Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347, USA
Author for correspondence (e-mail:
klym{at}spot.colorado.edu)
Accepted 12 August 2003
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SUMMARY |
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Key words: Xenopus, SOX3, Nodal-related protein, Xnr5, ß-Catenin, VegT
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Introduction |
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How the rotation-induced asymmetry in ß-catenin and other cytoplasmic
components interacts with the pre-existing animal-vegetal asymmetries
generated during oogenesis and meiotic maturation is the subject of intense
study. The earliest zygotic regulatory landmark identified to date is the
expression of the transforming growth factor ß (TGFß) family,
Nodal-related Xnr5 and Xnr6 genes. Both are first detected
at the 256-cell stage, well before the beginning of `general' transcription at
the mid-blastula transition (MBT), which occurs at stage 8.5
(Yang et al., 2002). Takahashi
et al. (Takahashi et al.,
2000
) reported that Xnr5 and Xnr6 RNAs are
present throughout the vegetal hemisphere in a shallow dorsal-vegetal
gradient, although they did not see expression of either gene before the MBT.
The activation of Xnr5 and Xnr6 expression in animal caps
(Rex et al., 2002
) and whole
embryos (Yang et al., 2002
) is
dependent upon the activation of ß-catenin and the vegetally localized
maternal T-box transcription factor VegT
(Zhang et al., 1998
;
Zhang and King, 1996
), also
known as Xombi (Lustig et al.,
1996
), Antipodean (Stennard et
al., 1996
) and Brat (Horb and
Thomsen, 1997
).
Studies of Xnr5 and Xnr6 expression
(Yang et al., 2002) and the
isolation of a minimal Xnr5 promoter (Hilton et al., 2003) suggest
that Xnr5 is directly regulated by ß-catenin and maternal TCFs.
As such, it joins Siamois (Brannon
et al., 1997
) and Twin
(Laurent et al., 1997
), which
encode homeobox-containing proteins expressed in the dorsal endoderm or
Nieuwkoop center (Carnac et al.,
1996
; Lemaire et al.,
1995
), Xbra (Vonica
and Gumbiner, 2002
), which encodes a T-box containing protein
expressed in mesoderm, and Xnr3
(McKendry et al., 1997
), which
encodes a Nodal-related protein expressed within the Spemann organizer, as
targets of ß-catenin/TCF regulation in the early Xenopus
embryo.
Another family of maternal and early zygotic factors that might influence
ß-catenin-regulated genes are the SOX proteins. SOXs and LEF/TCF proteins
are part of a larger family of sequence specific DNA binding proteins that
contain a single high mobility group (HMG) box DNA binding motif
(Bowles et al., 2000)
(Klymkowsky, 2004
). The HMG
boxes of SOX proteins share at least 50% identity with the HMG box of the
mammalian male sex determining polypeptide SRY. There are over 20 different
SOXs in mammals, and these have been divided into ten subgroups based on
similarities within their HMG box regions. Outside of the HMG box, SOX
proteins of the same subgroup share little primary sequence similarity
(Bowles et al., 2000
). As in
the case of LEF/TCF, the binding of SOX proteins to their target sites induces
DNA bending (Bewley et al.,
1998
; Giese et al.,
1992
; Love et al.,
1995
). Inhibitory interactions between ß-catenin-regulated
gene expression and SOX3, SOX17
and SOX17ß were first described by
Zorn et al. (Zorn et al.,
1999
). In Xenopus, ectopic expression of these SOXs
ventralizes embryos, blocks ß-catenin-mediated axis duplication and
inhibits ß-catenin-induced activation of LEF/TCF-responsive reporters in
cultured cells.
XSOX3 was described initially by two groups. Koyano et al.
(Koyano et al., 1997) reported
that XSOX3 was expressed in oocytes but that both RNA and polypeptide
disappeared in mature eggs and early embryos. Penzel et al.
(Penzel et al., 1997
) reported
that XSOX3 RNA was present maternally and expressed within the neural
plate. XSOX17
and ß are expressed zygotically, regulated by VegT
(Engleka et al., 2001
) and
required for endodermal differentiation
(Hudson et al., 1997
). Based
on DNA gel shift and in vitro protein binding studies, Zorn et al.
(Zorn et al., 1999
) concluded
that SOX3, SOX17
and SOX17ß inhibited ß-catenin-signaling by
competing directly with TCFs for binding to ß-catenin.
XSOX3 is a member of the B subgroup of SOX proteins, which have been
further subdivided into B1 (1, 2 and 3) and B2 (14 and 21) subgroups. The B1
SOXs are thought to act as transcriptional activators and the B2 SOXs as
transcriptional repressors (Uchikawa et
al., 1999). Studies in the mouse suggest that the B1 SOXs are
functionally redundant and that differences in phenotypes associated with
mutations in these genes are due largely to regulatory differences
(Avilion et al., 2003
).
The maternal nature of XSOX3 suggests that it could be directly involved in patterning the early embryo. We have extended our previous studies explore this possibility and to define further the mechanism by which XSOX3 modulates ß-catenin-mediated gene regulation. Using an affinity-purified antibody directed against the C-terminus of XSOX3 and point mutations in the HMG box region of the polypeptide, we find that XSOX3 binds to sites within the Xnr5 promoter, distinct from TCF-binding sites. At these sites, it unexpectedly acts as a repressor. In addition to its apparently direct effects on Xnr5, injection of XSOX3 RNA leads decreased levels of Siamois, Twin, Xnr3 and Xbra RNAs. Depletion of XSOX3 by morpholino injection, expression of an activating form of XSOX3 or injection of an anti-XSOX3 antibody leads to increased accumulation of Xnr5 RNA, suggesting that the normal function of maternal XSOX3 is to restrict Xnr5 expression to the vegetal hemisphere of the embryo.
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Materials and methods |
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Promoter reporter reagents
The Siamois-promoter/firefly-luciferase and
Siamois-null/luciferase plasmids
(Brannon et al., 1997) were
supplied by R. Moon and D. Kimelman (University of Washington), wild-type and
TcfA, TcfB and TcfA/TcfB mutated
Xnr5-promoter/luciferase plasmids (Hilton et al., 2003) were supplied
by E. Hilton and R. Old (University of Warwick, Warwick, UK). A mutated
version of the Xnr5 reporter in which the two SOX3 binding sites
upstream of the distal TCF site were removed was generated using the
Quickchange site-directed mutagenesis kit. The optimized TOPFLASH and FOPFLASH
reporters (Korinek et al.,
1997
) were supplied by R. Vogelstein (Johns Hopkins University).
The pRL-TK plasmid was used to normalize both embryonic and cell cultured
experiments using a Promega Dual Luciferase Assay system.
Embryonic and axis duplication studies
Eggs were obtained from hormone-stimulated female X. laevis,
fertilized, dejellied and injected following established lab procedures
(Bachant and Klymkowsky, 1997).
Embryonic stages were defined according to Nieuwkoop and Faber
(Nieuwkoop and Faber, 1967
).
Ultraviolet (UV) ventralization of fertilized eggs was performed as described
previously (Zorn et al.,
1999
). Animal caps were generated using a GastromasterTM
apparatus (Xenotek Engineering) and healed in 1xMMR; after healing, they
were maintained in 20% MMR. Injected embryos were cultured at 16°C and
analysed by immunoblot, immunoprecipitation or whole-mount
immunocytochemistry.
Antibodies and immunocytochemistry
Mouse monoclonal anti-V5-epitope antibody was purchased from Invitrogen. An
affinity purified rabbit antibody against Xenopus ß-catenin was
raised by Bethyl Laboratories, using purified His6-ß-catenin
polypeptide isolated from baculovirus infected cells. Affinity purified rabbit
polyclonal antibodies were raised against the N-terminal 20 amino acids of
XTCF3 (anti-XTCF3n), the C-terminal 20 amino acids of XTCF3 (anti-XTCF3c) or
the C-terminal 20 amino acids of XSOX3 (anti-XSOX3c) by Bethyl Laboratories.
The mouse monoclonal anti-Myc antibody 9E10
(Evan et al., 1985) was used
to visualize Myc-tagged polypeptides.
Immunochemical analyses
For immunoblot and immunoprecipitation studies, embryos were washed with
lysis buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 0.05% NP-40, 0.5% Triton
X-100, 1 mM EGTA, 5 mM NaF and protease inhibitors (Roche)] and homogenized in
20 µl lysis buffer per embryo, typically in groups of 20-25. Homogenates
were extracted with Freon and the aqueous layer was recovered and either used
immediately or stored frozen at 80°C. Alternatively, embryos were
recovered, the excess liquid removed, and the embryos stored at
80°C until used to generate lysates. For SDS-PAGE/immunoblot
analysis, 20 µl of lysate (approximately one embryo equivalent) was mixed
with 5 µl of 5xsample buffer, heated at 90°C for 10 minutes. For
immunoprecipitation analysis, from 100-300 µl of lysate (5-15 embryo
equivalents) were incubated with 0.5-1.0 µl of affinity-purified antibody
for 1-2 hours. Then, 25 µl of protein-A/agarose (Sigma) was added and
incubated overnight at 4°C. Agarose beads were recovered by low speed
centrifugation, washed sequentially in lysis buffer, high salt (50 mM Tris-HCl
pH 7.5, 500 mM NaCl, 0.1% NP-40, 0.05% sodium deoxycholate) and low salt (50
mM Tris-HCl pH 7.5, 0.05% NP-40, 0.05% sodium deoxycholate) buffers, recovered
by centrifugation, and resuspended in 1xSDS-PAGE sample buffer. After
electrophoresis, polypeptides were transferred to membranes. After Ponceau S
visualization, blots were blocked with 5% nonfat dry milk in Tris-buffered
saline with 0.5% Tween 20 (NFTT) for at least 20 minutes. Blots were incubated
for at least 1 hour with primary antibodies diluted into NFTT. The following
dilutions were used: anti-Xß-catenin, 1:2500; antiXTC3c, 1:2000;
anti-XTCF3n, 1:5000; anti-XSOX3c, 1:5000; antiV5, 1:5000 and anti-myc
supernatant, 1:5. Blots were washed three times in 0.1% Tween-20 TBS and then
incubated in goat anti-rabbit/horseradish-peroxidase (HRP) or goat
anti-mouse/HRP secondary antibodies (BioRad) diluted 1:20,000 in TBST, and
washed 3x in TBS-Tween. Bound antibodies were visualized using the
Pierce PicoWestern ECL reagent on Kodak XL1 film. For immunocytochemistry,
embryos were stained following established laboratory protocols
(Dent et al., 1989) (our
current protocol can be found at
http://spot.colorado.edu/~klym/Methods/wholemount.htm).
Cell transfection/luciferase assays
HeLa cells grown in Dulbecco's modified Eagle's Medium (DMEM) (Invitrogen)
supplemented with 10% fetal calf serum and antibiotics were transfected using
FuGene6 (Roche) following the manufacturer's protocol. Typically, 1 µg
pCS2-SOX-expressing plasmid was transfected along with 0.2 µg
pCS2mt-G-Xß-catenin plasmid, 0.2 µg of TOPFLASH plasmid and 0.2
µg pRL-TK plasmid, which expresses Renilla luciferase under the
control of a thymidine kinase promoter (Promega) as a normalization control.
`Empty' pCS2mt plasmid DNA was then added so that, in each experiment, a total
of 2 µg plasmid DNA was transfected. Cultures were lysed 18-24 hours after
transfection in 100 µl of chilled passive lysis buffer (Promega)
supplemented with protease inhibitor cocktail (Roche). 10 µl of lysate was
added to 100 µl of luciferase assay reagent II (Promega) and read for 10
seconds using a Turner TD-20/20 luminometer to obtain the firefly luciferase
reading. 100 µl of Stop-N-Glo (Promega) substrate was then added and a 10
second reading was made to quantify the level of Renilla luciferase.
Data was normalized by dividing the firefly by the Renilla luciferase
readings. All readings were made in duplicate and each assay repeated at least
twice.
Electrophoretic mobility shift assays
Proteins were synthesized using a TnTTM in vitro transcription and
translation kit (Promega) according to the manufacturer's directions. Protein
yield was verified by anti-V5 SDS-PAGE/immunoblot. Labeled DNA probes were
prepared by annealing complementary oligonucleotides followed by end labeling
with 32P-ATP using T4 polynucleotide kinase. Electrophoretic
mobility shift assays were performed using the protocol of Kamachi and Kondoh
(Kamachi and Kondoh, 1993). 2
µl of TnT reaction was incubated with probe in binding buffer (20 mM Hepes
pH 7.9, 100 µM KCl, 16% glycerol, 1 mM DTT, 1 mM EDTA, 0.5 µg salmon
sperm DNA) in a final volume of 12 µl. In antibody supershift experiments,
0.5 µl anti-V5 antibody was added to the reaction. Products were separated
on 4% native polyacrylamide gels, dried and visualized by autoradiography.
Biotinylated-DNA `fishing' analysis
For each biotinylated DNA target, one oligonucleotide was synthesized with
a 5' biotin group, the other was unmodified. For short sequences (such
as DC5, 5'Biot-catggtaggtgagcaacAACAATgaatattt-3'; TCF, 5'
Biot-gtgtcatcagaatcATCAAAGgacctccct-3'; and the distal Xnr5 site,
5'Biot-gtcacctgacattgttgtattGTTTGATgttgc-3') the two
oligonucleotides were annealed together. In DC5 and TCF, SOX/TCF sites are
capitalized; in the distal Xnr5 oligonucleotide, the two SOX sites are
underlined and the TCF site is capitalized. For the longer Siamois
and Xnr5 promoter fragments (200-400 base pairs), the desired
regions were amplified by PCR using a biotinylated and unbiotinylated primer
pair and Vent polymerase (New England Biolabs). All primers were synthesized
by Invitrogen.
Biotinylated DNAs were incubated with streptavidin-agarose beads (Sigma) in
coupling buffer (150 mM NaCl, 25 mM sodium phosphate, pH 6.9) for 1 hour
at room temperature with constant mixing. The beads were then washed twice
with binding buffer (20 mM HEPES, pH 7.9, 50 mM KCl, 5 mM MgCl2,
12% glycerol, 0.5 mM EDTA and 0.1% Triton X-100). 100-300 µl lysate (5-15
embryo equivalents) was made 1x in binding buffer, 1 mM DTT and 0.5
µg ml1 herring sperm DNA. After a 10 minute incubation at
room temperature, 50 µl of DNA-streptavidin-agarose beads were incubated
with the lysate for 10 to 20 minutes at room temperature with constant gentle
mixing. The beads were then recovered by centrifugation and washed twice with
binding buffer, and bound protein was eluted with 2xSDS sample buffer,
denatured and analysed by SDS-PAGE and immunoblot.
Quantitative real time PCR analysis
Total RNA was prepared from groups of five embryos homogenized in 1 ml of
Trizol reagent (Invitrogen). Homogenates were extracted with 200 µl of
chloroform and the upper layer was precipitated in two-thirds of a volume of
isopropanol at 20°C for at least 1 hour. After centrifugation at
4°C, 16,000 g for 15 minutes, the pellet was washed with
75% ethanol, dried and dissolved in 50 µl RNase-free H2O.
Samples were treated with RNase-free DNase I (Ambion) at 37°C for 1 hour
and then purified again using a RNeasy Kit (Qiagen) according to
manufacturer's instructions.
cDNA synthesis was performed from 1 µg purified RNA using random primers
and ImProm-II Reverse Transcription System (Promega) according to the
manufacturer's directions. Real time PCR was carried out using a DNA Engine
Opticon System (M J Research). A 20 µl PCR reaction contains 1xSYBR
Green I nucleic acid gel stain (Molecular Probes), used to quantify amplified
DNA, 1 µl cDNA, 1 µM each upstream and downstream primer, 2 mM
MgCl2, 0.2 mM dNTPs and 1 unit Taq DNA polymerase (Promega). A
standard curve was generated as described in Kofron et al.
(Kofron et al., 2001). A
dilution series (cDNA: H2O=100%, 75%, 50%, 25% and 10%) was made
from uninjected (control) embryo cDNA samples. Each sample was normalized to
the expression level of elongation factor 1
(EF1
). Melting curve
analysis was performed on each specific product. The primer sets used are
listed in the Table 1. The
cycling conditions used are as follows: step 1, 94°C for 4 minutes; step
2, 94°C for 30 seconds; step 3, 55°C or 60°C for 30 seconds
(primer dependent); step 4, 72°C for 30 seconds; step 5, 83°C for 1
second; step 6, plate read; step 7, go to step 2 (34 more times); step 8,
72°C for 10 minutes; step 9, melting curve analysis (60-95°C in
0.5°C increments, 1 second hold for each step); step 10, end.
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Results |
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The level of 35 kDa anti-XSOX3c reactive polypeptide remains stable
throughout early development and declines upon the onset of gastrulation
(Fig. 1F). In unfertilized
eggs, anti-XSOX3c recognizes a set of slower migrating bands
(Fig. 1F); these bands
disappear and are replaced by a 35 kDa band within 20 minutes of fertilization
or following the prick activation of the egg (data not shown). The faster of
these slower-migrating, anti-XSOX3c-reactive bands can sometimes be resolved
in cleavage-stage embryos (Fig.
1F). DNA binding studies indicate that the slower,
anti-XSOX3c-reactive polypeptide bind a SOX3-DNA target sequence (data not
shown). Whether these slower migrating bands are due to the CDK-mediated
phosphorylation of XSOX3 described by Stukenberg et al.
(Stukenberg et al., 1997) has
not yet been determined.
In situ hybridization analysis reveals that the maternal XSOX3
mRNA is concentrated in the animal hemisphere of early cleavage stage embryos
(Penzel et al., 1997)
(Fig. 2A). Whole-mount
immunocytochemistry with anti-XSOX3c revealed intense staining of the animal
hemisphere that was abolished by pre-incubating the antibody with the
peptide-conjugate against which it was raised
(Fig. 2B). XSOX3 appears to be
primarily cytoplasmic in early embryos
(Fig. 2B). By the 64/128-cell
stage, staining is clearly nuclear as well as cytoplasmic and its nuclear
localization becomes increasingly pronounced as development proceeds
(Fig. 2C,F). Cytoplasmic
staining can be see in mitotic cells throughout development
(Fig. 2F). The initial
cleavages that separate animal from vegetal blastomeres occur within the
animal hemisphere (Nieuwkoop and Faber,
1967
), leading to the partitioning of XSOX3 protein to vegetal
blastomeres (Fig. 2C). The
anti-XTCF3 antibodies, anti-XTCF3c (Fig.
2D) and anti-TCF3n (Fig.
2E), produced staining patterns that were similar to each other,
and to the pattern seen with anti-XSOX3c. Anti-XSOX3c stained nuclei are found
in all regions of the embryo through gastrulation, including the most vegetal
cells located within the yolk plug of the gastrula stage embryo
(Fig. 2G).
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LEF/TCF and SOX proteins bind to the core consensus binding site
5'-(A/T)(A/T)CAA(A/T)G-3', although optimal binding sites are
likely to be 10-12 base pairs long
(Klymkowsky, 2004)
(Mertin et al., 1999
;
van Beest et al., 2000
) (see
below). We examined the effects of the mutations on the binding of XSOX3 to
the consensus sequence 5'-ATTGTT-3' found within DC5, an enhancer
element found in the chick
-crystalline promoter
(Kamachi et al., 1998
;
Kamachi et al., 1999
). All
mutated versions of XSOX3, except m55, abolished the polypeptide's affinity
for the DC5 oligonucleotide in gel shift assays
(Fig. 3D,E). There was no
apparently binding of wild-type or mutated XSOX3 to the TCF binding sequence
5'-ATCAAAG-3' or to the TCF sites present in the Siamois
promoter (Brannon et al., 1997
)
under these conditions (data not shown).
In vivo analyses of mutated XSOX3 polypeptides
The TOPFLASH reporter (Korinek et al.,
1997) is widely used to assay Wnt and ß-catenin-regulated TCF
transcriptional activation (Williams et
al., 2000
). In HeLa cells, a mutationally stabilized form of
Xenopus ß-catenin strongly activated TOPFLASH; this activation
was suppressed by the coexpression of wild-type XSOX3-V5H6
(Fig. 4A). ß-Catenin did
not activate the FOPFLASH reporter, which lacks TCF-binding sites (data not
shown). All mutated forms of XSOX3 suppressed the ß-catenin-induced
activation of TOPFLASH (Fig.
4A). Western blot analysis revealed that these plasmids lead to
similar levels of exogenous protein accumulation (data not shown). This result
supported the hypothesis that XSOX3 acts to suppress ß-catenin-mediated
activation of TOPFLASH by binding to ß-catenin.
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Xnr5 as a target of XSOX3 regulation
Yang et al. (Yang et al.,
2002) reported that zygotic expression of the Nodal-related genes
Xnr5 and Xnr6 was detectable at the 256-cell stage, well
before the mid-blastula transition and significantly before other known
ß-catenin-regulated dorsalizing genes (e.g. Siamois, Twin and
Xnr3). Xnr5 is expressed throughout the vegetal region of
early to mid-blastula stage embryo
(Takahashi et al., 2000
) in a
VegT- and ß-catenin-dependent fashion
(Rex et al., 2002
;
Yang et al., 2002
). A
200bp minimal Xnr5 promoter sequence has been characterized
(Hilton et al., 2003). A reporter plasmid in which this Xnr5 promoter sequence
is used to drive firefly luciferase expression was generously supplied to us
by E. Hilton and R. Old. In our hands, the reporter was expressed somewhat
more actively in dorsal than in ventral blastomeres
(Fig. 6A) and was more active
in vegetal than in animal hemispheres (Fig.
6B). Both wild-type and m7 versions of XSOX3 RNAs
activated the Xnr5-luciferase reporter in dorsal, ventral
(Fig. 6C), animal and vegetal
(Fig. 6D) blastomeres.
Expression of the m8 version of XSOX3 had no effect on Xnr5 reporter
activity (Fig. 6C,D). This
pattern of activity correlates with the activity of these polypeptides in
ventralization and other in vivo assays (see above).
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To determine whether XSOX3 binds to the off-consensus TCF/LEFA site, we generated four mutated forms of the region (Fig. 6E). MUT1 removes the SOXa site but leaves the SOXb and TCF sites intact. MUT2 removes the SOXb and TCF sites while leaving the SOXa site intact. MUT3 and MUT4 remove both SOX sites. The orientation of the TCF site is ambiguous; it could be either 5'-TTGTTTG-3', which is similar to the TCF/LEFB site, or 5'-GTTTGAT-3'. MUT4 was designed to resolve its orientation. DNA fishing with these mutated SOXab-TCF/LEFA sequences (Fig. 6F) indicates that XSOX3 can bind to either SOXa or SOXb sites, although it is not clear whether both sites can be occupied simultaneously. XSOX3 does not appear to bind significantly to the TCF/LEFA site. Similarly, XTCF3 appears to bind to the TCF/LEFA site but not the SOX sites. The binding of XTCF3 to the MUT4 sequence suggests that the site it oriented 5'-GTTTGAT-3', although it is also possible that XTCF3 can bind to the TTGTTTGAT sequence in either orientation. Studies are ongoing to determine whether XSOX3 and XTCF3 can bind simultaneous to this DNA.
To confirm that the SOXab sites are responsible for XSOX3's effects on the Xnr5 reporter, we generated a mutant form of the reporter that carries the MUT4 sequence. The MUT4-Xnr5 reporter was no longer responsive to XSOX3, whereas removal of the TCF/LEFB site (ATGAAAG mutated to ATGCACG) had no effect on its activation by XSOX3 (Fig. 6G). Removal of the TCF/LEFA site (GTTTGAT mutated to GCTCGAT) also removed the SOXb site and produced a partial reduction in the reporter's response to XSOX3 (Fig. 6G). These studies indicate that the responsiveness of the Xnr5 reporter to XSOX3 is due solely to the presence of the SOXab sites.
We used DNA fishing to reexamine the relative binding affinities of wild-type, m7 and m8 forms of XSOX3 to the SOXab-TCF/LEFA Xnr5 promoter sequence (Fig. 7A). Fertilized eggs were injected with RNAs and lysates were prepared from stage 8 embryos. Wild-type and m7 polypeptides bound to DC5 and Xnr5-derived SOX-TCF sequences; under these condition, binding of the m8 polypeptide was clearly reduced compared with the binding of the wild-type and m7 polypeptides.
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Endogenous XSOX3 acts as a repressor of Xnr5
B1 type SOXs such as XSOX3 are commonly assumed to act as transcriptional
activators (Bowles et al.,
2000; but see Graves,
1998
). Injection of XSOX3 RNA decreases Xnr5 RNA
levels (Fig. 7B,
Fig. 8A,B). If endogenous XSOX3
represses Xnr5 expression, we would predict that depletion of XSOX3
protein would lead to an increase in Xnr5 RNA levels. Embryos
injected with a morpholino directed against the 5' untranslated region
and the translation initiation region of the XSOX3 mRNA
(Fig. 9A) show a decrease in
XSOX3 but not XTCF3 polypeptide levels, as determined by western blot
(Fig. 9B). A morpholino
directed against the analogous region of XSOX7 mRNA
(Fig. 9A) had no effect on
XSOX3 protein levels when injected into fertilized eggs
(Fig. 9B). Xnr5 RNA
levels were found to increase modestly in XSOX3 morpholino-injected embryos
and were unaltered by the injection of XSOX7 morpholino
(Fig. 9C).
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Discussion |
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The SOX/LEF/TCF protein family is phylogenetically ancient and appears to
have been present in the last common ancestor of the metazoans
(Bowles et al., 2000). There
are four known LEF/TCFs in vertebrates TCF1, LEF1, TCF3 and TCF4
three of which (TCF1, TCF3 and TCF4) are present as maternal RNAs in
Xenopus. LEF1 (Hovanes et al.,
2000
), TCF1 (van de
Wetering et al., 1996
) and TCF4
(Duval et al., 2000
;
Young et al., 2002
)
transcripts occur in alternatively spliced variants that produce polypeptide
variants. The activity of LEF/TCF appears to be determined by promoter context
and associations with accessory factors. For example, all LEF/TCFs associate
with Groucho-related co-repressors
(Brantjes et al., 2001
), as
well as with ß-catenin, whose C-terminal domain appears to act as a
co-activator (Vleminckx et al.,
1999
; Williams et al.,
2000
).
The SOX proteins are grouped based on the similarity of their HMG-box DNA
binding domain to that of the sex-related on the Y
(SRY) testis-determining gene of mammals
(Graves, 2001). Of these, the
B-type SOX proteins appear to be the most phylogenetically ancient and highly
conserved (Bowles et al.,
2000
). SRY has been suggested to have evolved from SOX3
(Foster and Graves, 1994
;
Stevanovic et al., 1993
). The
B-type SOXs have been divided into two subtypes, B1 and B2, which can be
distinguished by conserved amino acids at positions 2 and 79 of the 80 amino
acid long HMG box both types are present the arthropod Drosophila
melanogaster, the nematode Caenorhabditis elegans, the
hemichordate Ptychodera flava and the chordate Ciona
intestinalis (Bowles et al.,
2000
; Kirby et al.,
2002
; Taguchi et al.,
2002
; Yamada et al.,
2003
).
B-type SOX proteins are expressed early in the development of many
organisms. In the mouse, maternal SOX2 protein persists into the early
blastocyst; it is located cytoplasmically in cells of the trophectoderm and is
primarily nuclear in the cells of the inner cell mass. SOX2 mRNA
appears in morula stage embryos (2.5 days post-coitum) and is restricted to
cells of the inner cell mass (Avilion et
al., 2003). SOX3 is expressed together with SOX2 in the cell of
the epiblast at day 5.5 (Avilion et al.,
2003
; Wood and Episkopou,
1999
). SOX3 is expressed early during embryonic development in the
chick (Rex et al., 1997
).
Although Koyano et al. (Koyano et al.,
1997
) originally reported that XSOX3 was expressed during
oogenesis but was absent from eggs and early embryos, it is now clear that the
gene is expressed maternally and is present throughout early blastula stages
(Penzel et al., 1997
)
(Fig. 1F,
Fig. 2A-C). Preliminary studies
indicate that SOX3-like polypeptides are also supplied maternally in the
zebrafish (M.W.K. and K. B. Artinger, unpublished). In all vertebrates
examined to date, SOX3 is expressed zygotically in the developing neural tube
(Graves, 2001
).
Whole-mount immunocytochemistry reveals that the XSOX3 polypeptide is
initially cytoplasmic and becomes restricted to nuclei as development proceeds
(Fig. 2B,C). Cytoplasmic XSOX3
can be seen in cells captured in the process of mitosis
(Fig. 2F). Whether XSOX3
actively shuttles between cytoplasm and nuclei, as has recently been described
for mouse SOX10 (Rehberg et al.,
2002), remains to be seen, although we have seen evidence for
cytoplasmic XSOX3 in neurula stage embryos (data not shown). XSOX3
mRNA appears to be largely restricted to the animal hemisphere
(Fig. 2A) (Penzel et al., 1997
).
However, the nuclei generated during the first three cleavages lie within
animal hemisphere and all blastula stage nuclei, including the most vegetal
cells of the yolk plug, appear to contain SOX3 polypeptide
(Fig. 2G). We have not
quantified the amounts of XSOX3 per nuclei in animal and vegetal blastomeres,
although a superficial examination suggests that XSOX3 concentrations are
higher in the animal hemisphere. Immunocytochemical analyses with antibodies
directed against XTCF3 reveals a similar pattern of distribution through the
blastula stages of development (Fig.
2D,E).
Based on DNA and protein binding studies, Zorn et al.
(Zorn et al., 1999) concluded
that SOX exerted its ability to inhibit ß-catenin-mediated dorsal axis
formation by competing with endogenous TCFs for binding to ß-catenin. We
choose to extend those studies by formally eliminating the possibility the
XSOX3 was acting through its ability to bind to DNA. We generated a series of
six mutations in the XSOX3 HMG box domain
(Fig. 3A-C). We saw no obvious
effect of these mutations on the interaction between XSOX3 and ß-catenin
(data not shown) (see Fig. S1 at
http://dev.biologists.org/supplemental/).
When tested by conventional electrophoretic mobility gel shift assay, DNA
binding to the DC5 SOX consensus sequence was abolished by five of six
mutations and was reduced in the sixth (m55)
(Fig. 3E).
The TOPFLASH reporter is widely used as an assay for
ß-catenin-regulated TCF-mediated gene expression; for example, it was
used by Takash et al. (Takash et al.,
2001) as evidence for the ability of human SOX7 to modulate
ß-catenin activity. In the course of our studies of SOX/catenin
interactions, we have found several examples in which activity in the TOPFLASH
assay does not correlate with activity in the Xenopus embryonic
ventralization assay. For example, even though XSOXD inhibits TOPFLASH
activation by ß-catenin in cultured mammalian cells, it does not suppress
ß-catenin-induced axis duplication, nor can it ventralize
Xenopus embryos (Fig.
8H) (Klymkowsky,
2004
), suggesting that the two assays measure distinct facets of
the interaction between ß-catenin, SOXs and target genes.
Although all of the mutated forms of XSOX3 we analysed in this study
inhibited the ß-catenin activation of the TOPFLASH reporter
(Fig. 4A), they differed
dramatically in their ability to ventralize embryos
(Fig. 4D;
Table 2). Because they lie
adjacent to one another, we focused our analysis on the m7 and m8 mutations
m7 behaves very much like the wild-type XSOX3 polypeptide, whereas m8
appears to be inactive, although it accumulates to levels similar to that seen
for wild-type and m7 polypeptides in embryos, cultured mammalian cells and in
vitro protein synthesis extracts. The XSOX3 m7 and m8 mutations are analogous
to mutations made in mouse SOX2 by Scaffidi and Bianchi
(Scaffidi and Bianchi, 2001).
Their m7-like M47I mutation had little effect on DNA binding affinity or
bending, whereas the m8-like N48G mutation reduced DNA binding affinity more
than tenfold and DNA bending by
40°. When tested for binding to
target sequences in embryonic lysates, a similar difference in apparent
DNA-binding affinity was seen for the m7 and m8 mutant forms of XSOX3
(Fig. 7A). Based on these
differences, we conclude that differences in their DNA binding affinity are
responsible for the differences in the ventralizing activities of the two
polypeptides.
Dorsal-determination system and XSOX3
We began our analysis of the mode of XSOX3 action with the knowledge that
its overexpression ventralized embryos
(Fig. 4C,D) and inhibited
ß-catenin dorsal axis duplication
(Zorn et al., 1999). However,
where along the dorsalization pathway XSOX3 acts was unclear. It is known that
the cortical rotation establishes a cytoplasmic asymmetry within the
fertilized eggs that manifests itself in the blastula-stage embryo as
asymmetries in gene expression that underlie the initial
dorsal-ventral/organizer-contraorganizer axis. The best established of these
rotation-induced cytoplasmic asymmetries is the asymmetry in ß-catenin.
Over the past few years, several target genes regulated by ß-catenin
asymmetry have been identified. In the case of Siamois, Twin, Xnr3
and Xbra, the initial expression of these genes begins following the
mid-blastula transition, when embryos consist of
4000 cells. We found no
evidence, however, for the binding of XSOX3 to sites within the
Siamois promoter (Fig.
5D). Because the sequences of the TCF binding sites in
Siamois are similar to those found in Twin, Xnr3 and
Xbra, and are distinct from the sequences recognized by SOX3
(Klymkowsky, 2004
)
(Fig. 6E), we were unable to
explain the difference between the activity of m7 and m8 forms of XSOX3 in
terms of DNA binding to this specific set of target genes. We therefore
suspect that these effects are indirect, but it remains a formal possibility
that, in the context of intact chromatin, XSOX3 is more promiscuous in its DNA
binding than it is on the naked DNA probes used in our studies. We are
currently exploring this possibility using chromatin immunoprecipitation.
It was in this light that the observation that Xnr5 and
Xnr6 are expressed in a ß-catenin/TCF dependent manner as early
as the 256-cell stage (Yang et al.,
2002) was particularly resonant. Xnr5 and Xnr6
encode Nodal-related proteins, members of the TGFß family of secreted
signaling molecules (Agius et al.,
2000
; Jones et al.,
1995
; Whitman,
2001
; Zhou et al.,
1993
). A network of Nodal-related proteins is involved in the
patterning of the early embryo and the determination of left-right asymmetry
(Branford and Yost, 2002
;
Levin and Mercola, 1998
;
Onuma et al., 2002
;
Osada and Wright, 1999
;
Rex et al., 2002
;
Takahashi et al., 2000
).
Our immunochemical studies (Figs 1, 2) indicate that XSOX3 is abundant in 256-cell embryos. The connection between XSOX3 and Xnr5 was made possible by the isolation of a minimal promoter fragment of the Xnr5 gene (Hilton et al., 2003). The TCF/LEF sites within this promoter fragment differ from the conventional consensus TCF/LEF sequence (see above) and we originally hypothesized that XSOX3 might bind to these sites. However, a closer look at the Xnr5 promoter sequence (Fig. 6E) revealed the presence of two consensus SOX binding sites, which we termed SOXa and SOXb. DNA fishing experiments indicate that XSOX3 can bind to either of these sites but not to the TCF/LEF site (Fig. 6F). Removing the SOX sites abolished the Xnr5 reporter's responsiveness to exogenous XSOX3, whereas removing the TCF binding sites did not (Fig. 6G). Whether XSOX3 and XTCF3 can bind concurrently to this region of the Xnr5 promoter is currently under study.
Although both Siamois and Xnr5 reporters respond to the injection of XSOX3 RNA, the direction of the response is the opposite of what would be predicted based on the ventralizing activity of XSOX3. We currently have no compelling explanation for this anomalous behavior except to suggest that the promoter plasmids might form configurations distinctly different from those that occur within endogenous chromatin. Given that the binding of a SOX induces a dramatic 80-130° bend in DNA, subtle differences in DNA organization and accessory proteins could lead to dramatic differences between reporters and endogenous genes. Both exogenous and endogenous XSOX3 regulate endogenous genes in a manner consistent with their ability to ventralize embryos. XSOX3 overexpression inhibits the VegT-induced expression of Xnr5 in animal caps (Fig. 7B) and the ß-catenin-induced expression of Siamois in UV-ventralized embryos (Fig. 5C).
The action of XSOX3 on Xnr5 RNA accumulation is one of repression.
This conclusion is supported by the effects of chimeric forms of the XSOX3
polypeptide on Xnr5 RNA levels in the embryo
(Fig. 10C). Expression of a
chimeric form of XSOX3 that contains a viral transcription activation domain
leads to an increase in Xnr5 RNA accumulation, whereas a chimeric
form of XSOX3 that contains a transcription repressor domain behaves like the
wild-type protein (Fig. 10C).
B1-type SOX proteins such as SOX3 are often assumed to be transcriptional
activators (Uchikawa et al.,
1999; Bowles et al.,
2000
). SOX3 has been proposed to act as a transcriptional
repressor of SOX9 (Graves,
1998
), although no direct molecular data has been supplied to
support this contention.
Beginning with the observation that injection of int-1
(Wnt1) RNA induced axis duplication in Xenopus
(McMahon and Moon, 1989),
RNA-based overexpression studies have been invaluable in elucidating the
mechanism of axis formation in particular and signaling pathways in general.
At the same time, the relationship of such studies to the normal developmental
processes is not necessarily straightforward. It was in this light that the
results of downregulating XSOX3 activity (Figs
9,
10) are particularly crucial.
We injected a morpholino that suppressed the accumulation of XSOX3 protein
(Fig. 9A,B) and the anti-XSOX3c
antibody, which inhibits XSOX3 DNA binding
(Fig. 10A,B) to examine the
role of endogenous XSOX3. In each case, the results were consistent with the
hypothesis that endogenous XSOX3 acts to suppress the accumulation of
Xnr5 RNA (Fig. 9C,
Fig. 10C).
The embryonic phenotypes associated with these two reagents (XSOX3
morpholino and anti-XSOX3c antibody) are quite different. We find little if
any overt effect from the injection of the XSOX3 morpholino, even though it
produces a clear decrease in XSOX3 protein levels by late blastula stages
(Fig. 9B). We attribute this
result to the maternal nature of the SOX3 protein, the early expression of the
XSOX3-regulated target genes and, later in development, to the expression of
compensatory SOX proteins, particularly XSOX2
(Avilion et al., 2003) (A. A.
Avilion, unpublished). By contrast, injection of the anti-XSOX3c antibody
produces dramatic, complex phenotypes (C.Z. et al., unpublished). The effects
of the injected anti-XSOX3c antibody appears to involve a number of distinct
gene targets. XSOX3 appears to regulate eFGF/FGF4 RNA levels
positively (C.Z. et al., unpublished) while decreasing Xnr5 and
Xnr6 RNAs (as reported here). Nevertheless, the data presented here
clearly support a mechanism in which maternal XSOX3 inhibits the
ß-catenin-mediated process of dorsal axis specification by directly
repressing animal hemisphere expression of very early zygotic gene
Xnr5.
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ACKNOWLEDGMENTS |
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Footnotes |
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* These authors contributed equally to this work
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