Department of Molecular Genetics (D.J.H., P.L.N., H.V.)
Novo Nordisk A/S Novo Allè, DK-2880
Bagsvaerd,
Denmark
ZymoGenetics Inc. (J.H.) Seattle, Washington
98102
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ABSTRACT |
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INTRODUCTION |
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The estrogen receptor-related receptors, ERR and -ß, are two group
3 orphan receptors, which were initially identified in human testis,
kidney, and cardiac cDNA libraries by screening with probes derived
from the DNA-binding domain (DBD) of human ER
(7). Although these
receptors are related to ER at the amino acid level, functionally they
are quite distinct. ER
and ß bind as homodimers to estrogen
response elements (ERE), consisting of an inverted repeat of the common
NR hexameric half-sites separated by three nucleotides, AGGTCANNNTGACCT
(where N is any nucleotide) (2, 5). In contrast to the ERs, ERR
and
-ß can bind to DNA as monomers to an extended NR half-site,
TCAAGGTCA (half-site in bold) (8, 9, 10). This
sequence is also the response element for group 3 orphan receptors of
the SF-1/Ftz-F1 subfamily (2, 5).
Although discovered 10 yr ago, investigation of the transcriptional
activity of the ERR family of receptors is still ongoing. Until
recently, ERR and -ß were thought to activate transcription
constitutively (in the absence of ligand). However, two recent reports
suggest that ERR
may indeed be a ligand-activated receptor. Yang and
Chen (11) have shown that two organochlorine pesticides, toxaphene and
chlordane, appear to act as antagonists for ERR
activation in HepG2
cells, whereas these compounds are weak ER
agonists. Also,
activation by both ERR
and -ß may be regulated by an agonist found
in the serum which is removable by treatment with charcoal (12, 13). In
certain instances ERR
may also act as a repressor of transcription
as it inhibits expression from the SV40 major late promoter during the
switch from early to late gene expression (14). ERRß has been shown
to specifically repress transcription induced by the glucocorticoid
receptor (GR) in CV-1 but not HeLa cells perhaps via titration of
cofactors required for activation by GR (15).
The expression patterns of ERR and ß are quite different. Mouse
ERR
is highly expressed in adult heart, brain, skeletal muscle,
kidney, and testis and more weakly expressed in liver, lung, and vagina
(7, 16, 17, 18). ERR
is also known to be expressed in osteoblasts during
bone development (16) and in the uterus (endometrium carcinoma cell
lines) (8). During mouse development, ERR
mRNA can be detected in
embryonic stem (ES) cells, and by its expression pattern it has been
implicated in the development of heart and skeletal muscle, central and
peripheral nervous system, the epidermis, and the epithelium of the
intestine and urogenital tract (17). In contrast to ERR
, ERRß
expression is barely detectable in a few tissues of the adult rat
including kidney, heart, testis, brain, and prostate (7), whereas in
the mouse it is weakly expressed in adult kidney and heart (19). In the
mouse, ERRß has been shown to be involved in early placental
development and its expression appears to be restricted to a subset of
extraembryonic tissues in a small window between 5.5 days post coitum
(d.p.c.) and 8.5 d.p.c. (19, 20). In agreement with this
observation, homozygous ERRß-/- knockout
mouse embryos die by 9.5 d.p.c. due to abnormal chorion formation
(20). Therefore it appears that both members of the ERR subfamily of
receptors play important roles during development and that ERR
is
also involved in gene expression in a number of adult tissues.
In this work we describe the molecular cloning and characterization of
a human orphan receptor, ERR, which has 77% overall sequence
identity to its closest relative, ERRß. In contrast to ERRß, ERR
is highly expressed in a number of adult human tissues including brain,
skeletal muscle, heart, kidney, and retina. This receptor is also
expressed in several human fetal tissues including placenta, brain,
heart, skeletal muscle, kidney, and lung. These findings suggest a
possible role for ERR
in the differentiation and maintenance of
these tissues. Interestingly, there are multiple tissue-specific
alternatively spliced forms of the ERR
mRNA, some of which give rise
to different protein isoforms. Full-length ERR
binds as a monomer to
an extended NR hexamer half-site (TCAAGGTCA) in vitro,
suggesting potential redundancy of function with ERR
in tissues
where both receptors are expressed. The ligand-binding domain of
ERR
, when fused to the GAL4 DBD, activates transcription in a
constitutive manner, and this activity is not affected by estradiol.
Furthermore the activation potential of the GAL4-ERR
is much greater
than GAL4-ERR
or -ß in transfected HEK293 cells.
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RESULTS |
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To confirm the sequence of the existence of the various ERR
transcripts, oligonucleotides specific for the three different
5'-RACE-PCR clones (Fig. 1A
) and an oligonucleotide derived from the
3'-RACE-PCR clone (primer 5, see Fig. 1B
) were used to amplify the
full-length coding sequence from the appropriate tissues: ERR
2 was
amplified from a placenta library; ERR
1 from kidney cDNA, and
ERR
3 from a skeletal muscle library. The PCR products were cloned
into an expression vector giving rise to constructs pERR
1, -2, and
-3. From these sequences, predictions of the amino acid sequence and
size of the various protein isoforms were made. The ERR
2 isoform is
458 amino acids long and has a predicted molecular mass of 51.3
kDa. The ORF in the ERR
1 clone gives rise to a 435-amino acid
protein with a predicted molecular mass of 48.7 kDa. If the putative
GUG start codon present in the muscle-derived ERR
3 isoform is used,
the resulting protein would be 469 amino acids in length and have a
predicted molecular mass of 52.6 kDa. If this alternative start site is
not recognized, translation of this mRNA should start at the first
downstream AUG codon, which is the same as that in the pERR
1 clone
(see below).
Comparison of the predicted amino acid sequence of ERR with related
NRs revealed highest homology with the human ERRß receptor (Fig. 1C
).
The DNA binding domain (domain C) of ERR
is 99% identical to that
of ERRß and 93% identical to that of ERR
. The ligand-binding
domain (domain E/F) is less conserved between ERRß and
, only 77%
identical, and the hinge region (domain D) is 70% identical. The
N-terminal A/B domain is 59% identical between ERR
1 and ERRß.
There is no significant homology between the putative A/B domains
present in ERR
clones and other NRs. A number of differences exist
between the ERR
sequence of Eudy et al. (22) and the one
reported here. These include 10 base changes within the coding
sequence, 6 of which alter the amino acid sequence of ERR
1.
Sequencing and comparison of all independently derived RACE-PCR and
full-length clones (PCR amplified by Pfu polymerase) have
confirmed the validity of the coding region sequence presented here.
The sequence of the ERR
2 clone presented here is also 100%
identical to the amino acid sequence for ERR
2 described recently by
Chen et al. (21).
ERR Isoforms Are Expressed in a Tissue-Specific Manner in Adult
Tissues and during Development
Northern blot analysis of the expression pattern of ERR in
adult human tissues (MTN blots, CLONTECH Laboratories, Inc.) was performed using a probe derived from the N-terminal
region of ERR
2 but extending to the beginning of the DNA binding
domain (nucleotides 166569), therefore containing sequences common to
all spliced forms so far identified. Autoradiograms revealed that
ERR
is encoded by an mRNA of approximately 5.56.5 kb. ERR
mRNA
is highly expressed in heart and skeletal muscle, kidney, pancreas,
placenta, and brain. Weak expression is also seen in prostate, spleen,
testis, and small intestine (Fig. 2
).
This Northern blotting experiment was repeated with a second batch of
MTN blots with identical results (data not shown). To further confirm
our Northern results and analyze the expression patterns of the
different ERR
mRNAs, panels of human cDNA libraries (CLONTECH Laboratories, Inc.), corresponding to the Northern blots, were
screened by PCR using primers specific for the ligand-binding domain of
ERR
and primers designed to specifically amplify the ERR
1, -2, or
-3 cDNA (see Fig. 1
). A retina cDNA library (CLONTECH Laboratories, Inc.) was also included due to the fact that EST
W26275 originated from this tissue. Amplification of the ligand-binding
domain (LBD) of ERR
from multitissue cDNA panels using primers 2 and
4 (Fig. 1B
) indicates a pattern of expression very similar to that
observed on the Northern blots (compare Fig. 2
and Fig. 3A
). Also there appears to be strong
ERR
expression in the retina (lane 17). However, the signal observed
in muscle by PCR is rather weak compared with the Northern blot. This
may reflect the amount of muscle mRNA loaded on the Northern blot (Fig. 2
, compare the ß-actin signal in lane 7 to other lanes). Also, in
comparison to the Northern blot, there is only a weak amplification of
the ERR
LBD from small intestine cDNA, which may indicate a further
spliced form in this tissue (see Discussion). Interestingly,
this method indicates weak ERR
expression in lung, thymus, ovary,
and colon where no expression was observed in Northern blots.
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Analysis of ERR expression in human fetal tissues by PCR of the LBD
revealed that, reflecting the pattern seen in adult tissues, ERR
is
highly expressed in the fetal brain, kidney, heart, and skeletal muscle
(Fig. 3B
). Weak ERR
expression also seems to be present in spleen,
thymus, and lung whereas there is no detectable expression in these
tissues in the adult. Interestingly, the three ERR
isoform mRNAs are
also restricted to expression in specific tissues during development.
The ERR
2 signal is strong in heart but is also detectable in a
number of other fetal tissues including brain, kidney, lung, skeletal
muscle, and liver. ERR
1 appears to be most highly expressed in fetal
kidney and is also weakly expressed in skeletal muscle. As in the
adult, ERR
3 expression appears to be completely restricted to the
skeletal muscle (Fig. 3B
). Therefore, ERR
is highly expressed in
some tissues during development and as in the adult the three
different ERR
mRNAs demonstrate a tissue- restricted expression
pattern somewhat different from that seen in adult tissues.
In Vitro Translation of the ERR Transcripts
To determine whether the various ERR clones code for proteins
of the expected size, the cDNAs were translated in vitro in
the presence of 35S-labeled methionine and
products separated on a denaturing 412% gradient gel. The most
abundant protein species produced from the pERR
2 clone is migrating
at approximately 51 kDa, indicating that the first AUG in the ORF is
used preferentially as the translation start site in this mRNA (Fig. 4A
, band 1). The protein arising from the
ERR
1 clone is migrating just above the 46 kDa marker, suggesting
that the predicted molecular mass of 48.7 kDa is correct for this
isoform (band 2). The ERR
2 clone gives rise to a similar protein
band, suggesting some read-through of the upstream AUG in the
reticulocyte lysate. The majority of protein produced from the pERR
3
construct also migrates at approximately 48 kDa (band 2), suggesting
that the translation start site is not at the putative alternative
start codon located upstream of this GUG. Another labeled polypeptide
arising from the ERR
3 clone runs at a molecular weight higher than
expected if the GUG codon had been used (band 3); therefore the
identity of this polypeptide is unclear. Interestingly, in comparison
to the 48 kDa band produced by the ERR
1 clone, the polypeptide
migrating at 48 kDa arising from ERR
3 appears more abundant, giving
a thicker more diffuse band. It seems for the ERR
3 mRNA that the
majority of translation initiates at the first AUG but that the
sequence upstream of this site has an influence on the efficiency of
translation. Finally, a polypeptide of approximately 44 kDa is produced
by all clones (band 4). This corresponds to the polypeptide expected if
translation began at the next downstream AUG, 72 residues downstream of
the ERR
2 start site (Fig. 1B
), which is in a better Kozak sequence
environment than the AUG at codon position 24 in the ERR
2 sequence
(see Fig. 1B
).
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The ERR Ligand-Binding Domain Has a Transcription Activation
Function
Both ERR and -ß have been found to activate transcription
constitutively, in the absence of ligand, and their activity is
unmodified by estrogen. To test whether this is also true for ERR
,
the LBD (amino acids 189458, Fig. 1B
) was fused to the DBD of GAL4
(amino acids 1147). The ability of this fusion protein to activate
transcription of a reporter construct containing GAL4 binding sites was
determined in transiently transfected HEK293 cells (Fig. 5A
). The results indicate that, as
expected, the GAL4-ERR
fusion protein activates transcription more
than 50-fold over the signal with the GAL4 DBD alone. This activity is
not significantly altered in the presence of 5 x
10-7 M ß-estradiol (Fig. 5A
). A
control construct in which the GAL4 DBD is fused to the ER
LBD
demonstrates the activity of ß-estradiol in this system as
transcription activation by this fusion protein was strongly induced
upon addition of ß-estradiol (Fig. 5A
). Some squelching of
transcription is observed when 1 µg of the GAL4-ER
construct is
used. Note that in the presence of ligand, activation by the GAL4-ER
construct is more than 5-fold greater than that seen with the
GAL4-ERR
construct.
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DISCUSSION |
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Northern blots indicate that ERR is highly expressed in several
adult human tissues including heart, brain, placenta, skeletal muscle,
kidney, pancreas, and retina. ERR
expression is also observed in
prostate, testis, spleen, and small intestine. RT-PCR with ERR
LBD-specific primers also indicates weak ERR
expression in lung,
thymus, ovary, and colon, which were all negative in Northern blot
experiments even after long exposure (data not shown). PCR with primers
specific for the ERR
1 and 2 mRNAs supports the observation of weak
expression of ERR
in colon and ovary but not in lung and thymus,
perhaps suggesting the existence of further mRNAs coding for ERR
in
these tissues. Our Northern blot results differ from those published by
Eudy et al. (22), who reported high expression of human
(h)ERR
in lung where we see nothing on the Northern and only a trace
signal by RT-PCR with the LBD-specific primers. Further, these authors
found no expression of hERR
in skeletal muscle or kidney, whereas
our Northern and RT-PCR data indicate relatively high expression in
these tissues. The pattern of expression of hERR
also differs
somewhat from that of the mouse (m)ERR
as seen in a paper that
appeared while this manuscript was being revised (27). While these
authors also detected high mERR
expression in the mouse kidney using
a probe for the N-terminal region of the mRNA corresponding to the
hERR
2 transcript, it appears that, in contrast to humans, ERR
mRNA is not present in mouse skeletal muscle but is present in liver.
These differences may be explained in part by the large number of
tissue-specific alternative splicing events observed for the 5'-end of
the ERR
mRNA in human tissues. If the same is true in mouse, the
probe used by Hong et al. (27) may not detect other spliced
forms of the mRNA.
The ERR family of orphan NRs appear to be important transcription
factors involved in the differentiation of specific tissues during
mouse development. In the case of ERRß, this has been demonstrated by
production of a knockout mouse, which in homozygous mutants results in
lethality at 9.5 d.p.c. due to a defect in the development of
extraembryonic tissues (20). This phenotype correlates exactly with the
restricted expression pattern of ERRß in the developing embryo (19, 20). For ERR, a role in development is suggested by its highly
restricted expression pattern during mouse embryogenesis (16, 17). We
report here that ERR
is also expressed in a tissue-restricted manner
during development in humans, suggesting that this receptor may be an
important factor in the differentiation of tissues such as cardiac and
skeletal muscle, brain, liver, and kidney. Intriguingly, the different
ERR
mRNA species are also expressed in a tissue-specific manner
during development. As in the adult, ERR
2 is expressed in the fetal
heart and brain but is also expressed in kidney, muscle, lung, and
liver. ERR
1 is most highly expressed in the fetal kidney but is also
present in brain, muscle, lung, and spleen cDNA libraries. Like in the
adult, the ERR
3 mRNA is restricted to the developing skeletal
muscle. Extrapolating from studies of ERR
expression in the rat and
mouse (7, 17, 18), it appears that high expression of human ERR
and
-
could overlap in several tissues including heart, brain, skeletal
muscle, and kidney. Since results presented here demonstrate that,
similarly to ERR
and ß, ERR
binds as a monomer to an extended
hormone response element half-site of the ERRE type (TCAAGGTCA), this
suggests a possible functional redundancy of ERR
and
in these
tissues, and future gene knockout experiments should take this into
account.
A GAL4-ERR fusion protein increases reporter gene transcription up
to 60-fold over GAL4 alone, and this activity is unaffected by
ß-estradiol, indicating that the LBD of ERR
contains a
transcriptional activation domain that is unaffected by estrogen, as
reported previously for ERR
and -ß (13). The level of activation
by the GAL4-ER
construct in the presence of ß- estradiol is
more than 5-fold greater than that of ERR
. While ERR
may simply
have a weaker activation potential as compared with ER
, this result
may also indicate either that the full-length ERR
receptor is
required or that binding to a ligand is necessary to achieve full
activation. However, in comparison to other members of the ERR
subfamily of receptors, the LBD of ERR
appears to have the strongest
activation potential. Activation by the ERR
LBD is 10-fold over
background, which corresponds well to previous observations (12, 13),
but is 5- to 6-fold lower than the ERR
signal. In contrast to ERR
and -
, no detectable activation was observed with GAL4-ERRß
construct. Although full-length ERRß receptor has been shown to
result in activation levels similar to full-length ERR
in CV-1 cells
(13), transcription activation by GAL4-ERRß fusion proteins has not
been previously reported. The results presented here may indicate that
the ERRß LBD alone is insufficient for activation.
A 10- to 12-fold activation by the full-length mouse ERR receptor
was reported recently using a reporter construct containing two
estrogen response elements (ERE) in CV-1 cells (27). In our hands
full-length hERR
1 and
2 receptors showed only a 2-fold increase
over background expression in cotransfection with a reporter construct
containing a single WT ERRE (results not shown). In these experiments
background luciferase expression was 6-fold higher for reporter
constructs containing a single WT ERRE compared with those containing a
mutant ERRE (data not shown). Therefore, further work is necessary to
elucidate the functional properties of the ERR
1 and
2 isoforms
and their ability to activate transcription from an ERRE in
vivo.
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MATERIALS AND METHODS |
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Database Mining and Molecular Cloning of ERR cDNAs
The EST databases were screened by BLAST searches (29) for
sequences similar to the known NRs. An EST (GenEMBL Accession no.
W26275) was identified and RACE-PCR was used to amplify the 5'- and
3'-ends of the corresponding cDNA using Marathon-Ready cDNA libraries
(CLONTECH Laboratories, Inc.) following the manufacturers
recommended protocol. Fragments were cloned into the vector pCRII by
Topo-TA cloning (Invitrogen, San Diego, CA) and analyzed
by DNA sequencing. Sequences of RACE-PCR fragments containing the
original EST sequence were used to design oligonucleotides for PCR
amplification of the full- length sequence from appropriate cDNA
libraries by Pfu DNA polymerase (Stratagene, La
Jolla, CA). Full-length ERR PCR products were cloned into the
mammalian expression vector pcDNA3.1/V5/His by TA cloning
(Invitrogen).
Analysis of Gene Expression Patterns by Northern Blotting and
RT-PCR
Northern blots of Poly-A+ mRNA derived from a variety of human
tissues (MTN blots, CLONTECH Laboratories, Inc.) were
probed according to the manufacturers instructions. A PCR-amplified
fragment derived from the putative N-terminal A/B domain of ERR2
(corresponding to nucleotides 143546 of the full-length sequence; see
Fig. 1A
) was labeled with [
-32P]-dCTP by
random priming using the Prime-a-Gene kit (Promega Corp.,
Madison, WI). Briefly, the blots were prehybridized for 30 min at 68 C
and then hybridized to the radioactive probe for 2 h at 68 C in
Expresshyb hybridization solution (CLONTECH Laboratories, Inc.). Stringent washes were performed at 50 C in 0.1x SSC,
0.1% SDS. Blots were exposed either overnight to a PhosphorImager
screen (Molecular Dynamics, Inc.) or to x-ray film at -80
C using an intensifying screen for the appropriate length of time.
PCR was performed on multitissue cDNA panels (CLONTECH Laboratories, Inc.) according to the manufacturers instructions.
PCR conditions were 94 C, 30 sec; 55 C, 30 sec; 72 C, 2 min for 35
cycles. For analysis of tissue-specific expression of the different
ERR isoforms, a downstream primer localized in the ligand binding
domain (primer 3 for ERR
1 and 2 and primer 5 for ERR
-3; see Fig. 1B
) was combined with primers specific for each of the different
5'-sequences identified (Fig. 1A
). The ligand-binding domain was also
PCR amplified (primers 2 and 4; Fig. 1B
) as this portion of the
molecule should be present in all mRNAs. Glyceraldehyde-3-phosphate
dehydrogenase (G3PDH) cDNA was amplified as a control using primers
supplied by the manufacturer (CLONTECH Laboratories, Inc.).
In Vitro Translation and Electrophoretic Mobility
Shift Assays
ERR isoforms were synthesized in vitro in rabbit
reticulocyte lysates using the T7 transcription-coupled translation
system (Promega Corp.) in the presence of
35S-methionine (Amersham Pharmacia Biotech) following the manufacturers instructions. Proteins were
separated by denaturing 412% NuPAGE gradient gels using the
MOPS (3-[N-morpho- lino]propanesulfonic acid)
buffer system (Novex, San Diego, CA) and the gel was dried
and exposed to a PhosphorImager screen or x-ray film.
For the EMSA assays, ERR2 protein was synthesized as above in the
absence of 35S-methionine. Double-stranded (ds)
oligonucleotides containing an extended half-site sequence or ERRE
(5'-CCGGGGCTTTCAAGGTCATATGCA-3') or a mutation within this
sequence (5'-CCGGGGCTTTCAAccTCATATGCA-3') (8) were labeled
with [
-32P]-dCTP by filling in the
XmaI and BglII sites at the 5'- and 3'-ends of
the ds oligos using the Klenow fragment of DNA polymerase I. DNA
binding was performed as described by Kliewer et al. (30).
Protein-DNA complexes were separated by electrophoresis through a 6%
DNA retardation gel (Novex) at 4 C using 0.5xTBE as a
buffer. The gel was dried and exposed to a PhosphorImager screen
(Molecular Dynamics, Inc., Sunnyvale, CA) for 30 min.
GAL4 Fusions and Transient Transfection Assays
The DEF regions of ERR (amino acids 189458 of ERR
2; see
Fig. 1B
), human ER
[amino acids 263595 (31)], human ERR
[amino acids 237521 (7)], and human ERRß [amino acids 164438
(21)] were PCR amplified using Pfu DNA polymerase such that
a BamHI site was engineered into the 5'-end of the PCR
products. PCR products were digested with BamHI and fused
in-frame downstream of the DBD of the yeast GAL4 transcription factor
[amino acids 1147 (32)] which was cloned previously into the
HindIII and BamHI sites of the vector pcDNA3.1(+)
(Invitrogen). These constructs, along with a reporter
construct containing five GAL4 binding sites upstream of the firefly
luciferase gene in the pGL2-Promoter vector (Promega Corp.), were cotransfected into HEK293 cells plated in 96-well
plates using FuGene transfection reagent (Roche Molecular Biochemicals). After 24 h the transfected cells were
treated with 5 x 10x7 M 17ß-estradiol
or ethanol vehicle and the cells incubated for a further 1624 h.
Luciferase activity was measured in a luminometer (Packard Instruments,
Meriden, CT) using the FireLite detection system (Packard).
Results were normalized by comparison to the RenLite signal from the
cotransfected pRL-CMV construct (Promega Corp.). The
amount of DNA was kept constant in each transfection by the addition of
empty vector (pcDNA3.1).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Current Address: Exonhit Therapeutics SA, 65 Boulevard Masséna,
F-75013 Paris, France.
P.L.N. was supported by the European Commission DG XII Biomed 2 Grant PL962433.
Received for publication April 12, 1999. Revision received November 8, 1999. Accepted for publication December 1, 1999.
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REFERENCES |
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