SmNR1 contains an autonomous transacti-vation function AF1 in the A⁄ B domain as demonstrated in a yeast one-hybrid assay; it interacts with SmRXR1 in a yeast two-hybrid assay and in a g
Trang 1subfamily I member in the Platyhelminth Schistosoma
mansoni (SmNR1)
Wenjie Wu1,*, Edward G Niles1, Hirohisa Hirai2and Philip T LoVerde1
1 Department of Microbiology and Immunology, School of Medicine and Biomedical Science, State University of New York, Buffalo, NY, USA
2 Primate Research Institute, Kyoto University, Inuyama, Japan
Nuclear receptors (NRs) belong to a superfamily of
transcriptional factors that regulate homeostasis,
dif-ferentiation, metamorphosis and reproduction in
meta-zoans Members of the nuclear receptor superfamily
are characterized by a modular structure: a conserved
DNA-binding domain (DBD) that contains two zinc
finger motifs binding to the cis-regulatory region of a
target gene, and a conserved ligand-binding domain
(LBD) that is involved in transcriptional activation of
the target gene via ligand and coregulator binding Some NRs have no known ligand and are called orphan receptors [1,2] A DNA core motif recognized
by a NR is known as a hormone response element The typical hormone response element is a consensus hexameric sequence AGGTCA, which is called a half-site NRs can bind to the half-site in different orientations or repeats either as a monomer, a homo-dimer or a heterohomo-dimer [2] For heterohomo-dimer binding,
Keywords
nuclear receptors; Schistosoma mansoni;
SmNR1 ⁄ SmRXR1 interactions
Correspondence
P T LoVerde, Southwest Foundation for
Biomedical Research, PO Box 760549,
San Antonio, TX, 78245–0549, USA
Fax: +1 210 6703322
Tel: +1 210 2589852
E-mail: ploverde@sfbr.org
*Present address
Southwest Foundation for Biomedical
Research, PO Box 760549, San Antonio,
Texas 78245-0549, USA
Note
The nucleotide sequences reported in this
paper have been submitted to the GenBank
under accession number: AY395037,
AY395051-AY395057
(Received 25 September 2006, revised
6 November 2006, accepted 9 November
2006)
doi:10.1111/j.1742-4658.2006.05587.x
A cDNA encoding a nuclear receptor subfamily I member in the platy-helminth Schistosoma mansoni (SmNR1) was identified and characterized SmNR1 cDNA is 2406 bp long and contains an open reading frame encoding a 715 residue protein Phylogenetic analysis demonstrates that SmNR1 is a divergent member of nuclear receptor subfamily I with no known orthologue SmNR1 was localized to S mansoni chromosome 1 by fluorescent in situ hybridization Gene structure of SmNR1 was deter-mined showing it to consist of eight exons spanning more than 14 kb Quantitative real-time RT-PCR showed that SmNR1 was expressed throughout schistosome development with a higher expression in eggs, sporocysts and 21-day worms SmNR1 contains an autonomous transacti-vation function (AF1) in the A⁄ B domain as demonstrated in a yeast one-hybrid assay; it interacts with SmRXR1 in a yeast two-hybrid assay and in a glutathione S-transferase pull-down assay Electrophoretic mobil-ity shift assay showed that SmNR1 could form a heterodimer with SmRXR1 to bind to DNA elements containing the half-site AGGTCA, a direct repeat of the half-site separated by 0–5 nucleotides (DR1-DR5) and a palindrome repeat of the half-site not separated by nucleic acids (Pal0) Transient transfection in mammalian COS-7 cells showed that SmNR1⁄ SmRXR1 could enhance the transcriptional activation of a DR2-dependent reporter gene Our results demonstrate that SmNR1 is a partner of SmRXR1
Abbreviations
BAC, bacterial artificial chromosome; DBD, DNA-binding domain; GST, glutathione S-transferase; LBD, ligand-binding domain; NR, nuclear receptor; RAR, retinoic acid receptor; SD, synthetic dropout.
Trang 2the nuclear receptor Retinoic X Receptor (RXR) acts
as a critical partner and thus plays a central role in a
variety of nuclear signaling pathways [3–6]
Schistosoma mansoni is a multicellular eukaryotic
parasite with a complex life cycle that involves
mam-malian and snail hosts Study of schistosome NRs
enables us to understand how they regulate signaling
pathways in the schistosome itself and to understand
the molecular relationship between the schistosome
and vertebrate and snail hosts Recently two S
man-soni RXR homologues, SmRXR1 [7] and SmRXR2
[8,9], have been identified and characterized SmRXR1
demonstrated that it may have an important role in
regulation female-specific p14 genes [7] SmRXR2 also
showed a pattern of recognition of cis-sequences
pre-sent in the p14 gene [10] SmRXR1 and SmRXR2 are
expressed throughout schistosome development
sug-gesting that they play a pleiotropic role in the
regula-tion of a number of genes [7–10] Study of SmRXR
partners will add to our knowledge of nuclear receptor
gene regulation in schistosomes and to an
understand-ing of the evolution of RXR’s function We present
herein the characterization of a nuclear receptor
sub-family I member from S mansoni (SmNR1) and
dem-onstrate its interaction with SmRXR1
Results
cDNA isolation
A 2343 bp cDNA containing the 5¢-UTR, entire open
reading frame, 3¢UTR and poly A tail was isolated by
PCR An additional 62 bp 5¢ UTR was extended by 5¢
RACE generating a 2406 bp cDNA The sequence was
confirmed as belonging to a single mRNA species by
sequencing the products of PCR on single-stranded
cDNA using primers within the 5¢-and 3¢ UTR
The cDNA of SmNR1 encodes an open reading
frame of 2145 bp corresponding to a 715 amino acid
protein The DNA binding domain (DBD) is highly
conserved, the P-box (EGCKG), which is involved in
determining DNA binding specificity, is identical to
most members of nuclear receptor subfamily I, for
instance retinoic acid receptor (RAR) and vitamin D3
receptor In a C-terminal extension of the DBD, the
T-box which corresponds to a dimerization interface is
highly conserved, but the A-box showed less
conserva-tion (for example, 33.3% similarity to hRAR gamma
and 22.2% to dHR3) (Fig 1A) The hinge region (D
domain) of SmNR1 is unusually long, similar to other
reported schistosome nuclear receptors [7–9,11–14]
The precise length of the D domain was not
deter-mined due to the highly divergent helices 1–2 in the
LBD However, the DBD terminates at amino acid
332 and the signature sequence of the LBD (Ts) starts
at amino acid 513 (Fig 1B) The end of the hinge region to Ts is usually 40 amino acids in most NRs, and the length of the hinge in SmNR1 thus can be estimated to be 140 amino acids The role of the large hinge in schistosome NRs remains unknown The degree of conservation of the LBD in SmNR1 is lower, helices 1–2 are highly divergent as mentioned above, like that in other S mansoni NRs [7–9,11–14] Although the LBD of SmNR1 is less conserved, the consensus signature of LBD (F,WY)(A,SI)(K,R,E,G) XXX(F,L)XX(L,V,IXXX(D,S) (Q,K)XX(L,V)(L,I,F) [15,16] (from the C-terminus of helix 3 to the middle
of helix 5) and the consensus motif II EFXXXLXXLX LDXXEXALLKAIXLFSXDRXGLXXXXXVEXLQE XXXXALXXY [17] (from helix 7 to helix 9) is highly conserved (Fig 1B) One amino acid in helix 10 has been demonstrated to have an important role in het-erodimer formation with RXRs In SmNR1, a methi-onine that occurs at position 668 may be an amino acid that corresponds to the amino acids found in hRARc and LXRa [18] This suggests that helix 10 of SmNR1 is probably involved in forming a dimer with SmRXR (Fig 1B) A putative AF2 activating domain core (AF2-AD) is present in SmNR1 (Fig 1B); it exhibits a high degree of conservation (represented by CLKEFL) in comparison with the common consensus AF2-AD core structure ofFFXEFF, where F denotes
a hydrophobic residue [19,20]
Phylogenetic analysis
A phylogenetic tree was constructed using the maximum likelihood method under the Jones–Taylor– Thornton substitution model, with a gamma distribu-tion of rates between sites (eight categories, parameter alpha) Support values for the tree were obtained by bootstrapping 100 replicates (Fig 2) The result shows that SmNR1 is a divergent member belonging to NR subfamily I The same result was obtained by Bayesian inference and neighbor-joining distance analysis (sup-plementary Figs S1 and S2) Even though SmNR1 was clustered with Onchocerca volvulus NR1 on the maxi-mum likelihood tree, the low bootstrap value (29%) did not support SmNR1 to be an orthologue to
O volvulusNR1 (Fig 2)
Chromosome localization and gene organization
A bacterial artificial chromosome (BAC) library of
S mansoni [21] was screened with a SmNR1-specific probe, and three positive clones (SmBAC1 28A22,
Trang 3SmBAC1 121N20 and SmBAC1 41A19) were
identi-fied SmBAC1 41A19 was used as a probe for
fluores-cent in situ hybridization and SmNR1 was localized to
chromosome 1 (Fig 3)
Gene organization of SmNR1 was determined by
sequencing BAC DNA (SmBAC1 41A19) and by
cDNA alignment with a 24 kb genomic DNA contig
(Contig_0012771) obtained from WTSI S mansoni
WGS database (ftp://ftp.sanger.ac.uk/pub/databases/
Trematode/S.mansoni/genome) The SmNR1 gene
con-sists of eight exons spanning over 14 kb (Fig 4A), and
all splice donor and acceptor sites fit the GT-AG rule
(supplementary Table S1) The 5¢-UTR is encoded by two exons, A⁄ B, C (DBD), hinge and E–F domain (LBD) are each encoded by 2–3 exons, respectively (Fig 4B)
We previously demonstrated that the splice junction
in the DBD encoding region was conserved in SmNR1 [22] In vertebrate NRs, two conserved splice sites were identified in the LBD encoding region, one is in motif
I (also known as signature sequence of LBD) and the other is in motif II [17] The splice junction of motif I
in SmNR1 is at the same position as that found only
in RARs (NR1B) [17] (Fig 4C) The splice site of
A DNA binding domain
B Ligand binding domain
Fig 1 Sequence alignment (A) Alignment
of DNA binding domain (C domain) and its C-terminal extension (B) Alignment of ligand binding domain (E domain) (after helix 2) Helices as described in [60] are boxed The putative autonomous activation domain (AF2-AD) is also indicated The number at the end of each line indicates residue position in the original sequence H3-H12, helices 3–12.
Trang 4motif II in SmNR1 is located at the same conserved
position as found in all analyzed NRs [17] The
con-served splice junctions in SmNR1 suggest that the gene
structure of SmNR1 is ancient and has been main-tained through out evolution of NRs
Developmental expression Quantitative real-time RT-PCR was performed to evaluate mRNA expression of SmNR1 Normalized gene expression [23] was standardized to the relative quantities of S mansoni a-tubulin SmNR1 was expressed in all tested stages, with a higher expression
in eggs (19.9-fold greater than male worms), sporocysts (13.6-fold greater than male worms) and 21-day worms (6.8-fold greater than male worms) It was expressed in
a similar manner in the other developmental stages tested (Fig 5) The results suggest that SmNR1 is expressed throughout development but may have a more significant role in the development of eggs, sporocysts and 21-day worms
Determination of transactivation
A yeast one-hybrid assay was employed to determine whether ligand-independent autonomous transactiva-tion functransactiva-tion was present in SmNR1 Yeast strain AH109 was transformed with pGBKT7-SmNR1, pGBKT7-SmNR1(A⁄ B) (containing the A ⁄ B domain) and pGBKT7-SmNR1(CF) (without the A⁄ B domain), respectively, spread on synthetic dropout (SD)⁄ –Leu media and SD⁄ –Leu ⁄ –His medium plus 3 mm 3-AT Yeasts transformed with SmNR1 or pGBK-SmNR1(A⁄ B) grew on both SD ⁄ –Leu medium and
Fig 2 Phylogenetic tree of SmNR1 A maximum likelihood tree
showing that SmNR1 (in black) is a member of the NR subfamily I.
The phylogenetic tree was constructed by maximum likelihood
method under the Jones–Taylor–Thornton substitution model with a
gamma distribution of rates between sites (eight categories,
parame-ter alpha) Support values for the tree were obtained by
boot-strapping, 100 replicates The subfamilies are according to the
nomenclature system for the nuclear receptor (for nuclear receptor
nomenclature, see http://www.ens-lyon.fr/LBMC/laudet/nurebase/
nomenclature/Nomenclature.html) The GenBank accession numbers
of the analyzed sequences are provided in supplementary Table S2.
Fig 3 Fluorescent in situ hybridization mapping of SmNR1 Mitotic metaphase chromosomes (2n ¼ 16) of male schistosomes obtained from the S mansoni sporocyst stage SmBAC 41A19 BAC DNA was used as a probe and hybridized to chromosome 1 Scale bar ¼ 6 lm.
Trang 5SD⁄ –Trp ⁄ –His medium plus 3 mm 3-AT (Fig 6A),
while yeasts transformed with pGBK-SmNR1(CF)
grew on SD⁄ –Leu medium but not on SD ⁄ –Trp ⁄ –His
medium plus 3 mm 3-AT (Fig 6A) The results
sugges-ted that both full-length and the A⁄ B domain of
SmNR1 activated transcription of GAL4 reporter gene
in the absence of ligand, while the C-F domain did
not Thus the A⁄ B domain exhibits an autonomous
transactivation function (AF-1) element Yeast
trans-formed with the control plasmids grew as expected (see the legend to Fig 6A for a complete explanation)
SmNR1 interacts with SmRXR1
A yeast two-hybrid assay was performed to address whether SmNR1 interacted with SmRXR1 or SmRXR2, or acted as a homodimer in a yeast system
As the A⁄ B domain of SmNR1 (as demonstrated above) and SmRXR1 can activate transcription of GAL4 reporter [7], SmNR1(CF) and SmRXR1(CF) were used
in the DBD vectors Yeast transformed with pSV40⁄ p53 (positive control), pSV40⁄ pLamin C (negative control), pGBK-SmNR1(CF)⁄ pACT-SmRXR1, pAS-SmRXR1(CF)⁄ pGAD-SmNR1, pGBK-SmNR1(CF)⁄ pACT-SmRXR2, pAS-SmRXR2⁄ pGAD-SmNR1 and pGBK-SmNR1(CF)⁄ pGAD-SmNR1 grew on SD⁄ –Trp⁄ –Leu medium If SmNR1 interacts with SmRXR1
or SmRXR2, or acts as a homodimer, the Gal4 DNA binding domain fusion partner will bind to the Gal1 UAS element and the Gal4 activation domain will drive transcription of HIS reporter gene Yeasts cotrans-formed with pGBK-SmNR1(CF)⁄ pACT-SmRXR1
or pAS-SmRXR1(CF)⁄ pGAD-SmNR1 grew on SD ⁄ –Trp⁄ –His ⁄ –Leu medium plus 3 mm 3-AT, indicting that SmNR1 and SmRXR1 interacted Yeasts cotrans-formed with pGBK-SmNR1(CF)⁄ pACT-SmRXR2, pAS-SmRXR2⁄ pGAD-SmNR1 or pGBK-SmNR1 (CF)⁄ pGAD-SmNR1 did not grow on SD ⁄ –Trp ⁄ –His⁄ –Leu medium plus 3 mm 3-AT, indicating that SmNR1 did not interact with SmRXR2 or act as a homodimer The positive control yeast cotransformed with plasmids pSV40⁄ p53 grew on SD ⁄ –Trp ⁄ –His ⁄ –Leu
A
C
Show-ing exons and size of introns; roman numer-als indicate exons (B) Showing the size of exons and their correspondence to the dif-ferent NR domains A ⁄ B, A ⁄ B domain; C, C domain (DBD); D, D domain (hinge); Ts, sig-nature sequence of the LBD; E, E domain (LBD) after Ts (C) Showing the splice junc-tion of SmNR1 within motif I which is at the same position as that found only in RARs (NR1B) [17] hRARa, human retinoic acid receptor alpha (GenBank: AC018629); CiRAR, C intestinalis retinoic acid receptor (www.jgi.doe.gov, Genomic sequence Scaf-fold (v 1.0): (14) H4, helix 4; H5, helix 5.
Fig 5 Quantitative real-time RT-PCR shows mRNA expression of
SmNR1 Gene expression [23] of SmNR1 was normalized to the
relative quantities of S mansoni a-tubulin For graphical
representa-tion of fold of expression, the normalized expression was
recalcu-lated by dividing the expression level of each stage by the lowest
expression stage (male worms) Egg, eggs; Sp, secondary
sporo-cysts in 30-day infected snail; Cer, Cercariae; 15d, 15-day
schistos-omules; 21d, 21-day schistosschistos-omules; 28d, 28-day worms; 35d,
35-day worms; Pair, adult worm pairs; Female, adult female
worms; Male, adult male worms.
Trang 6medium plus 3 mm 3-AT, yeast cotransformed with the
negative control plasmids pSV40⁄ pLamin C did not
grow on SD⁄ –Trp ⁄ –His ⁄ –Leu medium plus 3 mm 3-AT
as expected (Fig 6B)
A glutathione S-transferase (GST) pull-down assay
was performed to verify the interaction of SmNR1 and
SmRXR1 in vitro To address whether the heterodimer
interface is located in the EF domain, both SmNR1
and SmNR1(EF) were employed GST-SmNR1 and
GST-SmNR1(EF) fusion proteins were immobilized on
glutathione beads.35S-labeled SmRXR1 was produced
in a rabbit reticulocyte system GST protein was
used as a negative control The pull-down results
showed that both SmNR1 and SmNR1(EF) interacted
with SmRXR1 (Fig 7A) SmNR1(EF) interacted with
SmRXR1 at a level similar to that of SmNR1 suggest-ing that there was a heterodimer interface located in SmNR1 EF domain (Fig 7B)
DNA binding assays with SmNR1⁄ SmRXR1 heterodimers
Electrophoretic mobility shift assays were performed
to determine DNA binding specificity of SmNR1 A DNA element containing the half-site AGGTCA, a direct repeat of the half-site spaced with 0–5 nucleic acids (DR0-DR5) and palindrome repeat of the half-site not separated by nucleic acids (Pal0) were employed No gel shift was observed when c-32 P-labe-led half-site DR0-DR5 or Pal0 were added to SmNR1 alone (Fig 8) Smears were observed when the same oligonucleotides were added to SmRXR1, and strong shifts were observed when the oligonucleo-tides were added to SmNR1⁄ SmRXR1 (Fig 8) A weak shift was observed when labeled half-site was added to SmNR1⁄ SmRXR1 (Fig 8) The results indi-cated that SmNR1 did not bind to the tested oligonu-cleotides, SmRXR1 bound to the oligonucleotides in
an unstable state and SmNR1⁄ SmRXR1 heterodimer strongly bound to the target oligonucleotides The results suggest that SmNR1 requires heterodimeriza-tion with SmRXR1 to bind to the tested DNA ele-ments
The preference for SmNR1⁄ SmRXR1 heterodimer binding to oligonucleotides was determined by
competi-A
B
Fig 6 Yeast one and two-hybrid assays (A) Yeast one hybrid
assay showing that SmNR1 contains an autonomous transactivation
function in A ⁄ B domain Individual AH109 yeast colonies obtained
from an initial transformation were re-streaked on SD ⁄ –Trp medium
and on SD ⁄ –Trp ⁄ –His medium plus 3 m M 3-AT (a) Diagram of
reporter system used in yeast one-hybrid assay The HIS3 reporter
gene is controlled by binding of the Gal4 DNA binding domain
(GAL4DB) to the GAL4 response elements When a GAL4BD
fusion protein contains an activation domain, it will transactivate
the expression of the reporter genes (b) On SD ⁄ –Trp medium,
yeasts transformed with SmNR1 (streak 1),
pGBKT7-SmNR1(A ⁄ B) (streak 2), pGBKT7-SmNR1(CF) (streak 3), P53 (streak
4), PSV40 ⁄ P53 (streak 6), pLamin C (streak 7) and pLamin
C ⁄ PSV40 (streak 8) grew, because pGBKT7, P53 and pLamin C
plasmids expressed trp gene, yeasts transformed with PSV40 did
not grow because PSV40 plasmid did not express trp gene (streak
5, negative control) (c) On SD ⁄ –Trp ⁄ –His medium plus 3 m M 3-AT,
yeasts transformed with P53 (streak 4), PSV40 (streak 5), pLamin
C (streak 7) and pLamin C ⁄ PSV40 (streak 8) did not grow, because
P53 and pLamin C plasmids did not express the trp gene Yeasts
transformed with pGBKT7-SmNR1(CF) (streak 3) did not grow,
indi-cating that the C-F domain of SmNR1 could not active transcription
of the His reporter gene Yeasts transformed with pGBKT7-SmNR1
(streak 1) and pGBKT7-SmNR1(A ⁄ B) (streak 2) grew indicating that
the A ⁄ B domain contains an activation function to active
transcrip-tion of His reporter gene PSV40 ⁄ P53 (streak 6, positive control)
grew as expected (B) Yeast two hybrid assay showing SmNR1
interaction with SmRXR1 (a) Diagram of the system used in yeast
two hybridization If protein 1 (P1) interacts with protein 2 (P2), the
Gal4 DNA binding domain fusion partner will bind to the Gal1 UAS
element and the Gal4 activation domain will drive transcription of
the expression of the reporter gene Individual AH109 yeast
colonies obtained from initial transformation were re-streaked
on SD ⁄ –Trp ⁄ –Leu medium (b) and on SD ⁄ –Trp ⁄ –Leu ⁄ –His
medium plus 3 m M 3-AT (c) Streak 1, pSV40 ⁄ p53 (positive
control); streak 2, pSV40 ⁄ pLamin C (negative control); streak 3,
pGBK-SmNR1(CF) ⁄ pGAD-SmNR1; streak 4, pAS-SmRXR1(CF) ⁄
pGAD-SmNR1; streak 5, pGBK-SmNR1(CF) ⁄ pACT-SmRXR1; streak
6, pAS-SmRXR2 ⁄ pGAD-SmNR1; and streak 7, pGBK-SmNR1(CF) ⁄
pACT-SmRXR2.
Trang 7tion of unlabeled DR0-DR5 and Pal0 with c-32P-labeled
DR4 (Fig 9) A 10-, 50- and 200-fold molar excess of
unlabelled oligonucleotides was used for competition
The results showed that a 10-fold excess of unlabelled specific oligonucleotides led to a reduction in the signal, while a 50- and 200-fold excess of unlabelled specific competitors completely abolished the binding of the labeled DR4 No reduction of binding was observed when unlabelled nonspecific oligonucleotides were used (Fig 9) The order of preference for SmNR1⁄ SmRXR1 heterodimer binding to DNA elements was thus determined by competition of a 10-fold excess of unlabe-led specific oligonucleotides to be DR2 > DR5 > DR3 > DR4 > DR1 > DR0 > Pal0
To determine the role of the A⁄ B domain of SmNR1 in DNA binding, SmNR1(CF)⁄ SmRXR1 binding to DR1 and DR2 were employed Although SmNR1(EF) can form a heterodimer with SmRXR1 (demonstrated by pull-down experiment, Fig 7), no shifts were observed when c-32P-labeled DR1 and DR2 were added to SmNR1(CF)⁄ SmRXR1, while strong shifts were observed when same oligonucleotides were added to SmNR1⁄ SmRXR1 (Figs 8 and 10) The results suggested that the A⁄ B domain of SmNR1 was necessary for SmNR1⁄ SmRXR1 heterodimer to bind
to the tested DNA elements To determine the role of the C-terminal extension of SmNR1 and SmRXR1 in binding to DNA elements, in vitro synthesized SmNR1 (Ile247 to Ser372) (containing 20 amino acids at the 5¢ end of the DBD, the DBD and 40 amino acids at 3¢ end of the DBD) and SmRXR1 (Glu251 to Asn376) (containing 20 amino acids at 5¢ end of the DBD, the DBD and 40 amino acids at the 3¢ end of the DBD) were tested Both SmNR1 (Ile247 to Ser372) and SmRXR1 (Glu251 to Asn376) bound to half-site, and
Fig 8 DNA binding of SmNR1 and SmRXR1 in vitro A single protein or a combination of two proteins were synthesized in a TNT quick cou-pled transcription ⁄ translation system (Promega) and allowed to bind to c- 32 P-labeled DNA elements containing a half-site, DR0-DR5 and Pal0 Lanes 1, 5, 9, 13, 17, 21, 25 and 29 contain lysate from the control transcription-translation reaction as negative controls Lanes 2, 6, 10, 14,
18, 22, 26 and 30 contain lysate with in vitro translated SmNR1 Lanes 3, 7, 11, 15, 19, 23, 27 and 31 contain lysate with in vitro translated SmNR1 and SmRXR1 Lanes 4, 8, 12, 16, 20, 24, 28 and 32 contain lysate with in vitro translated SmRXR1 NS, nonspecific binding.
A
B
Fig 7 GST pull-down assay showing SmNR1 interaction with
SmRXR1 in vitro (A)35S-labeled SmRXR1 was synthesized in vitro
using pCITE-SmRXR1 as template and then incubated with
GST-SmNR1, GST-SmNR1(EF) or GST (negative control) protein affixed
to glutathione-Sepharose beads The beads were collected, washed
and the bound protein was resolved on 10% SDS acrylamide gel
and visualized by autoradiography Each experiment was repeated
three times (a) GST-SmNR1 ⁄ pCITE-SmRXR1 reaction (b)
GST-SmNR1(EF) ⁄ pCITE-SmRXR1 reaction (B) Bar graph representation
of the relative band intensities of SmNR1 ⁄ SmRXR1 and
SmNR1(EF) ⁄ SmRXR1 reaction and compared with SmRXR1 input.
(a) GST-SmNR1 ⁄ pCITE-SmRXR1 reaction (b) GST-SmNR1(EF) ⁄
pCITE-SmRXR1 reaction The diagram explains the reactions.
Trang 8SmNR1 (Ile247 to Ser372) bound to DR2, weakly to
DR1, DR4 and DR5 SmRXR1 (Glu251 to Asn376)
bound to DR1, DR2, DR4 and DR5 (Fig 11)
SmNR1 (Ile247 to Ser372) and SmRXR1 (Glu251 to
Asn376) did not form a heterodimer to bind to the
DNA elements The results suggest that although there
is a dimer interface located in DBD and C-terminal
extension [24–26], the D-E domain has an important
role in SmNR1⁄ SmRXR1 heterodimer binding to the
DNA elements Figure 10 demonstrated that SmNR1
CF could not bind to DR1 or DR2 elements, while
SmNR1 missing both the A⁄ B and E ⁄ F domains
(Fig 11) was capable of binding to a half-site and
to several DR elements We suggest that the E⁄ F
domains of SmNR1 might prevent the interaction
between the C domain of SmNR1 and DNA response
elements, as previously demonstrated for S mansoni
RXR2 [8]
Transcriptional activation of a DR2
element-dependent reporter gene
Electrophoretic mobility shift assay results showed
that SmNR1⁄ SmRXR1 heterodimer could bind to DNA
element DR2 strongly A pUTK-3xDR2 reporter plasmid
was constructed to test the ability of SmNR1⁄ SmRXR1
to transactivate DR2-dependent reporter gene in
mammalian COS-7 cells The results showed that SmNR1⁄ SmRXR1 activated the reporter gene with a significant difference to control plasmid PcDNA [degrees
of freedom (d.f.)¼ 9, p ¼ 0.03] (Fig 12)
Discussion
Phylogenetic analysis shows that SmNR1 is a divergent member of NR subfamily I with no known orthologue This suggests that other unknown NR groups may be expected to be present in invertebrate lineages as their sequences become available for analysis SmNR1 is a new NR group which does not exist in Drosophila, Caenorhabditis or vertebrates whose NR complement
is well studied
Recently an alternative splice variant of SmNR1 was identified (DQ439962) Our 5¢ sequence (nt 1–84) aligns to nt 3397–3480 on the genomic DNA Con-tig_0012771, while the first exon of DQ439962 runs from nt 4069–4166 Both variants encode the same protein sequence; this is therefore a case of alternative splicing in the noncoding region similar to what was found for the S mansoni nuclear receptor, SmFtz-F1 [11] Whether the corresponding mRNAs interact differently with the translational machinery or have different stabilities as proposed for SmFTZ-F1 [11] is yet to be determined
Fig 9 Competition of DNA binding to SmNR1 ⁄ SmRXR1 heterodimer in vitro Combination of SmNR1 and SmRXR1 proteins were synthes-ized in vitro, added to c- 32 P-labeled DR4 Unlabelled DR0-DR5, Pal0 or unrelated oligonucleotides (·10, ·50 and ·200 fold, respectively) were added to compete with labeled DR4 Lanes 1, 11 and 21 contain lysate from the control transcription-translation reaction as negative con-trols Lanes 2, 12 and 22 contain no competitor Lanes 3, 13 and 23 contain nonspecific competitor Lanes 4, 14 and 24 contain DR0 as competitor Lanes 5, 15 and 25 contain DR1 as competitor Lanes 6, 16 and 26 contain DR2 as competitor Lanes 7, 17 and 27 contain DR3
as competitor Lanes 8, 18 and 28 contain DR4 as competitor Lanes 9, 19 and 29 contain DR5 as competitor Lanes 10, 20 and 30 contain Pal0 as competitor NS, nonspecific binding.
Trang 9Most NRs which can form a heterodimer with RXR
are from subfamily I, for example, thyroid hormone
receptor and RAR [2] Our studies show that SmNR1
exhibits similarity to RAR, PPAR and EcR, which
need RXR to form a heterodimer to confer hormone
response element binding [25,27–31] RXRs have been
characterized in a wide variety of metazoans, including
in Cnidaria [32], Platyhelminths [7–9], Mollusca [33],
Nematoda [34] and Arthropoda [35,36], and
verte-brates [37,38] The functional relationship between
vertebrate RXR with other NRs was described as the
1-2-3-4-5 rule [39,40] and was extended to insect
RXR⁄ EcR heterodimers [28] For example, vertebrate
RXR⁄ RAR can bind to DR1, DR2 and DR5 but
not to DR3 or DR4, RXR⁄ vitamin D3 receptor
heterodimer can bind to DR3 but not to DR1, DR2,
DR4 or DR5 [25,27,29,31,41] In insects, Drosophila USP (RXR homologue) forms a heterodimer with EcR that can bind to DR0-DR5 [28] and to an imperfect palindromic structure [42] The DNA binding
specifici-ty of RXR⁄ NR heterodimer in most invertebrates is not well known A recent study showed that S man-soni SmRXR1⁄ SmFtz-F1 heterodimer could bind to SF-1 element (which contains a conserved half-site AGGTCA) via SmFtz-F1 physical binding to the DNA element, while SmRXR1 did not bind to the DNA [43] In the mollusk, Biomphalaria glabrata RXR (BgRXR) was shown to bind to DR1 as a homodimer
or as a heterodimer with mammalian RARa, LXR, FXR or PPARa [33] In this study, we showed that SmNR1⁄ SmRXR1 heterodimer could bind to DR0-DR5, as such it is similar to the Drosophila USP⁄ EcR heterodimer [28] but with a different preference order (Fig 9) The results suggest that RXR⁄ NR heterodi-mer obtained the ability to bind to conserved half-site repeats before the split of Arthopods and Platyhelm-inths, but has not subsequently evolved a strict spacing between half-sites as it can bind to all of DR1 to DR5 elements This lack of binding specificity is different from the vertebrate RXR⁄ RAR interaction that can bind to DR1, DR2 and DR5 but not to DR3 or DR4 [25,31] SmRXR1 alone was known to bind to a non-conserved direct repeat in the promoter region of
S mansoni p14 gene [7] In this report, we demonstra-ted that SmRXR1 alone could also bind to a con-served half-site and direct repeats of half-site (Fig 8)
In addition, we showed that SmNR1⁄ SmRXR1 het-erodimer could bind in vitro to a perfect palindrome (Pal0) containing element (Figs 8 and 9) Further stud-ies of SmNR1 will help us to understand the mechan-ism of RXR⁄ NR signal pathway in invertebrates and its evolutionary role
A yeast one-hybrid assay was employed and a ligand-independent autonomous transactivation function (AF1) was determined to be present in the A⁄ B domain
of SmNR1 Furthermore, we demonstrated that the
A⁄ B domain has an important role in determining SmNR1⁄ SmRXR1 heterodimer binding to the DNA element That amino acids in the A⁄ B domain can affect DNA binding and dimerization has previ-ously been reported in the chicken thyroid hormone receptor [44]
Our data shows that SmNR1⁄ SmRXR1 can activate transcription from a DR2-dependent reporter plasmid
in mammalian cells (Fig 12) Future studies will exam-ine transcriptional activation in detail Although the full-length of SmNR1 could not bind to the response element without the presence of SmRXR1 in vitro (Fig 8), SmNR1 alone enhanced transactivation of
Fig 10 DNA binding of SmNR1(CF) ⁄ SmRXR1 in vitro A single
pro-tein or a combination of two propro-teins were synthesized in a TNT
quick coupled transcription ⁄ translation system and allowed to bind to
c-32P-labeled DR1 and DR2, respectively Lane 1, lysate from the
control transcription-translation reaction with labeled DR1 as negative
control; lanes 2–4, SmNR1(CF) ⁄ SmRXR1 with labeled DR1 plus
indi-cated amounts of unlabeled DR1 as competitor; lanes 5–7,
SmNR1 ⁄ SmRXR1 plus indicated amounts of unlabeled DR1 as
com-petitor (positive control); lane 8, lysate from the control
transcription-translation reaction with labeled DR2 as negative control; lanes 9–11,
SmNR1(CF) ⁄ SmRXR1 with labeled DR2 plus indicated amounts of
unlabeled DR2 as competitor; lanes 12–14, SmNR1 ⁄ SmRXR1 with
labeled DR2 plus indicated amounts of unlabeled DR2 as competitor
(positive control) NS, nonspecific binding.
Trang 10transcription in mammalian cells Whether SmNR1
can dimerize with mammalian RXR or whether a low
level of homodimer formation of SmNR1 is needed is
unknown However, our yeast two-hybrid and
pull-down assays show no evidence for homodimerization
of SmNR1 (Fig 6, unpublished data) The ability of SmNR1⁄ SmRXR1 to transactivate DR2-dependent reporter gene in mammalian cells suggests that SmNR1⁄ SmRXR1 can interact with mammalian coac-tivators of transcription, and S mansoni coaccoac-tivators
of transcription may have a similar mechanism to SmNR1⁄ SmRXR1 Recently four NR coactivators, SmGCN5, SmPRMT1, SmCBP1 and SmCBP2 were isolated from S mansoni [45–47] It was shown that they could interact with schistosome NRs For exam-ple, SmCBP1 interacted with S mansoni nuclear recep-tor SmFTZ-F1 and exhibit transcriptional activity in mammalian cells [47] Importantly, the interaction of SmNR1⁄ SmRXR1 is demonstrated by in vitro (GST pull-down assays) and in vivo (yeast two-hybrid and mammal cell assays) results Likewise, the ability of the heterodimer to bind DNA is shown by in vitro (electrophoretic mobility shift assay) assays and to bind DNA and drive transcription in a mammalian cell reporter gene assay in in vivo results
Experimental procedures
Parasites
The NMRI strain of S mansoni was maintained in snails (Biomphalaria glabrata) and Syrian golden hamsters (Mesocricetus auratus) Cercariae were released from
infec-Fig 11 DNA binding of SmNR1(Ile247-Ser372) and SmRXR1(Glu251-Asn376) in vitro DNA binding of a protein containing 20 amino acids at the 5¢ end of the DBD, the DBD and the 40 amino acids at the 3¢ end of the DBD of SmNR1 (Ile247-Ser372) and SmRXR1 (Glu251-Asn376) were tested in vitro Lanes 1, 5, 9, 13, 17, 21, 25 and 29, lysate from the control transcription-translation reaction as negative control; lanes
2, 6, 10, 14, 18, 22, 26 and 30 contain with lysate with in vitro translated SmNR1(Ile247-Ser372); lanes 3, 7, 11, 15, 19, 23, 27 and 31, lysate with in vitro translated SmNR1(Ile247-Ser372) and SmRXR1(Glu251-Asn376); lanes 4, 8, 12, 16, 20, 24, 28 and 32, lysate with in vitro trans-lated SmRXR1(Glu251-Asn376) NS, nonspecific binding.
0
1
2
3
4
5
6
*
Fig 12 SmNR1 ⁄ SmRXR1 transactivated DR2-dependent reporter
gene in vivo Mammalian COS-7 cells were transfected with
pUTK-DR2 reporter plasmids, pRL4.74 and various expression plasmids
for pcDNA-3.1, SmNR1, SmRXR1 and SmNR1 ⁄ SmRXR1 Cells
were lysed and luciferase activities were measured 48 h after
transfection Results are expressed in fold activation (relative to the
pcDNA-3.1 vector control) Each experiment was repeated at least
three times The statistical significance of increase in luciferase
activities of cells transfected with SmNR1, SmRXR1 and
SmNR1 ⁄ SmRXR1 compared to cells transfected with pCDNA-3.1
was determined using Student’s t-test ( d.f ¼ 9, *P < 0.05).