Transition from Monomeric to Homodimeric DNA Binding by Nuclear Receptors: Identification of RevErbA
Determinants Required for ROR
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
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ABSTRACT
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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
and RevErbA
, 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
also binds as a homodimer to an extended DR2 element.
Gain-of-function experiments using point mutations and subdomain swaps
between ROR
and RevErbA
identify four amino acids within
RevErbA
sufficient to confer ROR
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.
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INTRODUCTION
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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
RXRs partner as well as by distinct usage of dimerization
determinants in the first and second zinc finger modules of RXR
(16).
ROR
is an orphan nuclear receptor that was initially cloned based on
its similarity to the retinoic acid receptor (5). ROR
is a monomeric
DNA-binding receptor that constitutively activates genes harboring
ROR
response elements (ROREs). Mouse genetic studies have shown
ROR
to be encoded by the staggerer locus and essential
for cerebellar development (23, 24, 25, 26, 27). The ROR
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
provides intramolecular interactions necessary to stabilize
receptor-DNA interactions (29).
RevErbA
, an orphan member of the superfamily of nuclear receptors,
is encoded on the opposite strand of the c-ErbA (T3R
)
gene (30, 31). DNA-binding studies have independently shown that
ROR
, RevErbA
, 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
lacks a typical activation function (AF2)
within the LBD, and competition for common binding sites results in
down-regulation of ROR
-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
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
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
dimer, but
not the monomeric form, can recruit corepressors and act as an active
repressor. Recently, the crystal structure of the RevErbA
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
and RevErbA
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
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.
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RESULTS
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Experimental Model
Figure 1
schematically represents
the structure of the ROR
DBD peptide used in this study and its
similarity to the RevErbA
DBD. The DBD is subdivided into three
domains referred to as zinc finger module 1 and 2 and the CTE. The
minimal ROR
and Rev-ErbA
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
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
and RevErbA
constituted targets for our
mutagenesis study.
To validate our experimental model, we first tested the binding of
full-length ROR
1 and RevErbA
, synthesized in vitro, to
oligonucleotides encoding the RORE and extended DR2 elements. As
expected, both ROR
1 and RevErbA
bind the RORE as monomers (Fig. 2A
, lanes 2 and 3, respectively). In
contrast, ROR
1 still binds as a monomer on an extended DR2 whereas
RevErbA
forms homodimers on this element (Fig. 2A
, 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
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
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. 2B
, 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. 2B
, 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. 2C
, 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. 2C
).
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.
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. 3A
). 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. 3B
). 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. 3B
, lane 10).
In contrast, a D-box swap (RORm2) has no significant effect on either
DNA-binding affinity or homodimer formation (Fig. 3B
, lane 11). A
switch in both zinc finger modules represented by RORm3 does not
increase homodimer binding beyond that observed with RORm1 (Fig. 3B
, compare lane 10 to 12). Results presented in Fig. 3C
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.
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. 4A
). 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. 4
, 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. 4
, 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. 4
, 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 s Zinc Finger
Module 1 Are Sufficient to Provide ROR 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.
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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
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. 5A
). The
Thr120Ile mutation (RORm9) increases considerably the
amount of dimer complexes formed (Fig. 5B
). 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 , ROR , 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).
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Providing Full-Length ROR
with a Dimerization Interface in the
DBD Is Sufficient for Cooperative Homodimerization
We tested whether the introduction of a dimerization
interface in the ROR
1 DBD would be sufficient to allow the
full-length receptor to form homodimers on an extended DR2 element.
ROR
1 constructs encoding the RevErbA
dimerization determinants of
the first and second zinc finger, ROR
1m6 and ROR
1m9,
respectively, were constructed and assayed by electrophoretic mobility
shift assay (EMSA) (Fig. 6
). At highest
protein concentrations, both ROR
1m6 and ROR
1m9 mutants form two
times more homodimer complexes than wild-type ROR
1 on an extended
DR2 element. The result obtained with ROR
1m9 confirms and extends
the finding reported by Zhao et al. (21). The ROR
1m10
mutant encoding both the dimerization determinants of the first and
second zinc finger modules forms homodimeric complexes slightly less
efficiently than RevErbA
.
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DISCUSSION
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On the basis of their DNA-binding properties, nuclear receptors
can be classified into two major groups: monomers, exemplified by
orphan nuclear receptors ROR
, RevErbA
, SF-1, and NGFI-B, and
dimers, which include homodimers and heterodimers (1). Some receptors
belong to more than one group. RevErbA
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
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, RXRs
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
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
DBD was published (21). This study showed that, in
contrast to RXR heterodimer complexes bound to direct-repeat HREs,
homodimer formation of RevErbA
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
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. 7
). 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 1
(marked by an asterisk) are highlighted.
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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
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
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
-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
and other monomeric
receptors to stably and precisely make contacts with their cognate
site.
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MATERIALS AND METHODS
|
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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
1 (5)
and pCMXhRev-ErbA
(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
and RevErbA
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
and RevErbA
and the
truncated DBD peptides from pCMX-based plasmids according to the
manufacturers 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
|
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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.
 |
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