1 Department of Oncology, The Hutchison/MRC Research Centre, University of
Cambridge, Hills Road, Cambridge CB2 2XZ, UK
2 Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge,
Tennis Court Road, Cambridge CB2 1QR, UK
Author for correspondence (e-mail:
so218{at}cam.ac.uk)
Accepted 10 September 2004
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SUMMARY |
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Key words: Smad2, Mesoderm induction, PIASy, Xenopus
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Introduction |
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The importance of Smad2 in mesoderm induction and patterning was
demonstrated in Xenopus embryos by observations that inhibition of
Xenopus Smad2 (XSmad2) at the dorsal marginal zone (DMZ) results in
loss of expression of mesodermal markers in association with a defect in
dorsal structure. Furthermore, activation of XSmad2 induces mesoderm markers
in a dose-dependent manner and the formation of a secondary axis
(Graff et al., 1996;
Hemmati-Brivanlou and Melton,
1992
; Hoodless et al.,
1999
). During mesoderm induction and patterning, the Smad2 mRNA
and protein are widely distributed mainly in the animal half of
Xenopus embryos. However, interestingly, activation of endogenous
XSmad2, which is monitored by phosphorylation of XSmad2, occurs at the
marginal zone in a dorsal-to-ventral direction
(Faure et al., 2000
;
Lee et al., 2001
;
Schohl and Fagotto, 2002
). To
explain this localized activation, a morphogen model of activin-like ligands
is widely accepted (Green et al.,
1992
; Gurdon and Bourillot,
2001
; McDowell and Gurdon,
1999
). In this model, locally activated secreted factors diffuse
in the embryo and induce specific fates and proper patterning of embryos in a
concentration-dependent manner. However, the activation of endogenous XSmad2
is excluded from the prospective ectoderm at the animal pole. This spatial
exclusion permits formation of ectoderm at the animal pole, by a mechanism
that is not well understood.Application of activin-like ligands to animal caps
between late blastula and early gastrula stages converts prospective ectoderm
into mesodermal fate by activation of Smad2
(Eppert et al., 1996
;
Graff et al., 1996
). However,
if activin-like ligands are applied to animal caps before or after these
stages, their application does not induce mesoderm, suggesting that competence
towards XSmad2 is temporally and spatially regulated
(Grimm and Gurdon, 2002
;
Lee et al., 2001
). Despite the
well-characterized mechanism of mesoderm induction and patterning, it is not
yet clear how this temporally and spatially restricted competence, which is
required for the precise patterning of germ layers, is regulated. Nor is it
understood what prevents the vegetally produced activin-like signal from
inducing mesoderm all the way up into the animal hemisphere.
The Smad2 pathway is activated by activin-like molecules of the
transforming growth factor ß (TGFß) superfamily
(Kofron et al., 1999;
Osada and Wright, 1999
;
Thomsen and Melton, 1993
)
through their receptor-mediated phosphorylation of Smad2. The activated Smad2
makes a complex with Smad4 and then translocates from the cytoplasm to the
nucleus. By recruiting other transcription activators such as FAST1, the
Smad2-Smad4 complex activates transcription of target genes
(Baker and Harland, 1996
;
Chen et al., 1997
;
Graff et al., 1996
;
Green et al., 1992
;
Harland and Gerhart, 1997
;
Horb and Thomsen, 1997
;
LaBonne and Whitman, 1994
;
Nomura and Li, 1998
). Smad2
protein has three major domains: MH1, linker and MH2 domains (reviewed by
Fortuno et al., 2001
;
Lutz and Knaus, 2002
). The MH1
and MH2 domains are highly conserved in members of the Smad family. The MH1
region has a role in autoinhibition by physically interacting with the MH2
domain (Kim et al., 1997
). The
linker regions among Smad proteins have diverse structures but are well
conserved through evolution. The linker domain of Smad2 has three serine
phosphorylation sites, which regulate its nuclear exclusion and contribute to
the competence of precursor cells to activin-mediated mesoderm induction
(Grimm and Gurdon, 2002
). The
MH2 domain is crucial for regulation of its activity and has three
phosphorylation sites for ligand-mediated activation
(Abdollah et al., 1997
). So
far, many Smad2-interacting proteins, including receptors
(Ro et al., 1995
), other Smad
proteins (Wu et al., 1997
),
and many positive and negative transcription factors [such as FAST and Mixer
(Germain et al., 2000
;
Watanabe and Whitman, 1999
;
Yeo et al., 1999
), Swift
(Shimizu et al., 2001
),
p300/CBP (Janknecht et al.,
1998
; Pouponnot et al.,
1998
), Ski and SnoN
(Macias-Silva et al., 2002
;
Stroschein et al., 1999
)] have
been identified mainly from studies using mammalian cell lines. Each protein
interacts with a specific domain of Smad2 and functions at a specific position
in the Smad2 signalling pathway. However, their regulatory mechanisms during
early development still remain to be solved.
In order to understand the regulation of the Smad2 complex and the
importance of Smad2 regulation in early embryogenesis, we performed a yeast
two-hybrid screen using XSmad2 as a bait and have identified Xenopus
PIASy (protein inhibitors of activated STAT y), which is a member of the PIAS
family. Recently, members of the PIAS family have been shown to interact with
several transcription factors, including Smad1, Smad2, Smad4, Lef1 and
androgen receptors, and to be involved in their modification with SUMO (small
ubiquitin-like modifier) and transcriptional regulation of the interacting
proteins in mammalian cell lines
(Jimenez-Lara et al., 2002;
Kahyo et al., 2001
;
Lin et al., 2003a
;
Long et al., 2003
;
Sachdev et al., 2001
).
However, its role and physiological targets in development remain to be
elucidated. Therefore, in this paper, we have analyzed the role of XPIASy in
mesoderm induction and patterning using Xenopus embryos by gain- and
loss-of-function approaches. Our analysis has revealed that XPIASy negatively
regulates transcription activity of XSmad2 as a main physiological target
during mesoderm induction by their direct interaction, but not by its
SUMOylation activity. Moreover, we have found that transcription of XPIASy is
positively and negatively regulated by XSmad2 and ß-catenin,
respectively, and, consistent with this regulation, endogenous XPIASy
expression is largely overlapping with that of Smad2 in the animal half of
embryos, but its expression in the DMZ is significantly reduced at the stage
of dorsal mesoderm induction. These observations provide a possible mechanism
by which XPIASy ensures the zone of Smad2 activation required for mesoderm
induction and patterning: by its developmentally regulated expression and by
the inhibition of Smad2 activity in appropriate regions and with appropriate
timing.
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Materials and methods |
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The yeast two-hybrid screen
The C-terminal region of XSmad2 (amino acids 180 to 432) was subcloned into
a pBTM116 bait vector (Vojtek et
al., 1993). The yeast two-hybrid screening was performed as
described (Shimizu et al.,
2001
) using a Xenopus egg cDNA library as many components
of the TGFß signalling pathway are maternally expressed
(Horb and Thomsen, 1997
;
Koyano et al., 1997
;
Shimizu et al., 2001
).
Constructs
The XSmad2-myc construct and the XSmad1 cDNA are from Dr J. Graff. The
dominant-negative XSmad2 (P445H) was constructed by PCR mediated mutagenesis.
XSmad1-pCS2 construct was made by PCR. The XSmad4-myc constructs are from
Prof. E. Nishida, and T7-mPIASy is from Dr Grosschedl. The wild-type and
deletion constructs of XPIASy were made using pCS2+ derivatives or the pACTII
vector. For in situ hybridization, the cDNAs were subcloned into the pBSSK+
vector (Stratagene). The Xenopus ß-catenin and Tcf3 constructs
were gifts from Dr Van de Wetering. pLexA-Smad2 (amino acids 180 to 432),
pLexA-Ras (G12V), pACTII-HK-Swift and pVP-Raf were prepared as described
(Shimizu et al., 2001).
Flag-tagged SUMO-1 construct was made using Xenopus SUMO-1 cDNA
(AW767329) and pCS2+.
mRNA injection and animal cap assay
Capped mRNAs were produced from linearized constructs using the relevant
promoter according to the manufacturer's instructions (mMessage mMachine,
Ambion). The mRNAs were injected at the given concentrations into the
indicated regions. For the animal cap assay, the embryos were grown until
stage 8 in 0.1xMBS and the animal caps were dissected in 1xMBS.
The caps were then cultured in 0.5xMBS either up to stage 10.5 and used
for RT-PCR analysis or western blotting, or up to stage 27 to analyse animal
cap morphology.
In situ hybridization
Digoxigenin-labelled antisense RNA probes were produced from the
corresponding constructs. Hybridizations were performed on whole embryos
according to standard protocols (Harland,
1991).
Immunoprecipitation and western blotting
Lysate of injected embryos or animal caps in a modified RIPA buffer was
pre-cleared with Protein G fast flow agarose (Sigma) for 1 h at 4°C. The
supernatant was then subjected to incubation with anti-Flag (M2, Sigma),
anti-Myc (9E10) or T7 antibody (Novagen) for 2 hours at 4°C followed by a
1 hour incubation with protein G agarose. Immunoprecipitated samples were
separated by SDS-PAGE gel. The blotted membrane was probed with primary
antibodies [anti-Smad2/3 (BD transduction lab), anti-Myc, anti-T7 or anti-Flag
antibody] and then with secondary antibody (HRP-conjugated anti-mouse
IgG).
Semi-quantitative RT-PCR analysis
cDNAs were made from the extracted mRNA as described
(Daniels and Brown, 2001). All
primers, except for XSmad2, XPIASy and Xvent1, were used
according to previous publications
(Gawantka et al., 1998
;
Xanthos et al., 2002
). The
primer sequences for XSmad2 are: forward,
5'-agtcatcatgaactgaaagc-3'; reverse,
5'-ggttccgaataggtgacagg-3'. For XPIASy: forward,
5'-agcctatcacatcatgcacc-3'; reverse,
5-'caatctctgtaatagctcgg-3'. The primers for Xmsx1 are based
from
http://www.hhmi.ucla.edu/derobertis/index.html.
Quantitative ranges were determined before the final analysis. All reactions
were normalized against ODC product.
Luciferase assay
The reporter construct (50 pg) of pARE-luc or p3TP-lux and mRNAs indicated
were injected into both blastomeres at the two-cell stage. For analysis using
animal caps, the animal caps were dissected at stage 8. The luciferase
activity was measured using caps at stage 10.5 according to the manufacturer's
protocol (Dual-Luciferase, Promega).
Morpholino
The sequences of XPIASy related morpholino are indicated in
Fig. 5A. The control morpholino
is 5'-cctcttacctcagttacaatttata-3'.
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Results |
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Expression of XPIASy overlaps with that of XSmad2
The evidence that XPIASy binds with XSmad2 suggests that XPIASy may be
involved in XSmad2-mediated developmental events such as mesoderm induction.
However, the developmental roles of PIASy have not been studied well except
for that of Drosophila PIASy in eye development
(Betz et al., 2001;
Hari et al., 2001
). To obtain
an insight into the developmental role of XPIASy, its expression pattern was
analyzed by RT-PCR and in situ hybridization, and compared with that of
XSmad2. First, semi-quantitative RT-PCR of whole embryos at different
stages was performed (Fig. 2A).
Before mid-blastula translation (MBT), maternal mRNA of XPIASy is
strongly observed. After MBT, the level of XPIASy mRNA is quickly
downregulated, while the level of XSmad2 is more gradually downregulated
(Howell et al., 1999
;
Howell et al., 2001
;
Schohl and Fagotto, 2002
). The
expression of XPIASy remains constant throughout late development
until stage 34 and then is activated again after stage 38. Next, to analyze
the spatial distribution of XPIASy at early gastrulation, we
dissected out the animal pole, vegetal pole, DMZ and ventral marginal zone
(VMZ) from stage 10 embryos, and XPIASy expression was analyzed by
semi-quantitative RT-PCR (Fig.
2B). The analysis revealed that XPIASy is largely
distributed in the animal side and the VMZ with less expression in the vegetal
side and the DMZ (Fig. 2B).
XSmad2 is also highly expressed in the animal side but its expression
at the VMZ is slightly less than the DMZ
(Fig. 2B). In situ
hybridization of Xenopus embryos was carried out to elucidate in more
detail the temporal expression patterns of XSmad2 and XPIASy
(Fig. 2C). At early stages, the
maternal mRNA of XPIASy is detected to the animal side
(Fig. 2C, part a). At the
neurula stage, XPIASy exclusively distributes within the neural
ectoderm, with strong expression in the anterior region including the eye
primordium (Fig. 2C, parts
b,c). Later in the development, its expression in neural tissues continues and
the expression in the eye continues to be strong
(Fig. 2C, parts d-g). This
restricted expression pattern is very similar to that of XSmad2
(Fig. 2C, parts h-n). These
synchronized expression patterns of XPIASy and XSmad2
support the idea that they functionally interact with each other during
embryogenesis.
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Smad2 activates many downstream targets, most of which are important for
dorsal mesoderm induction. Thus, to confirm the activity of XPIASy as a
negative regulator of XSmad2, we analyzed its effect on expression of
downstream targets induced by 0.5 ng of XSmad2 mRNA co-injection in
animal caps using semi-quantitative RT-PCR analysis and the luciferase assay.
The downstream targets include Chordin (dorsal mesoderm marker)
(Sasai et al., 1995),
Xenopus nodal related 1 (Xnr1)
(Lowe et al., 1996
),
Xbrachyury 1 (Xbra1, pan-mesoderm marker)
(Cunliffe and Smith, 1992
) and
Mix.2 (early mesoderm and endoderm marker)
(Vize, 1996
). XSmad2 induces
expression of these mesoderm markers (Fig.
3D-a). As expected, XPIASy co-expression strongly inhibits the
induction of their transcriptional levels even at a relatively low
concentration of XPIASy, whereas target genes in the Smad1/5 pathway
(BMP4, Xvent1, and msx1) are not affected by XPIASy
overexpression (Fig. 3D, part
a) (Kim et al., 1998
;
Xanthos et al., 2002
). In
addition, activin-mediated activation of mesoderm markers is inhibited by
XPIASy (Fig. 3D, part b). Next,
the effect of XPIASy on transcriptional activity of XSmad2 was analyzed using
reporter constructs of p3TP-luciferase and pARE-luciferase (Mix.2
promoter region), which respond specifically to Smad2, Smad3 and Smad4
activities, but not to Smad1 or ß-catenin
(Carcamo et al., 1995
;
Yeo et al., 1999
). As shown
in Fig. 3E, XSmad2-mediated
activation of luciferase activity in whole embryos or animal caps was strongly
inhibited by XPIASy in both reporter constructs.
Overexpression of XPIASy in the DMZ suppresses dorsalization of embryos,
which is similar to the inhibition of XSmad2
(Fig. 3A, parts f,g) and can be
largely rescued by co-overexpression with XSmad2
(Fig. 3B, part b). However,
there is a small difference between embryos injected with XPIASy
(Fig. 3A, parts a-d) and
dominant-negative Smad2 (Fig.
3A, parts f,g). In XPIASy-injected embryos, head formation is
slightly inhibited, while Smad2 inhibition does not affect head formation.
Mouse PIASy was reported in cell culture experiments to inhibit activity of
LEF1, a downstream component of the canonical Wnt pathway
(Sachdev et al., 2001). It is
well known that the activation of the maternal Wnt pathway is required for
dorsal mesoderm induction, in particular for head induction
(Sokol et al., 1991
).
Therefore, in order to determine whether XPIASy also regulates the
transcriptional activity of the Wnt pathway, the expression of Wnt targets,
Siamois and Xnr3 was analyzed
(Xanthos et al., 2002
).
Injection of XPIASy itself cannot induce expression of these markers
in animal caps (data not shown). However, XPIASy weakly inhibits
ß-catenin induced expression of the Wnt targets Siamois, Xnr3
and Chordin (Fig. 3D,
part c), although much higher concentrations of XPIASy are required than that
for downregulation of XSmad2 target genes, suggesting that XSmad2 is the
primary target of XPIASy in Xenopus embryogenesis.
Even though a large amount of XPIASy was injected, the effect on the head
formation was much weaker than the phenotype induced by inhibition of Wnt
activity, which frequently shows a headless phenotype
(Brannon et al., 1999;
Heasman et al., 2000
). To
further confirm that the phenotype induced by XPIASy is mainly caused
by inhibition of XSmad2 and not by effect on Wnt signalling, we compared the
morphology of XPIASy injected embryos with those injected with a
ß-catenin morpholino (Heasman et al.,
2000
; Hino et al.,
2003
). The inhibition of ß-catenin at the DMZ results in
headless embryos with normal axes, which are different from the phenotype
induced by XPIASy (Fig. 3A,
parts a-d). Next, to determine the degree of involvement of the Wnt pathway, a
rescue experiment was performed using wild-type Xenopus
ß-catenin. As shown in Fig.
3B, parts c,d, and Table
2, these constructs could hardly rescue the XPIASy phenotype.
Moreover, in activin-treated animal caps, inhibition of the canonical Wnt
pathway, which includes TCF/LEF1 transcription factors, hardly inhibits the
induced elongation (Tada and Smith,
2000
). However, as discussed above, XPIASy does effectively
inhibit the elongation (Fig.
3C). These observations clearly indicate that XPIASy primarily
functions as an inhibitor of XSmad2 in association with a weak inhibitory
activity of the canonical Wnt pathway.
Zygotic expression of XPIASy is activated by XSmad2
The importance of positive and negative feedback has been reported in many
developmental systems to ensure the precise timing of the degree of
activation. For example, activation of activin-like signals induces expression
of negative regulators of the pathway such as antivin and cerberus
(Agius et al., 2000;
Cheng et al., 2000
;
Lee et al., 2001
;
Piccolo et al., 1999
).
Conversely, expression of Chordin, a BMP inhibitor, at the chick
organizer is inhibited by the activity of BMP4
(Streit et al., 1998
).
Therefore, the effects of XSmad2 and ß-catenin on the expression of
XPIASy were examined. As shown in
Fig. 3F, overexpression of
XSmad2 increased the expression level of XPIASy in a dose-dependent
manner, whereas overexpression of ß-catenin inhibits transcription of
XPIASy. The feedback induction of XPIASy by XSmad2
is consistent with the synchronized expression of XPIASy and
XSmad2 during development (Fig.
2C). However, the inhibition by ß-catenin may
explain the reason why XPIASy expression is downregulated at the
dorsal mesoderm compared to ventral side because ß-catenin is activated
at the dorsal side (Larabell et al.,
1997
).
How does XPIASy inhibit XSmad2 activity?
As mentioned above, PIASy has been identified as a SUMO E3 ligase for LEF1
and Smads (Imoto et al., 2003;
Sachdev et al., 2001
). SUMO
has been implicated in several mechanisms such as determining the
localization, stability and transcriptional activity of target proteins
(reviewed by Melchior, 2000
;
Seeler and Dejean, 2001
;
Seeler and Dejean, 2003
). In
cell culture experiments, mouse Smad2 has been shown to be modified by SUMO
(Lee et al., 2003
). This
suggests the possibility that SUMOylation of XSmad2 by XPIASy downregulates
its activity. Thus, we analyzed the degree of SUMOylation of XSmad2. The mRNAs
of XSmad2 and XPIASy were injected in the animal side of two-cell stage
embryos and SUMOylation of XSmad2 was analyzed by western blotting after
development until stage 10.5 (Fig.
4A). The molecular mass of XSmad2 is 58 kDa. It is known that
SUMOylation generally alters the size of the target protein by about 18 kDa on
SDS-PAGE gels (Melchior,
2000
). As shown in lane 2 and 3 of the top panel, XPIASy
co-overexpression with Smad2 and SUMO-1 showed a weak band around 73 kDa,
which was confirmed as SUMOylated XSmad2 by analysis of the immunoprecipitated
samples (lower panel). However, although under these conditions,
XSmad2-mediated mesoderm induction was strongly inhibited by XPIASy
(Fig. 3C,D), the SUMOylated
band is too weak to explain the downregulation of XSmad2 activity by its
SUMOylation. Analysis by densitometry showed that 98% of XSmad2 is the
non-modified form. Similar results were achieved using non-tagged XSmad2 (data
not shown). These indicate that if the XSmad2 activity is linearly correlated
with the amount of non-modified XSmad2 protein, the SUMOylation of XSmad2
cannot account for the downregulation of XSmad2 activity.
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Next, Mo-1 by itself was injected into Xenopus embryos, and development was examined. The embryos injected into the DMZ at the four-cell stage exhibited no obvious phenotype until the neurula stage. Interestingly, the embryos injected into the VMZ formed a low frequency of secondary axes (8.2%, n=98, Fig. 5D). This effect is similar to that observed when injected with wild-type XSmad2 (100%, n=36, data not shown). Moreover, animal cap assays revealed that Mo-1 at 40 ng slightly induces the elongation of animal caps (Fig. 5E). This phenotype, again, is similar to that injected with XSmad2. To determine if Mo-1 activates XSmad2 activity, in situ hybridization of Chordin was performed after injecting mRNA of XSmad2, XPIASy or Mo-1 into one side of the embryos. As shown in Fig. 5F, the expression of Chordin is largely enhanced by overexpressing XSmad2, while slightly reduced by XPIASy. Mo-1, as expected, induced expansion of Chordin expression. Next, after injection of Mo-1 into both blastomeres at the two-cell stage, the effect on mesoderm markers in animal caps was analyzed by semi-quantitative RT-PCR. As shown in Fig. 5G, Mo-1 clearly induces the expression of mesodermal markers in a dose-dependent manner although the induction level was weaker than that induced by XSmad2 overexpression, while expression of XSmad1 and ß-catenin targets were not affected. Finally, to confirm whether the effects of Mo-1 are specific, we designed a second morpholino (Mo-2) and a mutated Mo-1 (Mo-mut) that has five point mutations (Fig. 5A) and analyzed their function. Effects of Mo-2 on induction of mesoderm markers (Fig. 5F, part d; 5H) and animal cap elongation (data not shown) were almost identical to those by Mo-1, while Mo-mut did not show any effect on our analysis, including mesoderm marker induction (Fig. 5H, data not shown). Moreover, the induction of mesoderm markers by Mo-2 was completely suppressed by co-introduction of XPIASy mRNA, which does not have 5'-noncoding region (Fig. 5I). These observations demonstrate that endogenous XPIASy functions as a negative regulator of XSmad2 and that the XSmad1 and Wnt pathways are not main physiological targets of XPIASy. Furthermore, taken together with the XPIASy expression pattern and gain-of-function analysis, all observations clearly indicate that XPIASy functions as a gatekeeper in early embryonic patterning to avoid unscheduled activation of XSmad2 in inappropriate places.
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Discussion |
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How does XPIASy inhibit XSmad2 activity?
All examined members of the PIAS family show SUMOylation activity for
various types of proteins, including several transcription factors
(Schmidt and Muller, 2003).
Many members of the Smad family, such as Smad1, Smad2, Smad3 and Smad4, have
been reported to be SUMOylated by members of the PIAS family (PIASy, PIAS1,
PIASxß) or Ubc9 (Imoto et al.,
2003
; Lee et al.,
2003
; Lin et al.,
2003a
; Lin et al.,
2003b
; Long et al.,
2003
; Long et al.,
2004a
; Long et al.,
2004b
) and PIAS family members are localized in nucleus. These
observations suggested that XPIAS might regulate transcriptional activity of
XSmad2 through their SUMOylation activities. However, this possibility is
disputed by our observations: (1) the majority of XSmad2 is not SUMOylated
under conditions where XPIASy shows a developmental phenotype
(Fig. 4A); (2) in addition,
XPIASy still inhibits the activity of a constitutively active XSmad2 mutant,
which lacks the MH1 domain (Baker and
Harland, 1996
) and the putative consensus SUMOylation site
(Lysine-156) (data not shown); and (3) the XPIASy mutant without the RING
domain, P
R, which has lost its SUMOylation activity
(Fig. 4A), still binds to
XSmad2 (Fig. 1H) and inhibits
the activity of XSmad2 (Fig.
4B,C), although its activity was weaker than wild-type XPIASy.
These observations indicate that the SUMOylation activity through the RING
domain is not essential for the effect of inhibition of XSmad2 activity,
although it might attenuate the activity. This conclusion is further supported
by the following functional difference between PIAS family members in cell
culture experiments: PIAS1, PIAS3 and PIASxß activate Smad-mediated
activities (Long et al.,
2004b
; Ohshima and Shimotohno,
2003
), while PIASy inhibits them
(Imoto et al., 2003
;
Long et al., 2003
;
Long et al., 2004b
); however,
all members of the PIAS family show SUMOylation activity of Smad proteins in
overexpression experiments.
PIASy was originally reported to function as a transcriptional co-repressor
of Stat1 (Liu et al., 2001a).
This activity requires the LXXLL motif in the SAP domain. The LXXLL motif is
known to interact with histone deacetylases, HDACs, which function as
transcriptional repressors (Ahmad et al.,
2003
). Recently, PIASy has been reported to directly bind to
HDAC1, while PIAS3 binds to p300/CBP transcriptional activators
(Long et al., 2003
;
Long et al., 2004b
).
Interestingly,
1-94, a deletion construct of the SAP domain, which has
the RING domain and can bind to XSmad2
(Fig. 1F), did not show any
functions similar to the full-length XPIASy (data not shown), indicating the
importance of the SAP domain. All these observations strongly suggest that
XPIASy might inhibit XSmad2 activity by recruiting HDACs via the SAP domain
and not by modulating SUMOylation activity via the RING domain.
Function of XPIASy in the canonical Wnt pathway and the BMP pathway
Recent evidence suggests that the Wnt and TGFß pathways cooperate to
regulate embryonic axis formation and the organizer as ß-catenin and
Smad2 synergize to transcribe siamois and Xnr3
(Crease et al., 1998;
Hussein et al., 2003
;
Labbe et al., 2000
;
Letamendia et al., 2001
;
Nishita et al., 2000
;
Xanthos et al., 2002
). As
mentioned above, the SUMOylation activity of PIASy was originally identified
using LEF1, a downstream target of the canonical Wnt pathway, as a substrate
(Sachdev et al., 2001
). In
addition, the developmental role of SUMOylation has been reported in the
context of the Wnt pathway (Kadoya et al.,
2000
; Kadoya et al.,
2002
). Indeed, our gain-of-function analysis shows that XPIASy can
negatively regulate the canonical Wnt pathway
(Fig. 3A,D). However, this
activity seems not to be the primary function in Xenopus early
embryogenesis based on the following observations. (1) A much higher amount of
XPIASy is required for downregulation of gene expression induced by the Wnt
pathway compared with its effect on XSmad2 targets
(Fig. 3D, part b). (2)
ß-Catenin cannot rescue the defect in dorsal structure induced by XPIASy
(Fig. 3B, parts c,d;
Table 2), although the high
dose of ß-catenin (2 ng) can only rescue head formation (data not shown).
Moreover, Tcf3, another binding partner of PIASy and negative regulator of the
Wnt pathway, cannot activate the ventralization phenotype induced by XPIASy
(Table 2). (3) It has been
reported that inhibition of the canonical Wnt pathway does not inhibit
activin-mediated convergent extension of animal caps
(Vonica and Gumbiner, 2002
).
However, XPIASy clearly inhibits the extension
(Fig. 3C). (4) The XPIASy
morpholinos do not induce expression of targets of the Wnt pathway
(Fig. 5E,F). Thus, XPIASy is
likely to primarily regulate the Xsmad2 pathway but probably secondarily
regulates the Wnt pathway during mesoderm formation and patterning. However,
we showed that the zygotic expression of XPIASy is negatively regulated by
ß-catenin, while positively regulated by XSmad2
(Fig. 3F). These observations
suggest that XPIASy may monitor and coordinate relative activities of the Wnt
and Smad2 pathways to ensure their proper activities during developmental
events.
In addition to the Smad2 and Wnt pathways, mouse PIASy has been reported to bind to Smad1, Smad4, Smad6 and Smad7, and the overexpression of PIASy influences the activities of Smads other than Smad2. However, our binding assay in the Xenopus system showed that XPIASy preferentially binds to XSmad2 and does not affect expression of targets of the XSmad1/XSmad4 complex. In addition, phenotypes produced by XPIASy modulation are largely different from those expected by modulation of XSmad1 activity. These observations suggest that other Smads are not likely to be physiological targets of XPIASy in early embryogenesis.
The role of XPIASy in mesoderm induction and patterning
How is XPIASy involved in the process of mesoderm induction and patterning?
Based on our observations, we propose a `gatekeeper' model
(Fig. 6). XPIASy morpholinos
can induce expression of mesoderm markers in the animal cap and formation of a
secondary axis by their injection into the VMZ, although these inductive
activities are not as strong as observed with XSmad2 overexpression
(Fig. 5). In addition, XPIASy
is expressed in the appropriate region and at the appropriate time to play a
key role in mesoderm induction (Fig.
2). These observations strongly suggest that XPIASy has an
essential role in mesoderm induction and patterning. PIASy largely distributes
in the nucleus (Sachdev et al.,
2001) and SUMOylation of Smad4 in cell culture was suggested to
occur in the nucleus (Lin et al.,
2003a
). Moreover, as mentioned previously, XPIASy is likely to
inhibit XSmad2 activity in the nucleus by recruiting HDACs. These observations
suggest that XPIASy regulates mesoderm induction and patterning by acting at
the end of the activin-like ligands/Smad2 pathway in nucleus. Therefore, in
the gatekeeper model, XPIASy functions as an essential transcriptional
regulator (gatekeeper) to ensure the proper initiation (opening) of
transcription (gate) of the Smad2 target genes at the end of the signal. This
gate is opened by the tightly regulated activity of the gatekeeper (see
below).
|
How is the activity of gatekeeper regulated in mesoderm induction and
patterning? As semi-quantitative RT-PCR analysis revealed, the ratio of
expression level of XPIASy to XSmad2 is well synchronized during embryogenesis
(Fig. 2). However, after stage
8, when XSmad2 is activated for mesoderm formation, the total amount of
XPIASy mRNA is largely reduced compared with that of XSmad2
(Fig. 2A). The spatial
expression pattern in stage 10 embryos shows that the XPIASy
expression is downregulated in the DMZ but is still expressed in the animal
half of embryo (Fig. 2B;
Fig. 6A). Our loss-of-function
analysis indicates that the animally expressed XPIASy is likely to inhibit
ectopic mesoderm induction in ectoderm and that its downregulation in the DMZ
promotes organizer formation. Moreover, our analysis of XPIASy expression has
revealed that the XPIASy expression is positively and negatively regulated by
activities of the Smad2 and Wnt pathways, respectively. These observations
suggest that the localized regulation of XPIASy expression is likely to be
produced by a combination of local XSmad2 and Wnt activities; the Wnt pathway
is activated from the vegetal-dorsal side and XSmad2 is expressed mainly in
the animal half including the marginal zone. In addition to XPIASy, other
negative regulators of the Smad2 pathway have been identified such as Smad7,
Ski and Sno (Liu et al.,
2001b; Nakao et al.,
1997
). Interestingly, these negative regulators as well as XPIASy
are induced by activated Smad2 (Nakao et
al., 1997
; Stroschein et al.,
1999
). These observations suggest that the precise temporal and
spatial regulation of XSmad2 activation seems to be controlled by complex
feedback mechanisms including several negative factors such as XPIASy.
Collectively, our data indicate that XPIASy functions as a final `gatekeeper' at the end of the complex XSmad2 pathway in Xenopus early embryogenesis and that this gate is opened with appropriate timing and in appropriate regions by the combination of mesoderm induction signals such as the Wnt and Smad2 pathways (Fig. 6).
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
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Present address: Cincinnati Children's Hospital Medical Center, Division of
Developmental Biology, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA
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