Transition from Monomeric to Homodimeric DNA Binding by Nuclear Receptors: Identification of RevErbA{alpha} Determinants Required for ROR{alpha} Homodimer Complex Formation

Anna N. Moraitis and Vincent Giguère

Molecular Oncology Group McGill University Health Centre Montréal, Québec, Canada H3A 1A1
Departments of Biochemistry, Medicine, and Oncology McGill University Montréal, Québec, Canada H3A 1A1


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nuclear hormone receptors belong to a class of transcription factors that recognize specific DNA sequences either as monomers, homodimers, or heterodimers with the common partner retinoic X receptor. In vitro mutagenesis studies, as well as determination of the crystal structure of several complexes formed by the DNA-binding domain of receptors bound to their cognate response elements, have begun to explain the molecular basis for protein-DNA and protein-protein interactions essential for high-affinity and specific DNA binding by nuclear receptors. In this study, we have used the related orphan nuclear receptors, ROR{alpha} and RevErbA{alpha}, to study the molecular determinants involved in the transition from monomeric to homodimeric modes of DNA binding by nuclear receptors. While both receptors bind DNA as monomers to a response element containing a core AGGTCA half-site preceded by a 5'-A/T-rich flanking sequence, RevErbA{alpha} also binds as a homodimer to an extended DR2 element. Gain-of-function experiments using point mutations and subdomain swaps between ROR{alpha} and RevErbA{alpha} identify four amino acids within RevErbA{alpha} sufficient to confer ROR{alpha} with the ability to form cooperative homodimer complexes on an extended DR2. This study reveals how the transition from monomer to homodimer DNA binding by members of the nuclear receptor superfamily could be achieved from relatively few amino acid substitutions.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The nuclear receptor superfamily consists of transcription factors whose activity is regulated by small lipophilic molecules that include sterols, steroid hormones, vitamin D, thyroid hormone, retinoids, prostanoids, and fatty acids (1). Superfamily members also embody a large group of related proteins, termed orphan nuclear receptors, for which ligands have not yet been identified (2). Nuclear receptors transduce the effects of their ligands mostly through binding to short DNA sequences, referred to as hormone response elements (HREs). HREs are composed of consensus hexameric sequences arranged in tandem as inverted, everted, and direct repeats upon which nuclear receptors can bind as homodimers or heterodimers with the ubiquitous partner RXR (3). In addition, a subset of nuclear receptors bind DNA as monomers to a single consensus half-site preceded by a 5'-A/T-rich flanking sequence (4, 5, 6). Functional analysis of mutant receptors coupled with the determination of the crystal structure of several complexes formed by the DNA-binding domain (DBD) of receptors bound to their cognate response elements have begun to explain the molecular basis for protein-DNA and protein-protein interactions essential for high-affinity and specific DNA binding by nuclear receptors. Specific recognition of the core half-site sequence is provided by three amino acid residues at the base of the first zinc finger module (the P box) (7, 8, 9, 10), while recognition of the 5'-A/T-rich flanking sequence present in monomeric HREs is mediated by contacts between DNA and amino acid residues located in the carboxy-terminal extension (CTE) of the core DBD (11, 12, 13). On the other hand, binding specificity for a given homodimer or heterodimer complex is dictated by DNA-dependent dimerization of the two DBD subunits. Spacing specificity is regulated by motifs contained in determinants located in the first and second zinc finger modules as well as in the CTE, and the importance of an individual motif in determining half-site specificity depends on the configuration of the HRE (14, 15, 16, 17, 18, 19, 20, 21). For example, steroid receptors homodimerize on inverted repeats, and strict half-site spacing by 3 bp is regulated by determinants located at the base of the second zinc finger module of the DBD (the D box) (7, 9, 22). On the other hand, nuclear receptors that heterodimerize with RXR bind with highest affinity to direct repeats (DR) separated by a characteristic number of nucleotides, and spacer discrimination is provided by the CTE of the RXR’s partner as well as by distinct usage of dimerization determinants in the first and second zinc finger modules of RXR (16).

ROR{alpha} is an orphan nuclear receptor that was initially cloned based on its similarity to the retinoic acid receptor (5). ROR{alpha} is a monomeric DNA-binding receptor that constitutively activates genes harboring ROR{alpha} response elements (ROREs). Mouse genetic studies have shown ROR{alpha} to be encoded by the staggerer locus and essential for cerebellar development (23, 24, 25, 26, 27). The ROR{alpha} gene generates at least four distinct isoforms that share common DBDs and ligand-binding domains (LBDs) but have distinct amino-terminal domains (NTDs) (5, 28). Detailed in vitro mutagenesis studies has determined that the CTE is required for high-affinity DNA binding and that the distinct NTDs influence how the CTE recognizes the extended 5'-A/T-rich flanking sequence present in ROREs (13), leading to the proposal that the NTD of ROR{alpha} provides intramolecular interactions necessary to stabilize receptor-DNA interactions (29).

RevErbA{alpha}, an orphan member of the superfamily of nuclear receptors, is encoded on the opposite strand of the c-ErbA (T3R{alpha}) gene (30, 31). DNA-binding studies have independently shown that ROR{alpha}, RevErbA{alpha}, and its close relative RVR/BD73 (also known as RevErbAß) recognize the same monomeric binding site consisting of a half-site AGGTCA motif preceded by a 5'-A/T-rich sequence (6, 32, 33). However, RevErbA{alpha} lacks a typical activation function (AF2) within the LBD, and competition for common binding sites results in down-regulation of ROR{alpha}-induced gene expression (32, 34). The physiological importance of the monomeric binding site has been demonstrated through the characterization of a functional RORE within the N-myc protooncogene transcription unit (35). ROR{alpha} and RVR have opposite transcriptional effects on the N-myc gene, and mutation of the RORE increases the oncogenic potential of the N-myc gene in a rat embryonic fibroblast transformation assay, suggesting that deregulation of the activity of members of the ROR and RevErbA family could contribute to the initiation and progression of certain types of neoplasia (35). However, RevErbA{alpha} has also been shown to bind DNA as a homodimer to an extended DR2 containing the 5'-A/T-rich flanking sequence present in ROREs (36). The biological importance of the dimeric interaction has been reinforced by the study of Zamir et al. (37), which provides evidence that the RevErbA{alpha} dimer, but not the monomeric form, can recruit corepressors and act as an active repressor. Recently, the crystal structure of the RevErbA{alpha} DBD bound to an extended DR2 was solved (21). The crystal structure demonstrated that the CTE plays an important role in making direct contacts with the 5'-A/T-rich flanking sequence of an extended DR2 and confirmed that contacts between the CTE and the core DBD are necessary to stabilize receptor dimers.

Taken together, our current knowledge of ROR{alpha} and RevErbA{alpha} DNA-binding activities demonstrates that these related orphan nuclear receptors can be used as an experimental model to investigate the molecular basis involved in the transition from monomeric to homodimeric modes of DNA binding by nuclear receptors. In this study, we have used in vitro mutagenesis to produce chimeric receptors to dissect the molecular determinants of monomeric and homodimeric DNA binding within the DBD. We demonstrate that by changing a minimum of four amino acid residues, we are able to confer to the ROR{alpha} DBD the ability to homodimerize on an extended DR2 element. These results identify structural determinants necessary for transition from monomer to homodimer DNA binding by members of the nuclear receptor superfamily and reveal that this transition can be achieved from relatively few amino acid substitutions.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Experimental Model
Figure 1Go schematically represents the structure of the ROR{alpha} DBD peptide used in this study and its similarity to the RevErbA{alpha} DBD. The DBD is subdivided into three domains referred to as zinc finger module 1 and 2 and the CTE. The minimal ROR{alpha} and Rev-ErbA{alpha} DBD constructs used in this study (referred to as ROR and Rev) include the core DBD encoding the two zinc finger modules flanked by 10 amino acids at the N-terminal end and the entire CTE as previously defined (13). The illustration also depicts determinants previously shown by mutagenesis and crystallographic studies to be required for the formation of homodimeric RevErbA{alpha} or heterodimeric RXR/T3R complexes (16, 21). Specific amino acid residues that mediate subunit dimerization in these complexes are identified. Residues present in the dimerization determinants and distinct in ROR{alpha} and RevErbA{alpha} constituted targets for our mutagenesis study.



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Figure 1. Schematic Representation of the ROR{alpha} DBD Used in This Study and Known Nuclear Receptor Dimerization Determinants

The DBD of human ROR{alpha} is divided in three functional subdomains referred to as zinc finger module 1 and 2 and the CTE. Numbering is according to the full-length ROR{alpha}1 (5 ). Residues that are identical in the ROR{alpha}1 and RevErbA{alpha} sequences are shown in white circles; nonconserved residues are represented by gray circles. The Ser residue represented by a black circle is not conserved within the ROR family. Closed and open symbols linked to amino acid residues represent residues that have been shown to mediate dimerization between partners in the RevErbA{alpha} homodimer (21 ) and RXR-T3R heterodimer (16 ) complexes, respectively. Arrows point to four amino acid substitutions (stippled circles) that together confer the ROR DBD peptide with the ability to bind as a dimer to an extended DR2 element.

 
To validate our experimental model, we first tested the binding of full-length ROR{alpha}1 and RevErbA{alpha}, synthesized in vitro, to oligonucleotides encoding the RORE and extended DR2 elements. As expected, both ROR{alpha}1 and RevErbA{alpha} bind the RORE as monomers (Fig. 2AGo, lanes 2 and 3, respectively). In contrast, ROR{alpha}1 still binds as a monomer on an extended DR2 whereas RevErbA{alpha} forms homodimers on this element (Fig. 2AGo, lanes 5 and 6, respectively). Nuclear receptors that bind DNA as dimers possess dimerization interfaces in both the LBD and DBD. However, the LBD dimerization interface plays no role in binding site selectivity. Likewise, the dimerization interface in the LBD of RevErbA{alpha} is not essential for DR2 recognition, and the minimal region required for cooperative homodimer formation on this element is the DBD (36). As the DBD appears to play a dominant role in determining RevErbA{alpha} DNA binding specificity, we chose to study the properties of the isolated DBDs. As expected, both ROR and Rev DBDs form monomers on a RORE (Fig. 2BGo, lanes 2 and 3, respectively). In contrast, the ROR DBD binds as a monomer, and the Rev DBD preferentially forms homodimer complexes on an extended DR2 element (Fig. 2BGo, lanes 5 and 6, respectively). To be able to monitor the monomer-to-homodimer transition by ROR DBD mutants in future experiments, we determined the fraction (%) of total bound probe that is contained in the monomer and dimer complexes for a range of protein concentrations using a Bio-Imaging Analyzer (Fuji Bas 1000 MacBAS, Fuji Medical Systems, Stamford, CT). As ROR DBD concentration increases, a slower migrating homodimeric complex appears (data not shown). As shown in Fig. 2CGo, homodimer binding of the ROR DBD to the DR2 is noncooperative, suggesting that the DR2 half-sites are progressively filled by protein monomers as previously observed (38). Similar results were obtained when DNA binding activity of the ROR DBD was tested on DR1, DR3, DR4, and DR5 elements containing RORE-like 5'-A/T-rich sequences (data not shown). For the Rev DBD, the increase in dimer complex formation was more rapid than could be accounted for by additivity alone, demonstrating that the Rev DBD possesses determinants necessary to achieve cooperative DNA binding (Fig. 2CGo). Therefore, starting with the premise that a homotypic phenotype would be achieved if both of the required dimerization interfaces are present in the same molecule, we decided to progressively introduce amino acid residues present in the Rev DBD into the ROR DBD and monitor the ability of ROR DBD to homodimerize on an extended DR2.



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Figure 2. ROR{alpha} Is a Monomeric Binding Orphan Nuclear Receptor

EMSA using the RORE and extended DR2 response elements and in vitro translated full-length ROR{alpha}1 and RevErbA{alpha} receptors (A) and DBD peptides (ROR and Rev) (B). C, Quantification of dimer and monomer complexes bound to an extended DR2 element for increasing concentrations of ROR and Rev DBDs programmed rabbit reticulocyte lysate (RRL) from a representative experiment. D, Homodimer; M, monomer; free, unbound probe.

 
Three Amino Acid Residues in Zinc Finger Module 1 Participate in the Monomer-to- Homodimer Transition by ROR Mutants
We first studied the first zinc finger module and the base of the second zinc finger module [previously referred to as the D-box (14)] as these determinants were shown to play important roles in dimer formation and HRE recognition in both homodimeric and heterodimeric DNA-receptor complexes. We engineered complete subdomain swaps between ROR and Rev DBDs by introducing five and four amino acid changes in the first and second zinc finger modules, respectively (Fig. 3AGo). As expected, the three chimeric ROR/Rev DBD peptides, RORm1, RORm2, and RORm3, retain their ability to bind as monomers to the RORE, although a reduction in total binding is observed when the first zinc module is swaped alone (RORm1) or in combination (RORm3) with the D-box of Rev DBD (Fig. 3BGo). On an extended DR2 element, introducing the first zinc finger module of the Rev DBD in the ROR DBD also reduces binding efficacy: interestingly, the chimeric RORm1 peptide efficiently binds DNA as a homodimer (Fig. 3BGo, lane 10). In contrast, a D-box swap (RORm2) has no significant effect on either DNA-binding affinity or homodimer formation (Fig. 3BGo, lane 11). A switch in both zinc finger modules represented by RORm3 does not increase homodimer binding beyond that observed with RORm1 (Fig. 3BGo, compare lane 10 to 12). Results presented in Fig. 3CGo demonstrate that a mutated zinc finger module 1 provides the ROR DBD with a dimerization interface and demonstrate that the D-box does not play an important role in Rev DBD homodimer formation.



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Figure 3. ROR{alpha} DBD Peptide Encoding RevErbA{alpha}’s Zinc Finger Module 1 Homodimerizes on an Extended DR2

A, The primary sequences of the Rev and ROR DBD core comprising the two zinc finger modules beginning with the first cysteine of the first zinc finger as well as those of chimeric ROR/Rev constructs are shown. Asterisks indicate the amino acids that are not conserved between ROR{alpha} and RevErbA{alpha}. B, EMSA of in vitro translated ROR, Rev, and ROR DBD mutants using RORE and an extended DR2 as probes. C, Quantification of dimer and monomer complexes formed by ROR, Rev, and ROR mutants on an extended DR2 element. Results of a representative experiment are presented as the fraction (%) of probe bound by receptor dimers formed on an extended DR2 element.

 
A series of mutations in the ROR DBD were generated to identify specific amino acid residues participating in the dimerization determinants within the first zinc finger module (RORm4 to RORm8, Fig. 4AGo). Ile83 is of particular interest as all dimeric receptors surveyed possess either a Phe or a Tyr residue at this position. In particular, these residues were shown to be directly involved in the formation of the heterodimeric T3R/RXR complex (16). On the other hand, the four other divergent amino acid residues between ROR and Rev DBDs have not been shown to be directly involved in making protein-protein contact in any structure solved so far. Surprisingly, introducing either Lys79Val and Ser80Ala mutations simultaneously (RORm4) or Ile83Phe alone (RORm5) both considerably increase dimer formation by the ROR DBD (Fig. 4Go, B and C). Combining both changes in a single mutant (RORm6) further increases the ability of the ROR DBD to bind as a homodimer (Fig. 4Go, B and C). The less efficient homodimer formation by RORm6 than the Rev DBD suggests that additional determinants are needed for protein-protein interactions. Changing the last two amino acid residues of that module (Ile88His and Thr89Ala) in mutant RORm7 has no significant effect on homodimer formation but lowers binding affinity for the extended DR2, as judged by the intensity of the complex relative to the ROR DBD and other mutants (Fig. 4Go, B and C). Combination of the Lys79Val, Ser80Ala, Ile88His, and Thr89Ala mutations in RORm8 demonstrates that while the chimeric DBD peptide has a lower binding affinity, it retains the ability to bind as a homodimer.



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Figure 4. Three Amino Acids in RevErbA{alpha}’s Zinc Finger Module 1 Are Sufficient to Provide ROR{alpha} DBD with the Ability to Form Homodimers

A, The primary sequences of the first zinc finger module beginning with the first cysteine of the first zinc finger of Rev and ROR as well as those of chimeric ROR/Rev DBD constructs are shown. B, EMSA of in vitro translated ROR DBD wild type and mutants using an extended DR2 probe. C, Quantification of the amount of ROR DBD dimer complexes. The mean of three independent experiments is presented as the fraction (%) of probe bound by receptor dimers formed on an extended DR2 element.

 
A Single Amino Acid Residue in Zinc Finger Module 2 Participates in Monomer-to-Homodimer Transition by ROR Mutants
Determination of the crystal structures of T3R-RXR DBD heterodimer and RevErbA{alpha} DBD homodimer complexes revealed that, of the four amino acid residues involved in protein-protein interactions, only Thr120 is divergent between the ROR and Rev DBDs (16, 21). We therefore decided to target this amino acid for site-directed mutagenesis of the ROR DBD (Fig. 5AGo). The Thr120Ile mutation (RORm9) increases considerably the amount of dimer complexes formed (Fig. 5BGo). This mutation was then combined with the three amino acids of the first zinc finger module previously shown to be important for the monomer-to-homodimer transition. The resulting construct (RORm10) strongly homodimerizes on an extended DR2 with a dimer ratio equivalent to that of the Rev DBD. Therefore, a minimum of four amino acid changes, three in the first zinc finger module (Lys79Val, Ser80Ala, Ile88Phe) and one in the second zinc finger module (Thr120Ile), are required to provide the ROR DBD with the ability to homodimerize on an extended DR2.



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Figure 5. Four Amino Acids Are the Key Dimerization Determinants

A, Amino acid sequences of the two zinc finger modules of RevErbA{alpha}, ROR{alpha}, and chimera and their respective fraction (%) of probe bound by receptor dimers formed on an extended DR2 element represents the mean of three independent experiments (B).

 
Providing Full-Length ROR{alpha} with a Dimerization Interface in the DBD Is Sufficient for Cooperative Homodimerization
We tested whether the introduction of a dimerization interface in the ROR{alpha}1 DBD would be sufficient to allow the full-length receptor to form homodimers on an extended DR2 element. ROR{alpha}1 constructs encoding the RevErbA{alpha} dimerization determinants of the first and second zinc finger, ROR{alpha}1m6 and ROR{alpha}1m9, respectively, were constructed and assayed by electrophoretic mobility shift assay (EMSA) (Fig. 6Go). At highest protein concentrations, both ROR{alpha}1m6 and ROR{alpha}1m9 mutants form two times more homodimer complexes than wild-type ROR{alpha}1 on an extended DR2 element. The result obtained with ROR{alpha}1m9 confirms and extends the finding reported by Zhao et al. (21). The ROR{alpha}1m10 mutant encoding both the dimerization determinants of the first and second zinc finger modules forms homodimeric complexes slightly less efficiently than RevErbA{alpha}.



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Figure 6. Full-Length ROR{alpha}1 Encoding the Four Dimerization Determinants Forms Cooperative Homodimers on an Extended DR2 Element

The fraction (%) of probe bound by receptor dimers formed on an extended DR2 element was determined for increasing concentrations of receptor protein synthesized in programmed rabbit reticulocyte lysate (RRL) for ROR{alpha}1 (white circles), RevErbA{alpha} (black circles), ROR{alpha}1m6 (gray squares), ROR{alpha}1m9 (gray triangles), and ROR{alpha}1m10 (gray diamonds). The graph displays the results of one representative experiment.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
On the basis of their DNA-binding properties, nuclear receptors can be classified into two major groups: monomers, exemplified by orphan nuclear receptors ROR{alpha}, RevErbA{alpha}, SF-1, and NGFI-B, and dimers, which include homodimers and heterodimers (1). Some receptors belong to more than one group. RevErbA{alpha} binds DNA both as a monomer and as a homodimer (36), NGFI-B binds as both a monomer and a heterodimer (39, 40), whereas T3R can bind DNA as a monomer, homodimer, and heterodimer with RXR (for references, see Ref. 3). Homodimeric orphan nuclear receptors such as RevErbA{alpha} and hepatic nuclear factor 4 bind to direct repeat HREs (36, 41), whereas steroid hormone receptors form homodimers on inverted HREs (42). Heterodimeric complexes always involve RXR, and interestingly, RXR’s partner is usually associated with a known ligand (43). The flexibility observed in the DNA-binding properties of nuclear receptors suggests that, as previously observed for the determinants required for discrimination of HRE sequences (7, 8, 14), few changes would be required for a receptor to acquire novel DNA-binding characteristics and thus provides a simple mechanism for receptor evolution. The results of this study clearly demonstrate that this may be the case since, by changing only four amino acids, the DNA-binding mode of the orphan nuclear receptor ROR{alpha}1 can be converted from monomer to homodimer.

Transition from monomeric to homodimeric DNA binding by nuclear receptors is facilitated by the dual role played by the CTE in DNA binding. As described in Introduction, the CTE contains essential determinants for recognition of the 5'-A/T-rich flanking sequence of monomeric HRE and, in addition, participates in the formation of the dimer interface of homodimeric and heterodimeric receptor complexes (11, 13, 16, 21). Thus, one can hypothesize that while keeping intact the highly conserved CTE required for monomeric DNA binding, progressive evolutionary changes in the zinc finger modules of the DBD could allow nuclear receptors to acquire the ability to bind DNA as homodimers. In fact, significant homodimer binding can be observed with single amino acid changes without significant loss of monomeric DNA binding (data not shown), indicating that the transition from monomer to homodimer binding could be done progressively without engendering a nonfunctional receptor. This process, which expands the repertoire of target genes regulated by nuclear receptors, parallels the previously observed nondisruptive changes in the P-box that allow for progressive acquisition of new binding specificities by nuclear receptors (7). Alterations in the CTE and zinc finger modules could lead to recognition of novel HREs with distinct half-site spacing. Taken together, these studies illustrate how few changes in common determinants could lead to a wide variety of DNA-binding mechanisms used by members of the nuclear receptor superfamily.

While this paper was in preparation, the crystal structure of the RevErbA{alpha} DBD was published (21). This study showed that, in contrast to RXR heterodimer complexes bound to direct-repeat HREs, homodimer formation of RevErbA{alpha} DBD subunits to an extended DR2 element involves direct contact between residues in the second zinc finger module of the first subunit with the CTE of the second subunit, but does not involve residues within the first zinc finger module. While our study supports the importance of amino acid residues within the second zinc finger module for homodimerization, it also demonstrates that amino acids within the first zinc finger module are equally important for dimer formation by chimeric ROR DBD peptides. It is possible that the amino acids encoded in the first zinc finger module are not directly involved in protein-protein contacts of the dimerization interface but rather are involved in intramolecular interactions necessary for the proper positioning of other residues involved in forming the dimer interface. It is interesting to note that while position 88 in the first zinc finger module of ROR{alpha}1 is occupied by an Ile or Val residue within the ROR family, including the Drosophila orphan receptor DHR3 shown to bind DNA as a monomer (44), the corresponding position in nuclear receptors belonging to group 1 of the nuclear receptor superfamily (45) is occupied by residues containing aromatic rings (Fig. 7Go). Members of this subgroup that have a residue with a hydrocarbon sidechain instead of an aromatic ring at this position could be predicted to bind DNA exclusively as monomers. So far, the only nuclear receptor outside of the ROR family to possess this characteristic is Onchocerca volvulus NHR-1 (46), but its DNA-binding characteristics have not been investigated. If this observation is supported by future studies, the prediction will be that very few members of the nuclear receptor superfamily would bind DNA exclusively as monomers.



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Figure 7. First Zinc Finger Module Sequence Alignment of Group 1 Nuclear Receptors

The amino acid residues with a hydrocarbon sidechain in place of an aromatic ring at the position corresponding to residue 88 in ROR{alpha}1 (marked by an asterisk) are highlighted.

 
Dimerization is usually required for DNA binding by nuclear receptors as it orients and stabilizes adjacent DBDs that are unable to interact in the absence of DNA (3). Since ROR{alpha} lacks key DBD dimerization determinants but nonetheless binds to DNA with high affinity, it must do so in a way that is different from other nuclear receptors. Our previous biochemical and mutagenesis analyses showed that a ROR{alpha} monomer binds a RORE in a bipartite manner, placing the first zinc finger module into the major groove at the 3'-AGGTCA element, and the CTE interacting with the adjacent minor groove at the 5'-A/T-rich extension of the RORE (13). More importantly, these experiments have also demonstrated that intramolecular interactions stabilize the ROR{alpha}-DNA monomer complex, as the NTD and the nonconserved hinge region cooperate to properly align the zinc finger modules and the CTE with respect to each other (29). While the molecular basis for high-affinity monomeric DNA binding (and for the transition to dimeric DNA binding) begin to be unraveled, application of direct structural approaches will be required to understand fully the complex intramolecular interactions necessary for ROR{alpha} and other monomeric receptors to stably and precisely make contacts with their cognate site.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
DBD peptides were generated by using pairs of oligonucleotide primers, one containing the antisense strand encoding the end of the CTE with a 5'-tail containing a stop codon and a BamHI site, and the other containing the sense sequence beginning 10 amino acids N-terminal to the first cysteine of the core DBD and an Asp718 site, for PCR using 50 ng of pCMXhROR{alpha}1 (5) and pCMXhRev-ErbA{alpha} (31) DNA as templates. The amplified fragments were digested with Asp718 and BamHI and then reintroduced into the Asp718 and BamHI sites of pCMX. The DBD peptides generated are 102 amino acids (ROR) and 103 amino acids (Rev) long. ROR{alpha} and RevErbA{alpha} DBD mutants used in this study were generated using site-directed mutagenesis as described by the Quick Change Site-Directed mutagenesis kit protocol (Stratagene, La Jolla, CA). The nucleotide sequences of all constructs described above were confirmed by sequencing.

EMSA
Coupled in vitro transcription and translation with T7 RNA polymerase and TNT rabbit reticulocyte lysate (Promega, Madison, WI) was used to synthesize full-length ROR{alpha} and RevErbA{alpha} and the truncated DBD peptides from pCMX-based plasmids according to the manufacturer’s instructions. Between 1 and 10 µl programmed rabbit reticulocyte lysate was used in DNA-binding reactions as previously described (13). Samples were loaded onto a 5% nondenaturing polyacrylamide gel for full-length receptors or 8% for DBD peptides and electrophoresed at 150 V at room temperature. Quantification of dimer and monomer complexes was done using a Bio-Image Analyzer Bas1000 (Fuji). Each experiment was performed in triplicate. The following oligonucleotides and their complements were used as probes: RORE, 5'-TCGACTCGTATAACTAGGTCAAGCGTG-3', DR2, 5'-TCGACTCGTCTAATT-AGGTCAGTAGGTCAGCGCTG-3'; both probes are derived from consensus sequences obtained from binding site selection experiments (5).


    FOOTNOTES
 
Address requests for reprints to: Dr. Vincent Giguère, Molecular Oncology Group, McGill University Health Centre, 687 Pine Avenue West, Montréal, Québec, Canada H3A 1A1. E-mail: vgiguere{at}dir.molonc.mcgill.ca

Financial support was provided by the Medical Research Council of Canada (MRCC), the National Cancer Institute of Canada, and the Cancer Research Society Inc. to V.G. A.N.M. was the recipient of a training grant from the Fonds de Recherches en Santé du Québec and V.G. holds a Scientist Award from the MRCC.

Received for publication September 2, 1998. Revision received October 28, 1998. Accepted for publication November 30, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schütz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM 1995 The nuclear receptor superfamily: the second decade. Cell 83:835–839[Medline]
  2. Willy PJ, Mangelsdorf DJ 1998 Nuclear orphan receptors: the search for novel ligands and signaling pathways. Hormones and Signaling. San Diego, Academic Press. 307–358
  3. Glass CK 1994 Differential recognition of target genes by nuclear receptors monomers, dimers, and heterodimers. Endocr Rev 15:391–407[Medline]
  4. Wilson TE, Fahrner TJ, Johnson M, Milbrandt J 1991 Identification of the DNA binding site for NGFI-B by genetic selection in yeast. Science 252:1296–1300[Medline]
  5. Giguère V, Tini M, Flock G, Ong ES, Evans RM, Otulakowski G 1994 Isoform-specific amino-terminal domains dictate DNA-binding properties of ROR{alpha}, a novel family of orphan nuclear receptors. Genes Dev 8:538–553[Abstract]
  6. Harding HP, Lazar MA 1993 The orphan receptor Rev-ErbA{alpha} activates transcription via a novel response element. Mol Cell Biol 13:3113–3121[Abstract]
  7. Umesono K, Evans RM 1989 Determinants of target gene specificity for steroid/thyroid hormone receptors. Cell 57:1139–1146[Medline]
  8. Mader S, Kumar V, de Verneuil H, Chambon P 1989 Three amino acids of the oestrogen receptor are essential to its ability to distinguish an oestrogen from a glucocorticoid-responsive element. Nature 338:271–274[CrossRef][Medline]
  9. Luisi BF, Xu WX, Otwinowski Z, Freedman LP, Yamamoto KR, Sigler P 1991 Crystallographic analysis of the interaction of the glucocorticoid receptor with DNA. Nature 352:497–505[CrossRef][Medline]
  10. Danielsen M, Hinck L, Ringold GM 1989 Two amino acids within the knuckle of the first zinc finger specify DNA response element activation by the glucocorticoid receptor. Cell 57:1131–1138[Medline]
  11. Wilson TE, Paulsen RE, Padgett KA, Milbrandt J 1992 Participation of non-zinc finger residues in DNA binding by two nuclear orphan receptors. Science 256:107–110[Medline]
  12. Wilson TE, Fahrner TJ, Milbrandt J 1993 The orphan receptors NGFI-B and steroidogenic factor 1 establish monomer binding as a third paradigm of nuclear receptor-DNA interaction. Mol Cell Biol 13:5794–5804[Abstract]
  13. Giguère V, McBroom LDB, Flock G 1995 Determinants of target gene specificity for ROR{alpha}1: monomeric DNA-binding by an orphan nuclear receptor. Mol Cell Biol 15:2517–2526[Abstract]
  14. Umesono K, Murakami KK, Thompson CC, Evans RM 1991 Direct repeats as selective response elements for the thyroid hormone, retinoic acid, and vitamin D3 receptors. Cell 65:1255–1266[Medline]
  15. Perlmann T, Rangarajan PN, Umesono K, Evans RM 1993 Determinants for selective RAR and TR recognition of direct repeat HREs. Genes Dev 7:1411–1422[Abstract]
  16. Rastinejad F, Perlmann T, Evans RM, Sigler PB 1995 Structural determinants of nuclear receptor assembly on DNA direct repeats. Nature 375:203–211[CrossRef][Medline]
  17. Zechel C, Shen X-Q, Chambon P, Gronemeyer H 1994 Dimerization interfaces formed between the DNA binding domains determine the cooperative binding of RXR/RAR and RXR/TR heterodimers to DR5 and DR4 elements. EMBO J 13:1414–1424[Abstract]
  18. Zechel C, Shen X-Q, Chen J-Y, Chen Z-P, Chambon P, Gronemeyer H 1994 The dimerization interfaces formed between the DNA binding domains of RXR, RAR and TR determine the binding specificity and polarity of the full-length receptors to direct repeats. EMBO J 13:1425–1433[Abstract]
  19. Predki PF, Zamble D, Sarkar B, Giguère V 1994 Ordered binding of retinoic acid and retinoid X receptors to asymmetric response elements involves determinants adjacent to the DNA-binding domain. Mol Endocrinol 8:31–39[Abstract]
  20. Kurokawa R, Yu V, Näär A, Kyakumoto S, Han Z, Silverman S, Rosenfeld MG, Glass CK 1993 Differential orientations of the DNA binding domain and C-terminal dimerization interface regulate binding site selection by nuclear receptor heterodimers. Genes Dev 7:1423–1435[Abstract]
  21. Zhao Q, Khorasanizadeh S, Miyoshi Y, Lazar MA, Rastinejad F 1998 Structural elements of an orphan nuclear receptor-DNA complex. Mol Cell 1:849–861[Medline]
  22. Schwabe JWR, Chapman L, Finch JT, Rhodes D 1993 The crystal structure of the estrogen receptor DNA-binding domain bound to DNA: how receptors discriminate between their response elements. Cell 75:567–578[Medline]
  23. Dussault I, Fawcett D, Matthyssen A, Bader J-A, Giguère V 1998 Orphan nuclear receptor ROR{alpha}-deficient mice display the cerebellar defects of staggerer. Mech Dev 70:147–153[CrossRef][Medline]
  24. Hamilton BA, Frankel WN, Kerrebrock AW, Hawkins TL, FitzHugh W, Kusumi K, Russell LB, Mueller KL, van Berkel V, Birren BW, Kruglyak L, Lander ES 1996 Disruption of nuclear hormone receptor ROR{alpha} in staggerer mice. Nature 379:736–739[CrossRef][Medline]
  25. Giguère V, Beatty B, Squire J, Copeland NG, Jenkins NA 1995 The orphan nuclear receptor ROR{alpha} (RORA) maps to a conserved region of homology of human chromosome 15q21–q22 and mouse chromosome 9. Genomics 28:596–598[CrossRef][Medline]
  26. Steinmayr M, Andre E, Conquet F, Rondi-Reig L, Delhaye-Bouchaud N, Auclair N, Daniel H, Crepel F, Mariani J, Sotelo C, Becker-Andre M 1998 Staggerer phenotype in retinoid-related orphan receptor {alpha}-deficient mice. Proc Natl Acad Sci USA 95:3960–3965[Abstract/Free Full Text]
  27. Matysiak-Scholze U, Nehls M 1997 The structural integrity of ROR{alpha} isoforms is mutated in staggerer mice: cerebellar coexpression of ROR{alpha}1 and ROR{alpha}4. Genomics 43:78–84[CrossRef][Medline]
  28. Carlberg C, van Huijsduijnen R, Staple JK, DeLamarter JF, Becker-André M 1994 RZRs, a new family of retinoid-related orphan receptors that function as both monomers and homodimers. Mol Endocrinol 8:757–770[Abstract]
  29. McBroom LDB, Flock G, Giguère V 1995 The non-conserved hinge region and distinct amino-terminal domains of the ROR{alpha} orphan nuclear receptor isoforms are required for proper DNA bending and ROR{alpha}-DNA interactions. Mol Cell Biol 15:796–808[Abstract]
  30. Miyajima N, Horiuchi R, Shibuya Y, Fukushige S-i, Matsubara K-i, Toyoshima K, Yamamoto T 1989 Two erbA homologs encoding proteins with different T3 binding capacities are transcribed from opposite DNA strands of the same genetic locus. Cell 57:31–39[Medline]
  31. Lazar MA, Hodin RA, Darling DS, Chin WW 1989 A novel member of the thyroid/steroid hormone receptor family is encoded by the opposite strand of the rat c-erbA{alpha} transcriptional unit. Mol Cell Biol 9:1128–1136[Medline]
  32. Retnakaran R, Flock G, Giguère V 1994 Identification of RVR, a novel orphan nuclear receptor that acts as a negative transcriptional regulator. Mol Endocrinol 8:1234–1244[Abstract]
  33. Dumas B, Harding HP, Choi H-S, Lehman KA, Chung M, Lazar MA, Moore DD 1994 A new orphan member of the nuclear hormone receptor superfamily closely related to Rev-Erb. Mol Endocrinol 8:996–1005[Abstract]
  34. Forman B, Chen J, Blumberg B, Kliewer SA, Henshaw R, Ong ES, Evans RM 1994 Cross-talk among ROR{alpha}1 and the Rev-erb family of orphan nuclear receptor. Mol Endocrinol 8:1253–1261[Abstract]
  35. Dussault I, Giguère V 1997 Differential regulation of the N-myc proto-oncogene by ROR{alpha} and RVR, two orphan members of the superfamily of nuclear hormone receptors. Mol Cell Biol 17:1860–1867[Abstract]
  36. Harding HP, Lazar MA 1995 The monomer-binding orphan receptor Rev-erb represses transcription as a dimer on a novel direct repeat. Mol Cell Biol 15:4791–4802[Abstract]
  37. Zamir I, Zhang JS, Lazar MA 1997 Stoichiometric and steric principles governing repression by nuclear hormone receptors. Genes Dev 11:835–846[Abstract]
  38. Harding HP, Atkins GB, Jaffe AB, Seo WJ, Lazar MA 1997 Transcriptional activation and repression by ROR{alpha}, an orphan nuclear receptor required for cerebellar development. Mol Endocrinol 11:1737–1746[Abstract/Free Full Text]
  39. Perlmann T, Jansson L 1995 A novel pathway for vitamin A signaling mediated by RXR heterodimerization with NGFI-B and NURR1. Genes Dev 9:769–782[Abstract]
  40. Forman BM, Umesono K, Chen J, Evans RM 1995 Unique response pathway are established by allosteric interactions among nuclear hormone receptors. Cell 81:541–550[Medline]
  41. Jiang G, Nepomuceno L, Hopkins K, Sladek FM 1995 Exclusive homodimerization of the orphan receptor hepatocyte nuclear factor 4 defines a new subclass of nuclear receptors. Mol Cell Biol 15:5131–5143[Abstract]
  42. Beato M, Herrlich P, Schütz G 1995 Steroid hormone receptors: many actors in search of a plot. Cell 83:851–857[Medline]
  43. Mangelsdorf DJ, Evans RM 1995 The RXR heterodimers and orphan receptors. Cell 83:841–850[Medline]
  44. Horner MA, Chen T, Thummel CS 1995 Ecdysteroid regulation and DNA binding properties of Drosophila nuclear hormone receptor superfamily members. Dev Biol 168:490–502[CrossRef][Medline]
  45. Laudet V 1997 Evolution of the nuclear receptor superfamily: early diversification from an ancestral orphan receptor. J Mol Endocrinol 19:207–226[Abstract/Free Full Text]
  46. Yates RA, Tuan RS, Shepley KJ, Unnasch TR 1995 Characterization of genes encoding members of the nuclear hormone receptor superfamily from Onchocerca volvulus. Mol Biochem Parasitol 70:19–31[CrossRef][Medline]