(Received for publication, May 30, 1996, and in revised form, October 4, 1996)
From the Department of Medicine and the Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110
To investigate the mechanisms involved in the
transcriptional control of retinoid X receptor (RXR) gene expression,
the 5-flanking region of the human RXR
2 isoform was characterized.
An imperfect hexamer repeat (
retinoid X response element;
RXRE)
with a single nucleotide spacer (GGTTGAaAGGTCA) was identified
immediately upstream of the RXR
2 gene transcription start site.
Cotransfection studies in CV-1 cells with expression vectors for the
retinoid receptors RXR
and retinoic acid receptor
(RAR
)
demonstrated that the
RXRE confers retinoid-mediated transcriptional
activation with preferential activation by RXR in the presence of its
cognate ligand, 9-cis-retinoic acid (RA). Electrophoretic
mobility shift assays demonstrated that RXR homodimer binding to
RXRE is markedly enhanced by 9-cis-RA, whereas RAR·RXR
heterodimer binding is ligand-independent. DNA binding studies and cell
cotransfection experiments also demonstrated that the nuclear receptor,
chicken ovalbumin upstream promoter transcription factor (COUP-TF),
repressed transcription via the
RXRE. Cotransfection experiments
revealed that COUP-TF and RXR
compete at the
RXRE to modulate
transcription bidirectionally over a wide range. These results
demonstrate that the human RXR
2 gene promoter contains a novel
imperfect repeat element capable of mediating RXR-dependent
transcriptional autoactivation and COUP-TF-dependent
repression.
The retinoid derivatives of vitamin A regulate a wide variety of
biological processes, including development, differentiation, and
cellular metabolism. The retinoids exert influence at the level of gene
transcription by serving as ligands for the retinoid receptor families
of transcription factors. The retinoid X receptors (RXR, -
, and
-
)1 play a distinctly unique role within
the nuclear receptor superfamily in that they may
trans-activate not only as 9-cis-retinoic acid (9-cis-RA) activated homodimers but also as obligate
heterodimeric partners for retinoic acid receptor (RAR), thyroid
hormone receptor (TR), vitamin D receptor, peroxisome
proliferator-activated receptor, and several "orphan" receptors
(Refs. 1-6; reviewed in Ref. 7). RXR has thus been described as a
"master regulator" of a subset of nuclear receptor signaling
pathways.
RXR and RXR
exhibit ubiquitous expression patterns during murine
development and in adult tissues (8, 9). In contrast, RXR
expression
is restricted both in fetal and adult tissues (8-10). The mouse RXR
gene has two known mRNA isoforms (RXR
1 and RXR
2), produced
via alternative exon splicing and differential promoter utilization
(10). The RXR
isoforms exhibit a distinct tissue-restricted
expression pattern; RXR
1 is enriched in neural tissue, whereas
RXR
2 is cardiac enriched (9, 10). Both transcripts are relatively
abundant in skeletal muscle (9, 10). During embryologic development,
RXR
transcripts are expressed in distinct temporal patterns (8, 9,
11). The RXR
gene is therefore unique among the RXR gene family
members in that its expression is spatially and temporally restricted,
suggesting the possibility that the function of this nuclear receptor
is distinct from the other RXRs. Little is known about the mechanisms
involved in the control of RXR gene expression or if cross-signaling
occurs between members of the RXR gene family.
As an initial step in the investigation of the transcriptional
regulatory mechanisms involved in the restricted pattern of expression
of RXR isoforms, we have cloned the 5
-flanking region of the human
RXR
2 gene. In this report, we describe a novel autoregulatory retinoid X response element (
RXRE) located within this promoter and
present evidence that this element is capable of conferring transcriptional activation via retinoid pathways and transcriptional repression via the orphan receptor COUP-TF.
Human
RXR cDNA fragments were produced via polymerase chain reaction
from a human heart cDNA library template (Clontech) using
oligonucleotide primers that were designed based on cross-species homology between mouse (8, 10), chicken (12), and Xenopus (13) RXR
cDNA sequences. Several overlapping human RXR
partial cDNA clones were obtained, encompassing sequences
representing most of the coding region and having an overall nucleotide
identity of greater than 90% with the mouse RXR
cDNA.2 One partial human cDNA clone
(hRX45), representing a fragment colinear with bp 451-669 relative to
the published mouse RXR
1 sequence (10), was generated by primers rx4
(5
-ATCAggatccCTTCTGCCATGGGTCCACCCTCA-3
) and rx5
(5
-TCTGggatccTCCCCACAGATGGCACAGATGTG-3
). hRX45 was used as a probe to
screen a human genomic EMBL3 phage library (Clontech). Three genomic
clones were isolated, two of which were characterized (G1SH10 and
G2SH1). Restriction endonuclease and Southern blot analysis confirmed
that these genomic clones were overlapping and contained sequences
recognized by the hRX45 probe. Additional Southern blot analysis using
a polymerase chain reaction-generated mouse RXR
exon 1b probe
(corresponding to the 5
-untranslated region of the RXR
2 cDNA
sequence (10)) identified overlapping genomic DNA restriction fragments
containing the putative human homolog of exon 1b. Primer extension
analysis using total human heart RNA and 32P-end-labeled
rx20 (5
-AATCTGCCCATGCGATCCAGAGTC-3
) and rx15
(5
-GCCTTTTTTCCAGTGTCATC-3
) primers from within the putative human
exon 1b mapped the transcription start site to 322 bp upstream of the
ATG start codon located in exon 3. To compare the genomic sequence with
cDNA sequence, 5
-RACE was performed using a human heart
5
-RACE-Ready cDNA library (Clontech) and primers from within the
putative human exons 3 (rx5) and 1b (rx16, 5
-
ATCGggatccCATGGGCAGATTATTCC-3
, rx20, and rx15). Comparison of the DNA
sequence of G1SH10 and G2SH1 confirmed (i) there was 100% nucleotide
identity in the 5
-untranslated region region between the 5
-RACE
clones and genomic clones G1SH10 and G2SH1, (ii) the human RXR
exon
1b was spliced to human exon 3 in a manner identical to that found in
mice (10), and (iii) no intervening sequences existed between exon 1b
and the transcription start site.
hRXR2.luc.
1140 was constructed by cloning a
BamHI-PstI fragment of the hRXR
2 5
-flanking
region from
1140 bp upstream to +71 bp downstream of the
transcription start site into the luciferase reporter plasmid pGL2
basic (Promega). The mutated human RXR
2 promoter reporter plasmid
(hRXR
2.luc.
1140.m1) was constructed by replacing the native
sequence from
121 to +71 (an NdeI-PstI
fragment) with a polymerase chain reaction-generated fragment
containing point mutations in the
RXRE
(G
TTGAAA
GTCA; underlined nucleotides
represent substitution mutations, compared with Fig. 1). The
construction of pTKLuc has been described (14).
RXRE.TKluc and
RXREm2.TKluc were each constructed by ligating two copies of the
corresponding double-stranded oligonucleotide fragments in sense
orientation into the BamHI site of pTKLuc
(5
-gatccTGGGGTTGAAAGGTCAGATGGAtc-3
for
RXRE.TKluc and
5
-gatccTGGG
TTGAAA
GTCAGATGGAtc-3
for
RXREm2.TKluc (underlined nucleotides represent mutations)). Dideoxy
DNA sequencing confirmed the location and orientation of the inserts.
The construction of eukaryotic expression vectors for use in cell
culture transfection experiments (pCDMRXR
, pCDMRAR
, and pCDMCOUP)
have been described (14) and were the generous gifts of Drs. Tod Gulick
and David Moore (Harvard University). The murine expression vectors
pSG5RXR
, -
, and -
were generously provided by Dr. Pierre
Chambon, Institut de Genetique et de Biologie Moleculaire et
Cellulaire, Strasbourg, France.
Mammalian Cell Transfections
Simian CV-1 cells were
employed for all transfection experiments. Cells were maintained and
transient cotransfections performed as described (15). In brief,
transient transfections were performed by the calcium phosphate
coprecipitation method in 12-well tissue culture plates (Falcon) with 4 µg of reporter construct and 1 µg of receptor expression plasmid
(as indicated in the figure legends) or an equivalent amount of pCDM
without insert (pCDM()). Cells were harvested 48 h after
transfection. One µg of a Rous sarcoma virus
-galactosidase
expression vector (RSV
gal) was included to correct for transfection
efficiency with the exception of experiments involving pCDMCOUP.
pCDMCOUP was noted to exert a modest repressive effect on the
transcriptional activity of the thymidine kinase (TK) promoter.
Accordingly, activities were adjusted to the effect of COUP-TF on the
TK promoter based on the results of parallel experiments using pTKLuc
without insert. In experiments involving the addition of ligand,
9-cis-RA or all-trans-RA was added 48 h
prior to harvest, and vehicle was added at the same concentration to
control wells. Luciferase activity was measured using the standard
luciferin-ATP assay (16), and
-galactosidase activity was measured
using the Galacto-Light chemiluminescence assay (Tropix) in an
Analytical Luminescence Monolight 2010 luminometer.
EMSAs were
performed as described (14, 17). The pT7lac-RXR and
pT7lac-myc-COUP-TF bacterial expression vectors
(14) were generously provided by Dr. Tod Gulick. The nuclear receptors were overproduced in bacterial cells and partially purified as described previously (14). Antibody supershift experiments were performed with monoclonal antibodies to RXR (4RX1D12, directed against
the D or E domain of all three murine RXRs; a generous gift of Dr.
Pierre Chambon) and a monoclonal antibody directed against an epitope
in the c-Myc protein (9E10, Oncogene Science).
A human EMBL phage genomic library was
screened with a partial human RXR cDNA probe (hRX45; see
"Materials and Methods") encoding a portion of human RXR
gene
exon 2 and all of exon 3 (nucleotides 451-669 relative to the
published mouse RXR
1 cDNA sequence (10)). The human RXR
gene
exon 3 was identified by the high degree of cross-species nucleotide
identity with the murine exon 3 (>90%) (10). To determine whether the
5
-flanking region of the human RXR
2 gene was contained within
either of two genomic clones, Southern blot analysis was performed with a polymerase chain reaction-generated murine RXR
2-specific cDNA probe containing only the 5
-untranslated region sequence encoded by
the murine exon 1b (10). A single 4.0-kilobase pair BamHI restriction fragment was identified with this exon 1b probe and DNA
sequence analysis defined a 250-bp region with over 70% nucleotide identity with the murine RXR
exon 1b sequence. 5
-RACE clones from a
human heart library confirmed that, as in mouse, the human exon 1b
sequence is spliced to exon 3 (10). Comparison of the genomic DNA
sequence with that of multiple 5
-RACE clones revealed 100% nucleotide
identity, confirming that no intervening sequences existed between exon
1b and the transcription start site. The 5
-RACE sequence data and
primer extension analysis with human heart total RNA using two
different antisense oligonucleotides from within the human exon 1b
sequence (data not shown) localized the transcription start site to 322 bp upstream of the start codon. Of note, in addition to a high degree
of identity, the human and mouse exon 1b nucleotide sequences are
nearly colinear, diverging by no more than 4 consecutive nucleotides
over the entire length of both sequences (data not shown). These
results confirmed that the 4-kilobase pair BamHI genomic
fragment contained 1.14 kilobase pairs of RXR
2 gene 5
-flanking
sequence, the 250 bp of 5
-untranslated region sequence encoded by the
human homolog of murine RXR
exon 1b, and approximately 2.5 kilobase
pairs of downstream sequence (Fig. 1).
Analysis of the DNA sequence of the RXR2 5
-flanking region revealed
a putative TATA sequence (TATATTA) at bp
16 (relative to the
transcription start site, +1), numerous potential E boxes (18), and
several putative CArG sites (19) (Fig. 1), consistent with the muscle-
and cardiac enriched expression of RXR
2. An imperfect repeat
sequence located at
100 to
86 conformed to the binding consensus
for class II and class III nuclear receptors (Fig. 1). This sequence
contains two potential hexamer binding sites separated by a single
nucleotide and thus conforms to the direct repeat-1 (DR-1) group of
elements known to confer transcriptional regulation by retinoid
receptors (20).
To test the possibility that the putative nuclear receptor response
element was retinoid-responsive and to characterize the transcriptional
activity of the RXR2 gene 5
-flanking region from
1140 to +71,
transient cell transfection studies were performed with this DNA
fragment fused to a luciferase reporter (hRXR
2.luc.
1140). A series
of cotransfection studies was performed in simian CV-1 cells with
eukaryotic expression vectors for human RXR
(pCDMRXR
) and human
RAR
(pCDMRAR
) in the presence and absence of the retinoid ligands
9-cis-RA and all-trans-RA. As shown in Fig.
2A, the transcriptional activity of
hRXR
2.luc.
1140 was minimally increased in the presence of
9-cis-RA or RXR
alone but was induced 7-13-fold upon the
addition of both 9-cis-RA and RXR
, indicating that this
promoter fragment was activated by RXR
in a
ligand-dependent manner. In contrast, hRXR
2.luc.
1140
transcription was only minimally activated by all-trans-RA
or 9-cis-RA in the presence of RXR
and RAR
(Fig. 2A). These results suggest that the RXR
2 promoter is
preferentially activated by RXR homodimers rather than RXR·RAR
heterodimers.
To localize the region of retinoid responsiveness and to determine
whether the imperfect repeat sequence located at 100 bp was indeed an
RXR-responsive element, cotransfections were repeated with a
5
-deletion series of hRXR
2.luc constructs. The results of these
experiments (data not shown) revealed that the sequences conferring
9-cis-RA-mediated response resided primarily within the
fragment flanked by NdeI (
121 bp) and PstI (+71
bp) sites (see Fig. 1), which contained the putative RXR response
element. Cotransfection studies were repeated with a mutated
hRXR
2.luc.
1140 construct containing cytidine substitutions for the
invariant second position guanine within each hexameric half-site of
the imperfect repeat sequence (hRXR
2.luc.
1140.m1; Fig.
2B). The 9-cis-RA/RXR
-mediated activation of
hRXR
2.luc.
1140.m1 was markedly lower (>75%) than that of
hRXR
2.luc.
1140, confirming that the imperfect repeat conferred the
majority of the 9-cis-RA/RXR-mediated response (Fig.
2B). This retinoid-responsive element is here referred to as
the
RXRE.
To test whether the RXRE could confer retinoid responsiveness to a
heterologous promoter and to examine its transcriptional regulatory
properties further, including its potential to interact with other
class II and class III nuclear receptors, two copies of the
RXRE
were cloned upstream of the herpes simplex TK promoter fused to a
luciferase reporter (
RXRE.TKluc). Cotransfection studies showed that
RXRE.TKluc was activated 8-10-fold by 9-cis-RA in the
presence of RXR
(Fig. 3A). Significant
RXR-mediated activation of
RXRE.TKluc occurred only in the presence
of its ligand, 9-cis-RA, as was observed with the homologous
promoter (hRXR
2.luc.
1140). When point mutations identical to those
present in hRXR
2.luc.
1140.m1 were introduced into both copies of
the
RXRE in the context of TKluc (
RXREm2.TKluc),
9-cis-RA-mediated responsiveness was abolished (Fig.
3A). The cotransfection experiments were repeated with
expression vectors for murine RXR
, -
, and -
to determine
whether the
RXRE was capable of conferring
9-cis-RA-mediated transcriptional activation via all known
RXRs. All three RXRs mediated 9-cis-RA-dependent activation to a similar level (data not shown).
Previous studies have demonstrated that RXR·RAR heterodimers may
confer transcriptional activation via DR-1 elements in the presence of
either 9-cis-RA or all-trans-RA (21, 22). In
contrast to RXR homodimer-mediated activation, the ligand-mediated
activation of RXR·RAR heterodimers on a DR-1 element occurs mainly or
solely via RAR (23, 24). Accordingly, RXR·RAR heterodimers may
function as transcriptional inhibitors of RXR homodimer activation on
DR-1 elements. In fact, the transfection studies shown above (Fig. 2A) revealed that, in the context of the homologous
promoter, RXR-mediated activation of
RXRE was reduced by the
presence of RAR
. To explore the activation of
RXRE in the context
of a heterologous promoter, cotransfection studies were performed with
RXRE.TKluc and pCDMRXR
and/or pCDMRAR
in the presence and
absence of either 9-cis-RA (a potential ligand for either RXR or RAR) or all-trans-RA (an RAR ligand) (Fig.
3B). Cotransfection of pCDMRAR
alone or pCDMRAR
plus
pCDMRXR
in the absence of ligand did not significantly alter the
transcriptional activity of
RXRE.TKluc. Activation of
RXRE.TKluc
by either all-trans RA or 9-cis-RA in the
presence of both RXR
and RAR
was lower than the induction
obtained with RXR
alone in the presence of 9-cis-RA (mean
of 4-5-fold versus 8-fold, respectively). These results and
the retinoid-mediated activation studies of the homologous promoter
(Fig. 2A) indicate that the
RXRE is preferentially
activated by RXR and its cognate ligand 9-cis-RA. The
retinoid-mediated transcriptional regulatory properties of the
RXRE
is similar to that of other DR-1 elements such as the cellular
retinol-binding protein II gene RXRE (23, 25) in which cotransfection
of RAR
blunts 9-cis-RA mediated transactivation by
RXR
. Additional experiments demonstrated that several other known
RXR partners, including TR
1, TR
1, or peroxisome
proliferator-activated receptor
, had no effect on
RXRE.TKluc
transcriptional activity in the presence of appropriate hormone ligands
(thyroid hormone) or peroxisome proliferator-activated receptor
activators (fatty acids or clofibrate) with or without RXR (data not
shown).
To characterize the interaction of RXR
with the RXRE, EMSAs were performed with a
32P-radiolabeled
RXRE oligonucleotide probe and
bacterially overexpressed, partially purified RXR
. As shown in Fig.
4A, RXR
homodimers bound the
RXRE as a
single complex with an affinity that was significantly increased by the
addition of 9-cis-RA (Fig. 4A, lane 3)
compared with vehicle (Fig. 4A, lane 2). The
specificity of the RXR homodimer-
RXRE interaction was demonstrated
by competition studies showing complete inhibition of formation of the
complex by the addition of a 100-fold molar excess of unlabeled
RXRE but no reduction in complex formation with an equivalent molar amount
of unlabeled, unrelated double-stranded oligonucleotide (Fig.
4A; lanes 3-5). In addition, the specific
complex was "supershifted" with anti-RXR antisera (Fig.
4A; lanes 6 and 7). Finally, when a
mutated
RXRE probe, containing the same point mutations previously shown to abolish functional activity, was incubated with RXR
and
9-cis RA, no complex formed (Fig. 4A, lanes
8 and 9). These data confirm that RXR
homodimers can
interact directly and specifically with the
RXRE and that ligand
increases binding affinity.
To characterize RXR·RAR heterodimer binding to the RXRE, EMSA was
performed with bacterially overexpressed RAR
and RXR
(Fig.
4B). A minimal complex was observed when either RAR or RXR alone was added to
RXRE probe in the absence of ligand. In contrast, incubation of the probe with both receptors resulted in a marked increase in complex formation. Competition experiments confirmed that
this interaction was specific (Fig. 4B; lanes
4-6). Accordingly, RXR·RAR heterodimers bind the
RXRE in a
cooperative manner. In contrast to the interaction of
RXRE with RXR
homodimers, the RXR·RAR-
RXRE interaction was not influenced by the
addition of 9-cis-RA or all-trans-RA (data not
shown).
A significant body of evidence indicates that RXR and the
known orphan nuclear receptor COUP-TF often compete at a single DR-1-type element (26-31). To examine the potential binding of COUP-TF
to the RXRE, EMSAs were performed using COUP-TF tagged with an
NH2-terminal Myc peptide overproduced in bacteria
(COUP-TFMyc). COUP-TFMyc formed a specific complex with the
RXRE, as
demonstrated by competition studies (Fig. 5).
"Supershift" experiments with an anti-Myc antibody provided
additional evidence for the specificity of the COUP-TF-
RXRE
interaction. Thus, COUP-TF binds the
RXRE with high affinity.
Cotransfection mixing experiments were performed with RXRE.TKluc,
pCDMRXR
, and pCDMCOUP to determine whether these transcription factors could compete at the
RXRE to modulate transcription (Fig. 6). For these experiments, increasing amounts of
pCDMCOUP were transfected into CV-1 cells with a fixed amount of
pCDMRXR
in the presence of 9-cis-RA. Because parallel
experiments with pTKLuc alone demonstrated a modest repressive effect
of COUP-TF on TK transcription, all data presented for
RXRE.TKluc
have been corrected for the effect on the TK promoter. COUP-TF blunted
the 9-cis-RA-mediated RXR activation via the
RXRE in a
dose-dependent fashion (Fig. 6). With the highest amounts
of pCDMCOUP transfected, transcription of
RXRE.TKluc was repressed
below basal levels, indicating that, in addition to competing with RXR,
at higher levels COUP-TF actively represses transcription via the
RXRE, a property shown for most known COUP-TF response elements (14,
26-37). The transcriptional activity of
RXRE.TKluc varied over
50-fold in these cotransfection experiments. These results, together
with the binding studies, indicate that COUP-TF modulates
retinoid-mediated activation of
RXRE and suggest a mechanism whereby
transcriptional activity can be modulated over a wide range.
This report demonstrates that the human RXR2 gene promoter
contains a RXR response element, a mechanism for the regulation of
RXR
2 gene expression by retinoid-mediated pathways. The presence of
autoregulatory elements within the promoters of genes encoding other
nuclear receptor isoforms, including RAR
2, RAR
2, RAR
2, and
TR
1, suggests that the expression of a subset of nuclear receptor
genes are controlled by this mechanism (38-43).
The transcriptional regulatory properties of RXRE are similar to
those of previously reported DR-1-type retinoid response elements (21,
23, 25, 29-31, 44-46). A comparison of the
RXRE sequence with the
relatively few known natural DR-1 RXREs is shown in Fig.
7. Although the 5
-half-site sequence of the
RXRE is novel compared with other known elements, it conforms to the known consensus (PuG(G/T)TNA) for binding class II or class III nuclear receptors (reviewed in Ref. 7). Furthermore, the
RXRE sequence, including the extended heptamer of the 5
-half-site
(GGG
T
A) resembles the RXR
binding site
(GGGGTCAaAGGTCA) and the high affinity RXR
consensus (RGRNCAaAGGTCA)
determined by nonbiased random oligonucleotide selection (47-49).
Interestingly, comparison of the sequences shown in Fig. 7 reveals that
the 5
-half-site sequence often diverges from the idealized class
II/III sequence (AGGTCA). To our knowledge, the role, if any, of such
sequence differences in dictating transactivation properties of RXR
isoforms has not been established.
We show here that the interaction of RXRE with RXR homodimers but
not RXR·RAR heterodimers is induced by the ligand
9-cis-RA, a unique property. Although others have shown
ligand-dependent binding of RXR homodimers to DR-1 elements
with receptor produced in reticulocyte lysate expression systems, to
our knowledge this is the first example of ligand-induced binding with
receptor protein produced in bacteria. In fact, several other groups
have shown that RXR homodimer (produced in bacterial expression
systems) binding to perfect DR-1 elements is ligand-independent (25, 26, 48). We have also shown that binding of RXR
(produced in our
bacterial expression system) to an idealized DR-1 (AGGTCAaAGGTCA) is
not dependent on or induced by 9-cis-RA.2 Taken
together, these results suggest that the unique sequence of the
RXRE
dictates the relative affinity by which RXR homodimers bind this
element and thus require ligand for this interaction.
Our results also demonstrate that the orphan receptor COUP-TF competes
with RXR homodimers on the RXRE to repress transcription. This
finding is consistent with the known role of COUP-TF as a negative
modulator of RXR-mediated transcriptional regulatory pathways through
competition for DNA binding. This competitive interaction has been
demonstrated for a variety of natural and synthetic retinoid-responsive
elements, including DR-1 (26, 27, 29-31) and DR-5 elements (27, 33) as
well as complex retinoid-responsive elements (14, 27, 28, 33, 36, 50). Our data also indicate that, in addition to interference with RXR
homodimer binding to the
RXRE, COUP-TF represses transcription via
this element as it does on the majority of other COUP-TF response elements.
A major unanswered question in nuclear receptor biology involves the
specific biological roles of multiple RXR and RAR isoforms generated by
differential promoter utilization and/or alternative splicing. The
RXR2 isoform is an excellent focus for the study of the function of
nuclear receptor isoforms because of its tissue- and developmental
stage-restricted expression pattern (8-10). The recent
characterization of mice homozygous for targeted ablation of retinoid
receptors demonstrates the importance of retinoids in murine cardiac
development (51-55). In addition, recent studies by us and others
suggest that retinoids play a role in the control of postnatal cardiac
energy metabolism (56) and antagonize the cardiac hypertrophy program
(57). The known cardiac enriched expression of RXR
2 and our
identification of the
RXRE raises the intriguing possibility that
the cardiac specific effects of retinoids occur via retinoid signaling
pathways that converge on this gene. Given that RXR
and RXR
are
expressed prior to RXR
in the developing heart and somites, it
follows that the RXR
2 gene promoter could be a downstream target
during embryologic development. Cotransfection studies performed in our
laboratory indicate that the
RXRE is activated similarly by RXR
,
RXR
, or RXR
(data not shown), suggesting that the human RXR
2
promoter is a potential target for any of the known RXRs.
We especially thank Tod Gulick and David Moore for helpful discussions and Kelly Hall for expert secretarial assistance.