonate (DEPC)-interference studies of both strands are summarized in Fig. 3BGo. These experiments show that there was a fundamental difference between binding of the monomer and dimer, with the dimer contacting both half-sites and the monomer preferentially contacting the upstream half-site.



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Figure 3. Characteristics of ROR{alpha} Binding to the RevDR2

A, The dimer contacts both half-sites, whereas the monomer contacts the upstream half-site. Methylation interference analysis was described in Materials and Methods. The position of the half-sites and the A and G residues seen in the gel are shown. Arrows indicate the position of G residues protected by the dimer but not the monomer. B, Summary of contacts made by ROR{alpha} monomer and dimer using both methylation interference and DEPC-interference analysis. Filled circles indicate residues seen to be protected by both methylation interference (MI) and DEPC; open circles indicate residues protected from DEPC only. D indicates sites protected in the dimer in addition to the monomer. C, Monomeric binding by ROR{alpha} is more stable than dimeric binding. Off-rate analysis, in which cold oligonucleotide was added at the times indicated and loaded on running EMSA gel. Monomer (M) and Dimer (D) complexes are indicated.

 
We also explored the off-rates of the monomer and dimer from the RevDR2- binding site. Consistent with the apparently increased affinity of the monomer, Fig. 3CGo shows that the monomer binding to the RevDR2 was more stable than that of the dimer. Thus, as discussed later, factors other than DNA binding affinity or stability of the protein-DNA complex must be involved in the increased transcription from the RevDR2 site.

ROR{alpha} Contains Cell Type-Specific Activation and Repression Domains
To begin to understand the mechanism by which ROR{alpha} activates transcription, we took the approach of fusing the ROR{alpha} activation domain to the heterologous DNA-binding domain of the GAL4 transcription factor, thereby studying transcription independent of the effects of the DNA-binding site. This was particularly important for ROR{alpha} since the above results indicated that on ROR{alpha}-binding sites, DNA binding was necessary but not sufficient for transcriptional activation.

Figure 4Go shows that GAL4-ROR{alpha} (140–523) was a potent transcriptional activator in JEG-3 human choriocarcinoma cells. Interestingly, a region in the hinge domain of ROR{alpha} (amino acids 221–272) appeared to play an inhibitory role since deletion of these amino acids increased transcriptional activation by ROR{alpha} from 7-fold to more than 150-fold. Similar results were obtained at different concentrations of transfected GAL4-ROR{alpha} constructs (data not shown). Amino acids 221–272 by themselves were transcriptionally neutral when fused to GAL4 (data not shown), suggesting that this region is likely to be part of a larger domain that either determines the affinity of ROR{alpha} for potential coactivator proteins or recruits a corepressor protein. Deletion of the C terminus of ROR{alpha}, which contains the AF2 activation helix required for transcriptional activation by other members of the nuclear receptor superfamily (GAL4-ROR 140–491 and 140–451), not only abolished activation but revealed a modest repression function of these ROR{alpha} polypeptides.



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Figure 4. Transcriptional Activation and Repression Domains in ROR{alpha}

GAL4 constructs (or wild type ROR{alpha}) were transfected into JEG-3 and 293T cells, along with a reporter plasmid containing five GAL4-binding sites upstream of SV40-luciferase. The transcriptional activity in a representative experiment is shown. Similar results were obtained three to five for each construct.

 
We also tested the transcriptional activities of the identical GAL4-fusion proteins in 293T human kidney cells. Remarkably, Fig. 4AGo shows that none of the ROR{alpha} polypeptides, including the superactivating amino acids 272–523, were sufficient for transcriptional activation in these cells, although all of the GAL4 fusion proteins were expressed at comparable levels (not shown). In light of these results, it should be noted that in 293T cells full-length ROR{alpha} is also a much weaker activator of reporters containing either monomer or RevDR2 sites (data not shown). Indeed, ROR{alpha} fused to GAL4 DNA-binding domain (DBD) repressed transcription in 293T cells. The GAL4-fusion proteins containing the ROR{alpha} polypeptides that lacked the AF2 activation helix and were modest repressors in JEG-3 cells were the most potent repressors of transcription from the GAL4 binding site-containing reporter gene in 293T cells.

ROR{alpha}1 Interacts with Nuclear Receptor Corepressors N-CoR and SMRT, and This Interaction Correlates with Repression by ROR{alpha} Polypeptides
The transcriptional repression function of RevErb is mediated by a corepressor protein called N-CoR (33, 34). N-CoR as well as another corepressor called SMRT have also been implicated in repression by thyroid and retinoic acid receptors (6, 7). To investigate whether the repression function of ROR{alpha} was mediated by these corepressors, we tested the ability of in vitro translated ROR{alpha} to interact with N-CoR and SMRT. Figure 5AGo shows that ROR{alpha} interacted with the receptor-interacting domains of both N-CoR and SMRT fused to glutathione-S-transferase (GST), but not with GST alone, indicative of specificity. Furthermore, the GAL4-ROR{alpha} polypeptides whose repression function was demonstrated in Fig. 4Go interacted strongly with N-CoR and SMRT in vitro, as shown in Fig. 5BGo. Approximately 5% of the input ROR{alpha} protein was bound by the corepressors, which is about half of the binding that we rountinely observe between corepressors and RevErb or TR (33). By contrast, the GAL4 DBD alone did not interact with N-CoR or SMRT.



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Figure 5. Interactions between ROR{alpha} and Nuclear Receptor Corepressors N-CoR and SMRT

A, ROR{alpha} interacts with both N-CoR and SMRT in vitro. GST, GST-N-CoR (amino acids 1744–2453), and GST-SMRT (amino acids 1282–1495) were incubated with in vitro translated full-length ROR{alpha}. Input shown is 20% of the amount used in each interaction incubation. B,. The corepressor interaction domain of ROR{alpha} correlates with the functional repression domain. GAL4-ROR{alpha} fusion proteins as indicated were labeled during in vitro translation and assayed for interaction with GST, GST-N-CoR (amino acids 1944–2453), and GST-SMRT (amino acids 1282–1495). Input shown is 40% of the amount used in each interaction incubation.

 
The AF2 Activation Helix Inhibits ROR{alpha} Interaction with N-CoR on DNA
We have recently shown that in the case of RevErb, which is a constitutive repressor, as well as in the case of unliganded TR, the ability to repress transcription is best correlated with the ability to interact with nuclear receptor corepressors on DNA (34). It was therefore of interest to determine whether ROR{alpha}, a constitutive activator, could interact with corepressors on DNA. Figure 6Go shows that ROR{alpha} did not interact with the receptor interaction domain of either N-CoR or SMRT on the RevDR2. This result was consistent with the in vivo effects of ROR{alpha}, which functions as a transcriptional activator on this site. We hypothesized, however, that either due to presence of an endogenous ligand in the in vitro binding reaction or due to an intrinsically activated conformation, the ROR{alpha} AF2 helix was functionally in a state analogous to the liganded form of TR that cannot bind corepressor on DNA. To test this, C-terminal deletions of ROR{alpha} were tested for the ability to bind corepressor on the RevDR2. Similar mutations in TR allow binding to corepressor even in the presence of ligand (7, 35). Indeed, Fig. 6Go shows that C-terminal deletions that interacted with corepressors in the context of GAL4-fusions also interacted with N-CoR on the RevDR2 in the context of the native DBD of ROR{alpha}. It should be pointed out that the binding of N-CoR bound to ROR{alpha} on the RevDR2 considerably less well than to RevErb (34). In this context, we point out that the repressor activity of ROR{alpha} 1–452 and 1–491 on the RevDR2 site was less than would be predicted from the Gal4-ROR{alpha} fusion results (data not shown). We believe that this is because the proteins are not expressed well in the transfected cells, although it is also possible that the interaction between ROR{alpha} and N-CoR on the RevDR2 is too weak for an in vivo response. The latter could be due to the N terminus of ROR{alpha}, which is not present in the Gal4 constructs and is known to affect the protein’s conformation and function (1, 37). Interestingly, ROR{alpha} selectively interacted with N-CoR and did not interact with SMRT on this site, similar to what we have observed for RevErb (34).



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Figure 6. The AF2 Helix of ROR{alpha} Regulates Its Interaction with N-CoR on DNA

EMSA analysis of wild type and C-terminal deletion mutants of ROR{alpha} binding to RevDR2 in the presence or absence of GST, GST-N-CoR (amino acids 1944–2453), and GST-SMRT (amino acids 1282–1495). Binding of TRß to DR4 oligonucleotide under the same condition is also shown. The free probes were run off the gel.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The discovery that homozygous mutation of ROR{alpha} prevents normal cerebellar development has added greatly to the significance of understanding the function of this orphan receptor. Most of what is known about ROR{alpha} to date is the result of elegant studies of Giguère and colleagues (36, 37), which have emphasized the ability of ROR{alpha} to activate transcription on a monomer site and the role of the T/A boxes and N terminus in regulating this function. The present work indicates that ROR{alpha} activates transcription only on a subset of monomer-binding sites and is actually a better transcriptional activator on the RevDR2 site to which it binds as a homodimer. One potential explanation for the selective dimerization of ROR{alpha} on the RevDR2 is that there is protein-protein interaction between the two monomers on this site, as we have previously observed to be the case for RevErb (32). However, the lack of cooperativity of the binding suggests that this interaction is not energetically favored compared with monomer binding and that the lack of two separate ROR{alpha} monomers binding to direct repeats with spacing of 1, 3, 4, and 5 bp may reflect steric hindrance to adjacent binding of two monomers on these sites.

There is no evidence that homodimer binding is favored over monomer binding in vitro. On the contrary, monomer binding is quantitatively greater than dimer binding to the RevDR2. in addition, expression of ROR{alpha} in the transfected cells does not increase its homodimer binding and there is no evidence that ROR interacts with other cellular proteins such as RXR to explain such findings (data not shown). In spite of these findings, the correlation between binding of two molecules of ROR{alpha} and transactivation from the RevDR2 site is striking. Consistent with this, ROR{alpha} also activated transcription from the reporter containing two widely spaced monomer sites, where steric hindrance to two monomers binding simultaneously is unlikely. It is also formally possible that two molecules of ROR{alpha} bind to the reporters containing the transcriptionally active monomer sites, although this would not be predicted from the sequences of the reporter constructs. On the basis of the increased activity of the binding sites allowing binding by two ROR{alpha} molecules, it is tempting to speculate that both molecules of ROR{alpha} may be required to bind coactivator protein. The related orphan receptor RevErb behaves similarly, in that it binds to both monomer and RevDR2 sites but only functions (as a repressor) on the dimer site. In that case, we have shown that this correlates with the ability of RevErb to bind to N-CoR on the RevDR2 site but not on the monomer-binding site (34). The ability of ROR{alpha} to activate transcription well in JEG-3 cells, but to a much lesser extent in 293T cells, suggests that one or more coactivators may be cell type-specific or that cell-specific modification of ROR{alpha} affects its transactivation function. To address this we have used the activation domain of ROR{alpha} as bait in the yeast two-hybrid screen and are currently evaluating known coactivators as well as novel clones identified in this manner (G. B. Atkins and M. A. Lazar, preliminary findings). It is also possible that the difference between these two cell types is due to a putative activating ligand for ROR{alpha} that is not present in 293T cells or to a cell-specific posttranslational modification. Alternatively, the repressive function of ROR{alpha} might be dominant in 293T cells.

The repression domain within ROR{alpha} was unanticipated from previous work. While these studies were in progress, Greiner et al. (38) described a functional analysis of the related orphan RORß/RZRß that suggested that this protein has a repression domain (38). Our work significantly extends the study on RORß, in that we have not only shown for the first time that ROR{alpha} also has a repression domain, but have demonstrated and characterized interactions between ROR{alpha} and transcriptional corepressors N-CoR and SMRT. ROR{alpha} interacts with both N-CoR and SMRT in GST-pulldown assays, but only with N-CoR on DNA. This pattern is distinct from that of TR but similar to that which we previously observed for RevErb (34).

The AF2 helix of ROR{alpha} appeared to prevent interaction with N-CoR on DNA but not in the GST-pulldown assay. This suggests that DNA binding by ROR{alpha} alters the conformation of the protein. Indeed, the amino terminus of ROR{alpha}1 regulates its DNA binding (1), and ROR{alpha} bends its DNA-binding site (37). Furthermore, DNA binding allosterically regulates the conformation of a number of other nuclear receptors (39), and these conformational changes regulate interaction with corepressors (40). Thus, DNA binding by ROR{alpha} may alter the protein conformation to the extent that an additional destabilizing effect of the AF2 helix is sufficient to inhibit corepressor binding. The result is that on DNA the constitutively active ROR{alpha} orphan behaves like a liganded receptor, such as TR or RAR, for which deletion of AF2 abolishes the effect of ligand to induce corepressor dissociation. If ROR{alpha} indeed requires a ligand for its activity, this would imply that the ROR{alpha} ligand is present in transfected JEG-3 cells as well as in reticulocyte lysate. An alternative explanation is that native ROR{alpha} assumes a conformation similar to that of ligand-bound receptors, in which ligand-induced movement of the AF2 activation helix contributes to recruitment of coactivator proteins (41) and causes the dissociation of corepressors (7). Thus, ROR{alpha} could have evolved from a ligand-regulated precursor protein, and mutations causing the protein to achieve the activated conformation in the absence of ligand may have provided a competitive advantage. In this scenario, the ability of truncated ROR{alpha} to bind corepressor might simply be a vestige of the evolutionary relationship between ROR{alpha} and other nuclear receptors. However, the repression by GAL4-ROR{alpha} in 293T cells suggests that, in at least some cell types, ROR{alpha} can assume a conformation that allows functional interaction with corepressors. This could be due to stabilization of the ROR{alpha}-corepressor complex by a cell-specific ligand or posttranslational modification.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmid Constructs for Transfection
pCMX ROR{alpha} was kindly provided by V. Giguère. pCMX GAL4 was cloned by digesting pCDM GAL4 (26) with SmaI and HindIII and ligating the insert into pCMX digested with NheI, filled in with Klenow, and HindIII. pCMX ROR was digested with XhoI and with BamHI, DraI, and NheI for the construction of pCMX GAL4-ROR 140–523, 140–491, and 140–451, respectively. These sites were blunt-ended and cloned into the blunt-ended EcoRI site of pCMX GAL4. For the construction of pCMX GAL4-ROR 221–523 and 272–523, PCR fragments were cloned into the BamHI site of pCMX GAL4 using the following 5'-primers: 5'-CCGGGGATCCGCTTCTAC-3' (221–523) and 5'-CCGGGGATCCCAGAATTA-3' (272–523). These primers were used with the following 3'-primer to amplify DNA fragments from pCMX ROR: 5'-CTCTGTAGGTAGTTTGTCC-3'. For reporter constructs (monomer and RevDR series) double-stranded oligonucleotides containing a single copy of each binding site shown in Figs. 1AGo and 2AGo were inserted into the BglII site of pTK-luciferase using BamHI ends. The insert for the Rev-DR2 C-flanking reporter was 5'-CCCCCAGGTCACCAGGTCAAA-3', and the insert for monomer D x 2 was: 5'-TTTGACCTAGTTGGGGATCCTTTGACCTAGGTTGGAGGA-3'. The GAL4 reporter vector has been described (32).

Plasmid Constructs for in Vitro Transcription/Translation
To construct pCMX ROR 1–491, pCMX ROR was digested with DraI, filled in with Klenow, and HindIII. The insert was ligated into pCMX digested with NheI, filled in with Klenow, and HindIII. pCMX ROR{alpha} 1–452 was constructed by digesting pCMX ROR with NheI to remove a small fragment and religating the vector. pCMX-HA-TRß1 has been described (34). These constructs, in addition to ROR{alpha} and GAL4 constructs described earlier, were transcribed using T7 RNA polymerase and translated in reticulocyte lysates (Promega, Madison, WI) in the presence of [35S]methionine (for use in GST pulldown assays) or cold amino acids [for use in electrophoretic mobility shift assay (EMSA)].

Protein Interaction Assays Using GST Fusion Proteins
Plasmid constructs for GST fusion proteins have been described (34). GST fusion proteins were expressed in BL21 bacteria by induction with 0.5 mM isopropylthio-ß-O-galactoside at 30 C. Proteins were isolated by cell lysis with lysozyme and detergent followed by sonication. GST beads (50 µl) containing the fusion protein were incubated at room temperature in a buffer containing 50 mM KCl, 20 mM HEPES, pH 7.9, 2 mM EDTA, 0.1% NP-40, 10% glycerol, 0.5% nonfat dry milk, and 5 mM dithiothreitol. Five microliters of in vitro translated ROR{alpha}1, GAL4-ROR{alpha} 140–523, GAL4-ROR{alpha} 140–491, and GAL4 ROR{alpha} 140–451 proteins were added to the beads. Binding was allowed to proceed for 1 h and then the beads were washed four times in the same buffer. The bound proteins were eluted by boiling in 30 µl SDS-PAGE loading buffer and resolved by electrophoresis. In the experiments shown, the amount of input protein was 1 µl for ROR and 2 µl for GAL4 proteins. The GST fusion proteins were stained with Coomassie to ensure equal loading, and the bound proteins were visualized by autoradiography.

Cell Culture and Transfection
293T cells were maintained and transfected in DMEM high glucose with 10% FCS. JEG-3 cells were maintained and transfected in DMEM low glucose with 10% FCS. At 80% confluence, 60-mm dishes were transfected by the calcium phosphate precipitation method using 2 µg luciferase reporter, 0.5 µg ß-galactosidase (ß-gal) expression vector, and 2 µg receptor expression vector. Equivalent amounts of empty expression vector (pCMX) were included in cells transfected with submaximal amounts of receptor. Cells were lysed in Triton X-100 buffer and ß-gal and luciferase assays were carried out using standard protocols (32). The measured relative light units were normalized to ß-gal activity, which served as an internal control for transfection efficiency. Fold activation/repression was calculated as the activity of a given reporter after transfection with control expression vector divided by the activity of the same reporter in the presence of ROR expression vector. Figures show the results of representative experiments in which individual data points were assayed in duplicate, and the range or SEM are shown, respectively. Each experiment was repeated two to five times. The degree of activation/repression from a given site was highly consistent from experiment to experiment.

EMSA
The top strand of the DNA probes used for EMSA include those oligos described for use in reporter constructs for transfection (Monomer A-D, RevDR1–5) as well as DR4: 5'-GATCCTAAGGTCAAATAAGGTCAGAGG-3'. These oligos were annealed with complementary bottom oligos, thereby generating overhanging BamHI ends that were filled in with Klenow in the presence of [32P]dCTP. EMSA was carried out as described previously (32). In cases where in vitro translated receptors were mixed with 30 µg specified corepressor protein, protein was purified as described (34). The proteins were preincubated at room temperature for 15 min in the standard 30- µl binding reaction containing 1x binding buffer (10 mM HEPES, pH 7.9, 80 mM KCl, 5% glycerol, 0.01 M dithiothreitol), 200 µg/ul polydeoxyinosinic-deoxycytidylic acid, and 25 ng/ml salmon sperm DNA. Labeled probe (100,000 cpm) was added, and after incubation for 10 min at room temperature, reaction mixtures were loaded on a 5% polyacrylamide gel and separated in 0.5x Tris-borate-EDTA at room temperature. Gels were dried before autoradiography. For off-rate experiments, polyacrylamide gels (5%) were prerun for at least 2 h before loading. Proteins were preincubated with labeled probe for 1 h at room temperature and then the first lane was loaded into the running gel, 500-fold molar excess of cold DNA competitor was added to the remaining reaction mix, and equal aliquots were loaded onto the remaining lanes at the times indicated in the figure.

Methylation and DEPC Interference Assays
The RevDR2 site digested from the reporter construct was labeled on either strand by using Klenow to fill in one 3'-overhang XbaI digested before digestion with the second enzyme (HindIII). Methylation interference was performed essentially as described (32). Briefly, the gel-purified, labeled DNA was modified by treatment with dimethylsulfate. The modified probe (300,000 cpm) was incubated with 20 µl in vitro translated ROR{alpha} in a 40-µl reaction and subjected to EMSA. The protein-DNA complexes and free probe were eluted from the wet gel, and DNA was cleaved with piperidine and electrophoresed on a 8% sequencing gel. DEPC-interference assays were performed on the same DNA fragment as previously described (32).


    ACKNOWLEDGMENTS
 
We thank Iris Zamir for helpful discussions. We also thank V. Giguere for providing the ROR{alpha} cDNA.


    FOOTNOTES
 
Address requests for reprints to: Mitchell A. Lazar, M.D., Ph.D., University of Pennsylvania School of Medicine, 611 CRB, 415 Curie Boulevard, Philadelphia, Pennsylvania 19104-6149.

This work was supported by NIH Grant DK-45586 (to M.A.L.). G.B.A. was supported by the Medical Scientist Training Program.

1 The first two authors contributed equally to this work. Back

Received for publication May 13, 1997. Revision received June 26, 1997. Accepted for publication July 2, 1997.


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