From the Laboratory of Molecular and Developmental Biology, NEI, National Institutes of Health, Bethesda, Maryland 20892-2730
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ABSTRACT |
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Crystallins are a diverse group of abundant
soluble proteins that are responsible for the refractive properties of
the transparent eye lens. We showed previously that Pax-6 can activate
the B-crystallin/small heat shock protein promoter via the
lens-specific regulatory regions LSR1 (
147/
118) and LSR2
(
78/
46). Here we demonstrate that retinoic acid can induce the
accumulation of
B-crystallin in N/N1003A lens cells and that
retinoic acid receptor heterodimers (retinoic acid receptor/retinoid X
receptor; RAR/RXR) can transactivate LSR1 and LSR2 in cotransfection
experiments. DNase I footprinting experiments demonstrated that
purified RAR/RXR heterodimers will occupy sequences resembling retinoic
acid response elements within LSR1 and LSR2. Electrophoretic mobility
shift assays using antibodies indicated that LSR1 and LSR2 can interact
with endogenous RAR/RXR complexes in extracts of cultured lens cells.
Pax-6 and RAR/RXR together had an additive effect on the activation of
B-promoter in the transfected lens cells. Thus, the
B-crystallin gene is activated by Pax-6 and retinoic acid receptors,
making these transcription factors examples of proteins that have
critical roles in early development as well as in the expression
of proteins characterizing terminal differentiation.
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INTRODUCTION |
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The refractive properties of the transparent eye lens depend on a diverse group of globular proteins called crystallins that comprise approximately 90% of the water-soluble proteins of this tissue (1, 2). Despite their specialized function in the lens, crystallins are surprisingly diverse and may differ among species. Moreover, crystallins often play more than one biological role, a situation called gene sharing (3), with many being related or identical to metabolic enzymes or stress proteins (4-6). These multifunctional crystallins are expressed very highly in the lens and to a lesser extent in other tissues, where they have nonrefractive roles.
The molecular basis for the specialized expression of crystallin genes has been investigated for some time (7). While no one cis-control element or transcription factor is solely responsible for the high lens expression of the crystallin genes, Pax-6 (8-11) and retinoic acid (RA)1 (12-14) appear to have prominent roles. This is consistent with the critical use of these transcription factors for eye and lens development (15-28).
We have been studying mouse B-crystallin, a conserved small heat
shock protein (29, 30) that is constitutively expressed highly in the
lens and more moderately in many other tissues (31, 32).
B-crystallin is also induced by stress (33) and overexpressed in
numerous diseases (34, 35). The differential constitutive expression of
the murine
B-crystallin gene is developmentally and
transcriptionally controlled (32, 36, 37). Transgenic mouse experiments
have established that the sequences downstream of
164 are sufficient
to direct lens-specific gene expression (38). This 5'-flanking sequence
contains two lens-specific regulatory regions called LSR1 (
147/
118)
and LSR2 (
78/
46). Pax-6 can interact with both LSR1 and LSR2 and
activate the
B-crystallin promoter in transient transfection
experiments (39). In the present study, we show by DNase I
footprinting, antibody/electrophoretic mobility shift assay (EMSA),
site-directed mutagenesis, and transient-cotransfection experiments
that RAR/RXR heterodimers can interact at retinoic acid-responsive
elements (RAREs) within LSR1 and LSR2 and can activate the
B-crystallin promoter in lens cells either alone or collaboratively
with Pax-6.
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EXPERIMENTAL PROCEDURES |
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Nucleic Acid Isolation-- For transfection experiments, plasmid DNA was isolated and purified using the Qiagen plasmid kit according to the manufacturer's instructions (Qiagen Inc., Chatsworth, CA).
Northern (RNA) Analysis--
Total RNA was isolated from
N/N1003A cells (40) treated with RA (Sigma) by using the RNA Isolation
Kit (Stratagene, La Jolla, CA) and subsequently fractionated by
electrophoresis through a 1.5% agarose-formaldehyde gel. The RNA was
transferred to a Duralon membrane (Stratagene, La Jolla, CA) and
hybridized to a 230-base pair HindIII-BamHI
restriction fragment from exon 3 of the mouse B-crystallin gene
(32). The probe was labeled by using the Ready-To-Go Random Prime
Labeling System (Amersham Pharmacia Biotech). Prehybridizations were
performed at 60 °C for 30 min, and hybridizations were carried out
at 60 °C for 90 min by using QuickHyb (Stratagene, La Jolla, CA)
according to the manufacturer's instructions. Membranes were washed
and autoradiographed as described previously (41). Methylene blue
staining was performed as earlier (42) to monitor the integrity of RNA,
the relative amounts of RNA loaded on the gel, and the efficiency of
transfer to Duralon membranes. Membranes were exposed for
autoradiography on Kodak XAR5 film at
80 °C with an intensifying
screen for 12 h.
Nuclear Extracts, Oligonucleotides, and Antisera--
Nuclear
extracts (9) were prepared from TN4-1 (43) and N/N1003A lens cells.
Complementary oligodeoxynucleotides were synthesized (model 380A
synthesizer; Applied Biosystems) and annealed at a 1:1 molar ratio as
described previously (44). The oligodeoxynucleotides were labeled on
one strand using T4 polynucleotide kinase, and electrophoretic mobility
shift assays (EMSAs) were performed as described previously (44).
Double-stranded oligodeoxynucleotides LSR1, LSR2, and short LSR2
containing sequences
136 to
109,
78 to
28, and
73 to
48,
respectively, of the
B-crystallin promoter were used for EMSAs.
Anti-mouse RAR/RXR monoclonal antibodies (45) were generous gifts from
Drs. Pierre Chambon and Maria Gaub (Centre National de la Recherche
Scientifique, Strasbourg, France). Polyclonal antibodies were bought
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) (anti-RXR
,
catalog number sc 831; anti-RAR
, catalog number sc 551; anti-RAR
,
catalog number sc 552; anti-RAR
, catalog number sc 773; and
anti-RXR
, catalog number sc 774).
EMSA and DNase I Footprinting--
A polymerase chain
reaction-generated fragment corresponding to the 190 to +40 sequence
of the
B-crystallin gene was used for footprinting experiments with
purified mouse RAR/RXR receptors. DNA and protein were incubated and
treated with DNase I as described previously (44). The RAR/RXR proteins
were kindly provided by Drs. Keiko Ozato and Jorge Blanco (NICHD,
National Institutes of Health, Bethesda, MD). RAR
was obtained from
Santa Cruz Biotechnology. End-labeling, EMSA, and DNase I footprinting
were performed as described earlier (44).
Western (Protein) Analysis--
Nuclear extracts prepared from
TN4-1 and N/N1003A lens cells were fractionated by electrophoresis
in a Tris-glycine polyacrylamide gel; the separated proteins were
transferred to nitrocellulose membranes using a Trans-Blot (Bio-Rad).
Immunoblotting was performed according to the manufacturer's
instructions (Vector Labs, Burlingame, CA).
Site-directed Mutagenesis--
Plasmids containing mutations
generated previously (38) within the 164/+44
EcoRI/PstI fragment of the mouse
B-crystallin gene, cloned in pRD30A (36), were used for transient transfection experiments and EMSAs. In brief, site-specific mutations (Mu-9762 and
Mu-9763) (38) were introduced by using an oligodeoxynucleotide-directed mutagenesis kit (Sculptor in vitro mutagenesis kit, Amersham
Pharmacia Biotech). Mutated oligodeoxynucleotides contained the
substitution sequence TCTAGA (XbaI site) and 20 bases on
each side complementary to the
B-crystallin promoter sequence. The
resulting mutated restriction fragments were subcloned into pRD30A at
the unique BamHI site (36). All constructs were confirmed by
sequencing the ligated junctions and mutated regions.
Cell Culture, Transient Transfections, and CAT Assays--
Mouse
COP-8 fibroblasts (46) and N/N1003A lens cells were grown in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
containing 10% fetal calf serum and 50 µg/ml of gentamicin in 10%
CO2. The cells were propagated on 60-mm diameter plastic dishes. 10 µg of wild-type B-promoter-cat plasmids
(p65-7 and p11-3) (36) or mutated test plasmids (Mu-9762 and Mu-9763)
(38); increasing amounts (0.25-1 µg) of pSV40RAR
and pRSVRXR
(gifts from Drs. Keiko Ozato and Jorge Blanco), which express the
wild-type RAR
and RXR
, respectively (47); and 2 µg of internal
control pCH110, which expresses
-galactosidase (Amersham Pharmacia
Biotech), were cotransfected for 6 h by the calcium phosphate
method as described previously (44). Cells were treated with 100 µl
of 0.1 ng/ml of RA in the morning following transfection for 1 h. The cells were harvested, and extracts were prepared 48 h after transfection. CAT activities were determined by the biphasic assay (48), and
-galactosidase activities were determined as described previously (44). The transfection data represent the means of three
separate experiments, with each experiment being conducted in
duplicate.
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RESULTS |
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Northern Blot Hybridization of B-crystallin mRNA--
In
order to test whether retinoid signaling can induce endogenous
B-crystallin gene expression in lens cells, Northern blot hybridizations were performed with total RNA isolated from N/N1003A cells treated with increasing concentrations of RA (Fig.
1). The intensity of hybridization of the
labeled probe to
B-crystallin mRNA was approximately 3 times
greater in the cells treated with higher concentrations of RA (Fig. 1,
lanes 1-3). Control tests showed no increase in
glyceraldehyde-3-phosphate dehydrogenase and
-actin mRNAs after
treatment with RA (data not shown).
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Western Blot Analysis for RAR/RXR Receptors--
Nuclear extracts
from N/N1003A and TN4-1 lens cells were used to test for the
presence of RAR/RXR receptors. Western blot analysis using anti-RAR and
anti-RXR antibodies showed that both
TN4-1 and N/N1003A cells
express RAR (Fig. 2A,
lanes 3 and 4) and RXR receptors (Fig.
2B, lanes 3 and 4),
consistent with the induction of
B-crystallin gene expression by RA.
Purified RAR
(Fig. 2A, lane 2) and
RXR
(Fig. 2B, lane 1) were used as
positive controls in these tests.
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DNase I Footprinting with Lens Nuclear Extract and RAR/RXR--
We
next examined the possibility that heterodimers of retinoic acid
receptors can bind to the lens-specific sites LSR1 and LSR2. Three
retinoic acid receptor heterodimers (RAR/RAR
, RXR
/RAR
, and
RAR
/RXR
) were tested for the ability to protect the
190/+40 fragment of the
B-crystallin gene from digestion with DNase. Fig.
3 shows that three regions were protected
by each of the heterodimers tested, with the weakest footprint
generated by RXR
/RAR
. The protected regions comprised LSR1
(
132/
110), LSR2 (
73/
54), and a region between LSR1 and LSR2
(
106/
87). The LSR1 and LSR2 sequence was footprinted on both DNA
strands; however, the intervening region (
106/
87) was not
footprinted on the upper (sense) strand (data not shown).
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EMSAs Using Competitor Oligodeoxynucleotides and RAR/RXR
Antibodies--
Next we examined the binding of nuclear proteins
derived from the N/N1003A lens cells to the LSR1 and LSR2 regions of
the B-crystallin promoter. Incubation of double-stranded
oligodeoxynucleotide LSR2 (
78/
28) with the N/N1003A nuclear extract
resulted in the formation of three major complexes (Fig.
5A, lane
2, C1-3). These complexes were abolished by
competition with self-oligodeoxynucleotide LSR2 (Fig. 5A,
lane 3) and diminished with a double-stranded
oligodeoxynucleotide containing LSR1 (Fig. 5A,
lane 4). Double-stranded oligodeoxynucleotides RARE and RAR
, which both contain the consensus RARE site, competed for the formation of complexes 1 and 3 (Fig. 5A,
lanes 5 and 6, respectively). By
contrast, double-stranded oligodeoxynucleotide 9718/9719, which
contains the chicken
A-crystallin Pax-6 binding site, and
double-stranded oligodeoxynucleotide 926/927, which contains a
consensus binding sequence for Pax-6, competed for the formation of
complex 2 but not complexes 1 and 3 (Fig. 5A, lanes 7 and 8, respectively). Although
we cannot be certain, we believe that the two complexes migrating
faster than complex 3 are nonspecific, since they were not
significantly affected by competition with oligodeoxynucleotides
containing consensus RARE and Pax-6 binding sites.
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Functional Cotransfection Tests with RAR/RXR
and Pax-6
cDNA Expression Plasmids--
To test whether RAR
/RXR
receptors can activate the
B-crystallin promoter, transient
cotransfection experiments were performed using cDNA expression
plasmids in N/N1003A lens cells. Vector alone (pRSV) did not activate
the
164/+44
B-crystallin promoter fused to the cat gene
(p65-7) in cotransfected N/N1003A cells (data not shown). p65-7
contains LSR1 and LSR2. By contrast, cotransfection with a mixture of
pSV40RAR
and pRSVRXR
caused a 5-6-fold RA-dependent stimulation of CAT activity in the cells transfected with p65-7 (Fig.
7A). Cotransfection with
either pSV40RAR
or pRSVRXR
alone, however, did not stimulate the
reporter gene expression to a significant level (data not shown).
pSV40RAR
and pRSVRXR
stimulated CAT expression approximately
3-fold in cotransfection experiments using p11-3, which contains LSR2
but lacks LSR1 (Fig. 7A). The absolute amount of CAT
activity produced from p11-3 was at least 3-fold lower than that
resulting from p65-7 (data not shown). Site-specific mutations Mu-9762
and Mu-9763 that were generated previously in the
147/
118 sequence
(38) of LSR1 were used to verify that RAR
/RXR
stimulates
B-promoter activity through LSR1. Thus, the mutated promoter
constructs (Mu-9760 and Mu-9761) were compared with the wild-type
construct (p65-7) for their ability to direct expression of the
cat gene in N/N1003A cells cotransfected with pSV40RAR
and pRSVRXR
. The
B-crystallin promoters containing the Mu-9762
and Mu-9763 mutations were only about half as responsive as the wild
type promoter in p65-7 to stimulation by pSV40RAR
and pRSVRXR
in
the cotransfected cells (Fig. 7B).
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DISCUSSION |
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We have shown previously in transgenic mice using reporter
transgenes that the high lens and lower nonlens expression of the mouse
B-crystallin gene are developmentally controlled at the transcriptional level (36, 37). Lens-specific expression has been
localized to LSR1 (
147/
118) and LSR2 (
78/
46), with the minimal
lens-specific
B-crystallin promoter fragment identified being the
115/+44 sequence (38, 39). The combined presence of LSR1 and LSR2 in
the
164/+44 promoter fragment is approximately 30 times more active
in the lens than is LSR2 alone in the minimal promoter fragment in
transgenic mice (39). Promoter activity is augmented approximately
7-fold in the transgenic mouse lens when the
426/+44 promoter
fragment is used, which includes an enhancer at positions
426/
259
(36, 38). Thus, high lens activity of the mouse
B-crystallin gene
depends on coupling multiple cis-control elements and their
cognate transcription factors.
We have shown earlier and confirm here that Pax-6 can activate the
B-crystallin promoter via both LSR1 and LSR2 in transient transfection experiments (39). Pax-6 also contributes to the lens
expression of the mouse (9) and chicken (8)
A-, the chicken
1-
(10), and the guinea pig
- (11) crystallin genes (7). In recent
transgenic mouse experiments, a mutant TATA-like sequence associated
with LSR2 preferentially reduced
B-crystallin promoter activity in
the lens in a Pax-6-independent fashion, indicating that, as expected,
multiple factors contribute to the specialized activity of the
B-crystallin promoter in the lens (50). The present investigation
shows that retinoic acid receptors, especially RAR
/RXR
heterodimers, can also bind LSR1 and LSR2 and activate the
B-crystallin promoter. The activation of the
B-crystallin
promoter by the simultaneous presence of RAR
/RXR
and Pax-6 is
additive rather than synergistic and leaves unresolved whether or not
these factors physically interact with each other or through
co-factors. Indeed, the mechanism of gene activation by retinoid
receptors has not been established and involves chromatin alterations
as well as direct interactions with DNA sequences (51). It remains to
be determined if retinoic acid receptors play a role in the nonlens or
stress-induction of the
B-crystallin/small heat shock protein
(35).
Retinoic acid receptors are members of the superfamily of nuclear
factors (thryroid hormone, steroid hormone, and vitamin D3 receptors)
and are involved in a wide array of developmental processes (52-56).
The importance of retinoid signaling for eye development in mice has
been established by application of exogenous RA (57) and by deleting
various combinations of RAR and RXR genes (18, 19, 58, 59). The
existence of retinoic acid receptors in cultured lens cells was shown
in our Western blots in the present experiments. A role of retinoid
signaling for lens differentiation is implied by the generation of
abnormal lens phenotypes by ectopic expression of cellular RA-binding
protein 1 (15) and RAR (60) and by the expression of reporter genes driven by RAREs of the RAR gene in the presumptive (61) and developing (20) lens of transgenic mice. It has also been demonstrated that a minimal promoter-lacZ reporter gene fused to the RARE
from the human RAR
-2 gene is expressed in the zebrafish as early as embryonic day 9.5 in specific embryonic regions including the optic cup
(62). Thus, retinoic acid receptors and Pax-6 are both examples of
general factors that play essential roles in the early development of
the lens as well as in the regulated expression of crystallin genes,
which encode the major proteins of the terminally differentiated lens
(1). This is consistent with the idea that one of the selective
mechanisms used for recruiting the multifunctional crystallins is their
responsiveness to transcription factors required for the development
and maintenance of the transparent lens (5, 6, 63).
So far our data show only that RXR and at least one of the RARs
(
,
, or
) are present in the N/N1003A and
TN4-1 lens cells. With respect to the intact lens, a broad complex forms with the
LSR2 oligodeoxynucleotide and lens nuclear extract; however, this
complex was unaffected by the addition of the set of RAR and RXR
antibodies used in the experiments with the cultured cells (data not
shown). Because of the overlap between the Pax-6 and RAR/RXR binding
sites, it is possible that Pax-6, RAR/RXR, and other factors present in
the lens nuclei bind simultaneously at LSR2 and leave the
antibody-interactive sites for the retinoic acid receptors unavailable.
In any case, further experiments are necessary to establish
unequivocally which retinoic acid receptors may be involved in the
activation of the
B-crystallin gene in the cultured lens cells and
in the intact lens.
The present results add to previous experiments indicating that RA and
its receptors play a critical role in the regulation of crystallin
genes in the lens. RA has been shown to activate the 1-crystallin
gene in stably transformed mouse teratocarcinoma stem cells (64) and in
cultured lens epithelial cells from newly hatched chickens (17). Recent
cotransfection experiments using reporter genes in recombinant plasmids
have provided more direct evidence implicating retinoic acid receptors
in the control of the chicken
1-crystallin gene (14). Unlike the
1-crystallin promoter/enhancer, the
2-crystallin
promoter/enhancer is not stimulated by RAR
in the cotransfected
primary lens epithelial cells. This differential responsiveness is
particularly interesting, since
2-crystallin, an active
argininosuccinate lysase, is present at a relatively low concentration
in the chicken lens, while enzymatically inactive
1-crystallin is
the major
-crystallin in the lens (3, 65).
Extensive experiments have demonstrated that the mouse F-crystallin
gene is controlled by retinoid signaling. It was first shown that
RAR/RXR heterodimers bind to a novel everted RARE (called
F-HRE)
consisting of two half-sites separated by eight base pairs in the
5'-flanking sequence (66). The regulation of the
F-crystallin gene
by retinoic acid receptors appears very complex, inasmuch as
F-HRE
is activated by T3R/RXR as well as RAR/RXR, yet is
repressed by T3R/RAR
(13). There is also an RAR-related
orphan receptor, ROR
1, that is expressed in the mouse lens and binds
as a monomer to the
F-HRE 3'-half site and spacer sequences (66).
ROR
1 stimulates
F-crystallin promoter activity in transfected
primary chicken lens epithelial cells. Moreover, ROR
1 occupancy and
promoter activation are blocked by competing RAR/RXR heterodimers in
the absence of RA; the blockage of ROR
1 activation of
F-HRE by
RAR
is dose-dependent and similar to the repression of
the T3 response from
F-HRE reporter plasmids. The
novelty of
F-HRE (12) and the involvement of ROR
1, which does not
compete for binding to
-RARE or TRE (66), raises the possibility
that the stimulation of the
F- and
B-crystallin promoters
operates by different pathways.
Many invertebrates have complex eyes with cellular lenses containing abundant crystallins (67). Virtually nothing is known about the developmental pathways controlling the development of these lens-containing invertebrate eyes or about the regulatory mechanisms used for expressing their crystallin genes. Recent experiments have raised the possibility that retinoid signaling may extend to crystallin gene expression in invertebrates. Two novel lens crystallin genes (J1A- and J1B-crystallin) cloned from the cubomedusan jellyfish (Tripedalia cystophora) (68) have RARE half-sites in their promoter regions, and these sequences bind a cloned RXR homologue derived from the same species.2 If a causal connection can be made between retinoic acid receptors and J-crystallin gene expression, it would provide strong evidence that retinoid signaling is a conserved pathway for crystallin gene expression throughout the animal kingdom.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. M. Busslinger (IMP, Vienna, Austria) for the Pax-6 expression plasmid, to Drs. K. Ozato and J. Blanco (NICHD, National Institutes of Health, Bethesda, MD) for RAR and RXR expression plasmids and recombinant proteins, and Drs. M. Gaub and P. Chambon (Centre National de la Recherche Scientifique) for the RAR and RXR antisera.
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FOOTNOTES |
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* 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.
Present address: National Cancer Institute, EPN, Room 609, 6130 Executive Blvd., Rockville, MD 20852.
§ To whom correspondence should be addressed: Bldg. 6, Rm. 201, National Institutes of Health, Bethesda, MD 20892-2730. Tel.: 301-496-9467; Fax: 301-402-0781; E-mail: joram{at}helix.nih.gov.
1 The abbreviations used are: RA, retinoic acid; EMSA, electrophoretic mobility shift assay; RAR, retinoic acid receptor; RXR, retinoid X receptor; RARE, retinoic acid-responsive element; CAT, chloramphenicol acetyltransferase.
2 Z. Kostrouch, M. Kostrouchova, W. Lowe, E. Jannini, J. Piatigorsky, and J. E. Rall, manuscript in preparation.
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REFERENCES |
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