Transcriptional Activation and Repression by ROR
, 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
|
---|
Mutation of the orphan nuclear receptor ROR
results in a severe impairment of cerebellar development by unknown
mechanisms. We have found that ROR
activates transcription from only
a subset of sites to which it binds strongly as a monomer. ROR
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
, we fused its C terminus
to a heterologous DNA-binding domain. Mutational analysis revealed that
ROR
contains both transcriptional activation and transcriptional
repression domains, with the repression domain being more active in
some cell types. The abilities of ROR
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
inhibits corepressor interaction on
DNA, consistent with the lack of repression by the full-length
receptor. Thus, transcriptional regulation by ROR
is complex and
likely to be regulated in a cell type- and target gene-specific manner.
 |
INTRODUCTION
|
---|
ROR
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
(3), which are encoded on
separate genes. ROR
is referred to as an orphan nuclear receptor
because it is not known whether it requires a ligand to activate gene
transcription (4). ROR
is particularly interesting because it has
been shown that mutation of the ROR
gene results in the failure of
normal cerebellar development in the Staggerer mouse (5).
However, the mechanism by which ROR
mutation impairs cerebellar
development is not known.
ROR
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
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
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
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
, we studied the
transcriptional activity of ROR
on a variety of binding sites.
Remarkably, although we confirmed that ROR
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
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
are complex and are
likely to vary in a manner that is dependent upon DNA-binding site and
cell type.
 |
RESULTS
|
---|
ROR
Activates Transcription on a Subset of Monomer-Binding
Sites
Although ROR
and RevErb have similar monomeric binding sites,
one difference between the proteins is that ROR
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
, we examined its DNA binding and
activation on multiple sites to which it binds as a monomer. Figure 1B
shows ROR
bound avidly as a monomer
to four different sites referred to as A through D in Fig. 1A
. However,
the function of the binding sites was qualitatively different. As shown
in Fig. 1C
, ROR
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
(data not shown).
ROR
Preferentially Activates Transcription on a Direct Repeat to
Which it Binds with a Stoichiometry of 2:1
The lack of transcriptional activity of ROR
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. 2A
) (32).
Remarkably, Fig. 2B
shows that ROR
, like RevErb, functioned most
potently on the RevDR2, and not on the other direct repeats shown in
Fig. 2A
. In the case of ROR
, the function was as an activator of
transcription. ROR
potently activated transcription from a reporter
gene containing one copy of the RevDR2 sequence (Fig. 2B
). In multiple
experiments this activity was 2- to 3-fold greater than that obtained
from Monomer A in Fig. 1
(data not shown). Furthermore, ROR
had
minimal effects on the other direct repeats, as well as on a DR2 site
in which the flanking sequences were replaced by Cs.
We next investigated whether DNA binding by ROR
correlated with its
function on these binding sites. Figure 2C
shows that ROR
bound
strongly as a monomer to all of the RevDR-binding sites. In contrast,
two ROR
molecules bound only to the RevDR2 site. Unlike RevErb
binding to the RevDR2, ROR
binding was not cooperative, in that
monomer binding was preferred at low concentrations of ROR
. This
result suggested that the failure of two ROR
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
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. 2D
, the presence in cis of two
monomer sites that were ineffective on their own allowed ROR
to
activate transcription even though the sites were widely spaced. Thus
the ability of two ROR
molecules to interact with an enhancer region
directly correlated with the ability of ROR
to activate
transcription, with the RevDR2 spacing being modestly more favorable.
ROR
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
Because of the striking increase in ROR
activity on the RevDR2
despite reduced binding of the dimer relative to the monomer, we
compared the characteristics of ROR
monomer and dimer binding to the
RevDR2. Figure 3A
shows the results of a
methylation interference study. As predicted, the ROR
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
dimer contacted both
half-sites, including the two G residues in the downstream half-site
(marked with arrowheads in Fig. 3A
), 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. 3B
. 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.
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. 3C
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
Contains Cell Type-Specific Activation and Repression
Domains
To begin to understand the mechanism by which ROR
activates
transcription, we took the approach of fusing the ROR
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
since
the above results indicated that on ROR
-binding sites, DNA binding
was necessary but not sufficient for transcriptional activation.
Figure 4
shows that GAL4-ROR
(140523) was a potent transcriptional activator in JEG-3 human
choriocarcinoma cells. Interestingly, a region in the hinge domain of
ROR
(amino acids 221272) appeared to play an inhibitory role since
deletion of these amino acids increased transcriptional activation by
ROR
from 7-fold to more than 150-fold. Similar results were obtained
at different concentrations of transfected GAL4-ROR
constructs (data
not shown). Amino acids 221272 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
for potential coactivator proteins or recruits a
corepressor protein. Deletion of the C terminus of ROR
, which
contains the AF2 activation helix required for transcriptional
activation by other members of the nuclear receptor superfamily
(GAL4-ROR 140491 and 140451), not only abolished activation but
revealed a modest repression function of these ROR
polypeptides.

View larger version (19K):
[in this window]
[in a new window]
|
Figure 4. Transcriptional Activation and Repression Domains
in ROR
GAL4 constructs (or wild type ROR ) 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. 4A
shows that none of the ROR
polypeptides, including the
superactivating amino acids 272523, 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
is also a much weaker activator of reporters
containing either monomer or RevDR2 sites (data not shown). Indeed,
ROR
fused to GAL4 DNA-binding domain (DBD) repressed transcription
in 293T cells. The GAL4-fusion proteins containing the ROR
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
1 Interacts with Nuclear Receptor Corepressors N-CoR and
SMRT, and This Interaction Correlates with Repression by ROR
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
was mediated by these corepressors, we
tested the ability of in vitro translated ROR
to interact
with N-CoR and SMRT. Figure 5A
shows that
ROR
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
polypeptides whose repression function was demonstrated in Fig. 4
interacted strongly with N-CoR and SMRT in vitro, as shown
in Fig. 5B
. Approximately 5% of the input ROR
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.
The AF2 Activation Helix Inhibits ROR
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
, a constitutive
activator, could interact with corepressors on DNA. Figure 6
shows that ROR
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
, 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
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
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. 6
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
. It should be pointed out that the binding of
N-CoR bound to ROR
on the RevDR2 considerably less well than to
RevErb (34). In this context, we point out that the repressor activity
of ROR
1452 and 1491 on the RevDR2 site was less than would be
predicted from the Gal4-ROR
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
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
, which is not present in the Gal4 constructs and is known to
affect the proteins conformation and function (1, 37). Interestingly,
ROR
selectively interacted with N-CoR and did not interact with SMRT
on this site, similar to what we have observed for RevErb (34).

View larger version (60K):
[in this window]
[in a new window]
|
Figure 6. The AF2 Helix of ROR Regulates Its Interaction
with N-CoR on DNA
EMSA analysis of wild type and C-terminal deletion mutants of ROR
binding to RevDR2 in the presence or absence of GST, GST-N-CoR (amino
acids 19442453), and GST-SMRT (amino acids 12821495). Binding of
TRß to DR4 oligonucleotide under the same condition is also shown.
The free probes were run off the gel.
|
|
 |
DISCUSSION
|
---|
The discovery that homozygous mutation of ROR
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
to date is the result of elegant studies of
Giguère and colleagues (36, 37), which have emphasized the
ability of ROR
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
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
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
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
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
and transactivation from the
RevDR2 site is striking. Consistent with this, ROR
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
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
molecules, it is tempting
to speculate that both molecules of ROR
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
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
affects its transactivation function. To address
this we have used the activation domain of ROR
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
that is not present in 293T cells or to a
cell-specific posttranslational modification. Alternatively, the
repressive function of ROR
might be dominant in 293T cells.
The repression domain within ROR
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
also has a
repression domain, but have demonstrated and characterized interactions
between ROR
and transcriptional corepressors N-CoR and SMRT. ROR
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
appeared to prevent interaction with N-CoR on
DNA but not in the GST-pulldown assay. This suggests that DNA binding
by ROR
alters the conformation of the protein. Indeed, the amino
terminus of ROR
1 regulates its DNA binding (1), and ROR
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
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
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
indeed
requires a ligand for its activity, this would imply that the ROR
ligand is present in transfected JEG-3 cells as well as in reticulocyte
lysate. An alternative explanation is that native ROR
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
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
to bind corepressor might simply be a vestige of the
evolutionary relationship between ROR
and other nuclear receptors.
However, the repression by GAL4-ROR
in 293T cells suggests that, in
at least some cell types, ROR
can assume a conformation that allows
functional interaction with corepressors. This could be due to
stabilization of the ROR
-corepressor complex by a cell-specific
ligand or posttranslational modification.
 |
MATERIALS AND METHODS
|
---|
Plasmid Constructs for Transfection
pCMX ROR
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 140523, 140491, and 140451, respectively. These sites
were blunt-ended and cloned into the blunt-ended EcoRI site
of pCMX GAL4. For the construction of pCMX GAL4-ROR 221523 and
272523, PCR fragments were cloned into the BamHI site of
pCMX GAL4 using the following 5'-primers: 5'-CCGGGGATCCGCTTCTAC-3'
(221523) and 5'-CCGGGGATCCCAGAATTA-3' (272523). 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. 1A
and 2A
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 1491, 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
1452 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
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
1, GAL4-ROR
140523, GAL4-ROR
140491, and
GAL4 ROR
140451 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, RevDR15) 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
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
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. 
Received for publication May 13, 1997.
Revision received June 26, 1997.
Accepted for publication July 2, 1997.
 |
REFERENCES
|
---|
-
Giguère V, Tini M, Flock G, Ong E, Evans RM,
Otulakowski G 1994 Isoform-specific amino-terminal domains dictate
DNA-binding properties of ROR staggerer mice. Nature 379:736739[CrossRef]
-
Carlberg C, Hooft van Huijsduijnen R, Staple JK,
DeLamarter JF, Becker-Andre M 1994 RZRs, a new family of
retinoid-related orphan receptors that function as both monomers and
homodimers. Mol. Endocrinol. 8:757770
-
Hirose T, Smith RJ, Jetten AM 1994 ROR
: the third member
of the ROR/RZR orphan receptor subfamily that is highly expressed in
skeletal muscle. Biochem. Biophys. Res. Commun. 205:19751983
-
Enmark E, Gustafsson J-Å 1996 Orphan nuclear receptorsthe
first eight years. Mol. Endocrinol 10:12931307
-
Hamilton BA, Frankel WY, Kerrebrock AW, Hawkins TL, FitzHugh
W, Kusumi K, Russell LB, Mueller KL, vanBerkel V, Birren BW, Kruglyak
L, Lander ES 1996 Disruption of the nuclear hormone receptor ROR
in
staggerer mice. Nature 379:736739[CrossRef][Medline]
-
Horlein AJ, Näär AM, Heinzel T, Torchia J, Gloss
B, Kurokawa R, Ryan A, Kamei Y, Soderstrom M, Glass CK, Rosenfeld MG 1995 Ligand-independent repression by the thyroid hormone receptor
mediated by a nuclear receptor co-repressor. Nature 377:397404[CrossRef][Medline]
-
Chen JD, Evans RM 1995 A transcriptional co-repressor that
interacts with nuclear hormone receptors. Nature 377:454457[CrossRef][Medline]
-
Lee JW, Ryan F, Swaffield JC, Johnston SA, Moore DD 1995 Interaction of thyroid-hormone receptor with a conserved
transcriptional mediator. Nature 374:9194[CrossRef][Medline]
-
Onate SA, Tsai SY, Tsai M-J, OMalley BW 1995 Sequence and
characterization of a coactivator for the steroid hormone receptor
superfamily. Science 270:13541357[Abstract]
-
Cavailles V, Dauvois S, LHorset F, Lopez G, Hoare S, Kushner
PJ, Parker MG 1995 Nuclear factor RIP140 modulates transcriptional
activation by the estrogen receptor. EMBO J 14:37413751[Abstract]
-
LeDouarin B, Zechel C, Garnier J-M, Lutz Y, Tora L, Pierrat B,
Heery D, Gronemeyer H, Chambon P, Losson R 1995 The N-terminal part of
TIF1, a putative mediator of the ligand-dependent activation function
(AF-2) of nuclear receptors, is fused to B-raf in the oncogenic protein
T18. EMBO J 14:20202033[Abstract]
-
Voegel JJ, Heine MJS, Zechel C, Chambon P, Gronemeyer H 1996 TIF2, a 160 kDa transcriptional mediator for the ligand-dependent
activation function AF-2 of nuclear receptors. EMBO J 15:36673675[Abstract]
-
Hong H, Kohli K, Trivedi A, Johnson DL, Stallcup MR 1996 GRIP1, a novel mouse protein that serves as a transcriptional
co-activator in yeast for the hormone binding domain of steroid
receptors. Proc Natl Acad Sci USA 93:49484952[Abstract/Free Full Text]
-
Hanstein B, Eckner R, DiRenzo J, Halachmi S, Liu H, Searcy B,
Kurokawa R, Brown M 1996 P300 is a component of an estrogen receptor
coactivator complex. Proc Natl Acad Sci USA 93:1154011545[Abstract/Free Full Text]
-
Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Lin
S-C, Heyman RA, Rose DW, Glass CK, Rosenfeld MG 1996 A CBP integrator
complex mediates transcriptional activation and AP-1 inhibition by
nuclear receptors. Cell 85:403414[Medline]
-
Chakravarti D, LaMorte VJ, Nelson NC, Nakajima T, Schulman IG,
Juguilon H, Montminy M, Evans RM 1996 Role of CBP/p300 in nuclear
receptor signaling. Nature 383:99103[CrossRef][Medline]
-
Wagner RL, Apriletti JW, McGrath ME, West BL, Baxter JD,
Fletterick RJ 1995 A structural role for hormone in the thyroid hormone
receptor. Nature 378:690697[CrossRef][Medline]
-
Renaud J-P, Rochel N, Ruff M, Vivat V, Chambon P, Gronemeyer
H, Moras D 1995 Crystal structure of the RAR
ligand-binding domain
bound to all-trans retinoic acid. Nature 378:681689[CrossRef][Medline]
-
Bourguet W, Ruff M, Chambon P, Gronmeyer H, Moras D 1995 Crystal structure of the ligand-binding domain of the human nuclear
receptor RXR-alpha. Nature 375:377382[CrossRef][Medline]
-
Forman BM, Casanova J, Raaka BM, Ghysdael J, Samuels HH 1992 Half-site spacing and orientation determines whether thyroid
hormone and retinoic acid receptors and related factors bind to DNA
response elements as monomers, homodimers, or heterodimers. Mol
Endocrinol 6:429442[Abstract]
-
Lazar MA, Berrodin TJ, Harding HP 1991 Differential DNA
binding by monomeric, homodimeric, and potentially heteromeric forms of
the the thyroid hormone receptor. Mol Cell Biol 11:50055015[Medline]
-
Katz RW, Koenig RJ 1993 Nonbiased identification of DNA
sequences that bind thyroid hormone receptor alpha 1 with high
affinity. J Biol Chem 268:1939219397[Abstract/Free Full Text]
-
Schrader M, Becker-Andre M, Carlberg C 1994 Thyroid hormone
receptr functions as monomeric ligand-induced transcription factor on
octameric half-sites. J Biol Chem 269:64446449[Abstract/Free Full Text]
-
Wilson TE, Paulsen RE, Padgett KA, Milbrandt J 1992 Participation of non-zinc finger residues in DNA binding by two nuclear
orphan receptors. Science 256:107110[Medline]
-
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 13:57945804[Abstract]
-
Harding HP, Lazar MA 1993 The orphan receptor Rev-ErbA
activates transcription via a novel response element. Mol Cell Biol 13:31133121[Abstract]
-
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
transcriptional unit. Mol Cell Biol 9:11281136[Medline]
-
Miyajima N, Horiuchi R, Shibuya Y, Fukushige S, Matsubara
K, 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 57:3139[Medline]
-
Forman BM, Chen J, Blumberg B, Kliewer SA, Henshaw R, Ong ES,
Evans RM 1994 Cross-talk among ROR
1 and the Rev-erb family of orphan
receptors. Mol Endocrinol 8:12531261[Abstract]
-
Dumas B, Harding H, Choi H-S, Lehmann K, Chung M, Lazar MA,
Moore DD 1994 A new orphan member of the nuclear hormone receptor
superfamily closely related to Rev-Erb. Mol Endocrinol 8:9961005[Abstract]
-
Retnakaran R, Flock G, Giguère V 1994 Identification of
RVR, a novel orphan nuclear receptor that acts as a negative
transcriptional regulator. Mol Endocrinol 8:12341244[Abstract]
-
Harding HP, Lazar MA 1995 The monomer-binding orphan rectptor
Rev-Erb represses transcription as a dimer on a novel direct repeat.
Mol Cell Biol 15:47914802[Abstract]
-
Zamir I, Harding HP, Atkins GB, Horlein A, Glass CK, Rosenfeld
MG, Lazar MA 1996 A nuclear hormone receptor corepressor mediates
transcriptional silencing by receptors with different repression
domains. Mol Cell Biol 16:54585465[Abstract]
-
Zamir I, Zhang J, Lazar MA 1997 Stoichiometric and steric
principles governing repression by nuclear hormone receptors. Genes Dev 11:835846[Abstract]
-
Sande S, Privalsky ML 1996 Identification of TRACs, a family
of co-factors that associate with and modulate the activity of nuclear
hormone receptors. Mol Endocrinol 10:813825[Abstract]
-
Giguère V, McBroom LDB, Flock G 1995 Determinants of
target gene specificity for ROR
1: monomeric DNA binding by an orphan
nuclear receptor. Mol Cell Biol 15:25172526[Abstract]
-
McBroom LDB, Flock G, Giguère V 1995 The nonconserved
hinge region and distinct amino-terminal domains of the ROR
orphan
nuclear receptor isoforms are required for proper DNA bending and
ROR
-DNA interactions. Mol Cell Biol 15:796808[Abstract]
-
Greiner EF, Kirfel J, Greschik H, Dorflinger U, Becker P,
Mercep A, Schule R 1996 Functional analysis of retinoid Z receptor
beta, a brain-specific nuclear orphan receptor. Proc Natl Acad Sci USA 93:1010510110[Abstract/Free Full Text]
-
Kurokawa R, DiRenzo J, Boehm M, Sugarman J, Gloss B, rosenfeld
MG, Glass CK 1995 Polarity-specific activities of retinoic acid
receptors determined by a co-repressor. Nature 377:451454[CrossRef][Medline]
-
Kurokawa R. Soderstrom M, Horlein A, Halachmi S, Brown M,
Rosenfeld MG, Glass CK 1995 Polarity-specific activities of retinoc
acid receptors determined by a co-repressor. Nature 377:451454[CrossRef][Medline]
-
Halachmi S, Marden E, Martin G, MacKay H, Abbondanza C, Brown
M 1994 Estrogen receptor-associated proteins: possible mediators of
hormone-induced transcription. Science 264:14551458[Medline]