Coactivators for the Orphan Nuclear Receptor ROR
G. Brandon Atkins,
Xiao Hu,
Matthew G. Guenther,
Christophe Rachez,
Leonard P. Freedman and
Mitchell A. Lazar
Division of Endocrinology (G.B.A., X.H., M.G.G., M.A.L.),
Diabetes, and Metabolism Departments of Medicine and Genetics,
and The Penn Diabetes Center University of Pennsylvania School
of Medicine Philadelphia, Pennsylvania 19104
Cell Biology
Program (C.R., L.P.F.) Memorial Sloan-Kettering Cancer Center
New York, New York 10021
 |
ABSTRACT
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A mutation in the nuclear orphan receptor ROR
results in a severe impairment of cerebellar development by unknown
mechanisms. We have shown previously that ROR
contains a strong
constitutive activation domain in its C terminus. We therefore searched
for mammalian ROR
coactivators using the minimal activation domain
as bait in a two-hybrid screen. Several known and putative coactivators
were isolated, including glucocorticoid receptor-interacting protein-1
(GRIP-1) and peroxisome proliferator-activated receptor (PPAR)-binding
protein (PBP/TRAP220/DRIP205). These interactions were confirmed
in vitro and require the intact activation domain of ROR
although different requirements for interaction with GRIP-1 and PBP
were detected. Even in the absence of exogenous ligand, ROR
interacts with a complex or complexes of endogenous proteins, similar
to those that bind to ligand-occupied thyroid hormone and vitamin D
receptors. Both PBP and GRIP-1 were shown to be present in these
complexes. Thus we have identified several potential ROR
coactivators that, in contrast to the interactions with hormone
receptors, interact with ROR
in yeast, in bacterial extracts, and in
mammalian cells in vivo and in vitro in the
absence of exogenous ligand. GRIP-1 functioned as a coactivator for the
ROR
both in yeast and in mammalian cells. Thus, GRIP-1 is the first
proven coactivator for ROR
.
 |
INTRODUCTION
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Members of the nuclear receptor superfamily are known to play
roles in development, cellular growth, differentiation, and homeostasis
(1). The nuclear orphan receptor ROR
has been shown to play an
important role in cerebellar development, as demonstrated with its
mutation in naturally occurring staggerer mice (2), as well as in
targeted knockout experiments (3). The exact mechanism by which ROR
mutation leads to this defect is not known. ROR
has also been
implicated in the transcriptional regulation of the N-myc protooncogene
(4) and in the apolipoprotein A-I gene (5), suggesting potential roles
in neoplastic and metabolic processes.
ROR
is categorized as an orphan nuclear receptor because it is not
known whether it binds a ligand or requires a ligand to activate gene
transcription (6). In the case of nuclear receptors with known ligands,
such as thyroid hormone receptor (TR) and retinoic acid
receptor, the biological ligands are not found in yeast, rabbit
reticulocyte lysate, or in most cultured mammalian cell lines. In these
settings, the unliganded receptors interact with corepressor proteins
N-CoR (nuclear receptor corepressor) and SMRT (silencing
mediator of retinoic acid and thyroid hormone receptor) (7, 8). In the
presence of ligand, these receptors undergo a conformational change,
which leads to dissociation of the corepressors and recruitment of
coactivators (9). Several putative nuclear receptor coactivator
proteins have been identified, including SRC-1/N-CoA-1,
GRIP-1/TIF-2/N-CoA-2, PCIP/ACTR/AIB1, RIP-140, TRIP-1, TIF-1, TRIP-230,
PCAF, TRIP-2/PBP/DRIP205/TRAP220, and CBP (reviewed in Ref. 7). Several
studies have shown that these coactivator proteins are in multiprotein
complexes that function, at least in part, by acetylating nucleosomal
histones, thereby opening chromatin structure in a manner favorable for
gene transcription (reviewed in Ref. 10).
We previously showed that ROR
contains a C-terminal activation
domain that functions both in the context of the native protein and
when fused to a heterologous DNA-binding domain (11). Here we describe
a yeast two-hybrid screen using the C-terminal activation domain of
ROR
as bait that identified interactions between ROR
and several
known coactivators. Two of these, glucocorticoid receptor-interacting
protein-1 (GRIP-1) (12, 13) [also known as hTIF-2 (14)]rsqb] and
peroxisome proliferator-activated receptor (PPAR)-binding protein (PBP)
(15) [also known as hDRIP205 (16), hTRAP220 (17), and RB18A (18)]
were studied in detail. GRIP-1 and PBP interactions correlated with the
ability of ROR
polypeptides to function as activation domains and
were demonstrated both in vitro and in vivo. In
addition, endogenous GRIP-1 and PBP were among a complex of proteins
that interact with ROR
. The GRIP-1 and PBP interactions occurred
with ROR
in a variety of settings including yeast, bacterial
extracts, rabbit reticulocyte lysates, and human-derived tissue culture
cells, raising the possibility that ROR
activation is truly
constitutive. Furthermore, GRIP-1 was shown to function as a
coactivator for ROR
in yeast as well as in mammalian cells.
 |
RESULTS
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The C Terminus of ROR
Contains a Strong Activation Domain
The C-terminal activation domain of ROR
was fused to the
DNA-binding domain of the Gal4 transcription factor and transiently
transfected into JEG-3 human choriocarcinoma cells. Figure 1
confirms that the region of ROR
containing amino acids 272523 contains a strong activation domain.
Moreover, amino acids 272385 were required for this activation. All
of the Gal4 fusion proteins were expressed at comparable levels (data
not shown). These results indicate that in addition to the AF2 helix,
which has been previously shown to be required for activation by ROR
(11, 19), these more N-terminal amino acids may be required for
interaction of ROR
with cellular coactivators.

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Figure 1. Transcriptional Activation Domains in ROR
Expression vectors encoding the indicated proteins were transfected
into JEG-3 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 times for each construct.
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Interaction of ROR
Activation Domain with Several Putative
Coactivators
The C-terminal activation domain of ROR
(amino acids 272523)
was used as bait to screen a 17-day mouse embryo library in yeast,
where this activation domain is not functional (data not shown). In
addition to double selection, interacting proteins were not considered
further if they also interacted with the transcriptionally inactive
ROR
polypeptide (amino acids 385523). Table 1
lists the cDNAs that fulfilled these
criteria. Remarkably, several of the proteins that interacted with
ROR
activation domain in the absence of exogenous ligand were
identical to proteins called TRIPs that were previously shown by Moore
and colleagues (20) to interact with thyroid hormone receptor in a
ligand-dependent manner (20). These included TRIP-1, TRIP-2, and
TRIP-11. TRIP-1 is a component of the 26S proteasome, whose
physiological role in nuclear receptor function is unclear (21). Full
length TRIP-11 was recently identified as a retinoblastoma
protein- and TR-binding protein, called TRIP230 (22).
Full-length TRIP-2 was cloned as a PBP (15), as well as a component of
transcriptionally active complexes that interact with liganded vitamin
D receptor (VDR) [DRIP205 (16) and liganded TR (TRAP220) (17)]. Other
coactivators isolated in this screen were TIF-1 (23) and GRIP-1 (12, 13). In addition, one novel partial cDNA was isolated, but this has
been difficult to characterize for technical reasons. In the remainder
of this paper, we focus on GRIP-1 and PBP and putative coactivators for
ROR
. GRIP-1 is representative of the p160/SRC-1 family of nuclear
receptor coactivators while PBP is a component of a large multiprotein
complex including the mediator proteins that play a role in
transcription by nuclear receptors as well as numerous other classes of
transcriptional activatiors (reviewed in Ref. 24).
ROR
Interacts with GRIP-1 and PBP in Vitro
The yeast in vivo interactions were next confirmed
in vitro. The nuclear receptor interaction domain of GRIP-1
(amino acids 624-1121, containing three LXXLL sequences, also known as
NR boxes) and PBP (amino acids 488739) were expressed as
glutathione-S-transferase (GST) fusion proteins in
Escherichia coli. Pulldown experiments were carried out
using in vitro translated nuclear receptors. Figure 2
demonstrates that ROR
interacts with
both GRIP-1 and PBP in vitro in the absence of any exogenous
ligand. GRIP-1 and PBP interacted with ROR
amino acids 272523 but
not with amino acids 385523, correlating with the transactivation
properties of these polypeptides and with the interactions observed in
yeast.

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Figure 2. Interactions between ROR and Nuclear Receptor
Coactivators GRIP-1 and PBP
A, Gal4-ROR fusion proteins were labeled during in
vitro translation and assayed for interaction with GST and
GST-GRIP-1 (amino acids 624-1121). Input shown is 20% of the amount
used in each interaction incubation. B, The identical fusion proteins
as in panel A were assayed for interaction with GST-PBP (amino acids
488739).
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Differential Requirements for ROR
Interaction with GRIP-1 and
PBP
We next studied the effects of mutations in helix 3, which is
contained within amino acids 272385, and helix 12 of ROR
. The
mutations used, V335R and LF510AA, are analogous to mutations in the
GRIP-1 interaction surface of TR that have been shown to abrogate
ligand-dependent TR activation and interaction with GRIP-1 (25). Both
mutations effectively blocked transcriptional activation by ROR
(Fig. 3A
). The helix 12 mutant actually
represses transcription, as has been previously described for a mutant
in which ROR
helix 12 is deleted (11). As expected from the
analogous mutations in TR, both ROR
mutants exhibited markedly
reduced interactions with GRIP-1 (Fig. 3B
). Consistent with the
importance of 272385 in ROR
interaction with PBP, the helix 3
mutation interacted poorly with ROR
. Interestingly, however, the
helix 12 mutant of ROR
, although not a transcriptional activator,
retained the ability to interact with PBP. This is consistent with the
observation that while VDR helix 12 is required for activation as well
as for interaction with the DRIP complex, not all conserved residues in
helix 12 are required for this interaction, which is dependent upon PBP
(16).
Endogenous GRIP-1 and PBP Interact with ROR
Recently, liganded nuclear receptors have been shown to interact
with endogenous protein complexes that can function as coactivators in
cell-free transcription (16, 17, 26). We compared the abilities of
ROR
and VDR to interact with endogenous proteins in Namalwa cell
nuclear extracts. Figure 4A
shows that
ligand-bound VDR interacts with a number of nuclear proteins, including
proteins in the 230- and 160-kDa range. PBP has previously been shown
to be present in this transcriptionally active multisubunit complex
(16). By contrast, unliganded VDR does not interact with these
proteins. Interestingly, in the absence of any exogenous ligand, ROR
interacts with many proteins, some of which comigrate with those in the
liganded VDR complexes (i.e. the DRIP coactivator complex).
We next tested whether endogenous GRIP-1 and PBP were in these
complexes. Western analysis shows that, indeed, both endogenous GRIP-1
and PBP were bound by ROR
(Fig. 4B
) and VDR (Fig. 4C
). The ROR
interactions occurred in the absence of exogenous ligand, whereas
1,25-(OH)2-vitamin D3 was required for
interaction with VDR. The efficiencies of the interactions, assessed
semiquantitatively by comparing the amounts of bound and input
proteins, were similar for ROR
and VDR, with GRIP-1 interacting
perhaps more efficiently with liganded VDR than with ROR
. It is
important to note that although both PBP and GRIP-1 are present in the
ROR
pulldowns, it is not yet clear whether they are in the same or
different complexes. In the case of the VDR, they appear to be in
different complexes (26).
GRIP-1 Is a Coactivator for ROR
in Yeast
We next sought to determine whether these ROR
-interacting
proteins can actually function as coactivators for ROR
. GRIP-1 has
previously been shown to function as coactivator in yeast for multiple
nuclear receptors, including glucocorticoid receptor (12, 13) and
TR (27). To date, such coactivator function has required cognate
ligand. Figure 5
clearly demonstrates
that full-length GRIP-1 (a generous gift of M. Stallcup) functioned as
a strong coactivator for ROR
, activating transcription 9-fold over
control. Importantly, and consistent with interaction data, full-length
GRIP-1 did not act as a coactivator for ROR
amino acids 385523
(data not shown). In contrast, PBP did not function similarly as a
coactivator in yeast, despite expression of the full-length protein
similar to that achieved for GRIP-1 (data not shown).
GRIP-1 Is a Coactivator for ROR
in Mammalian Cells
We next tested the ability of GRIP-1 to function as a coactivator
for ROR
in mammalian cells, utilizing 293T cells. Unlike yeast,
these cells already contain both PBP and GRIP-1, which interacted with
ROR
as shown in Fig. 6A
. ROR
interactions did not require any exogenous ligand, whereas TR
interacted with both GRIP-1 and PBP in the presence of added
T3 (Fig. 6A
). Figure 6B
shows that cotransfection of GRIP-1
potentiated transcriptional activation by ROR
(272523) by more
than 2-fold. This modest level of transcriptional potentiation
presumably reflects the basal abundance of GRIP-1, that contrasts
dramatically with the situation in yeast. Figure 6C
shows the
incremental effect of transfection on overall GRIP-1 abundance.
Transfection efficiency of 293T cells was determined to be 5070%,
indicating that the exogenous GRIP-1 was overexpressed by less than a
log order. No stimulation of transcription by ROR
(385523) was
observed, again confirming the interaction results. In contrast to the
ability of GRIP-1 to potentiate activation by ROR
, PBP had little or
no effect (data not shown) despite expression of PBP at levels that
were increased in the transfected levels by approximately the same fold
as was observed for transfected GRIP-1 (Fig. 6C
). PBP actually
inhibited ROR
and TR
transcription in some experiments (data not
shown). PBP also did not potentiate ROR
transcription in other cell
types, including JEG-3 cells. Since endogenous PBP is abundant in all
of the mammalian cell lines tested, we cannot rule out the possibility
that its basal expression is already in excess of what is required for
maximal transcription activity. However, even when cotransfected with
GRIP-1, PBP did not potentiate ROR
activity (data not shown).
 |
DISCUSSION
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The nuclear orphan receptor ROR
has been shown to play
important roles in brain development (2, 3), lipid metabolism (5), and
oncogenesis (28). Here we show that ROR
can interact with several
known and putative coactivators, including GRIP-1 and PBP. This is the
first demonstration that GRIP-1 can serve as a coactivator for ROR
.
We also demonstrate that a complex of proteins including GRIP-1 and PBP
can associate with ROR
. This observation further supports previous
data from other laboratories showing that a complex of coactivator
proteins may be involved in mediating nuclear receptor activation
function (16, 17, 29). At the same time, differences in the composition
of proteins that interact with ROR
suggests that the formation of
coactivation complexes may serve as another level of transcriptional
regulation.
The dramatic effects of GRIP-1 upon ROR
activity in yeast contrasts
with the modest potentiation observed in mammalian cells. The most
likely explanation is the absence of endogenous coactivator in yeast
and its relative abundance in mammalian cells. This could also explain
the lack of potentiation by PBP in mammalian cells; in earlier studies
PBP only weakly potentiated activation by PPAR (15) and had little or
no effect on other receptors (16, 17). This point is unresolved,
however, since while this paper was under review, Treuter et
al. (30) reported that PBP contains an activation domain and
potently coactivates TR in mammalian cells as well as in yeast. The
present observation that full-length PBP does not function as
coactivator for ROR
in yeast suggests that PBP may not be a
coactivator for ROR
in the same sense as GRIP-1. Indeed, mutations
in helix 12 that abolish activation and interaction with GRIP-1 did not
alter PBP interaction. Interestingly, helix 12 is also dispensable for
interaction of hepatocyte nuclear factor 4 (HNF4), another
ligand-independent nuclear receptor, with the coactivator CBP (31).
Unlike GRIP-1 or CBP, PBP does not possess intrinsic histone
acetyltransferase activity (data not shown). Rather, the role of PBP is
primarily to anchor a multisubunit complex to the nuclear receptor
ligand-binding domain (LBD) (16, 17), thus serving as a bridging
subunit of a much larger complex that has a transcriptional activation
function (16, 17).
The fact that exogenous ligand was not necessary to demonstrate
coactivator interaction or activation in systems as varied as yeast,
bacteria, rabbit reticulocyte, and human cells strongly suggests that
ROR
is a constitutively active nuclear receptor. The ability of
ROR
to interact with numerous other proteins that interact with TR
only in the presence of endogenous ligand is a further argument that
ROR
exists in the liganded conformation in the absence of exogenous
ligand. While this work was in progress, the constitutively active
nuclear receptors HNF4 and constitutive androstane receptor ß
(CARß) were also shown to interact with coactivators in the absence
of ligand (31, 32, 33). Interestingly, androstane ligands lead to
dissociation of coactivators from CARß (33). This raises the
possibility that, if there is a ligand for ROR
, it might inhibit its
activity. In this context it is noteworthy that deletion of the AF2
activation helix allows ROR
to repress transcription and interact
with corepressors (11), which is normally a characteristic of
unliganded hormone receptors (7, 8).
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MATERIALS AND METHODS
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Plasmids
The Gal4 reporter used in Fig. 1
has been described previously
(34). The Gal4 reporter construct used in Fig. 5
was kindly provided by
M. Brown. pCMX-Gal4 and pCMX-Gal4 ROR
272523 constructs have been
described previously (11). pCMX-Gal4 ROR
385523 was constructed
from PCR products of pCMX-ROR
using the primers
5'-CCGGGGATCCAGTATGCC-3' and 5'-CTCTGTAGGTAGTTTGTCC-3' and were cloned
into the BamHI site of pCMX-Gal4. pSG5 GRIP-1 full length
(FL) was kindly provided by M. Stallcup. pcDNA3 PBP will be described
elsewhere (26). ROR
272523 and 385523 were subcloned into pGBT9
(CLONTECH Laboratories, Inc., Palo Alto, CA) from the
corresponding Gal4 constructs. Helix 3 and Helix 12 mutations in ROR
272523 were created by PCR. pGAD424 GRIP-1 was kindly provided by M.
Stallcup. PBP was subcloned into pGAD424 by standard
restriction/ligation procedures. A DNA fragment encoding PBP 488739,
the yeast two-hybrid clone, was subcloned into pGEX-2TK
(Pharmacia Biotech, Piscataway, NJ) for preparing
GST-fusion protein. A DNA fragment encoding GRIP-1 624-1121 was
amplified from pSG5 GRIP-1 using the primers
5'-CAGGGATCCGACAGGGCTGAGGGACACAG-3' and
5'-GTTGGATCCGGACTCCTGACTTGAGAACT-3' and cloned into the
BamHI site of pGEX-2TK. Plasmids for production of GST-VDR
and GST-TR have been previously described (16, 35). The GST-ROR
expression plasmid was constructed by BamHI and
XhoI digestion of a PCR fragment amplified from pCMX-ROR
using the primers 5'-CACGGATCCGAGTCAGCTCCGGCAGCCCCCGAC-3' and
5'-GTCGGATCCCATCCGGTGTTTCTGTACTTC-3', which was ligated into the
BamHI site of pGEX-2TK with the 3' XhoI and
BamHI fragment of pCMX ROR
. All constructs were confirmed
by automated DNA sequencing.
Yeast Two-Hybrid Screen
Saccharomyces cerevisiae HF7c [MATa, ura352,
his3200, lys2801, ade2101, trp1901, leu23, 112, gal4542,
gal80538, LYS2::Gal1-HIS3, URA3::(Gal4
17-mers)3-CYC1-lacZ] containing pGBT9 ROR
272523 was transformed with a 17-day mouse embryo library in pGAD10
(CLONTECH Laboratories, Inc.) and plated on SD medium
lacking tryptophan, leucine, and histidine (containing 5 mM
3-aminotriazole). His+ colonies exhibiting positive
ß-galactosidase activity in the filter lift assay (CLONTECH Laboratories, Inc.) were further characterized. To recover
library plasmids, total yeast DNA was isolated, electroporated into
E. coli HB101, and isolated on minimal media lacking leucine
and containing ampicillin. Isolated plasmid was then cotransformed into
HF7c yeast with pGBT9, pGBT9 ROR 272523, or pGBT9 ROR 385523 and
those plasmids which only exhibited positive ß-galactosidase activity
with pGBT9 ROR 272523 were further characterized.
Yeast Liquid ß-Galactosidase Assay
The Y190 strain yeast [MATa, ura352, his3200,
lys2801, ade2101, trp1901, leu23, 112, gal4
, gal80
,
cychr2,
LYS2::Gal1UAS-HIS3TATA-HIS3,
URA3::Gal1UAS-Gal1TATA-lacZ]
was cotransformed with pGBT9 ROR 272523 or pGBT9 ROR 385523
and pGAD10, pGAD424 GRIP-1, or pGAD424 PBP as indicated. Five
independent colonies of each transformation were used to inoculate
overnight cultures grown in SD medium lacking tryptophan and leucine.
Yeast liquid ß-galactosidase assays were carried out with each
culture in triplicate, using the protocol provided by CLONTECH Laboratories, Inc.
Protein Interaction Assays Using GST Fusion Proteins
GST fusion proteins were expressed in BL21 cells induced 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 GST binding buffer (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). In vitro translated protein,
using the Promega Corp. TNT T7 Quick kit with
[35S]methionine, Namalwa nuclear extracts, or 293T
whole-cell extracts were added to the beads as indicated. Binding was
allowed to proceed for 1 h, and the beads were washed four times
in the same buffer. The bound proteins were eluted by boiling in 30
µl of SDS-PAGE loading buffer and resolved by electrophoresis.
SDS-PAGE gels performed with in vitro translated protein
were dried and visualized by autoradiography. SDS-PAGE gels performed
with cellular extract were silver stained or else the proteins were
transferred to PVDF membrane for Western blot analysis.
Immunoblotting
Western blots were performed with 1:750 dilution of rabbit
polyclonal antibody raised against GRIP-1 624-1121 or PBP 488739 and
a 1:5000 dilution of goat antirabbit IgG-peroxidase (Roche Molecular Biochemicals, Nutley, NJ) secondary as
previously described (36).
Nuclear and Whole-Cell Extract Preparation
293T whole-cell extracts were prepared by washing and harvesting
cells in PBS, and subsequent incubation at 4 C for 20 min in lysis
buffer or in GST binding buffer containing 1 mM
phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, 1 mg/ml aprotinin,
and 1 mg/ml pepstatin. Extracts were quantified using the Bio-Rad assay
(Bio-Rad Laboratories, Inc., Richmond, CA). Namalwa B
cells were cultured as previously described (16) and nuclear extracts
were prepared as previously described (37).
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. For JEG-3 cells, at 80% confluence, 60-mm
dishes were transfected by the calcium phosphate precipitation method
using 2 µg luciferase reporter, 0.5 µg ß-galactosidase (B-gal)
expression vector, and 2 µg receptor expression vector. For 293T
cells, at 80% confluence, 24-well plates were transfected by the
calcium phosphate precipitation method using 50 ng luciferase reporter,
50 ng ß-galactosidase (ß-gal) expression vector, and 15 ng receptor
expression vector and additional coactivator expression vector as
indicated. Equivalent amounts of empty expression vector (pCMX) were
included in cells transfected with submaximal amounts of receptor or
coactivator. Cells were lysed in Triton X-100 buffer, and ß-gal and
luciferase assays were carried out using standard protocols. The
measured relative light units were normalized to ß-gal activity,
which served as an internal control for transfection efficiency. Fold
activation 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 of the data is shown.
Each experiment was repeated two to five times. The degree of
activation from a given site was highly consistent from experiment to
experiment.
 |
FOOTNOTES
|
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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 a UNCF/Merck Graduate Science Research Dissertation
Fellowship as well as by NIH Training Grant (GM-0821612). Automated
DNA sequencing was supported in part by the Molecular Biology Core of
the Penn Center for Molecular Studies of Digestive Diseases (NIH Center
Grant P30 DK-50306).
Received for publication January 26, 1999.
Revision received May 10, 1999.
Accepted for publication June 1, 1999.
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