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First published online 21 January 2009
doi: 10.1242/dev.025577
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Research Report |
1 Human Genetics Unit, MRC, Western General Hospital, Edinburgh EH4 2XU,
UK.
2 Center `Bioengineering', 60-let Oktyabrya 7-1, Moscow, 117312, Russian
Federation.
3 Queen's Medical Research Institute, University of Edinburgh, Edinburgh EH16
4TJ, UK.
* Author for correspondence (e-mail: r.meehan{at}hgu.mrc.ac.uk)
Accepted 9 December 2008
SUMMARY
We demonstrate that a direct interaction between the methyl-CpG-dependent transcription repressor Kaiso and xTcf3, a transducer of the Wnt signalling pathway, results in their mutual disengagement from their respective DNA-binding sites. Thus, the transcription functions of xTcf3 can be inhibited by overexpression of Kaiso in cell lines and Xenopus embryos. The interaction of Kaiso with xTcf3 is highly conserved and is dependent on its zinc-finger domains (ZF1-3) and the corresponding HMG DNA-binding domain of TCF3/4 factors. Our data rule out a model suggesting that xKaiso is a direct repressor of Wnt signalling target genes in early Xenopus development via binding to promoter-proximal CTGCNA sequences as part of a xTcf3 repressor complex. Instead, we propose that mutual inhibition by Kaiso/TCF3 of their DNA-binding functions may be important in developmental or cancer contexts and acts as a regulatory node that integrates epigenetic and Wnt signalling pathways.
Key words: Cancer, DNA methylation, Kaiso, Siamois, TCF3, Chromatin
INTRODUCTION
The BTB/POZ transcriptional factor xKaiso is a bimodal DNA-binding protein
that is reported to specifically bind methyl-CpGs, or a CTGCNA consensus DNA
sequence (Ruzov et al., 2004
;
Park et al., 2005). Our
previous work has established the essential and global role of xKaiso in
regulating the timing of zygotic gene activation at the mid-blastula
transition (MBT) (Ruzov et al.,
2004). Other work proposes a model in which xKaiso specifically
binds CTGCNA sequences present in the promoter region of Siamois (and also
xWnt11) and interacts with the Wnt effector molecule xTcf3 to promote its
stable repression (Kim et al.,
2004; Park et al.,
2005).
In recent work that is not in keeping with the latter model, we
demonstrated that the CTGCNA motifs derived from the promoters of Siamois and
xWnt11 are not sequence specific xKaiso-binding sites and these genes are not
mis-expressed in xKaiso morphants (Ruzov
et al., 2009). Although our loss-of-function experiments did not
identify a role for xKaiso in regulating Wnt target genes, two observations
suggest a potential role for it in canonical Wnt signalling: the xKaiso
protein can be co-immunoprecipitated with xTcf3, and over-expression of xKaiso
can suppress axis-duplication that is induced by over-expression of
β-catenin in the ventral regions of a four-cell embryo
(Park et al., 2005). We wished
to determine the molecular basis of the Kaiso/Tcf3 interaction as it has
profound implications for the intersection of two important regulatory
pathways in amphibian development: regulation of transcriptional silencing in
pre-MBT Xenopus embryos and in Wnt signalling pathways
(Heasman, 2006). We find that
the interaction surfaces for both proteins correspond to their previously
identified DNA-binding domains. Our data squarely rules out a model for xKaiso
repression through stabilisation of xTcf3 binding to DNA. Instead, our
analysis suggests that the xKaiso and xTcf3/4 interaction results in their
mutual delocalisation from chromatin, a prediction that we demonstrate at the
cellular and the DNA levels.
MATERIALS AND METHODS
Reporter assays and expression constructs
The xKaiso expression constructs (XKaiso/pCS2+MT and HA-tagged)
(Kim et al., 2004) were
provided by Pierre McCrea. The dKaiso expression construct was from
(Ruzov et al., 2004). The
Myc-xTcf3, xTcf3dn and β-catenin expression vectors were provided by
Randall Moon. The VP16 fusions with the ZF1-3 region of xKaiso (amino acids
447-635) and the HMG domain of xTcf3 were from Ruzov et al.
(Ruzov et al., 2009). All
reporter assays using SuperTOP/FOP, Tex19 and Siamois luciferase reporters
were performed as described previously
(Houston et al., 2002;
Ruzov et al., 2009).
Embryos and microinjections
Embryos were manipulated as described previously
(Houston et al., 2002;
Ruzov et al., 2009). At the
two-cell stage, the embryos were injected into the animal half with 200-750 pg
of sense capped RNA (c-myc-xKaiso mRNA) synthesized in vitro (T3/T7 Cap-Scribe
kit, Boehringer).
GST pull-down assays, immunoprecipitation and EMSA
The Kaiso GST fusions were from Ruzov et al.
(Ruzov et al., 2009). The TCF4
constructs were provided by Vladimir Korinek. A coupled
transcription/translation kit (Promega) was used for in vitro
translation/labelling with 35S-Met. GST pull-down assays and
immunoprecipitations were performed according to standard protocols in the
presence of a 10- to 100-fold excess of recombinant full-length xKaiso or GST
where indicated. Samples were visualized by phosphoimaging. EMSA was performed
as described previously (Ruzov et al.,
2009).
Chromatin immunoprecipitation (ChIP) assay
The ChIP assay was performed as described previously
(Ruzov et al., 2009) using myc
tagged xTcf3.
Immunostaining
Immunostaining was performed according to standard techniques using
P53-/- (Trp53-/-) mouse embryonic
fibroblasts. Cells were analysed 24 hours after transfection. Mouse monoclonal
anti-T7 tag (Novagen), anti-HA-tag (Sigma), rabbit polyclonal anti-myc
(Upstate) and Alexa secondary antibodies were used.
Real-time RT-PCR
Quantitative real-time RT-PCR of Siamois was evaluated as described
previously (Houston et al.,
2002).
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Kaiso directly interacts with the HMG domain of TCF factors via ZF1-3
The TCF family proteins have a multi-domain organisation with a central HMG
box DNA-binding region recognizing the sequence A/TA/TCAAA; an N terminus
containing the β-catenin-interacting domain adjacent to a Groucho-binding
region; and a C-terminal CtBP1 interaction domain
(Roose et al., 1998). We
demonstrate using recombinant proteins that the DNA-binding, zinc-finger
domain of xKaiso (xZF1-3) is sufficient for direct interaction with
full-length xTcf3 and dominant-negative xTcf3 (xTcf3dn), which lacks the
β-catenin-interacting region (Fig.
1A,B). The ability of Kaiso to bind xTcf3 is conserved, as
comparable zinc-finger regions from zebrafish Kaiso (dZF1-3) and chicken Kaiso
(gZF1-3) can also interact with xTcf3 (Fig.
1B). The same pattern of interaction is seen between mouse TCF4
and xZF1-3. Deletion analysis suggests that the interaction occurs through the
HMG domain of TCF4 (Fig. 1C,D).
The experiment was performed in the presence of high concentrations of
ethidium bromide to exclude the possibility that the interaction was mediated
by non-specific binding to DNA. This suggests that the interaction between
Kaiso and TCF3/4 is mutually exclusive of their binding to DNA, and that
inhibition of β-catenin activation by Kaiso is not through competitive
binding of a shared interaction domain on xTcf3 and TCF4. We tested the first
possibility by performing an EMSA with xTcf3 and its target DNA binding site
(ATCAAA) in the presence of GST-Kaiso fusions. Binding of xTcf3 to its target
sequence was abolished in the presence of the full-length xKaiso, GST-xZF1-3
and GST-dZF1-3. GST alone or GST-xZF1-2 had no effect
(Fig. 1E). Increasing the
amount of xTcf3 overcomes the inhibitory effect of GST-xZF1-3. Moreover, the
interaction between xTcf3 and β-catenin in vitro is not compromised by
xZF1-3 (Fig. 2A). We also
examined the cellular location of myc-tagged xTcf3 in the absence and presence
of Kaiso. xTcf3 localisation in mouse cells is nuclear, with about 40% of
cells with a homogenous, as opposed to a speckled, pattern (60%)
(Fig. 2B); the latter is
reminiscent of the discrete foci observed for endogenous mouse TCF4
(Valenta et al., 2006). xKaiso
in mouse fibroblasts exhibits homogenous nuclear staining
(Fig. 2C). In the presence of
either xKaiso or dKaiso, the pattern of xTcf3 staining was uniformly
homogenous, suggesting they alter its nuclear sublocalisation
(Fig. 2D).
xKaiso displaces xTcf3 from its target promoters
The interaction data suggests that overexpression of xKaiso can displace
xTcf3 from its genomic binding sites. Using the chromatin immunoprecipitation
(ChIP) technique we could localise myc-xTcf3 to the Siamois promoter
in A6 cells (Fig. 3A). However,
myc-xTcf3 was delocalised in the presence of either Ha-xKaiso or T7tag-dKaiso
(Fig. 3A). In contrast to the
model of Park et al. (Park et al.,
2005), this suggests that Kaiso can disengage xTcf3 from its
target genes and alter their expression state. Under the same conditions, we
could not localize dKaiso or xKaiso alone to the Siamois promoter; this is not
surprising in view of the absence of high-affinity CTGCNA or methyl-CpG
binding sites in it (Ruzov et al.,
2009).
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)
(Fig. 3B). This activation was
inhibited between four- to sevenfold by co-expression of either xKaiso or
dKaiso (Fig. 3B). The
transcriptional activity of a control reporter with mutated TCF3-binding sites
is unresponsive to β-catenin induction and unaffected by the presence of
either Kaiso (Fig. 3B). An
obvious explanation of these results is that the Kaiso interaction with the
HMG domain of endogenous TCF3/4 blocks its ability to bind DNA and inhibits
β-catenin-dependent activation of transcription
(Fig. 3B). Conversely,
overexpression of xTcf3 interferes with transcriptional activation of a
methylated reporter template by an xKaisoZFVP16 fusion
(Fig. 3C) in a dose-dependent
manner suggesting the Kaiso/TCF3 interaction results in a mutual disengagement
of these factors from chromatin-binding sites. This model can account for the
inhibition of dorsal axis formation when Kaiso mRNA is co-injected with
β-catenin mRNA (Park et al.,
2005) as excess xKaiso will displace the xTcf3/β-catenin
complex and inhibit axis duplication. Similarly, xKaisoZFVP16 directly
inhibits xTcf3HMGVP16 activation of a Siamois reporter in a dose-dependent
manner (Fig. 3C).
|
). This repression is inactivated on the dorsal side by a
maternally encoded Wnt signalling pathway. Based on the above results, we
predicted that overexpression of xKaiso in developing Xenopus embryos
would mimic certain aspects of xTcf3 depletion such as ectopic Siamois
expression (Houston et al.,
2002). To test this, we injected xKaiso mRNA into two-cell embryos
and allowed them to develop until gastrulation (stage 10.5-11)
(Fig. 3D). In comparison with
controls, this results in an exogastrulae phenotype that is distinct from the
developmental delay phenotype observed in xKMO morphants, but is similar to
the phenotype of embryos that are maternally depleted of xTcf3
(Fig. 3D,E)
(Houston et al., 2002;
Ruzov et al., 2009). The
normalised (relative to histone H4 or GAPDH levels) expression levels of
Siamois are increased in the xKaiso mRNA-injected embryos
(Fig. 3F). This observation
suggests that high levels of xKaiso can interfere with xTcf3 function,
resulting in a relative relief of xTcf3/Groucho-mediated repression of Siamois
in gastrulating embryos.
These new results call for a revision of the model connecting the xKaiso
repressor function and the xTCF3 repression/activation of Wnt-target genes
(Kim et al., 2004;
Park et al., 2005). The mode of
interaction between xKaiso and xTCF3 mutually prevents binding to their
cognate DNA sites, which can potentially inhibit the two pathways in which
these transcription factors are major participants
(Heasman, 2006). However, the
recent observation that neither xWnt11 or Siamois expression is altered in
xKMO morphants suggests that there is no intersection between these pathways
during gastrulation (Ruzov et al.,
2009).
Is this model operative in other biological contexts?
The role of Kaiso expressed in adult somatic tissues is not clear as there
is no obvious mis-expression of normally silent genes in Kaiso-null mice
(Prokhortchouk et al., 2006).
In cancer cells, gene expression patterns are highly disturbed and this is
mirrored by alterations in the level and genomic distribution of DNA
methylation, as well as histone modifications
(Ohm et al., 2007).
Interestingly, Kaiso levels are increased in colon cancer cells and enhance
polyp formation in Min mice (ApcMin/+)
(Prokhortchouk et al., 2006).
This is juxtaposed by positional differences in TCF variant expression in
colon cancer (Clevers, 2006).
In this case, there is a possible intersection of Kaiso with Wnt signalling
pathways, where overexpression of Kaiso could attenuate constitutive Wnt
signalling, while at the same time promote cancer progression through
silencing of de novo methylated tumour suppressor genes. By the same token,
there is a potential for TCF3/4 to displace Kaiso from its cancer target genes
and promote their re-expression, which may be coincident with alterations in
the tumour environment, such as the transition to metastases phenotype
(Fig. 4E). There are two
related Kaiso-like proteins, Zbtb4 and Zbtb38, that may also mediate an
intersection between epigenetic and cellular signalling pathways in cancer via
protein-protein interactions (Filion et
al., 2006; Weber et al.,
2008). Zbt4 has been shown to inhibit MIZ1 regulation of p21CIP1
expression possibly by displacing this transcription factor from the p21CIP1
promoter (Weber et al., 2008).
This suggests that the distributions of these Kaiso-like proteins and their
relative abundance within nuclear sub-compartments has the potential to
determine their transcriptional output of many signalling pathways.
Footnotes
We thank Vladimir Korinek, Pierre McCrea and Randall Moon for reagents, and Nicola Grey and Bill Richardson for Xenopus embryos. Work in R.M.'s laboratory (R.M., D.D., A.R. and J.H.) is supported by the MRC. E.P. and A.P. were supported by Russian Academy of Sciences program grant `Molecular and Cellular Biology', RFBR 06-04-49216-a and CRDF RB-2808. Deposited in PMC for release after 6 months.
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