From the Departments of Microbiology and
§ Biochemistry, and Center for Microbial Pathogenesis,
School of Medicine and Biomedical Sciences, State University of New
York, Buffalo, New York 14214
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ABSTRACT |
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Schistosoma mansoni, a multicelluar
eukaryotic blood fluke, is a major cause of morbidity worldwide in
humans. The study of female parasite growth, development, and gene
regulation is important because the eggs produced are responsible for
the pathogenesis observed in schistosomiasis. p14, an
eggshell precursor gene expressed only in sexually mature females in
response to a male stimulus, is a model for female-specific gene
regulation. The upstream region of the p14 gene shares
sequences present in insect genes known to be regulated in a sex-,
temporal-, and tissue-specific manner by members of the steroid
receptor superfamily. Herein, we report the identification and
characterization of a cDNA that encodes the S. mansoni
(Sm) RXR homologue. Sequence analysis predicts and Western
blot analysis confirms the synthesis of a 74-kDa protein, the largest
member of the RXR family reported to date. We show by electrophoretic
mobility shift assay analysis that SmRXR binds to
cis-elements of the p14 gene including a
direct repeat that follows the "3-4-5" rule of binding elements
recognized by members of the steroid receptor superfamily. Furthermore,
we demonstrate that SmRXR can act as a transcription activator in the
yeast one-hybrid system. Through quantitative reverse
transcriptase-polymerase chain reaction, we show that the
SmRXR gene is constitutively expressed and thus must play
multiple roles throughout the schistosome life cycle.
Schistosomiasis is a major public health problem afflicting 300 million people in 76 countries (1). Egg production is not only
responsible for dissemination of the parasite but also for pathology of
the disease in humans. Therefore, studies of female schistosome
reproductive development and egg production may define targets useful
in disease control. Interestingly, female reproductive development is
regulated intimately by a stimulus from the male parasite (2). For many
years it has been known that female schistosomes from single sex
infections are stunted in size and sexually immature (2). This
observation indicates that schistosomes have an intriguing
developmental system requiring signaling from the male schistosome
leading to direct or indirect activation of a number of female-specific
genes (2-6). One gene, p14, encodes an eggshell precursor
expressed only in vitelline cells of mature females in response to
male stimulus (3, 7, 8). The region upstream of transcription
initiation of p14 contains elements similar to those found
in Drosophila and silkmoths that are known to regulate
chorion gene expression in a sex-, tissue-, and
temporal-specific manner (2, 7, 9, 10).
Ultraspiracle protein, a member of the
RXR1 subfamily of the steroid
receptor superfamily, binds to the s15 chorion gene of Drosophila recognizing an imperfect palindrome containing
the core sequence TCACGT (11, 12). Members of the steroid receptor superfamily are likely to play a role in female-specific gene regulation of schistosomes. A similar sequence is present as part of an
imperfect palindrome in the upstream region of p14 (2) (Fig.
1). Furthermore, a direct repeat that
follows the "3-4-5" rule of steroid response elements is present in
the p14 upstream region (13, 14). This latter sequence
consists of two identical half-sites, AACTATCA, spaced by five
nucleotides (Fig. 1). Presence of these cis-elements in the
p14 gene led to a search for schistosome homologues of
steroid hormone receptors. Herein, we report the identification and
characterization of Schistosoma mansoni RXR cDNA
(SmRXR), and we provide evidence for a possible role in
regulation of female-specific gene expression.
Identification of SmRXR cDNA--
Degenerate
oligonucleotides representing the conserved regions of RXR
genes with the amino acid sequences, CEGCKGFF (aa 288-295) and
GMKKEAVQEE (aa 335-344), were synthesized for use in PCR (Fig. 2). Using a cDNA pool derived from
schistosome worm pair mRNA as a template and the RXR
gene-specific primers, a 170-base pair fragment was amplified. This PCR
product was cloned into TOPO TA vector (Invitrogen), sequenced, and
shown to be related to RXRs by NCBI Blast search analysis.
The DNA fragment homologous to RXRs was radiolabeled with
[ Expression and Production of Recombinant SmRXR--
A fragment
of SmRXR encoding amino acids 200-561 was inserted into
pGEX 2T-1 to form pGEX-RXR. TOP 10 competent cells (Invitrogen) were
transformed with pGEX-RXR. Transformed cells were grown to 0.45 A600 at 37 °C and then cooled on ice for 20 min. After the addition of 0.5 mM
isopropyl-1-thio- Immunological Techniques--
Rabbit anti-RXR was purified from
serum of a rabbit immunized with recombinant GST-SmRXR by the method of
Beal and Mitchell (15). Extracts from adult worm pairs, mature male
worms, mature female worms, and 3-h schistosomules were loaded onto a
12.5% SDS-acrylamide gel in triplicate panels. One panel was stained with Coomassie Blue, and the other two panels were transferred onto
polyvinylidene difluoride membrane (Bio-Rad) for Western analysis. One
panel was probed with preimmunized rabbit serum (1:5000 dilution) and
the other panel with affinity purified rabbit anti-RXR antibody (2 µg/ml).
Quantification of SmRXR mRNA by RT-PCR--
RNA was
extracted from the following schistosome developmental stages: 45-day
worms, 44-day female (bisex infection), 44-day male (bisex infection),
44-day female (single sex infection), 44-day male (single sex
infection), 35-day worms, 32-day worms, 30-day worms, 28-day worms,
21-day, 15-day, 7-day, and 4-day schistosomule. RNA was isolated using
the RNA-STAT-60 Kit (Tel-Test) following the manufacturer's
instructions. To prepare total cDNA, 5 µg of total RNA was used
as template for each 50-µl reaction. A total reaction volume of 31 µl containing RNA, RNasin (Promega), 1 µg of oligo(dT), 0.1 µg of
random hexamers (Life Technologies, Inc.), and water were incubated for
10 min at 70 °C. After incubation, 1st strand buffer (Life
Technologies, Inc.), 0.01 M dithiothreitol, 0.4 mM dNTPs, and 400 units of Superscriptase (Life
Technologies, Inc.) were added to each reaction. After incubation at
37 °C for 1 h, 0.5 µl of reaction mix was used as template
for PCR. SmRXR-specific oligonucleotides were synthesized
for PCR: 5' AACAGATCTACCTAATTTAACATAC-OH (aa 434-441) as forward
primer and 5' GCACGTAGTAAAGCTAATTCAG-OH (aa 581-588) as reverse
primer. Construction of Yeast One-hybrid Target Reporter
Strains--
The Matchmaker One-hybrid System protocol
() was followed with a few exceptions. The
target-reporter construct pHisi-154, was made by inserting a 154-base
pair DNA fragment, delineating nucleotides Analysis of SmRXR Activity in Vivo, Yeast One-hybrid
Assay--
The competent reporter yeast strains were transformed with
either pACT-2 activation domain vector ()
containing the SmRXR gene downstream of the Gal4-AD
(pAC-RXR), pACT-2 vector, pADH1 expression vector containing the SmRXR
(pADH-RXR), or pADH1 vector. The pADH1 vector is a 2-µm expression
vector with a multi-cloning site upstream of a ADH1 promotor. Control
transformations without DNA were also performed. In a sterile 15-ml
conical tube, 10 µg of plasmid DNA, 200 µg of sheared salmon sperm
DNA, and 330 µl of component reporter strain yeast cells were added.
After mixing the DNA and yeast, 2 ml of LiAc/polyethylene glycol
solution (10 mM Tris, pH 7.5, 1 mM EDTA, pH
7.5, 0.1 M LiAc, and 40% polyethylene glycol) were added,
and the suspension was vortexed. The transformation suspension was
incubated while shaking at 200 rpm at 30 °C for 30 min. After
incubation, 230 µl of Me2SO were added and mixed by
inverting the tube gently. Cells were incubated for 15 min at 42 °C
and then placed on ice for 2 min. Cells were centrifuged at 1000 × g for 10 min, and the supernatant was aspirated. The pellet was resuspended in 2.3 ml of TE buffer. From the control tubes
that contained reporter strains lacking DNA, 10 µl of cells were
diluted 1:100 in TE and 200 µl per plate were spread onto SD/ Yeast Two-hybrid Assay--
Yeast two-hybrid assay, originally
developed by Fields and Song (17), was performed with the HybriZAP II
Two-hybrid System (Strategene) according to the directions supplied
from the manufacturer. SmRXR cDNA was ligated into the binding
domain vector, pAS2-1 () forming pAS-RXR.
Control DNA included pGal4, p53/pSV40, and pLaminC/pSV40 (Stratagene).
Yeast cells transformed with pAS-RXR, p53/pSV40, and pLaminC/pSV40 were
plated on SD/ In Vitro Transcription-Translation of
SmRXR--
SmRXR cDNA was inserted into the pCITE
vector (Novagen) forming pCITE-RXR and transcribed and translated using
the Single Tube Protein System (Novagen). The manufacturer's protocol
for [35S]methionine incorporation was followed except an
additional 1.25 mM cold methionine was added to each
50-µl reaction. Negative control reactions with circular pCITE DNA
were also performed. Samples were examined by SDS-polyacrylamide gel
electrophoresis, followed by autoradiography and then used immediately.
Analysis of SmRXR Activity in Vitro, EMSA--
A 250-bp sequence
of the p14 upstream region, nucleotides Identification of a Schistosome RXR Homologue cDNA--
A
170-base pair DNA fragment that showed homology to RXRs was amplified
using degenerate oligonucleotides representing conserved regions of RXR
genes as primers and adult schistosome cDNA as template in PCR
(Fig. 2). The sequence of the 5' primer used for PCR encodes for an
amino acid sequence found in the second zinc finger of all members of
retinoid receptors, RARs and RXRs (19-22). The sequence of the 3'
primer encodes for an amino acid sequence of the hinge region that is
identical in all identified RXRs and is not found in other members of
the steroid receptor superfamily. After screening 400,000 plaques of a
female adult worm Immunological Identification of SmRXR in Different Worm
Stages--
Using an affinity purified antibody produced to a
truncated form of recombinant GST-SmRXR, native SmRXR was identified in adult worm pair, mature male, mature female, and 3 h shistosomule extracts (Fig. 3). Native SmRXR has an
approximate molecular mass of 74 kDa as observed in the Western blot
(panel B). For a control, adult worm pair extract was probed
with preimmune rabbit serum (panel C). There are irrelevant
bands that appear on the Western blot, but none the 74-kDa size. These
bands are not present in panel B because the serum was
affinity purified.
Developmental Expression of the SmRXR Gene--
RT-PCR was
performed on total RNA isolated from various developmental stages to
evaluate developmental expression of the SmRXR gene. In
order to control for the expression of multiple schistosome RXR
homologues, primers to non-conserved regions of SmRXR were used in the
RT-PCR reactions. SmRXR Binds to the p14 Gene Upstream cis-Elements and Activates
Transcription--
To determine if SmRXR binds to
cis-elements of the p14 upstream region and drives
transcription of a female-specific gene, we employed the yeast
one-hybrid system. The reporter yeast strains, YTR-154, YTR-EL1, and
YTR-pHisi, were transformed with pAC-RXR and pAC-2. YTR-154 transformed
with salmon sperm DNA alone only grew on SD/
To define further the interaction between the p14 gene and
SmRXR-AD, YTR-EL1 that contained the direct repeat of the upstream region of p14 (Fig. 1) was used. As with the YTR-154 strain,
after transformation with pAC-RXR there were larger colonies at higher concentrations of AT. YTR-EL1 cells grew best on 45 mM AT
but did grow at 60 mM AT, at a slower rate. On the restreak
plates it only took 3 days for YTR-154 growth to appear on
SD/
Because RXR forms heterodimers in many species (42-50), we attempted
to employ the yeast two-hybrid system to obtain schistosome clones that
express proteins that interact with SmRXR. As a control, we transformed
Y190 yeast cells with pAS-RXR, the bait plasmid containing the GAL4 DNA
binding domain. SmRXR alone activated
YTR-EL1 cells were also transformed with pADH-RXR. Yeast colonies grew
on SD/ EMSA Analysis of SmRXR-DNA Interactions--
EMSA were performed
to study the in vitro interaction between p14 DNA
and SmRXR. A 250-bp sequence, nucleotides Schistosomes are the only trematodes with separate sexes. This
fact has driven the evolution of an interesting biological interplay
between the sexes, such that the male through an unknown stimulus is
able to regulate female-specific gene expression (2). A number of
female-specific genes have been isolated, but as of yet no factors
directly involved in the regulation of female-specific genes have been
identified. In this paper we report the identification of
SmRXR cDNA and provide evidence for its involvement in
female-specific gene expression.
Members of the RXR subfamily in other species are important
in the regulation of sex-, tissue-, and temporal- specific genes. For
example, USP of Drosophila is employed in the
regulation of chorion genes, embryogenesis, larval development, and
pupation (11, 12, 45). This pleiotrophy of function is representative of RXR subfamily members. RXRs are known to form
heterodimers with various partners that are essential for the
regulation of a number of genes involved in homeostasis and development
(45-50). Examples of RXR partners include the ecdysone receptor of
Drosophila, vitamin D receptor, thyroid hormone receptor,
and retinoic acid receptors (RAR) (42-50). When RXR forms
heterodimers, such as RXR/RAR and USP/ecdysone receptor, the
conformation of its binding partner changes allowing a greater affinity
not only for the DNA response element but also for the ligand (43-51).
RT-PCR results demonstrated that SmRXR mRNA is
constitutively expressed throughout schistosome development, not only
in mature females as are the female-specific genes (2, 3, 7, 9, 10).
Furthermore, Western blot analysis showed that the 74-kDa SmRXR protein
is present in both sexes, mature and immature. This demonstrates that
SmRXR is not female-specific and suggests that, like other members of
the RXR subfamily, SmRXR is important in the regulation of a number of genes. Future studies will entail identifying SmRXR-binding partners involved in the regulation of female-specific genes and defining the
putative role(s) SmRXR plays in the regulation of other genes.
To date, a genetic system to study the functions of schistosome genes
in vivo does not exist. Therefore, evidence for the involvement of SmRXR in female-specific gene regulation was obtained by
the yeast one-hybrid system and EMSA analysis. In these studies, we
showed that SmRXR binds to cis-elements of the
p14 gene and drives transcription of a reporter gene in
yeast. Members of the steroid receptor superfamily commonly bind to
cis-elements that consist of two half-sites spaced by 3, 4, or 5 nucleotides (13, 14). EMSA analysis demonstrates that the
preferential binding site for SmRXR, in vitro, is a sequence
containing the half-site AACTATCA spaced by five nucleotides (Fig. 1).
In the upstream region of s15, a Drosophila
chorion gene, the sequence TCACGT is an essential part of an imperfect
palindrome known to be a USP response element (12). Interestingly, a
similar sequence, TCACGCT, occupies the 3' end of the first half-site
and part of the spacer region of the direct repeat in the
p14 gene upstream region (Fig. 1). To define the region of
the p14 gene involved in SmRXR binding interaction and
trans-activation, the direct repeat sequence was used as a
target element in the one-hybrid assay. SmRXR most likely acts as a
homodimer to drive transcription of this chimeric reporter gene. It is
unlikely that SmRXR interacts with a yeast protein. RXRs have been
shown to form heterodimers with only members of the steroid receptor
family, and to date, steroid receptors have not been identified in
yeast (42-50).
An evolutionary study was performed to better understand the function
of SmRXR. Knowing the rate at which SmRXR has evolved as compared with
other RXRs and RARs may answer questions about how it functions in
regulating gene expression. Steroid receptors have recently been
identified in early metozoans, such as cnidarians and schistosomes
(52). Interestingly, in a nuclear receptor evolutionary study, the DNA
binding domain of a schistosome RXR homologue, which is not identical
to the SmRXR cDNA identified in our lab, was described (52). The
divergence of subfamilies of the steroid superfamily was previously
determined to have occurred before the divergence of the arthropods and
vertebrates (22, 53) (Fig. 7). From the
Escriva et al. (52) study and the phylogenetic tree designed
using full-length cDNA sequences of RXRs and RARs shown in this
report (Fig. 7), the above statement can be extended to note that the
divergence of subfamilies occurred before the divergence of cniderians
and platyhelminthes (52, 53). Because SmRXR is deeply rooted in
evolution (Fig. 7), comparison of this gene with other steroid
receptors may lead to the identification of the original ancestor of
the steroid receptor family.
INTRODUCTION
Top
Abstract
Introduction
References
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Fig. 1.
p14 upstream region. The
nucleotides in gray are similar to sequences found in insect
genes known to be important in regulation of sex-, tissue-, and
temporal-specific chorion gene expression. Underlined in
gray are cis-elements important for SmRXR
interaction as shown by the one-hybrid assay and gel shift assays. The
double underlined sequences represent half-sites of a direct
repeat or imperfect palindrome, respectively. The sequence of the probe
used in EMSA analysis extended from nucleotides 300 to
50. The
154-base pair fragment inserted into the target reporter plasmid mapped
from nucleotides
220 to
60. Underlined in
dots are consensus CAAAT and TATA boxes, respectively.
Nucleotide 1 designates the initiation site for transcription. The
start ATG codon is designated by MET (10).
EXPERIMENTAL PROCEDURES
-32P]dCTP by random priming and used as a probe to
screen a female adult worm
ZAP cDNA library. Seven positive
clones were identified, the phagemids were excised, and then the
cDNA inserts were shown to be identical in sequence. Amino acid
comparison of SmRXR to Drosophila USP and human RXR
was
performed using the Pile-Up program from the Wisconsin Package version
9. Phylogeny trees were constructed using the PAUPsearch and
PAUPdisplay program from the Wisconsin Package version 9. The following
parameters were used in construction of trees to confirm accuracy of
the study: heuristic with parsimony, branch-and-bound with parsimony, branch-and-bound with minimal evolution, and neighbor joining.
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Fig. 2.
Schematic of the functional domains of RXR
subfamily members (top). Comparison of the amino
acid sequences of SmRXR, dUSP, and human RXR (bottom).
Amino acids of SmRXR (accession number AF094759) and dUSP (accession
number P20153) that share identity to RXR
(accession number P19793)
in the highly conserved functional domains are shown in
gray. The solid box denotes the amino acids of
the DNA binding domain. Asterisks indicate the two zinc
fingers of the DNA binding domain. The conserved amino acids of the
hinge region are shown in dark gray. The amino acids
selected for the synthesis of the oligonucleotides used in PCR to clone
the 170-bp SmRXR fragment are underlined. The
amino acids of the dimerization and ligand binding domain are
boxed in dashed lines. SmRXR sub-domains,
i and
c, are located at the amino and
carboxyl terminus of the dimerization and ligand binding domain, amino
acids 457-512 and 656-676, respectively. Solid boxes
within the dimerization and ligand binding domain denote the heptad
repeats. The difference in size of SmRXR from other RXR family members
occurs at both the amino- and carboxyl-terminal ends of SmRXR by
approximately 130-170 amino acids and 50 amino acids,
respectively.
-D-galactopyranoside, cells were
incubated overnight at 20 °C. The cells were pelleted by centrifugation at 3000 × g for 20 min. Cells were
resuspended in lysis buffer (25 mM Tris, pH 8, 1 mM EDTA, 10% glycerol, 50 mM NaCl, 0.1%
sodium deoxycholate, 0.01% Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride, 10 µM leupeptin and 75 µM aprotinin), sonicated, and the supernatant fraction
collected after centrifugation at 18,000 × g for
1 h at 4 °C. The GST-SmRXR fusion protein in the S18 fraction was purified by passage over a glutathione- Sepharose column
pre-equilibrated with G-S binding buffer (25 mM Tris, pH 8, 1 mM EDTA, 50 mM NaCl, 10% glycerol).
GST-SmRXR was eluted by addition of 10 mM reduced glutathione in G-S binding buffer.
-Tubulin-specific primers (16) were used in each reaction as
a constitutively expressed gene control. To remain in the linear range
of PCR, 28 cycles were performed. The PCR products were size-separated
on a 1.5% agarose gel, visualized by ethidium bromide staining, and
quantified by the Molecular Analyst program, PC version 1.5 (Bio-Rad).
The amount of SmRXR PCR product was compared within each
reaction to
-tubulin product and then this value was compared among
the developmental stages.
220 to
60 of the
p14 gene (Fig. 1), repeated 3 times upstream of the His3
promotor in the pHISi-1 vector. Target-reporter construct, pHisi-EL1,
was made by inserting the DNA fragment, 5'
TTGACATTTTTAACTATCACGCTCAACTATCATT-OH (Fig. 1) repeated six
times upstream of the His3 promotor. After preparing competent YM4271
yeast cells as directed by , 10 µg of the
target-reporter construct was used for transformation. Yeast reporter
strains were analyzed by Southern blot to ensure that proper
integration of the target-reporter had occurred.
his
media containing 0, 15, 30, 45, and 60 mM
3-amino-1,2,4-triazole (AT, a HIS3 competitor). From each tube
containing reporter yeast strains transformed with plasmid DNA, 100 µl, were spread onto SD/
leu to test transformation efficiency. All
plasmids used contained the selection marker leu2 gene. From
each of these tubes, 200 µl per plate were spread onto SD/
his
leu
containing AT at concentrations of 0, 15, 30, 45, and 60 mM. The plates were incubated at 30 °C and checked each
day for growth. On the 7th day, selected colonies were restreaked on
SD/
his
leu plates containing AT.
trp plates. Yeast transformed with pGal4 were spread on
SD/
leu plates. After 1 week incubation at 30 °C, colonies were
assayed for
-galactosidase activity using the
protocol.
300 to
50 (Fig.
1), was radiolabeled by PCR amplification of the fragment using
[32P]dCTP. Every 20-µl reaction contained
ZnSO4 buffer (4 mM Tris, 80 mM
NaCl, 0.5 mM ZnSO4, 5% glycerol, 0.5 mM dithiothreitol, and 1 mM EDTA), 5 µg of
poly(dI·dC), 12% glycerol, and 5 fmol of radiolabeled p14
template (18). Protein-DNA binding reactions contained 1 µl of
transcription-translation lysate. In cold template competition
reactions, unlabeled p14 template (250-bp fragment) was
added in 10 and 100 × molar excess of the radiolabeled
p14 probe. In competition reactions with nonspecific DNA, a
250-bp fragment cut form pET32a DNA (Novagen), was added in 10, 100, and 250 × molar excess of the p14 probe. The following
DNA sequences were added in 10, 100, and 250 × molar excess for
competition reactions: 5' TTGACATTTTTAACTATCACGCTCAACTATCATT-OH
(direct repeat), 5' TTTAATAATGATGCACTTAGTGAGGCACAACTCTTC-OH (imperfect
palindrome), and 5' TTCTAACGGTAGAATCAAAATAGTGAGTAATTCGTG-OH (Fig. 1).
Binding reactions were incubated at 23 °C for 30 min and then loaded
onto a 16-cm 4% acrylamide gel (6.75 mM Tris, pH 8, 1 mM EDTA pH 8, 3.3 mM sodium acetate, 0.05%
bisacrylamide). Electrophoresis was performed with a 6.75 mM Tris, pH 8, 1 mM EDTA and 3.3 mM
sodium acetate running buffer at 4 °C.
RESULTS
ZAP cDNA library with the RXR homologous DNA
fragment, seven positive clones were obtained. Restriction digest and
sequencing demonstrated that all seven clones were identical. The
nucleotide sequence of SmRXR translates into a protein of 743 amino
acids with an approximate mass of 74 kDa. SmRXR is the largest reported
RXR to date. Sizes of RXRs range from 410 amino acids for mRXR
to 508 amino acids for dUSP (11, 23, 24). Comparison of the deduced amino
acid sequence of SmRXR to other RXRs reveals that this protein exhibits
significant homology to the DNA binding domain and the dimerization
domain of other members of the RXR family (Fig. 2). For example,
comparison of the amino acids of the DNA binding domain of SmRXR (aa
271-336) to those amino acids in the same domain of hRXR
(aa
135-200) and dUSP (aa 104-169) shows identities of 69 and 68%,
respectively (11, 22, 24, 25). The first zinc finger within the DNA
binding domain of SmRXR has only four amino acid changes from that of
hRXR
(25). The second zinc finger exhibits lower homology. Within
the dimerization and ligand binding domain are heptad repeats (Fig. 2)
containing hydrophobic amino acids at positions 1, 5, and 8 (21). These heptad repeats form a dimerization motif (21). The amino acids of the
dimerization motif of SmRXR (aa 531-680) share 48 and 35% identity to
those of hRXR
(aa 325-463) and dUSP (aa 357-506), respectively
(11, 21, 24, 25). Adjacent to the heptad repeats at the amino and
carboxyl ends of the dimerization and ligand binding domain are two
sub-domains,
i and
c, respectively, known
to be important for ligand-dependent transregulation in other members of the steroid hormone receptor superfamily (21, 26-32).
The
i sub-domain, thought to function in
hormone-relieved repression, has 20-45% conservation among steroid
receptors of vertebrates and arthropods (21, 33, 34). For example,
there is 38-40% conservation of the
i domains of
vertebrate RARs and RXRs (21, 23, 25). Steroid hormone receptors of
vertebrates within each subfamily, such as RXR, have at least 98%
identity in the
i domains (21-23). Homology of the
i domain of dUSP to that of vertebrate RXR is 50% (11,
21-25). The
i domain of SmRXR (aa 457-512) shares 13%
identity to the
i domain of hRXR
(aa 277-324) and
19% identity to the
i domain of dUSP (aa 282-338) (11,
21-25). The
c sub-domain, a small amphipathic helical
structure located at the carboxyl end of the dimerization and ligand
binding domain, is conserved only among steroid hormone receptors that share the same ligand (22, 23, 25-32). For example, the
c sub- domains of mRXR
, hRXR
, and hRXR
, which
bind 9-cis- retinoic acid, are identical to one another
except for a single amino acid change of the last carboxyl terminus
amino acid (23, 25, 27, 35). The
c sub-domains of RARs,
which bind all-trans-retinoic acid, are also identical to
one another but are dissimilar to the
c sub-domains of
the vertebrate RXRs (22, 23, 25-27, 36-41). The
c
domain of dUSP is not homologous to vertebrate RXR
c
domains and does not bind to 9-cis-retinoic acid (24).
Nine-cis-retinoic acid is not likely to be the ligand of
SmRXR due to the variant sequence of the
c domain.
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Fig. 3.
Identification of SmRXR in different worm
stages by Western blot analysis. Panel A, 12.5%
acrylamide gel stained with Coomassie Blue. Lane 1, adult
worm pair extract; lane 2, mature male worm extract;
lane 3, mature female worm extract; lane 4, 3-h
schistosomule extract. Panel B, Western blot probed with
affinity purified rabbit anti-SmRXR. Lanes 1-4 contain the
same amount of schistosome extract and in the same order as panel
A. An arrow points to the 74-kDa band in the extracts
that are specific for anti-SmRXR. Panel C is a Western blot
of adult worm extract probed with preimmune rabbit serum.
-Tubulin-specific primers that represent a
constitutive gene were added to each reaction. A 465-bp SmRXR cDNA fragment and a 500-bp
-tubulin product
were amplified (Fig. 4). By comparing the
amount of SmRXR PCR product to that of
-tubulin within
each reaction and then comparing this value among the different life
stages, we demonstrate that SmRXR is constitutively expressed
throughout development (Fig. 4).
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Fig. 4.
Quantification of SmRXR
mRNA in different developmental stages. RT-PCR products
were loaded onto a 1.5% agarose gel. RNA templates for the RT-PCR were
adult worm pairs (A), male worms from a bisex infection
(M), female worms from a bisex infection (F),
immature females from a single sex infection (Fi), males
from a single sex infection (Ms), and schistosomes collected
35, 32, 30, 28, 2, 15, 7, and 4 days post-infection, respectively. The
PCR products were visualized by ethidium bromide stain. The upper
bands are PCR products amplified using -tubulin primers (16).
The lower bands are PCR products amplified using
SmRXR specific primers.
his media. This is
expected because the His3 promotor is leaky. Therefore, growth of
YTR-154 transformed with pAC-RXR on SD/
his
leu media with even low
concentrations of aminotriazole would be a result of SmRXR-AD fusion
protein binding to cis-elements of p14 and
driving transcription of the HIS3 gene. The transformed YTR-154 cells were spread on SD/
his
leu plates with increasing amounts of AT. After 7 days of incubation, two classes of colonies grew
that ranged from 0.25 to 0.5 mm in size and from 1.5 to 2.5 mm in size
(data not shown). Colonies of both sizes were picked and streaked on
SD/
his
leu plates containing up to 60 mM aminotriazole. The smaller colonies did not grow. The larger colonies grew on SD/
his
leu containing 60 mM AT (Fig.
5A). YTR-154 cells transformed with pAC-2 grew as small colonies (0.25-0.5 mm) in the original spreads. These colonies were picked and streaked on SD/
his
leu containing a range of AT concentrations. At 15 mM AT these
colonies failed to grow on SD/
his
leu (Fig. 5A). The
negative control strain YTR-pHisi, transformed with either pAC-RXR or
pAC-2, grew as small colonies in the original spread but failed to grow
when restreaked on media containing AT (data not shown). Therefore, it
is evident that SmRXR fused to the Gal4 activation domain binds to
cis-elements of p14 and drives transcription of
the HIS3 reporter gene in the one-hybrid system.
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Fig. 5.
Demonstration of SmRXR activity expressed in
yeast. Panel A, individual YTR-154 colonies transformed
with pAC-RXR (1-5) and pAC-2 (6) were streaked
on SD/ his
leu plates containing 60 mM AT. Panel
B, individual YTR-EL1 colonies transformed with pAC-RXR
(1-5) and pAC-2 (6) were restreaked on
SD/
his
leu plates containing 60 mM AT. Panel
C, restreaks of individual YTR-154 transformed with pADH1-RXR
(1-5) and pADH1 (6) on SD/
his
leu containing
60 mM AT. Panel D, YTR-EL1 transformed
with pADH-RXR (1-5) and pADH1 (6) on
selective media with 60 mM AT. Above each plate is a
cartoon showing the target element inserted upstream of the
His3 reporter gene in the yeast strains. Whether or
not SmRXR, expressed in the yeast strains, is fused to the Gal4
activation domain is also depicted in the schematics.
his
leu containing 60 mM AT, whereas it took 5-6
days for growth of YTR-EL1 to appear on plates containing 60 mM AT (Fig. 5B). Growth of YTR-EL1 appeared on
day 4 on the plates with 45 mm AT. Fig. 5B shows growth of
five individual colonies of YTR-EL1 on 60 mm AT. The negative control
included in this experiment was YTR-EL1 transformed with pAC-2 alone.
Again there were small background colonies on the original spread that
did not grow on the restreak (Fig. 5B). Therefore SmRXR-AD
binds to the sequence including the direct repeat and enhances the
transcription of the HIS3 gene.
-galactosidase expression
(data not shown). This result indicated that SmRXR by itself may
activate gene transcription, perhaps as a homodimer, or by interacting
with yeast proteins. In order to determine if SmRXR by itself, or
perhaps with the help of a yeast protein, could drive transcription
when recognizing cis-elements of p14,
SmRXR was cloned into an expression vector that lacked the
Gal4 activation domain and DNA binding domain (pADH-RXR). When YTR-154
was transformed with pADH-RXR, the cells as in the case of
transformation with pAC-RXR (above) grew on SD/
his
leu media. There
were very small colonies, 0.25-0.05 mm in size, and larger colonies,
1.5-2.5 mm in size. Again the smaller colonies did not grow after
being restreaked on SD/
his
leu plates containing AT, but the larger
colonies grew on SD/
his
leu containing 60 mM AT (Fig.
5C). Growth of these colonies appeared at day 3 on the
restreak plates containing 60 mM AT. As in the above
experiment, YTR-154 transformed with salmon sperm alone grew only on
SD/
his media without AT. Therefore, SmRXR is binding to
cis-elements of the p14 upstream region and
driving transcription of the HIS3 gene without the Gal4
activation domain. Controls of this experiment included transforming
YTR-154 with pADH1. Again small colonies grew on all the plates of the
orginal spread but did not grow on SD/
his
leu media containing
aminotriazole after the restreak (Fig. 5C). Similar to the
above experiment YTR-pHisi, transformed with both pADH-RXR and pADH1,
grew on all plates of the original streak but not after the restreak
(data not shown).
his
leu containing 60 mM AT (Fig. 5D).
Growth of these colonies on media containing 60 mM AT was
slower than YTR-154 transformed with pADH-RXR and YTR-EL1 transformed
with pAC-RXR. Growth of YTR-EL1 transformed with pADH-RXR on the
restreak plates containing 60 mM AT appeared on days 6 and
7 depending on the clone. Small colonies grew on plates that contained
YTR-EL1 transformed with pADH1, but these colonies did not grow when
restreaked on SD/
his
leu containing AT. Indeed, SmRXR binds to the
sequence containing the direct repeat (Fig. 1) and drives transcription of HIS3 (Fig. 5D).
300 to
50, of the
p14 upstream region (Fig. 1) was used as a probe in gel shift assays to determine whether or not SmRXR, synthesized by in
vitro coupled transcription/translation, binds to RXR recognition sites. Shown in lane 1 of Fig.
6 is the probe without lysate from the
transcription-translation mix. In lane 2, the binding
reaction contains lysate from a transcription-translation reaction with pCITE vector used as template. There are a few irrelevant shifts of the
probe caused by contents of the lysate binding to the p14 probe. In lane 3 it is apparent that SmRXR causes unique
shifts of the radiolabeled p14 fragment. These shifts are
denoted by the two arrows (Fig. 6). When cold p14
DNA is added in 100 × molar amount (lanes 4 and
5), the upper shift is competed, whereas the lower shift
identified by the arrow is competed to a much lesser extent.
Nonspecific DNA (lanes 6-8) does not interfere with the changes in electrophoretic mobility. Therefore, the shifts observed in
lane 3 are specific for SmRXR-p14 interaction.
The direct repeat competes the shifts to a great extent (lanes
9-11). At 250 ×, the competitor completely competes the two
shifts denoted by both arrows. The DNA fragment that includes the
sequence GTAGAATCA, competes the upper shift slightly at 250 × molar amounts (lane 14). The sequence that includes the
imperfect palindrome (lanes 15-17) competes the upper shift
at 100 × and at 250 × competes both shifts. In
vitro studies demonstrate that the direct repeat sequence of the
p14 gene is a preferential binding site for SmRXR.
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Fig. 6.
Demonstration of SmRXR DNA binding activity
by EMSA. Lane 1, DNA alone. Lane 2, lysate
from the control transcription-translation reaction. Lane 3,
lysate containing in vitro translated SmRXR. Lanes
4 and 5, binding reactions contained cold
p14 competitor in 10 and 100 × amounts, respectively.
Lanes 6-8, binding reactions contained a 250-bp fragment of
a irrelevant DNA competitor in 10, 100, and 250 × amounts,
respectively. Lanes 9-17, binding reactions contained 10, 100, and 250 × amounts of three different DNA sequences, the
direct repeat, a sequence containing GTAGAATCA, and the imperfect
palindrome sequence, respectively (Fig. 1). Arrows denote
unique shifts caused by SmRXR-p14 interaction.
DISCUSSION
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Fig. 7.
Evolutionary tree showing the phylogenetic
relationship between SmRXR and RXRs and RARs of vertebrates and
arthropods. The node numbers are in bold. The assigned
branch numbers representing evolutionary distance are in
parentheses. Starting from node one, the common name and
GenBankTM accession numbers of the protein sequences used
in the PAUPsearch analysis are as follows: mRAR , mouse, P11416;
mRAR
, mouse, P22605; hRAR
, human, P10276; hRXR
, human, P28702;
mRXR
, mouse, P28704; hRXR
, human, P19793; mRXR
, mouse, P28700;
xenRXR
, Xenopus, P51128; hRXR
, human, P48443; mRXR
,
mouse, P28705; chRXR
, chicken, P28701; xenRXR
,
Xenopus, P51129; cf1bRXR, silkworm, P49700; manseUSP,
tobacco hornworm, P54779; dUSP, Drosophila, P20153; mHNF4,
mouse, P49698; smRXR, schistosome, AF094759. From top to
bottom, the vertebrate RARs are in one group, branching from
node 32; the vertebrate RXRs have node 26 in common. Mouse hepatocyte
nuclear receptor (mHNF), an ancient vertebrate steroid
receptor (53), is more deeply rooted than the arthropod RXRs that
branch from node 28. To our knowledge, SmRXR is the most ancient RXR of
which the full-length cDNA sequence has been identified.
The trans-activating function of steroid receptors is often
dependent on dimerization and ligand binding (22, 42-50). Ligand binding is thought to change the conformation of the steroid receptor into an active state (51). The ability of members of the RXR subfamily
to bind specific ligands appears to have evolved after the vertebrate
and arthopod lineages diverged, as of yet ligands have not been found
for arthropod RXRs (22, 45, 52, 53). The c sub-domain,
important for ligand binding specificity, of arthropod RXR
homologues and SmRXR shows minimal homology to one another and to
vertebrate RXRs (22, 23, 25-32). This suggests that if these more
ancient receptors have evolved to bind ligands, they do not bind the
same ligand. However, arthropod RXRs, like their vertebrate homologues,
do form dimers with other proteins. For example, dUSP dimerizes with
the ecdysone receptor (42-44). Upon sequence analysis of SmRXR, it is
evident that the heptad repeats that form dimerization interfaces on
one side of an
-helix exist in this protein (21). This suggests
that, like other steroid receptors, SmRXR forms dimers that function in
trans-activation (42-50). The fact that SmRXR and dUSP
putatively do not bind ligands, yet have the ability to form dimers,
suggests that
c and the dimerization interface have
evolved at different rates (22, 23, 25-32, 45, 51, 52). Comparing the
function of early metozoan, arthropod, and vertebrate RXRs will lead to
a greater understanding of the importance of these two sub-domains in
RXR facilitated trans-activation.
This report provides the first evidence that RXRs play a role in gene
regulation in primitive eukaryotic organisms such as schistosomes. Not
only does this study add to an increasing amount of information on the
evolution of steroid receptors but also has initiated the study of
steroid receptors involved in a very intriguing interplay between the
separate sexes of schistosomes and the regulation of female-specific
genes. The study of steroid receptors, such as SmRXR, involved in
female-specific gene regulation may lead to pharmacological compounds
that prevent female maturation and egg production, thereby controlling schistosomiasis.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Dan Kosman, State University of New York at Buffalo, for providing the pADH1 vector; Dr. Claude Mania, New England Biolabs, for sharing unpublished sequences of RXR family members; Dr. Stephen Elledge, Baylor College of Medicine, for the host yeast strain Y190; and Dr. Michael Garrick, SUNY at Buffalo, for providing advice on evolutionary studies.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant AI27219.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF094759.
¶ To whom correspondence should be addressed: Dept. of Microbiology, 138 Farber Hall, State University of New York, Buffalo, NY 14214. Tel.: 716-829-2459; Fax: 716-829-2169; E-mail: loverde{at}buffalo.edu.
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ABBREVIATIONS |
---|
The abbreviations used are:
RXR, retinoid X
receptor;
Sm, Schistosoma mansoni;
EMSA, electrophoretic
mobility shift assays;
aa, amino acids;
USP, ultraspiracle protein;
GST, glutathione S-transferase;
Gal4-AD, Gal4 activation
domain;
SD, synthetic dropout;
AT, 3-amino-1,2,4-triazole;
mRXR, mouse RXR
;
dUSP, Drosophila USP;
hRXR
, human RXR
;
RAR, retinoic acid receptor;
RT-PCR, reverse transcriptase-polymerase
chain reaction;
bp, base pair.
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REFERENCES |
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