(Received for publication, March 10, 1995)
From the
We have previously demonstrated that an everted repeat of the
hexamer PuGGTCA located within the F-crystallin promoter mediates
activation of the murine
F-crystallin gene by retinoic acid and
thyroid hormone receptors. Here, we show that the recently identified
retinoic acid receptor-related orphan nuclear receptor (ROR
) is
expressed in the murine lens and activates the
F-crystallin
promoter. In contrast to the retinoic acid and thyroid hormone
receptors, activation of the
F-crystallin promoter by ROR
requires binding to the single 3` half-site and spacer sequences of
F-crystallin hormone response element (
F-HRE). We further
demonstrate that ROR
-dependent activation is repressed by the
competitive binding of retinoic acid receptor/retinoid X receptor
heterodimers to the
F-HRE in the absence of
all-trans-retinoic acid. These studies suggest that the
interplay of retinoid receptors and ROR
on the
F-HRE may
constitute an important mechanism regulating
F-crystallin gene
expression in the murine lens.
The murine -crystallin gene locus contains six closely
related but differentially regulated genes encoding lens structural
proteins that are expressed concomitantly with lens cell
differentiation(1, 2, 3, 4) .
Lens-specific expression of the
F-crystallin gene is directed by
proximal promoter sequences located immediately upstream of the TATA
box which are conserved in all
-crystallin genes, while upstream
enhancer sequences are necessary for proper spatial expression within
the lens(5, 6) . We have previously shown that the
F-crystallin promoter is activated by retinoic acid (RA) (
)treatment and have characterized a complex hormone
response element (
F-HRE) located within the upstream enhancer
region(7) . The
F-HRE consists of an everted arrangement
of two nuclear receptor consensus half-sites motifs (PuGGTCA) separated
by 8 nucleotides (referred to as everted repeat-8 or ER-8) and confers
RA responsiveness when linked to a heterologous promoter(7) .
The
F-HRE is bound in vitro by RA (RAR) and retinoid X
(RXR) receptor heterodimers, and both classes of retinoid receptors
were shown to cooperate in vivo to trans-activate this
element. Recently, we have demonstrated that the
F-HRE also
mediates thyroid hormone (T
) responsiveness of the
F-crystallin promoter(8) . Although the
F-HRE is
bound by the T
receptor (T
R) in the form of
heterodimers with either RXR or RAR, only T
R
RXR
heterodimeric complexes appear to be transcriptionally active. These
results show that RAR
exerts a dominant role in the regulation of
transcription of the
F-crystallin gene and underscore the
complexity of the retinoid signal at the level of gene expression (for
review, see (9, 10, 11) ).
The retinoid
and T receptors are part of a large family of
ligand-dependent transcription factors that includes a growing class of
related proteins for which regulatory ligands have not been
identified(12) . These proteins are referred to as orphan
receptors. We have recently identified a novel family of orphan
receptors (ROR
, RAR-related orphan receptor) consisting of different isoforms that share common
DNA- and putative ligand-binding domains but possess distinct
amino-terminal domains which confer different DNA binding specificities (13) . ROR
activates transcription constitutively upon
binding as a monomer to response elements composed of the PuGGTCA core
binding motif preceded by a 6-nucleotide A/T-rich
sequence(13, 14, 15) . We have shown that the
F-HRE is bound by ROR
1 and acts as a strong HRE for this
orphan receptor(13) . In this report, we have investigated more
closely the interaction of ROR
with the
F-HRE and have
established that ROR
can activate the
F-crystallin promoter
in primary chick lens cell cultures. We demonstrate that RAR/RXR
heterodimers compete with ROR
for occupancy of the
F-HRE and
that, in the absence of RA, the retinoid receptor complexes block
ROR
activation. Since the retinoid receptors and ROR
are
expressed in the lens, we suggest that both classes of receptors are
likely to play important roles in the regulation of the
F-crystallin gene.
Figure 1:
Orphan receptor ROR is expressed in the
murine lens. 2 µg of total RNA extracted from 2-week-old
(postpartum) murine lens tissue were used in three parallel cDNA
synthesis reactions primed with either oligo(dT), a ROR-specific 3`
oligonucleotide, or a 5`-ROR-specific oligonucleotide. The products of
these reactions were amplified using the polymerase chain reaction
primed with ROR-specific primers. Amplified products were fractionated
on a agarose gel transferred to Hybond membrane and hybridized at high
stringency with a radiolabeled 107-bp human ROR1 probe. The size
of the amplified products was determined by comparison with known size
markers.
The
5`-flanking sequences of the murine F-crystallin gene between
nucleotides -226 to +47 are sufficient for optimal promoter
activity in primary cultures of chick lens cells and for proper
developmental regulation of a reporter gene in the lenses of transgenic
mice(6, 16) . The
F-HRE (located at -210 to
-185) mediates activation by both RAR and T
R and also
contributes to basal promoter activity since deletion of the 3`
half-site and spacer sequences (-202 to -185) decreases
promoter function(7, 8, 16) . In contrast,
mutation of the first half-site eliminates RA response but has minimal
effects on basal promoter activity(7) . These data suggest that
there are endogenous transcription factors in lens cells that
specifically recognize the downstream half-site and spacer of
F-HRE to stimulate basal promoter activity. The orphan receptor
ROR
interacts with a single PuGGTCA motif preceded by A/T-rich
sequences and binds to a number of characterized response elements
including the
F-HRE(13) . To determine whether ROR
could activate the
F-HRE within the natural promoter context we
performed transfections in chick lens cells using reporter gene
constructs containing the bacterial CAT gene under the control of the
mouse
F-crystallin promoter (-226 to +47). A mutant
reporter construct containing a deletion of spacer sequences and the 3`
half-site of
F-HRE (
-202/-185) was also tested.
Transfection of ROR
1 expression vector increased wild-type
promoter activity approximately 3.8-fold, while the
-202/-185 promoter mutant displayed reduced basal promoter
activity and was not significantly activated by transfected ROR
1 (Fig. 2).
Figure 2:
ROR1 activates the
F-crystallin
promoter. Chick primary lens cultures were transfected with 10 µg
of CAT gene reporter plasmids containing either wild type
F-crystallin 5`-flanking sequence -226 to + 47
(
F(-226/+47)) or an
equivalent segment containing a deletion of the
F-HRE 3` half-site
and spacer sequences (
-202/-185).
500 ng of an expression vector containing the human ROR
1 cDNA
under the control of the RSV-LTR were cotransfected together with 1
µg of RSV-
gal as a control for transfection efficiency. Cells
were harvested 48 h following transfection. The mean value of
duplicate determinations is plotted and dots indicate the
values of each determination.
Figure 3:
Competition analysis of hormone response
elements for binding of ROR1. A, sequences of response
elements used in this study. TREpal is an idealized response element
derived from the rat growth hormone gene(38) ,
RARE was
identified in the promoter of the human RAR
2 gene(39) ,
CRBPI-RARE was identified in the mouse cellular retinol binding protein
I gene(40) . B, radiolabeled
F-HRE was incubated
with reticulocyte lysate (2 µl) programmed with human ROR
1
mRNA and fractionated on polyacrylamide gel cast in 0.5
TBE.
Oligonucleotides corresponding to the different response elements were
included in the binding reactions at the indicated molar
excess.
We
next compared the transcriptional activity of ROR1 on
F-HRE
and TREpal which represent the highest affinity binding sites.
Luciferase gene reporter plasmids containing a single copy of either
element in front of the viral thymidine kinase promoter (-105 to
+51) were tested by transfection in P19 cells. Since both
F-HRE and TREpal mediate transcriptional responses to retinoid
receptors, duplicate transfected cells were treated with RA as a
control. As can be observed from Fig. 4, the TREpal reporter
plasmid was efficiently activated by RA treatment, but only the
F-HRE reporter could be activated by transfected ROR
1.
Figure 4:
ROR1 is transcriptionally inactive on
TREpal. Reporter plasmids (2 µg/plate) containing either the
F-HRE or TREpal in front of the thymidine kinase promoter linked
to the luciferase reporter gene were transfected into P19 cells
together with ROR
1 expression vector (0.5 µg/plate) and
plasmid RSV-
gal. Duplicate plates were treated with RA (100
nM). Cells were grown for 24 h following transfection. The
mean values of three independent experiments are plotted with the
standard error indicated.
Figure 5:
The 3` half-site and spacer are sufficient
for high affinity binding and transcriptional activation by ROR1.
Oligonucleotides containing either half-site and spacer sequences (A) were used as competitors in binding reactions (B)
at the indicated molar excess. (C) Luciferase reporter
plasmids (2 µg/plate) containing two copies of each oligonucleotide
in front of the thymidine kinase promoter were transfected in P19 cells
together with ROR
1 expression vectors (500 ng/plate). The mean
values of three independent experiments are plotted with the standard
error indicated.
Comparison of the spacer sequences preceding each half-site suggests
that the cytosines located at the first and second positions (-1
and -2) upstream of the 3` half-site may be required for
preferential binding of ROR1. As expected from previous analysis
of the ROR
1 binding properties(13) , deletion of
dinucleotide CC (m4) decreased binding, whereas replacement with AG
(m3) failed to do so (Fig. 6B). The importance of other
sequences within the spacer is confirmed by additional mutant
oligonucleotides (m5, m6, and m7) (Fig. 6C). In
accordance with the consensus RORE determined by binding site
selection(13, 14) , replacement of the dinucleotide AA
(positions -3 and -4) with thymidines does not affect
binding while replacement with CG causes a sharp reduction in binding
affinity. Mutation of TT at positions -5 and -6 causes a
slight reduction in binding.
Figure 6:
Analysis of spacer mutants for binding of
ROR1. Mutant oligonucleotides (A) were used as
competitors in binding reactions (B and C). Each
composite was derived from the same
experiment.
To investigate the functional relationship between retinoid
receptors and ROR on this element, we performed transfection
studies in CV-1 cells which contain low levels of endogenous retinoid
receptors. A sensitive reporter construct containing three copies of
F-HRE was activated approximately 20-fold by transfected ROR
1 (Fig. 7A). Additional RA-dependent stimulation was not
observed unless RAR expression vector was cotransfected. In the absence
of RA, transfected RAR represses ROR
mediated activation in a
dose-dependent manner. Thus, RAR can either repress ROR
activation
or cooperate in activation depending on the availability of its cognate
ligand. Repression in the absence of RA may be explained by competition
of retinoid receptor complexes with ROR
for occupancy of
F-HRE. To demonstrate this we performed EMSA with a limiting
concentration of radiolabeled
F-HRE, a constant amount of RAR and
RXR and increasing amounts of ROR
. An ROR
mutant with
truncated carboxyl terminus (ROR
235) used in these studies
forms a fast migrating complex due to its smaller size. As can be seen
in Fig. 7B, increasing the amount of ROR
235
in the binding reaction causes a dose-dependent decrease in the
formation of RAR
RXR complexes. Consistent with these results,
co-transfection of ROR
235 which is transcriptionally inactive
blocks both RA- and ROR
-dependent activation on
F-HRE
reporter plasmids (data not shown).
Figure 7:
Retinoid receptors and ROR compete for
binding to F-HRE. A, a constant amount of ROR
1
expression vector (1 µg) was transfected with the indicated amounts
of RAR
expression vector. A luciferase reporter plasmid was used
containing three copies of the
F-HRE. B, binding
reactions were carried out with a limiting amount of probe (0.01 ng), a
fixed amount of RAR/RXR and increasing amounts of a ROR mutant lacking
the carboxyl terminus (ROR
235). The RAR/RXR band
was quantified using an Molecular Dynamics PhosphorImager system.
Arbitrary values representing intensity of the RAR/RXR band was plotted
on the y axis and amount of ROR
235 added was plotted on
the x axis. Above is a representative experiment in duplicate
where the values of each determination did not vary by more than 30%.
The experiment was performed three times.
During lens development, differentiation of epithelial cells
into fiber cells is regulated by factors secreted from the
retina(24, 25, 26) . The F-crystallin
gene thus provides an excellent model to study the molecules and
transactivating factors that control differentiation processes in the
developing lens. Recently, we have shown that both RA and T
activate
F-crystallin gene expression through a novel HRE
(
F-HRE) located in the upstream enhancer element (-210 to
-185) of the
F-crystallin promoter (7, 8) and proposed that RA may serve as a regulatory
signal in lens development. This suggestion is supported by the
analysis of mice bearing disrupted RAR
and
genes which
display severe ocular defects including abnormal lens fibers and
agenesis of the lens(23) . In the present study, we report the
regulation of the
F-crystallin gene by ROR
1, an orphan member
of the superfamily of steroid-thyroid-retinoid receptors, via
interactions with the
F-HRE. We further demonstrate that
constitutive activation of the
F-HRE by ROR
1 is repressed by
the competitive binding of RAR/RXR heterodimeric complexes to the
element in the absence of RA.
The F-HRE consists of an everted
repeat of two consensus nuclear receptor half-sites (PuGGTCA) separated
by 8 nucleotides. This element is bound by all three heterodimeric
combinations of RAR, T
R, and RXR and these binding
activities require that both half-sites be
intact(7, 8) . In contrast, the 3` half-site and six
adjacent nucleotides (TTAACCAGGTCA) of the
F-HRE are sufficient
for proper recognition by ROR
1. Since transcriptional activation
by transfected ROR
1 is constitutive, these findings are consistent
with the mutational analyses of the
F-crystallin promoter.
Specifically, previous experiments indicated that alterations of the 5`
half-site of the
F-HRE abolished binding of lens nuclear factors
but had only a marginal effect on basal promoter activity while
deletion of the spacer sequences and 3` half-site significantly reduced
promoter function(7, 16) . Our mutational analysis
also demonstrates that the two A residues at positions -3 and
-4 relative to the 3` half-site are crucial for binding activity.
The A/T-rich sequences in ROR
binding sites may allow local
distortion of DNA upon binding which may optimize protein:DNA
interactions. Consistent with this notion, ROR
1 has been shown
recently to induce a large DNA bend (
130°) upon binding to a
consensus ROR binding site(15) .
The orphan receptor
ROR1 activates the
F-crystallin promoter in transfected lens
epithelial cells without addition of exogenous ligands suggesting
either that it functions in a ligand-independent manner or that a
putative ligand is present in the culture medium or metabolized by lens
cells. The identification of an ROR ligand would be important not just
in the regulation of
-crystallin genes and lens development but
probably in many developmental processes since ROR is widely
expressed(27, 28) . Recently, it has been reported
that human RZR
and RZR
(isoforms of ROR) can be activated by
melatonin and a synthetic ligand, CGP 52608(29, 30) .
Numerous attempts to demonstrate activation of ROR
1 by melatonin
on
F-HRE or consensus ROR
1 binding sites reporter plasmids
have failed. (
)It is clear from our results that additional
experiments are needed in order to ascribe a possible role for
melatonin in the activation of ROR
. Nonetheless, the regulation of
the
F-crystallin promoter by ligand activated transcription
factors (7, 8) suggests the possibility that their
ligands may constitute the signals originating from retina that control
lens development.
We have shown that in the absence of RA,
increasing levels of RAR blocks ROR
1 activation of
F-HRE
reporter genes in a dose-dependent manner. These results are similar to
those obtained in our study of the activation of the
F-crystallin
promoter by T
R/RXR heterodimers(8) . Transfected
RAR
potently represses T
response from
F-HRE
reporter plasmids apparently by competition for binding site occupancy.
Taken together, these data suggest that RAR/RXR heterodimers exert a
dominant role in the regulation of the
F-crystallin gene by
nuclear receptors. These results contrast to those observed for the
interactions of orphan receptors ARP-1/COUP-TFII, EAR3/COUP-TFI, and
RXR/RAR on the Oct-3/4 promoter where the orphan receptors completely
abolish promoter activation by RXR/RAR
heterodimers(31, 32, 33) . These findings
clearly demonstrate that the specificity of the retinoid signal is
dependent on the configuration of the response element as well as the
repertoire of competing orphan receptors.
A number of orphan
receptors have been identified which like ROR can regulate
transcription by binding as monomers to target sequences consisting of
a single half-site and a few adjoining
bases(14, 28, 34, 35, 36) .
Since response elements for RA, T
, and vitamin D
receptors consist of two or more half-sites, it is likely that
monomeric-binding orphan receptors interact with many of these response
elements possibly modifying hormonal responses(37) . The
combined action of ROR
and related nuclear receptors in the
regulation of the
F-crystallin gene is likely to be of primary
importance in the developmental expression in the lens. The
identification of putative ROR
binding sites in other
-crystallin genes (
)suggests a comprehensive role of
this orphan receptor in the regulation of
-crystallin gene family.