Heterodimeric Interactions between Chicken Ovalbumin Upstream
Promoter-Transcription Factor Family Members ARP1 and Ear2*
Dorina
Avram
,
Jane E.
Ishmael
,
Daniel J.
Nevrivy
§,
Valerie J.
Peterson
,
Suk-Hyung
Lee
,
Paul
Dowell
§¶, and
Mark
Leid
§
From the
Laboratory of Molecular Pharmacology,
College of Pharmacy and § Program in Molecular and Cellular
Biology, Oregon State University, Corvallis, Oregon 97331-3507
 |
ABSTRACT |
Members of the chicken ovalbumin upstream
promoter-transcription factor (COUP-TF) subfamily of orphan nuclear
receptors, which minimally includes COUP-TFI and ARP1, are highly
expressed in brain and are generally considered to be constitutive
repressors of transcription. We have used a yeast two-hybrid system to
isolate proteins expressed in brain that interact with ARP1. One of the proteins isolated in this screen was Ear2, another orphan receptor that
has been suggested to be a member of the COUP-TF subfamily. Here we
demonstrate that ARP1 and Ear2 form heterodimers in solution and on
directly repeated response elements with high efficiency and a
specificity differing from that of homodimeric complexes composed of
either receptor. ARP1 and Ear2 were observed to interact in mammalian
cells, and the tissue distribution of Ear2 transcripts was found to
overlap precisely with the expression pattern of ARP1 in several mouse
tissues and embryonal carcinoma cell lines. Heterodimeric interactions
between ARP1 and Ear2 may define a distinct pathway of orphan receptor signaling.
 |
INTRODUCTION |
COUP-TF1 orphan nuclear
receptors have been reported to interfere with the signaling pathways
of several nuclear receptors including retinoic acid (1-5), thyroid
hormone (3), estrogen (6-9), and vitamin D3 (4) receptors, as well as
peroxisome proliferator-activated receptor
(10, 11). In addition,
COUP-TFs negatively modulate hepatic nuclear factor 4- (12, 13),
RZR-/ROR- (14), and Nur77 (15)-mediated transcriptional activation. Four mechanisms have been postulated to underlie modulation of multiple
signaling pathways by COUP-TF subfamily members: 1) competition for DNA
response element binding, 2) inactive heterocomplex formation including
titration of retinoid X receptor (RXR), 3) active silencing of basal
transcription, and 4) transcriptional transrepression (16). Of these
potential mechanisms, competition for binding to response elements is
well documented and is based upon the ability of COUP-TF proteins to
bind a large variety of response elements (16). COUP-TFI and ARP1 are
known to form homodimers in solution and to bind promiscuously as such
to direct repeats (DR) of the canonical half-site AGGTCA exhibiting the
highest affinity for a DR spaced by 1 base pair (DR1; Refs. 1 and
3).
ARP1 and COUP-TFI share extensive identity, particularly in the ligand
and DNA binding domains (Refs. 12, 17, and 18; see also Fig. 1). Ear2
is less well conserved with either protein in these domains (Fig. 1),
which has led some to suggest that Ear2 may not be a member of COUP-TF
subfamily of nuclear receptors (16). However, based on compelling
evolutionary arguments, Escriva and co-workers (19) have placed Ear2
within the COUP-TF subfamily. Moreover, other groups consider Ear2 to
be a member of COUP-TF subfamily based on the capacity of this protein
to form homodimeric, DNA binding complexes and to repress
ligand-dependent activation of target genes mediated by
other nuclear receptors, such as retinoic acid (1, 20) and estrogen
(7-9) receptors, in a manner similar to that of COUP-TFI and ARP1.
COUP-TFI and ARP1 play important roles in development, particularly in
patterning of the nervous system in Xenopus (21), Drosophila (22, 23), and mammals (24, 25). COUP-TFI null animals exhibit perinatal lethality, possibly arising from
malformations of the glossopharyngeal ganglion and associated IXth
cranial nerve resulting in an inability to feed properly (25). Given
the overall similarity between COUP-TFI and ARP1, both in terms of
structural conservation and overlapping patterns of expression in the
developing central nervous system (24), it seems remarkable that ARP1
does not compensate for the lack of COUP-TFI expression in these
animals (25). Knock-out of the ARP1 gene in the mouse is
apparently embryonic lethal (16).
In this report, we demonstrate that Ear2 and ARP1 form heterodimers in
yeast and in vitro, both in solution and on directly repeated response elements. Moreover, these orphan receptors were shown
to interact in mammalian cells and to be coexpressed in the embryo and
in two embryonal carcinoma cell lines as well as in several adult
tissues suggesting that ARP1·Ear2 heterodimeric complexes may play a
role(s) in embryonic development and in the adult organism.
 |
MATERIALS AND METHODS |
Plasmids and Constructs--
The bait vectors (pBTM116 and pBL1)
for the yeast two-hybrid screen and the yeast reporter strains L40 (26)
and PL3 (27) were all generous gifts from Drs. R. Losson and P. Chambon
(IGBMC, Illkirch, France). Human ARP1 (ARP1, Ref. 28) was a kind gift from Dr. H. Nakshatri, and NR1 was a kind gift from Dr. S. Nakanishi. A
fragment corresponding to the hinge and putative ligand binding domain
of ARP1 (regions D and E, amino acids 144-414) was amplified by PCR
and cloned into both pBTM116 and pBL1 (26), encoding LexA DBD-ARP1 DE
and ER DBD-ARP1 DE fusion proteins, respectively. Receptor bait
constructs for mouse RXR
(amino acids 132-467) and human RAR
(amino acids 90-454) have been described previously (29). An unrelated
bait corresponding to the carboxyl tail (amino acids 835-938) of the
NR1 subunit of the N-methyl-D-aspartate receptor
(30) was similarly prepared for these studies. All fragments amplified
by PCR were verified by sequence analysis.
A mammalian expression vector encoding an ER DBD/ARP1 DE fusion protein
was constructed by inserting the EcoRI fragment of pBL1-ARP1
DE into the EcoRI site of pTL1 (31). A mammalian expression vector encoding the DE region of mouse Ear2 (amino acids 122-390) fused to the transcriptional activation domain of GAL4 (GAL4 AD; amino
acids 768-881) was prepared by insertion of the corresponding Ear2
fragment into pACT2 (CLONTECH, Palo Alto, CA),
yielding pACT2-Ear2 DE. This plasmid was then digested with
HindIII, excising the GAL4 AD-Ear2 DE fragment, which was
subsequently cloned into the eukaryotic expression vector, pTL1. An
amino-terminal truncation mutant of ARP1, mycARP1
AB, with a myc
epitope tag at the amino terminus was prepared by PCR amplification of
an appropriate fragment and insertion into pTL1. Full-length Ear2,
fused to an HA epitope tag and the GAL 4 AD, was excised from the
library plasmid (pACT2) by digestion with HindIII, and the
resulting fragment was ligated into pTL1 creating HA-Ear2. The latter
two constructs were prepared for use in DNA binding experiments
designed to distinguish between heterodimeric complexes of intermediate
electrophoretic mobility and homodimeric complexes composed of either
receptor. The ER-responsive CAT reporter, 17-mer-ERE-globin-CAT, has
been described previously (32).
GST fusion proteins were prepared for ARP1 DE (GST-ARP1 DE) and Ear2 DE
(GST-Ear2 DE) by PCR amplification of the corresponding fragments and
subcloning into pGEX-2T (Amersham Pharmacia Biotech, Uppsala, Sweden)
that had been previously digested with BamHI and
EcoRI. The integrity of all constructs was verified by
sequence analysis.
Yeast Two-hybrid Screen--
Saccharomyces cerevisiae
L40 reporter strain expressing ARP1 (D and E regions) was transformed
with a mouse brain cDNA library (CLONTECH)
fused to the GAL4 AD on a leucine-selectable vector, pACT2. The screen
for ARP1-interacting clones was conducted in synthetic medium lacking
tryptophan, leucine, and histidine and supplemented with 20 mM 3-aminotriazole. The plasmids from positive clones
containing the brain cDNAs were rescued, shuttled in
Escherichia coli, and sequenced using the dideoxynucleotide
dye terminator method on an Applied Biosystems, Inc., model 373 or 377 sequencer (Applied Biosystems, Inc., Foster City, CA) at the Central
Services Laboratory of the Oregon State University Center for Gene
Research and Biotechnology.
-Galactosidase Assays--
Filter assays were utilized to
confirm the positive clones. For quantitative purposes the positive
clones were grown in synthetic medium to an A600
of approximately 2 units, and
-galactosidase activity was assayed as
described by Kippert (33), and modified by Avram and Bakalinsky (34).
Ligand-dependent assays were carried out by inclusion of 1 µM 9-cis-RA or identical amounts of vehicle in
the culture medium.
Protein-Protein Interaction Assays--
GST pull-down
experiments were conducted as described previously (29, 35).
Electrophoretic Mobility Shift Assays--
EMSAs were conducted
essentially as described previously (31). ARP1
AB and HA-Ear2 were
expressed by in vitro transcription/translation in rabbit
reticulocyte lysates (Promega). In each case the receptors used for gel
retardation assays were translated in parallel in the presence of
[35S]methionine for the purpose of quantification. The
DR1(G) response element upper strand had the following sequence
(canonical half-sites are bold and underlined):
5'-TCGAGGGTAGAGGTCAGAGGTCACTCG-3'; DR2(G) through DR5(G) were synthesized with identical flanking and
repeated hexanucleotide sequence but with the following inter-repeat spacers (5' to 3'): GA (DR2), CGA (DR3), CGAA (DR4), and CCGAA (DR5).
The DR(T) series of oligonucleotides were identical to the DR(G) series
except that the repeated hexanucleotide had the sequence AGTTCA.
Cell Culture and Transfections--
Human embryonic kidney (HEK)
293 cells (ATCC CRL 1573) were cultured in minimum essential medium
with Earle's salts (Sigma) supplemented with 10% fetal bovine serum
(Summit, Boulder, CO) and 4 mM glutamine (Life
Technologies, Inc.). Cells were grown to 50-60% confluence and
transiently transfected using the calcium phosphate method. Each 100-mm
plate was cotransfected with 1 µg of expression vector encoding the
ER DBD or ER DBD/ARP1 DE, 1 µg of GAL4 AD, or GAL4 AD/Ear2 DE, 2 µg
of the 17-mer-ERE-Globin-CAT reporter construct, and 10 ng of
pCMV-SPORT-
gal (Life Technologies, Inc.) encoding
-galactosidase
which was used to normalize for transfection efficiency. Cells were
harvested 48 h after transfection, and extracts were prepared
using standard techniques.
-Galactosidase activity in cellular
extracts was quantified using a colorimetric assay, and CAT activity
was determined by thin layer chromatography.
Reverse Transcription-Polymerase Chain Reaction--
First
strand cDNA was synthesized using 1 µg of RNA (Ambion, Austin,
TX) and 100 ng of random hexamer primers (Promega) in a buffer
containing 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 100 µM concentrations of each dNTP, and 200 units of Moloney
murine leukemia virus reverse transcriptase (Life Technologies, Inc.)
in a total volume of 25 µl, and the reactions were carried out for 30 min at 37 °C. One µl of each reverse transcription reaction was
subjected to PCR amplification using ARP1-, Ear2-, and 36B4
(36)-specific primers. ARP1 and Ear2 amplifications were carried out
for 25 cycles, whereas amplification of 36B4 was conducted for 19 cycles. Amplification of 36B4 was used as a control for the quantity of
cDNA present in each sample as well in normalization of gel loading
and Southern blotting. Note that, in general, one cycle consisted of
the following steps: 94 °C × 30 s, 50 °C for 45 s, 72 °C for 60 s; however, the annealing temperature was
varied for each set of primers to optimize amplification. Amplification
products were separated on a 2% agarose gel and then transferred to
ZetaProbe membranes (Bio-Rad). The blots were probed with specific,
end-labeled oligonucleotides corresponding to internal sequence in the
PCR products. RT-PCR analyses of ARP1 and Ear2 expression in F9 and P19
embryonal carcinoma cells treated in monolayers with either vehicle or
trans-retinoic acid for 24 h were carried out as
described above.
 |
RESULTS |
Ear2 Interacts with ARP1 in a Yeast Two-hybrid System--
A
fragment encoding the ARP1 hinge region and putative ligand binding
domain was used as a bait in a yeast two-hybrid screen to identify
proteins expressed in brain that interact with ARP1, which is highly
expressed in brain (Refs. 18, 24, 37, and this report). A protein found
to interact with ARP1 was Ear2, another member of steroid hormone
receptor superfamily, which is closely related to COUP-TF subfamily,
that includes COUP-TFI and ARP1 (Fig.
1).

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Fig. 1.
Relationship between COUP-TF family
members. The amino acid identities between domains of COUP-TFI and
both ARP1 and Ear2 are indicated as percentages within each schematic
domain. The corresponding identities between ARP1 and Ear2 are
indicated within double-headed arrows. Alignments were
carried out using Clustal X (version 1.63b) and the following
GenBankTM accession numbers: COUP-TFI, gi466468; ARP1,
gi482927; Ear2, gi482930.
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The Ear2 clone isolated in our screen contains the entire open reading
frame identified by Jonk and co-workers (18). In addition, the present
clone contains 183 base pairs upstream of the putative initiator
methionine previously described (18). However, as there is no in frame
stop codon(s) in this upstream sequence, it is possible that the actual
Ear2 protein is extended in the amino terminus beyond the previously
identified initiator methionine (18).
To evaluate the specificity of ARP1-Ear2 interaction, we examined
interactions of Ear2 with RAR, RXR, and an unrelated bait, the carboxyl
terminus of the NR1 subunit of the
N-methyl-D-aspartate receptor (30). The
interaction of Ear2 with ARP1 was specific inasmuch as Ear2 did not
interact with RXR
or RAR
in the presence or absence of
9-cis-retinoic acid (Fig. 2).
Similarly, Ear2 did not interact with the carboxyl terminus of NR1
(Fig. 2).

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Fig. 2.
Interaction between Ear2 and ARP1,
RXR , RAR , and NR1 in
yeast. LexA DBD/receptor fusion baits (ARP1, RXR , RAR , and
NR1) were coexpressed together with Gal4 AD/Ear2 (pACT2:Ear2) or GAL4
AD (pACT2) and examined for the ability to activate
LexAop-lacZ reporter in the yeast strain L40.
Ligand-dependent interactions were examined in the presence
of 1 µM 9-cis-retinoic acid (9cRA).
The results shown represent the mean ± S.E. of three independent
experiments in which individual transformants were assayed.
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Ear2 and ARP1 Interact in Vitro--
A qualitative, in
vitro protein-protein interaction study was conducted to confirm
the ARP1-Ear2 interaction observed in yeast and to investigate the
ability of each protein to self-associate. The hinge and putative LDB
regions of each protein were fused to glutathione
S-transferase (GST), creating GST-ARP1 DE and GST-Ear2 DE,
and both proteins were expressed in bacteria for use in standard GST
pull-down experiments (29, 35). [35S]Met-labeled Ear2
interacted with GST-ARP1 DE (Fig.
3A, lane 3) confirming the
interaction observed in yeast. [35S]Met-labeled Ear2 was
also found to interact with itself (GST-Ear2 DE, lane 4)
with efficiency nearly identical to that with which it interacted with
GST-ARP1 DE (lane 3) suggesting that Ear2 may form
heterodimeric and homodimeric complexes with equal facility. Similarly,
[35S]Met-labeled ARP1
AB interacted with both GST-Ear2
DE and -ARP1 DE fusion proteins (Fig. 3B, lanes 3 and 4, respectively) but not with glutathione charged with
GST alone (lane 2). These in vitro findings
corroborate results obtained in yeast (Fig. 2) and also suggest that
ARP1 and Ear2 participate in heterodimeric interactions in solution
with an apparent efficiency comparable to that of the homodimerization
involving either receptor.

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Fig. 3.
In vitro interaction between ARP1
and Ear2. GST/ARP1 DE and GST/EAR2 DE were bound to
glutathione-Sepharose and used as affinity matrices to examine the
interaction with the in vitro translated
[35S]methionine-labeled Ear2 (A) and ARP1
AB (B). Approximately 10% of input
[35S]methionine-labeled protein was retained on GST
fusion proteins in all cases. Shown are representative experiments that
were replicated 4-6 times.
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ARP1 and Ear2 Form Heterodimers on Directly Repeated Response
Elements--
COUP-TFI, ARP1, and Ear2 form homodimers in solution and
bind promiscuously to direct repeats (DR) of the canonical half-site, AGGTCA, with varied inter-repeat spacing. However, all of these receptors exhibit a degree of selectivity for DRs spaced by a single
base pair (DR1) (1, 3). In addition, COUP-TFI and ARP1 bind as a
heterodimeric complex to DR elements composed of varied spacing (3).
Based on our observation that ARP1 and Ear2 form heterodimers in yeast
(Fig. 2) and in solution (Fig. 3), we conducted electrophoretic
mobility shift assays (EMSAs) to determine if ARP1 and Ear2 bind to
directly repeated response elements as a heterodimeric complex.
mycARP1
AB homodimeric complexes bound in a protein
concentration-dependent manner to the DR1 probe (Fig.
4, lanes 1-3; complex C5).
HA-Ear2 formed two complexes, C1 and C2, that exhibited differential
electrophoretic mobility on this probe (Fig. 4, lanes 4 and
5). The two species of HA-Ear2, which bound to this probe in
a protein concentration-dependent manner, most likely arose
from correct and internal initiation of HA-Ear2 translation in rabbit
reticulocyte lysates (data not shown). At comparable amounts of
protein, HA-Ear2 (C1 and C2) clearly bound to the DR1 probe less
efficiently than mycARP1
AB (C5; compare lanes
1-3 to 4-6 of Fig. 4). However, incubation of a fixed
amount of mycARP1
AB with increasing amounts of HA-Ear2 resulted in
the complete titration of the mycARP1
AB homodimeric complex (C5)
into mycARP1
AB·HA-Ear2 heterodimeric complexes, C3 and C4 (Fig. 4,
lanes 7-9). When excessive amounts of HA-Ear2 were used,
the appearance of HA-Ear2 homodimeric complexes (C1 and C2) were also
evident (Fig. 4, lanes 8-9). Similarly, titration of a
fixed amount of HA-Ear2 with increasing amounts of mycARP1
AB quantitatively shifted HA-Ear2 homodimeric complexes (C1 and C2) into
mycARP1
AB·HA-Ear2 heterodimeric complexes (C3 and
C4; Fig. 4, lanes 10-12). The homodimeric
mycARP1
AB complex (C5) was apparent when this protein was used in
excess (Fig. 4, lanes 11 and 12). Complexes C1/C2
and C5 were supershifted with anti-HA and anti-myc antibodies,
respectively, whereas both antibodies supershifted complexes C3 and C4,
confirming the identity of each complex (data not shown).

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Fig. 4.
Formation of ARP1 and Ear2 homodimers
and heterodimers on the DR1(G) probe. Lanes
1-3 and 4-6 contain increasing amounts of
mycARP1 AB and HA-Ear2 as indicated. Lanes 7-9
represent titration of a fixed amount of mycARP1 AB with increasing
amounts of HA-Ear2, and lanes 10-12 correspond
to the converse titration. The position of homodimeric HA-Ear2
(C1 and C2) and mycARP1 AB (C5)
complexes as well as heterodimeric mycARP1 AB·HA-Ear2 complexes
(C3 and C4) are indicated to the right
of this EMSA gel. Electrophoresis was carried out for about 1 h
after the free probe ran out of the gel to maximize resolution of the
multiple complexes. A representative experiment is shown which was
replicated five times. Note that the amount of reticulocyte lysate in
each lane was held constant by addition of unprogrammed lysate.
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ARP1·Ear2 Heterodimeric Complexes Bind Directly Repeated Response
Elements with Specificity Differing from Homodimers of Either
Receptor--
We next sought to determine if the capacity of ARP1 and
Ear2 to bind directly repeated response elements as homodimeric and/or heterodimeric complexes was a function of the length of the
inter-repeat spacer. mycARP1
AB homodimers bound to probes with
spacing ranging from 1 to 5 base pairs (complex C5; Fig.
5A, lanes 1, 4, 7, 10, and
13) with somewhat varied efficiency (DR2
DR1 > DR5 > DR3 > DR4). Conversely, HA-Ear2 did not appear to
bind any of these probes with the exception of DR1, to which HA-Ear2
homodimers bound weakly at the relatively low amount of HA-Ear2 protein
used in these experiments (Fig. 5B, lanes 2, 5, 8, 11, and
14). mycARP1
AB·HA-Ear2 heterodimeric complexes (C3 and
C4) bound to probes of all spacings with a distinct preference for DR1
and DR2 (Fig. 5A, lanes 3, 6, 9, 12, and 15).
Thus, mycARP1
AB·HA-Ear2 heterodimeric complexes exhibited a DNA
binding specificity (DR1
DR2 > DR4 > DR5 > DR3) that was clearly distinct from that of HA-Ear2 homodimers (which bound weakly to DR1 only) and subtly distinct from mycARP1
AB homodimeric complexes (see above).

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Fig. 5.
ARP1 and Ear2 homodimer and heterodimer
formation on DR(G) (A) and DR(T) (B)
series of probes of varied spacing. The amounts of mycARP1 AB
and HA-Ear2 used in these EMSA experiments correspond to 1 and 2 arbitrary units, respectively (see Fig. 4). Identification of complexes
is as described in the legend to Fig. 4. The length of the inter-repeat
spacer is given by n, and the sequence of this spacer is
given under "Materials and Methods." Note that the autoradiograph
corresponding to B was exposed to film longer than that of
A to allow visualization of weak complexes. Shown is a
representative experiment that was replicated five (A) or
three (B) times.
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Methylation interference studies have implicated a role for the
guanines present in each half-site of a DR1-type response element in
the specific interaction of COUP-TFI with DNA (38). To address the
potential role of the second guanine (G2) in each
AGGTCA half-site in DNA recognition by ARP1·ARP1,
ARP1·Ear2, and Ear2·Ear2 complexes, EMSAs were carried out using a
series of probes in which G2 in each half-site was mutated
to thymidine (see Fig. 5B). This G2
T
mutation had a dramatic effect on the efficiency of both mycARP1
AB
homodimer and mycARP1
AB·HA-Ear2 heterodimer binding to probes of
all spacing (Fig. 5B). However, mycARP1
AB homodimers did
bind to the "T" series probe with a single base pair spacer, DR1(T)
(Fig. 5B, lane 1), and with lower efficiency to DR5(T) (lane 13) and DR3(T) (lane 7). In contrast,
mycARP1
AB·HA-Ear2 heterodimer binding was not evident on any of
the "T" series probes with the possible exception of DR1(T) (Fig.
5B, lane 3). However, HA-Ear2 appeared to inhibit
mycARP1
AB homodimer binding to DR1(T), DR3(T), and DR5(T) (Fig.
5B, lanes 3, 9, and 15, respectively), the latter
of which is identical to the RARE in the RAR
promoter (39). These findings suggest that mycARP1
AB and HA-Ear2 may form a
heteromeric complex in solution that is not competent for DNA binding
to the T series of response elements. Consistent with this hypothesis,
mycARP1
AB and HA-Ear2 interacted in DNA-binding independent, GST
pull-down experiments (Fig. 3, A and B).
ARP1 and Ear2 Interact in Mammalian Cells--
Although ARP1 and
Ear2 interact in yeast and in vitro, both in solution and on
various directly repeated response elements, a physiologically relevant
interaction between these two proteins would only occur in cells in
which both proteins were expressed. Thus, cotransfection experiments
were carried out to determine if the two proteins are capable of
interacting in cells using a mammalian two-hybrid system. HEK 293 cells
were cotransfected with a plasmid (ER DBD-ARP1 DE) encoding ARP1 DE
fused to ER DBD together with a second plasmid (Ear2 DE-GAL4 AD)
encoding Ear2 DE fused to GAL4 AD. The previously described
17-mer-ERE-globin-CAT (32) was used as a reporter construct in these
studies. ER DBD alone (Fig. 6, lane
4) or when cotransfected with the GAL4 AD (lane 5)
failed to activate the reporter construct. Similarly, Ear2 DE-GAL4 AD
did not activate the reporter plasmid when cotransfected with the ER
DBD (Fig. 6, lane 6). However, cotransfection of ER DBD-ARP1
DE together with Ear2 DE-GAL4 AD resulted in strong induction of the
reporter suggesting that Ear2 and ARP1 are capable of interaction in
mammalian cells.

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Fig. 6.
ARP1 and Ear2 interact in HEK 293 cells.
Cells were cotransfected with the indicated mammalian expression
vectors and the 17-mer-ERE-globin-CAT reporter. Cells were harvested
24 h after transfection, and extracts were prepared using standard
techniques. Transfection efficiency was normalized across treatments
using a cotransfected -galactosidase expression vector, and CAT
activity was determined using [14C]chloramphenicol and
acetyl-CoA. Acetylated and unacetylated
[14C]chloramphenicol were separated by thin layer
chromatography and visualized by autoradiography. A representative
experiment is shown that was replicated three times.
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Ear2 and ARP1 Expression Patterns Overlap in Several Mouse
Tissues--
Based on the observation that ARP1 and Ear2 interact in
yeast, in solution, on DNA, and in mammalian cells, we next determined if the corresponding genes, Ear2 and Arp1, may
exhibit overlapping patterns of expression in several mouse tissues
that would facilitate a biologically relevant interaction between the
two proteins.
Jonk and co-workers (18) have previously observed, by in
situ hybridization, high level Arp1 expression in
embryonal brain, lung, and kidney, whereas transcripts derived from the
Ear2 gene were found to be present at lower levels and in
several tissues in the developing embryo (8.5-14.5 d.p.c.). We have
examined Arp1 and Ear2 expression in several
tissues of the adult mouse by RT-PCR. The expression patterns of both
genes were found to overlap precisely with the highest level of
expression in brain, lung, kidney, and in the embryo (10-12 d.p.c.;
Fig. 7A). Neither gene
appeared to be expressed in thymus, spleen, and testis (Fig.
7A). Although these studies demonstrate that Arp1
and Ear2 genes are expressed in similar tissues, one cannot
conclude from these data that the two orphan receptors are expressed in
the same cell type(s) that would obviously be required for a
physiologically relevant interaction to occur. To address the
possibility that Arp1 and Ear2 may be coexpressed
in a single cell type, RT-PCR analyses were conducted using RNA derived
from two embryonal carcinoma cell lines, F9 and P19. These analyses
revealed that Ear2 was constitutively expressed in both P19
and F9 cells and was unaffected by treatment of either cell line with
all-trans-retinoic acid (RA; Fig. 7B), consistent with the finding of Jonk and co-workers (18).
Arp1 was also found to be constitutively expressed in both
cells lines and did not appear to be induced by treatment with RA (Fig.
7C), in contrast to previous studies (18). However, the
treatment protocol employed in the current studies involved exposure to RA for only 24 h, whereas Jonk and co-workers (18) observed maximal induction of Arp1 in P19 cells by Northern analysis
after a 72-h treatment with RA.

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Fig. 7.
Tissue distribution of ARP1 and Ear2
transcripts. A, ARP1 and Ear2 transcripts were detected
in the embryo (10-12 d.p.c.) and the indicated adult mouse tissues by
RT-PCR and Southern blotting as described under "Materials and
Methods." An amplified fragment of the ubiquitously expressed
36B4 gene was used for normalization purposes (36).
B and C, RT-PCR/Southern analyses of Arp1,
Ear2, and 36B4 expression in F9 and P19 embryonal
carcinoma cells, respectively. Each cell type was treated with vehicle
(0.1% v/v ethanol) or 1 µM all-trans-RA as
indicated for 24 h prior to preparation of RNA.
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DISCUSSION |
ARP1 and Ear2 were shown to interact in yeast, in vitro
(both in solution and on various directly repeated response elements), and in mammalian cells. The two orphan receptors exhibit overlapping patterns of tissue expression and are coexpressed in two pluripotent, embryonal carcinoma cell lines. ARP1 and Ear2 clearly are capable of
interaction and, at least in the cell lines and possibly the tissues
examined, these two proteins have the opportunity to do so.
In addition to the ARP1·Ear2 complex described in this report, we
have also observed the formation of COUP-TFI·ARP1 as well as
COUP-TFI·Ear2 complexes on a DR1-type response element (data not
shown). Considered together, these finding suggest that the dimerization interfaces of all COUP-TF family members are competent for
both homodimerization and heterodimerization within the subfamily as
well as with other nuclear receptors (1, 3, 4). Heterodimerization within the COUP-TF subfamily has been previously demonstrated between
COUP-TFI and ARP1 (3). However, COUP-TFI·ARP1 heterodimeric complexes
apparently did not exhibit differences with regard to either DNA
binding specificity or efficiency of formation on various response
elements when compared with homodimers of either receptor (3). In
contrast, the present studies demonstrated that ARP1·Ear2 heterodimeric complexes exhibited a response element specificity that
was distinct from that of homodimers composed of either receptor. This
was most clearly evident in the case of Ear2 homodimers that bound
weakly to a DR1(G) probe and did not interact detectably with any other
probe tested. ARP1·Ear2 heterodimeric complexes bound to DR1(G) and
DR2(G) strongly but also bound to the DR4(G) and DR5(G) probes and, to
a lesser extent, the heterodimer bound to DR3(G). ARP1 homodimers bound
very efficiently to all direct repeats of the "G" series with a
modest preference for DR2(G) and DR1(G), a DNA binding specificity that
was quite similar to that of ARP1·Ear2 heterodimeric complexes.
However, ARP1 homodimers and ARP1·Ear2 heterodimers exhibited
differential capacities to bind to direct repeats of the T series. For
example, ARP1 homodimers bound efficiently to a DR1(T) probe and more
weakly to DR5(T) and DR3(T), whereas ARP1·Ear2 heterodimeric
complexes bound weakly to the DR1(T) probe only. Ear2 strongly
inhibited ARP1 homodimer binding to the T series of probes suggesting
that ARP1·Ear2 heterodimerization resulted in a complex that was
incapable of binding to T series probes. This "inactive"
heterodimeric complex may be speculated to have important implications
in the signaling pathways of both receptors. For example, one may
envision that ARP1 homodimeric complexes are capable of regulating the
expression of promoters containing either DR(G)-type response elements
or DR(T)-type response elements with inter-repeat spacing of 1, 3, or
5, the latter of which corresponds to the classical retinoic acid
response element (39). In contrast, both ARP1·Ear2 heterodimers and
Ear2 homodimeric complexes may only be capable of regulating DR(G)-type
response elements albeit with differential capacities. However,
relative overexpression of Ear2 in a given cell type at a given time
may disrupt ARP1 homodimer-mediated regulation of the aforementioned DR(T)-containing promoters by the formation of ARP1·Ear2
heterodimeric complexes that are inactive with respect to DR(T)-type
response elements. In addition, such an event could conceivably
represent a gain of function with respect to Ear2 as ARP1·Ear2
heterodimeric complexes bind efficiently to DR(G)-type response
elements with inter-repeat spacing ranging from at least 1 to 5, whereas Ear2 homodimers appear to bind efficiently only to DR1(G)-type
elements. Thus, external and/or autocrine factors that act to perturb
the stoichiometric balance of COUP-TF orphan receptors within a single cell may alter this regulatory dynamic and provide a mechanism by which
these proteins may regulate the expression of a broader network of
genes in a combinatorial fashion. Two such stimuli have been reported
and may be relevant to this example as follows: expression of both ARP1
and COUP-TFI has been demonstrated to be positively regulated by
treatment with retinoic acid (18), whereas Sonic Hedgehog has been
shown to induce expression of APR1 (40). Either of these treatments
could alter the balance of COUP-TF family members expressed in a
particular cell type, and it will be of interest to determine if such
stoichiometric alterations ultimately dictate the scope of target genes
subject to transcriptional regulation by these proteins.
Finally, members of the COUP-TF subfamily of nuclear receptors are
generally considered to be repressors of transcription, possibly
through interactions with silencing mediator of retinoid and thyroid
hormone receptors (SMRT, see Ref. 41), nuclear receptor corepressor
(NCoR, see Ref. 41), and/or splice variants of nuclear receptor
corepressor (42). However, it is important to note that COUP-TF
proteins may also activate transcription in some promoter and/or
cellular contexts. Indeed, COUP-TFI, purified from HeLa cells, was
originally characterized as a positive factor in the regulation of the
ovalbumin promoter by O'Malley and colleagues (43-46), who have also
demonstrated that COUP-TFI, presumably in a phosphorylated form, was a
transcriptional activator when fused to a heterologous DNA binding
domain (47). Other groups have subsequently demonstrated that COUP-TF
can also function as a transcriptional activator in transient
transfection experiments (48, 49) and in vitro (50). These
findings, when considered together with the possibility that an
activating ligand(s) for COUP-TFs may exist, raise the possibility that
homodimeric or heterodimeric complexes composed of COUP-TF family
members may positively regulate transcription of target genes in a
cell-specific manner. Although the potential role of APR1·Ear2
complexes in either transcriptional activation or repression remains to
be established, the results presented herein demonstrate that such heterodimeric complexes may play a role in the COUP-TF signaling pathways.
 |
ACKNOWLEDGEMENTS |
We gratefully thank H. Nakshatri, P. Chambon,
R. Losson, P. Kastner, A. Krust, T. Lufkin, and S. Nakanishi for
plasmid constructs and reagents and A. Fields, A. Son, and J. Webster
for expert technical assistance.
 |
FOOTNOTES |
*
This work was supported in part by the American Heart
Association Grant 9640219N (to M. L.), the Oregon State University
College of Pharmacy, and the Laboratory of Molecular Pharmacology.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Supported by a predoctoral fellowship from the American
Foundation for Pharmaceutical Education. Current address: The Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD 21205.
Established investigator of the American Heart
Association. To whom correspondence should be addressed: Laboratory of
Molecular Pharmacology, College of Pharmacy, Oregon State University,
Corvallis, OR 97331. Tel.: 541-737-5809; Fax: 541-737-3999; E-mail:
Mark.Leid{at}orst.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
COUP-TF, chicken
ovalbumin upstream promoter-transcription factor;
AD, activation
domain;
RXR, retinoid X receptor;
DR, direct repeat;
RAR, retinoic acid
receptor;
9-cis-RA, 9-cis-retinoic acid;
RT, reverse transcription;
PCR, polymerase chain reaction;
NR1, NR1 subunit
of N-methyl-D-aspartate receptor;
HEK, human
embryonic kidney;
CAT, chloramphenicol acetyltransferase;
ER, human
estrogen receptor;
ERE, estrogen response element;
DBD, DNA binding
domain;
d.p.c., days postcoitum;
GST, glutathione
S-transferase;
HA, hemagglutinin;
EMSA, electrophoretic
mobility shift assays.
 |
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