Archbold Et Al. 2011

Acta Physiol 2012, 204, 74–109
REVIEW
How do they do Wnt they do?: regulation of transcription by
the Wnt/b-catenin pathway
H. C. Archbold,1 Y. X. Yang,2 L. Chen2 and K. M. Cadigan1,2

1 Program in Cell and Molecular Biology, University of Michigan, Ann Arbor, MI, USA
2 Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, USA
Received 24 January 2011, Abstract

revision requested 15 February Wnt/b-catenin signalling is known to play many roles in metazoan devel-
2011,
opment and tissue homeostasis. Misregulation of the pathway has also been
revision received 17 March 2011,
accepted 19 March 2011 linked to many human diseases. In this review, specific aspects of the path-
Correspondence: K. M. Cadigan, way’s involvement in these processes are discussed, with an emphasis on how
Department of Molecular, Cellular Wnt/b-catenin signalling regulates gene expression in a cell and temporally
and Developmental Biology, specific manner. The T-cell factor (TCF) family of transcription factors,
University of Michigan, Ann Arbor,
which mediate a large portion of Wnt/b-catenin signalling, will be discussed
MI 48109-1048, USA.
E-mail: cadigan@umich.edu
in detail. Invertebrates contain a single TCF gene that contains two DNA-
binding domains, the high mobility group (HMG) domain and the C-clamp,
which increases the specificity of DNA binding. In vertebrates, the situation
is more complex, with four TCF genes producing many isoforms that contain
the HMG domain, but only some of which possess a C-clamp. Vertebrate
TCFs have been reported to act in concert with many other transcription
factors, which may explain how they obtain sufficient specificity for specific
DNA sequences, as well as how they achieve a wide diversity of transcrip-
tional outputs in different cells.
Keywords C-clamp, high mobility group domain, lymphoid enhancer-
binding factor 1, T-cell factor, Wnt, b-catenin.
Wnts are a family of secreted proteins that can exert several human diseases, most notably (but not restricted
profound influences on cell behaviour through activa- to) several cancers (Clevers 2006, Polakis 2007). This
tion of several signalling pathways. In this review, we review covers a few examples of Wnt/b-catenin signal-
focus on the best-understood Wnt signalling pathway, ling in normal and pathological contexts, where some
sometimes called ‘canonical’ Wnt signalling but here- information is known about the transcriptional targets.
after referred to as Wnt/b-catenin signalling. This The ability of this pathway to activate diverse tran-
particular Wnt pathway acts by increasing levels of scriptional programs in different contexts is remarkable,
nuclear b-catenin, which then serves as a co-regulator and we discuss some of the mechanisms that contribute
for transcription factors that can recruit b-catenin to to this diversity of transcriptional output.
specific regulatory elements (Cadigan 2008, Cadigan & Most of our attention will focus on the T-cell factor/
Peifer 2009, MacDonald et al. 2009). This pathway is lymphoid enhancer-binding factor 1 (TCF/LEF1) (TCF)
known to play many pivotal roles in animal develop- family of high mobility group (HMG) domain proteins,
ment (Logan & Nusse 2004, Grigoryan et al. 2008, which act with b-catenin to regulate numerous Wnt
Petersen & Reddien 2009, Niehrs 2010) and adult targets. But we also discuss other DNA-binding proteins
tissue maintenance (Polakis 2007, Nusse et al. 2008, that utilize b-catenin to regulate gene expression. It is
Haegebarth & Clevers 2009, Wend et al. 2010). In unlikely that TCFs possess enough DNA-binding spec-
addition, aberrant Wnt signalling has been linked to ificity to account for their ability to find specific Wnt
2011 The Authors

74 Acta Physiologica 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02293.x
17481716, 2012, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1748-1716.2011.02293.x by Cochrane Netherlands, Wiley Online Library on [07/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Acta Physiol 2012, 204, 74–109 H C Archbold et al. Æ Wnt/b-catenin transcriptional regulation
response elements (WREs) among the huge excess of Without Wnt signalling, the b-catenin destruction
genomic DNA sequences. How TCFs interact with other complex keeps the level of b-catenin low, restricting
transcription factors to increase DNA-binding specificity b-catenin to its essential role in supporting cadherin-
will be discussed. These interactions likely contribute to mediated cell adhesion (Stepniak et al. 2009).
the differences in target gene expression that the Wnt/b- When Wnt protein is recognized at the cell surface by
catenin pathway achieves in different cellular contexts. members of the Frizzled (Fz) family of proteins and low
b-catenin is most commonly thought of as a tran- density lipoprotein receptor related protein 5 or 6 (LRP
scriptional co-activator, and this is reflected in our 5/6), a large complex termed the ‘Wnt signalosome’ is
current knowledge of direct targets of Wnt/b-catenin formed (Cadigan & Peifer 2009, MacDonald et al.
regulation, which are predominately activated in 2009). The signalosome interacts with the b-catenin
response to Wnt signalling. But we also discuss reports destruction complex, inhibiting its activity. This results
that link b-catenin to direct transcriptional repression, a in the accumulation of b-catenin, some of which enters
less common form of Wnt regulation, but one that might the nucleus (Fig. 1).
be currently underappreciated among Wnt researchers. Once in the nucleus, b-catenin can bind to several
DNA-binding proteins, the best understood of which
are the TCFs. Some TCFs are thought to act as a
Overview of Wnt/b-signalling
transcriptional switch, repressing Wnt target gene
The overall stability and nuclear localization of b-cate- expression in the absence of signalling and activating
nin is thought to play a central role in determining the transcription upon forming a complex with b-catenin
level of Wnt/b catenin signalling. In the absence of Wnt (Fig. 1). There is also a wealth of information on other
stimulation, b-catenin is constitutively inhibited by a transcriptional co-factors that contribute to TCF repres-
complex (termed the b-catenin destruction complex) sion in the absence of Wnt signalling or are required for
containing glycogen synthase kinase 3 (GSK3) and b-catenin-dependent activation of Wnt targets. These
casein kinase I (CKI), as well as the scaffolding proteins factors, several of which alter the state of Wnt target
Axin and adenomatous polyposis coli (APC) protein gene chromatin, will not be discussed in detail here, but
(Cadigan & Peifer 2009, Kennell & Cadigan 2009). can be explored in several recent reviews (Arce et al.
Phosphorylation of specific residues in the N-terminus 2006, Willert & Jones 2006, Cadigan & Peifer 2009,
of b-catenin by CKI and GSK3 is followed by ubiqui- Mosimann et al. 2009).
tination and proteosomal degradation (Cadigan &
Peifer 2009, MacDonald et al. 2009), (Fig. 1). In
The TCF family: a historical perspective
addition, Axin and APC are thought to sequester
b-catenin in the cytosol, and/or to promote b-catenin The TCF family of transcription factors was first
efflux from the nucleus (Brocardo & Henderson 2008). discovered by researchers interested in lymphocyte gene
(a) (b)
Figure 1 Basic outline of the Wnt/b-catenin signalling pathway. (a) In the absence of the Wnt ligand, b-catenin is phosphory-
lated by a ‘destruction’ complex containing APC, Axin and the CKI and GSK3 kinases. These results in ubiquitylation and
proteosomal degradation of b-catenin. In the nucleus, TCF can recruit co-repressors to Wnt targets, keeping their rate of
transcription very low. (b) When Wnt ligand binds to the Fz and LRP5/6 co-receptors, the destruction complex moves to the plasma
membrane through multiple protein–protein interactions with the receptor complex and Dvl. b-catenin is no longer phosphorylated/
degraded and newly synthesized/b-catenin accumulates in the cytosol and the nucleus. Nuclear b-catenin binds to TCFs,
displacing co-repressors and recruiting co-activators to increase expression of Wnt targets. See the text for more details. APC,
adenomatous polyposis coli; CKI, casein kinase I; GSK3, glycogen synthase kinase 3; TCF, T-cell factor 1; Fz, Frizzled; LRP,
lipoprotein receptor related protein; Dvl, Dishevelled.
2011 The Authors

Acta Physiologica 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02293.x 75
Wnt/b-catenin transcriptional regulation Æ H C Archbold et al. Acta Physiol 2012, 204, 74–109
regulation. A protein originally called TCF1 a or up to 130, (Giese et al. 1992) which was confirmed by
lymphoid enhancer-binding factor 1 (LEF1) was highly solving the crystal structure of a LEF1–DNA-binding
expressed in pre-B and pre-T cells and bound a specific site complex (Love et al. 1995). This bending has been
DNA sequence in an enhancer controlling the T-cell proposed to play an architectural role in coordinating
receptor a (TCRa) gene (Waterman & Jones 1990, the binding of several other factors to the TCRa
Waterman et al. 1991) (Travis et al. 1991). Another enhancer (Carlsson et al. 1993, Giese & Grosschedl
protein enriched in immature T cells called TCF1 bound 1993, Giese et al. 1995). This protein–enhancer com-
to a similar sequence in a CD3a enhancer (van de plex is sometimes referred to as the ‘T cell enhanceo-
Wetering et al. 1991). Both LEF1 and TCF1 were found some’ (Balmelle et al. 2004). The high degree of
to contain a single HMG domain, which was sufficient conservation between the HMG domains of the TCF
for DNA-specific binding (Giese et al. 1991, Oosterwe- family (Fig. 2) suggests that all members have the ability
gel et al. 1991, Waterman et al. 1991). HMG domains to bend DNA, though this remains to be tested directly.
are found in animals, plants and fungi, and TCF1 and Although interest in LEF1 and TCF1 was initially
LEF1 belong to a subgroup within this family, most focused on lymphocytes, the finding that mice lacking a
closely related to the HMG domains of SOX proteins functional LEF1 gene displayed defects to several organ
(e.g. SRY) and fungal mating type proteins (e.g. STE11) systems indicated a much broader role in developmental
(Laudet et al. 1993). biology (van Genderen et al. 1994). This connection
In addition to sequence specific DNA binding, the was further solidified by the findings that LEF1 and
HMG domain of LEF1 has been shown to bend DNA TCF3 (another member of the family) could bind to
(a)
(b)
Figure 2 (a) Cartoon depicting the Drosophila TCF/Pan (the PanA isoform; 751 aa) showing the location of the b-catenin binding
domain (green), the HMG domain (red), the basic tail (aqua) and the C-clamp (blue). (b) Alignment of the HMG domains, basic tails
and C-clamps among metazoan TCFs. Non-conserved residues are not coloured in the alignment. The positions of the three a-helices
of the HMG domain, based on the structure of LEF1 (Love et al. 1995) are indicated at the top of the figure. The six invertebrate
TCFs possess all three domains, while only the E box isoforms of vertebrate TCF1 and TCF4 possess C-clamps. The degree of
conservation in the HMG domain is quite high, e.g. the TCF of Suberities domuncula and human TCF4E are 79.5% identical, 85.9%
conserved. The C-clamp is less conserved (55.2% identity; 58.6% for the S. domuncula-human TCF4E comparison). The number of
non-conserved residues between the basic tail and C-clamps are highly variable. The GenBank accession number of each protein
sequence is in parentheses: S. domuncula (CAH04889.1); Amphimedon queenslandica (ADO16566.1); Mnemiopsis leidyi
(ADO34164.1); Hydra magnipapillata (XP_002159974.1); Caenorhabditis elegans (NP_491053.3); Drosophila melanogaster
(isoform A; NP_726522); human TCF1E (EAW62279.1); TCF4E (CAB97213.1); LEF1 (NP_001124185) and TCF3
(NP_112573.1). TCF, T-cell factor 1; HMG, high mobility group; LEF1, lymphoid enhancer-binding factor 1.
2011 The Authors

b-catenin (Behrens et al. 1996, Huber et al. 1996, discusses the field’s progress towards realizing this
Molenaar 1996). It was known that mutants in the fly challenging goal.
homolog of b-catenin, armadillo (arm) caused defects
very similar to wingless (wg) mutants, a Wnt gene
Wnt/b-catenin signalling in establishing the primary body
important in many aspects of fly development
axes of metazoans
(Riggleman et al. 1989, Peifer et al. 1991, Noordermeer
et al. 1994, Siegfried et al. 1994). In addition, mis- Wnts and b-catenin are not found in choanoflagellates
expression of b-catenin in ventral blastomeres of (King et al. 2008), but are present in all metazoans.
Xenopus embryos induced a secondary body axis Three Wnt genes are found in the demosponge
(Heasman et al. 1994), reminiscent of misexpression Amphimedon queenslandica (Adamska et al. 2010)
of several Wnt genes (McMahon & Moon 1989, Smith and four Wnt genes are present in the Mnemiopsis
& Harland 1991, Sokol et al. 1991). leidyi genome (Pang et al. 1999). The sea anemone,
The N-terminus of TCFs are required for binding to Nematostella vectensis has 14 Wnt genes, including 12
b-catenin, and deletion of this portion of a TCF gene of the 13 subgroups found in mammals (Kusserow et al.
produces a protein that can dominantly inhibit Wnt 2005). This suggests a radiation of the Wnt family after
signalling in several organisms (Behrens et al. 1996, the cnidarian/bilaterian common ancestor diverged
Molenaar 1996, van de Wetering et al. 1997, Kratoch- from sponges and ctenophores. For a more comprehen-
wil et al. 2002). In addition, placing multiple copies of sive list of Wnt gene number across metazoan evolution
high affinity TCF binding sites upstream of a minimal see (Lengfeld et al. 2009).
promoter-reporter gene cassette results in reporter gene The expression patterns of Wnts in simple metazoans
expression that is highly activated by Wnt/b-catenin are highly suggestive of important roles in development.
signalling (Molenaar 1996, Korinek et al. 1997, van de In Amphimedon larvae Wnt is expressed at the posterior
Wetering et al. 1997). These now classic observations end (Adamska et al. 2010). In Hydra, Wnt3 is expressed
contribute to the current working model of Wnt target at the prospective oral pole of embryos and larvae,
gene activation depicted in (Fig. 1). which will give rise to the head of the adult (Hobmayer
et al. 2000, Plickert et al. 2006, Duffy et al. 2010). In
Nematostella embryos, Wnts are expressed in overlap-
Wnt/b-catenin signalling in development
ping patterns along the anterior/posterior (A/P) axis,
Stimulation of the Wnt/b-catenin pathway can result in a suggesting that they provide positional information
diverse array of cellular outcomes, depending on the along the primary body axis (Kusserow et al. 2005).
context. For example, in Drosophila, the fly Wnt protein The expression of the four ctenophore Wnts in com-
Wg acts through Arm to promote cell stem identity (Lin plementary dynamic patterns during embryogenesis also
et al. 2008, Takashima et al. 2008, Sinenko et al. 2009). suggests important developmental roles in this organism
While Wg/Arm signalling stimulates proliferation in (Pang et al. 1999).
several tissues (Baker 2007, Herranz & Milan 2008), the In addition to the presence of Wnts, simple metazoans
pathway can also repress the cell cycle (Duman-Scheel possess most of the downstream signalling components
et al. 2004). The same is true for apoptosis, where Wg/ identified in bilaterians. For example, sponges, cteno-
Arm signalling can activate (Cox et al. 2000, Lin et al. phores and cnidarians all contain one gene encoding
2004) or repress (Cox et al. 2000, Giraldez & Cohen b-catenin, GSK3 and TCF, which are well conserved
2003) programmed cell death. In some contexts, such as with those in other metazoans. Consistent with the
the developing wing, Wg acts as a morphogen, activat- existence of a functional Wnt/b-catenin pathway, appli-
ing distinct targets in a concentration dependent manner cation of GSK3 inhibitors, well known to stabilize
(Zecca et al. 1996, Neumann & Cohen 1997). Since b-catenin and TCF transcriptional readouts in mamma-
most transcriptional targets of Wg/Arm signalling are lian cell culture (Cohen & Goedert 2004), produce
regulated in a cell-type (Lee & Frasch 2000, Knirr & dramatic phenotypes in these organisms. In sponges,
Frasch 2001) or temporally restricted manner (Heems- GSK3 inhibition leads to ectopic formation of ostia
kerk et al. 1991), it is not surprising the the pathway can (canal openings), which can disrupt feeding (Lapebie
effect different cells in dramatically different ways. et al. 2009, Windsor & Leys 2010). GSK3 inhibition in
The following sections summarize some of the vast Hydra produces multiple head and tentacles along the
literature covering Wnt/b-catenin signalling in develop- body (Broun et al. 2005, Muller et al. 2007, Duffy et al.
ment, spanning the entire metazoan clade. Understand- 2010). In the marine cnidarian Hydractina echinata, this
ing the molecular basis for the specificity of multiple head phenotype is suppressed by RNAi deple-
transcriptional outcome in these different systems tion of TCF (Duffy et al. 2010). In Hydra, depletion of
requires a detailed understanding of how stabilized b-catenin results in loss of head structures, opposite to
b-catenin regulates gene expression, and this review the phenotype obtained with GSK3 inhibition (Gee
2011 The Authors

et al. 2010). These results indicate that the basic pathway, in which two b-catenin proteins (WRM-1 and
outlines of the pathway in these organisms are likely SYS-1) act through distinct mechanisms to regulate the
operating in a similar fashion to that outlined in Fig. 1. TCF family member POP-1. WRM-1 promotes POP-1
The direct transcriptional targets of Wnt/b-catenin nuclear efflux while SYS-1 acts in a similar manner as
signalling in these simple systems are currently not b-catenin/Arm (Mizumoto & Sawa 2007). It is not clear
known but one candidate in cnidarians is the Brachyury how the five C. elegans Wnts can provide positional
gene, known to be a direct target of Wnt3a signalling in information to regulate all the asymmetric cell divisions
mouse embryos (Yamaguchi et al. 1999). In Hydracti- controlled by POP-1, but there is evidence that tran-
na, Brachyury is expressed at the oral pole (like Wnt3a) siently expressed Wnt (MOM-2) in posterior blasto-
and is upregulated by GSK3 inhibition and downregu- meres can maintain A/P polarity in other cells through a
lated by Wnt3a and TCF depletion (Duffy et al. 2010). Wnt-dependent relay mechanism (Bischoff & Schnabel
In addition, this report provided evidence that Wnt3 2006). While the rapid generation time and stripped
and TCF are positively regulated by the pathway in down genomes of Drosophila and C. elegans have made
Hydractina (Duffy et al. 2010). Regulation of TCFs by them powerful genetic systems for understanding the
the Wnt/b-catenin pathway is also found in mammalian molecular basis of development (including Wnt/b-catenin
systems (Hovanes et al. 2001). Autoregulation of Wnt signalling) it appears that their overall developmental
gene expression by the pathway is also likely in Hydra, strategies for axial patterning have diverged from the
where in addition to Wnt3 (Hobmayer et al. 2000) six general metazoan scheme.
other Wnt genes are expressed at the oral end of the
organism (Lengfeld et al. 2009). During head regener-
Wnt/b-catenin signalling in establishing the A/P axis of
ation, expression of Wnt3 precedes the other Wnts,
vertebrates
suggesting that they may be activated by Wnt3 signal-
ling (Lengfeld et al. 2009). In addition to the Wnt/b-catenin pathway forming a
In bilaterians, there is also abundant evidence that gradient of Wnt signalling along the A/P axis, a gradient
Wnt/b-catenin signalling is critical for axial patterning. of BMP signalling along the dorsal/ventral (D/V) axis is
In Planaria, depletion of b-catenin results in additional found in a wide array of bilaterians. Niehrs (2010) has
head structures in posterior regions (Gurley et al. 2008, proposed a Cartesian coordinate system of Wnt and
Iglesias et al. 2008, Petersen & Reddien 2008). The BMP signalling to control bilaterian body axes. In
pathway is also required for the formation of posterior Xenopus embryogenesis, these perpendicular gradients
structures in mouse embryos (Liu et al. 1999, Yamag- are initiated by the Spemann organizer (De Robertis &
uchi et al. 1999, Huelsken et al. 2000, Kelly et al. Kuroda 2004, Vonica & Gumbiner 2007). Maternally
2004) and Wnt/b-catenin signalling plays a similar role provided Wnt5a and Wnt11 (Tao et al. 2005, Cha et al.
in specifying cell identities along the A/P axis of the 2008) and other Wnt signalling components (White &
CNS of Xenopus (Niehrs 2010). When one considers Heasman 2008) are relocated to the future dorsal side
that the oral pole of cnidarians likely corresponds to the of the embryo opposite to sperm entry. Wnt/b-catenin
posterior end of the body plan (Meinhardt 2002, Guder signalling then activates the expression of two homeo-
et al. 2006, Niehrs 2010), it appears that the role of domain transcription factors, Siamois and Twin, which
high levels of Wnt/b-catenin signalling in promoting establish organizer identity (Ishibashi et al. 2008).
posterior identity may be very ancient and predate the Analysis of the regions upstream of the twin and
split between cnidarians and bilaterians (Petersen & siamois transcription start sites (TSSs) demonstrated the
Reddien 2009, Niehrs 2010) (Fig. 3). existence of functionally important binding sites for
Two notable exceptions to the global role for Wnt/ TCFs (Brannon et al. 1997, Laurent et al. 1997, Fan
b-catenin signalling in establishing the A/P body axis et al. 1998), which is supported by more recent data
are Drosophila and Caenorhabditis elegans. In flies, with chromatin immunoprecipitation (ChIP) of TCF3
Wg/Arm signalling is involved in establishing A/P (Hikasa et al. 2010). Siamois, Twin and Wnt/b-catenin
identity in each segment (Sanson 2001), while the signalling then act together to activate expression of a
maternally provided transcription factor Bicoid is a variety of BMP antagonists, which emanate from the
major determinant of global A/P patterning (Porcher & organizer to set up a gradient of BMP4 signalling
Dostatni 2010). This is likely the result of the high activity across the D/V axis (De Robertis & Kuroda
degree of specialization that has occurred in dipteran 2004, Vonica & Gumbiner 2007).
evolution (Riechmann & Ephrussi 2001) and the role of In addition to expressing BMP antagonists, the
Wnt/b-catenin signalling in other insects is similar Spemann organizer also expresses antagonists of Wnt/
to other metazoans [reviewed in (Niehrs 2010)]. In b-catenin signalling, such as Dickkopf1 (Dkk1), Cer-
C. elegans, asymmetric cell divisions (mostly along the A/P berus and secreted frizzled related protein 2 (sFRP2).
axis) are controlled by the Wnt/b-catenin/asymmetry Dkk1 is thought to be directly activated by TCF and
2011 The Authors

(a)
(b)
Figure 3 Wnt/b-catenin signalling in establishing the metazoan A/P axis. (a) Some of the circuitry involved in establishing the A/P
axis in Xenopus. Maternal Wnt11 and Wnt5a act through b-catenin to directly activate the Spreman organizer genes siamois and
twin (Brannon et al. 1997, Laurent et al. 1997, Fan et al. 1998, Tao et al. 2005, Cha et al. 2008). Wnt/b-catenin signalling also
activates expression of Dkk1 (Chamorro et al. 2005), and Siamois and Twin are thought to activate cerberus and sFRP expression
(Engleka & Kessler 2001, Yamamoto et al. 2003a). These three Wnt antagonist suppress pathway activation in dorsal/anterior
structures, allowing anterior structures to form (Niehrs 2006). See the text for more details. (b) Cartoon of an amphibian tadpole or
regenerating Planaria, illustrating the Inhibition of Wnt/b-catenin signalling promotes an anterior identity, while expression of
Wnt3a promotes a posterior identity (centre column). Expression of Wnt antagonists in Xenopus or reduction of b-catenin activity
in Planaria causes an expansion of anterior structures at the expense of posterior structures (Niehrs 2006, Petersen & Reddien
2009). Conversely, inappropriate activation of Wnt/b-catenin signalling caused loss of anterior structures. See text for further
explanation. A/P, anterior/posterior.
b-catenin (Chamorro et al. 2005), while Cerberus and In Xenopus, low BMP4 signalling and other signals in
sFRP2 are Siamois-dependent (Engleka & Kessler 2001, the dorsal portion of the embryo are required for
Yamamoto et al. 2003a). These Wnt antagonists con- induction of the neural plate (Stern 2005). After the
tribute to a Wnt/b-catenin signalling gradient with the neural plate invaginates to form the neural tube, NC
highest Wnt signalling in the posterior (De Robertis & cells are specified near the dorsal–lateral portion of the
Kuroda 2004, Vonica & Gumbiner 2007). Overexpres- neural tube (Barembaum & Bronner-Fraser 2005). Wnt/
sion of Dkk1 (decreasing signalling levels) expands b-catenin signalling is required for this induction, and
anterior structures and morpholino depletion of Dkk1 the Wnt3a expressed in the overlying epidermis appears
(increasing signalling levels) results in reduced anterior to be the ligand (McGrew et al. 1997, Li et al. 2009).
identity (Niehrs 2006). Consistent with this, disruption The gradient of Wnt signalling from the posterior of the
of the mouse Dkk1 gene results in embryos with a loss embryo is required to prevent NC induction in the
of head (Mukhopadhyay et al. 2001). In Hydra, Wnt/b- anterior neural fold (Li et al. 2009). After specification,
catenin signalling inhibits Dkk expression, indicating NC cells migrate to different locations to differentiate
the existence of an ancient Wnt-Dkk axial patterning into a diverse array of tissues, including facial cartilage,
circuit (Guder et al. 2006, Niehrs 2010). bones and smooth muscle cells of the heart (Sauka-
Spengler & Bronner-Fraser 2006).
How Wnt/b-catenin signalling induces the NC is
Neural crest: cross-regulation facilitates Wnt-dependent
being elucidated in detail. There are over a dozen
context specificity
transcription factors that have been shown to be
One outcome of the Wnt/b-catenin signalling gradient required for specification of the NC cell fate (Barem-
in vertebrates is the induction of the neural crest (NC). baum & Bronner-Fraser 2005, Sauka-Spengler &
2011 The Authors

Bronner-Fraser 2006). The expression of many of these The cross-regulation of Wnt targets Meis3 and Gbx2
genes has been shown to be Wnt-dependent, but they is one illustration of the complex regulatory networks
also cross-regulate each other, making it difficult to that are found in development. Such cross-regulation
identify the direct targets of the Wnt/b-catenin pathway. can also occur between the TCF-b-catenin complex and
Recently, two reports have used a combination of its targets. Wnt3a is essential for posterior axial growth
approaches to provide evidence that Gbx2 and Meis3 in mice (Yamaguchi et al. 1999, Aulehla et al. 2003,
are important direct Wnt targets in NC induction in Dunty et al. 2008). A similar loss of caudal structures is
Xenopus (Li et al. 2009, Elkouby et al. 2010). Hor- observed when combinations of the caudal homeobox
mone-inducible versions of TCF or b-catenin (fused to genes (Cdx1, Cdx2 or Cdx4) are mutated in mice or
the glucocorticoid receptor) demonstrated activation in zebrafish (Shimizu et al. 2005, Davidson & Zon 2006,
the absence of protein synthesis. An enrichment of Young et al. 2009). Cdx genes are activated by Wnt/
b-catenin at the regulatory regions of these genes was b-catenin signalling (Ikeya & Takada 2001, Gaunt
demonstrated via ChIP. Finally, predicted TCF sites in et al. 2003) and in some cases this regulation is direct
the regulatory regions were mutated, leading to a loss of (Lickert et al. 2000, Ikeya & Takada 2001, Pilon et al.
reporter gene expression (Li et al. 2009, Elkouby et al. 2006). However, Wnt3a expression also requires Cdx
2010). These results argue that these genes are direct activity, and loss of function Cdx2 and Cdx4 pheno-
targets of the pathway. types can be rescued by an activated version of LEF1
Consistent with the above data, Gbx2 morphants (Young et al. 2009). A similar positive feedback loop
display a loss of many NC markers (Li et al. 2009). between Wnt and Cdx has also been reported to be
Depletion of Meis3 has a similar phenotype, including required for posterior development in Xenopus (Faas &
a reduction in Gbx2 expression (Elkouby et al. 2010). Isaacs 2009). Interestingly, Cdx1 autoregulation has
Meis3 is clearly a major target of Wnt/b-catenin been reported to require a physical interaction between
signalling, because exogenous addition of the Meis3 LEF1 and Cdx1 (Beland et al. 2004), providing a clue as
gene can rescue the loss of NC seen in Wnt3a to how TCF family members and Cdx proteins act
morphants (Elkouby et al. 2010). These studies suggest together. A similar relationship has been demonstrated
that Wnt/b-catenin signalling initially induces both between the Wnt target Brachyury and the Wnt/
Meis3 and Gbx2, and Meis3 contributes to Gbx2 b-catenin pathway in promoting posterior mesoderm
expression. In a similar manner, Slug 2 is directly development (Martin & Kimelman 2008).
activated by the Wnt/b-catenin pathway (Vallin et al.
2001) but also requires Meis3 and Gbx2 for expres-
Cardiogenesis: reiterative Wnt/b-catenin signalling
sion (Li et al. 2009, Elkouby et al. 2010). In addition,
required
Gbx2 is a transcription repressor, which inhibits the
expression of anterior neural fold markers such as Beyond helping establish the basic body plan, Wnt/
Six1, restricting NC induction to posterior regions b-catenin signalling is important for a multitude of
(Fig. 4). developmental decisions. These include limb formation,
bone, hair and teeth development as well as formation
of every major organ (see (Grigoryan et al. 2008) for a
comprehensive review of b-catenin-dependent develop-
mental processes in mice). In this review, we will focus
on heart formation, which provides a good example of
how the Wnt/b-catenin pathway is used repeatedly to
achieve different outcomes in different developmental
contexts, even in the same tissue. Further information
on the role of canonical and non-canonical Wnt
signalling in heart development can be found in
additional reviews (Cohen et al. 2008, Kwon et al.
2008, Gessert & Kuhl 2010).
Although the tube-like insect heart is morphologically
different from the multichambered vertebrate heart,
Figure 4 Wnt/b-catenin signalling induces neural crest. Wnt3a
they appear to share a common ancestry. One striking
from the overlying epidermis induces expression of Meis3 and
Gbx2 are induced in the neural tube (Li et al. 2009, Elkouby example supporting this view is provided by the tinman/
et al. 2010). Meis3 also cross regulates Gbx2 (Elkouby et al. Nkx2.5 gene, which is required for heart formation in
2010) and Slug2 is activated by a combination of Wnt3a, Gbx2 Drosophila and several vertebrate systems (Bodmer &
and Meis3 (Vallin et al. 2001, Li et al. 2009, Elkouby et al. Venkatesh 1998, Evans 1999). In flies, Wg/Arm signal-
2010). See text for further information. ling is required for tinman expression and heart
2011 The Authors

formation in general (Wu et al. 1995, Park et al. 1996). indicate that the activation of Islet1 transcription by
In contrast, the first studies in vertebrates found that Wnt/b-catenin signalling is direct (Lin et al. 2007).
Wnt/b-catenin signalling is required to restrict specifi- Later removal of b-catenin in the developing SHF
cation of cardiac mesoderm (Marvin et al. 2001, resulted in normal Islet1 expression, but the right
Schneider & Mercola 2001, Lickert et al. 2002). ventricle and outflow tracts still failed to form (Ai et al.
This paradox between invertebrate and vertebrates 2007, Kwon et al. 2007). These results indicate multiple
has been resolved by the realization that the Wnt/ roles for Wnt/b-catenin signalling in SHF development,
b-catenin pathway plays two opposing roles in early both in establishing the SHF and subsequent differen-
vertebrate cardiogenesis. Temporal control of Wnt tiation of heart tissue.
expression revealed that before gastrulation, the path- The complexity of target gene regulation by the Wnt/
way activates Nkx2.5 expression in zebrafish embryos b-catenin pathway in the development of the secondary
(Ueno et al. 2007). After gastrulation, the previously heart filed is evidenced by the findings that Wnt/b-
described inhibitory role was evident. This biphasic catenin signalling represses Islet1 expression in differ-
relationship was also observed in mouse ES cells, which entiating cardiomyocytes (Kwon et al. 2009). Likewise,
spontaneously differentiate into cardiomyocytes. Acti- GATA6, which is repressed by Wnt/b-catenin signalling
vation of Wnt/b-catenin signalling in early cultures in early heart development in Xenopus (Afouda et al.
dramatically enhanced cardiomyoctes differentiation 2008), is directly activated by Wnt/b-catenin signalling
but pathway activation in later cultures reduced car- in the posterior SHF (Tian et al. 2010). The factors that
diogenesis (Ueno et al. 2007). These data suggest that enable the same pathway to both activate and repress
the initial pro-cardiogenic effect of the Wnt/b-catenin the same targets during the cardiomyocyte cell lineage
pathway in vertebrates is analogous to the positive are not known.
effect that Wg/Arm signalling has on heart development There are several additional events in heart develop-
in the fly. ment where Wnt/b-catenin signalling is also required.
What are some of the direct targets in the initial Mutations lowering pathway activity in cardiac NC cells
regulation of cardiogenesis by the pathway? In flies, the result in defects to the cardiac outflow tract (Hamblet
presence of functional TCF binding sites in the enhanc- et al. 2002, Kioussi et al. 2002). This phenotype is
ers that drive the expression of the transcription factors similar to that observed when the gene encoding the
Sloppy paired 1 (Slp1) and Even-skipped (Eve) in the bicoid homeodomain protein PitX2 is mutated (Kioussi
cardiac precursors provides strong evidence that these et al. 2002). PitX2 is directly activated by LEF1 and b-
genes are directly activated by Wg/Arm signalling catenin and then PitX2 subsequently recruits b-catenin
(Halfon et al. 2000, Lee & Frasch 2000, Knirr & to the Cyclin D2 regulatory region, activating this cell
Frasch 2001, Han et al. 2002). In mouse ES cells, gene cycle regulator and promoting proliferation of the
profiling revealed many potential targets, including cardiac NC cells (Kioussi et al. 2002). The Wnt/b-
Brachyury, Mesp1 and Sox17 (Liu et al. 2007, Ueno catenin pathway also promotes endocardial cell prolif-
et al. 2007). In regard to repression of cardiogenesis, the eration, which contributes to heart valve formation
homeodomain protein Hex is an important Wnt target (Gitler et al. 2003, Hurlstone et al. 2003, Liebner et al.
that is repressed in the presumptive cardiac mesoderm 2004, Alfieri et al. 2010). Likewise, loss of b-catenin in
(Foley & Mercola 2005). In Xenopus, GATA6 expres- the developing epicardium results in defects in coronary
sion is repressed by Wnt/b-catenin signalling, and forced artery formation (Zamora et al. 2007).
expression of GATA6 is sufficient to rescue many Heart development highlights the multiple roles that
aspects of heart development that are disrupted by the Wnt/b-catenin pathway plays in regulating cell fate
ectopic Wnt pathway activation (Afouda et al. 2008). and organogenesis (Fig. 5). How can one signalling
The mechanism by which these genes are repressed by pathway be utilized so many times to regulate different
Wnt/b-catenin signalling is not known. genes in the cardiac cell lineage? Understanding how
After the initial specification of the presumptive heart Wnt transcriptional output diversity is generated
field, a population of cardiomyocytes known as the requires a more detailed understanding of how TCF
secondary heart field (SHF) will give rise to the future family members and other transcription factors that
right ventricle and inflow and outflow tracts of the heart mediate Wnt/b-catenin-dependent gene regulation func-
(Dyer & Kirby 2009). Conditional knockout of b-cate- tion on target gene chromatin.
nin causes a significant reduction in these structures (Ai
et al. 2007, Cohen et al. 2007, Klaus et al. 2007, Kwon
Wnt/b-catenin signalling in stem cell biology
et al. 2007, Tian et al. 2010). Removal of b-catenin
and regeneration
before SHF specification resulted in a loss of Islet1 (a
LIM homeodomain) expression, a marker of SHF In addition to the myriad roles that the Wnt/b-catenin
cells (Cai et al. 2003). There is some evidence to pathway plays in normal development, it is also a key
2011 The Authors

orphan receptor Lgr5, which has received a great deal

of attention, since it marks a population of multipotent
cells in the crypt that give rise to all the specialized cells
of the intestinal epithelia (Barker & Clevers 2010).
Another biologically important Wnt target is the bHLH
transcription factor Achaete scute-like 2 (Ascl2), which
is required for the maintenance of the Lgr5-positive
stem cells and can induce crypt hyperplasia upon forced
expression (van der Flier et al. 2009). Both Lgr5 and
Ascl2 were identified in a genome-wide survey of
chromatin enriched for TCF4 binding (Hatzis et al.
2008), suggesting they may be direct targets of Wnt/b-
catenin signalling. In addition to intestinal stem cells,
Lgr5 and the related protein Lgr6 also appear to mark
stem cells populations in several other organs as well
(Haegebarth & Clevers 2009, Snippert et al. 2010).
Wnt/b-catenin signalling has also been linked to the
Figure 5 Wnt/b-catenin signalling (Wnt/b-cat) has multiple ability to replace damaged cells or to regenerate deleted
roles in heart development. This schematic depicts a few of the tissues in a wide array of metazoans. In Hydra and
roles the pathway plays in mammalian heart development, Planaria, organisms renowned for their ability to
both as an activator and as a repressor of gene activity. Prior to regenerate large portions of their bodies after bisection,
gastrulation, nkx2.5 is positively activated by Wnt/b-cat
the pathway promotes posterior cell fates as described
signalling, while it is repressed after gastrulation, as are
in the previous section. In Hydra, decapitation of the
cardiogenic factors GATA6 and Hex (Foley & Mercola 2005,
head results in rapid induction of Wnt3 expression in
Ueno et al. 2007, Afouda et al. 2008). Activation of a number
of Wnt target genes early in development, such as Islet1 have the epithelial cells which is required for head regener-
been linked to the proliferation of cardiac progenitor cells (Lin ation (Hobmayer et al. 2000, Chera et al. 2009, Leng-
et al. 2007). These Islet1+ cells contribute to the second heart feld et al. 2009). When Hydra is bisected in the
field (SHF), inflow tract (IFT) and outflow tract (OFT). The midgastric region, a wave of apoptosis among the
SHF is required for proper formation of the atria and the right interstitial cells is coupled to release of Wnt3 from these
ventricle. In the posterior SHF, Wnt activates GATA6 (Tian dying cells, which results in subsequent activation of
et al. 2010), and represses Islet1 expression (Kwon et al. Wnt3 transcription in epithelial and head regeneration
2009). Wnt/b-cat signalling is also instrumental in driving (Chera et al. 2009, Galliot & Chera 2010).
proliferation of cardiac neural crest cells (Kioussi et al. 2002),
The coupling of apoptosis to generation of a Wnt
which migrate to the heart tube and OFT, and is important in
signal in regenerating Hydra is reminiscent of a similar
valve and artery formation (Zamora et al. 2007) along with
phenomenon described in Drosophila imaginal discs,
endocardial proliferation (Gitler et al. 2003, Hurlstone et al.
2003, Liebner et al. 2004, Alfieri et al. 2010). See text for more where apoptotic cells express Wg, which is thought to
information. FHF, first heart field. promote compensatory proliferation of neighbouring
cells to maintain the size of the tissue (Fan & Bergmann
2008, Martin et al. 2009). While Wg is dispensable for
regulator of adult tissue homeostasis. The role of Wnt/ disc repair in response to irradiation (Perez-Garijo et al.
b-catenin signalling in stem cell maintenance has been 2009), a functional role for Wg has been reported in
well documented in the intestine (Pinto & Clevers 2005, disc regeneration following expression of a pro-apop-
Barker & Clevers 2010), hair follicles and in the skin totic signal, where Wg induced expression of myc and
(Blanpain et al. 2007, Blanpain & Fuchs 2009, Haege- cyclin E to promote proliferation (Smith-Bolton et al.
barth & Clevers 2009). In the mouse small intestine, 2009).
TCF4 and b-catenin are required for maintenance of the In vertebrates, Wnt/b-catenin signalling has been
crypt stem cells (Pinto & Clevers 2005). Microarray shown to be required for tail fin regeneration in zebrafish
profiling has identified many transcriptional targets of (Stoick-Cooper et al. 2007). Wnt10a is induced after fin
the Wnt/b-catenin pathway (van de Wetering et al. cutting, and blocking the pathway prevents induction of
2002, Van der Flier et al. 2007). One biologically fibroblast growth factor 20a (fgf20a) expression, which
important target is c-myc, which is required for normal is required for regeneration of this tissue (Wills et al.
intestinal crypt development (Muncan et al. 2006). As 2008). Interestingly, fgf20a is directly activated by TCF-
will be discussed in the following section, c-myc appears b-catenin in cultured human cells (Chamorro et al.
to be a direct target of the pathway. Another gene that is 2005), though it is not clear whether this is the case in
activated by Wnt/b-catenin signalling encodes the regenerating zebrafish tails. The pathway is also thought
2011 The Authors

to play a role in the initial step of limb regeneration in indicating that c-myc is a major factor in mediating the
Xenopus (Yokoyama et al. 2007), though the molecular effect of elevated Wnt/b-catenin signalling in the intes-
targets remain to be identified. tinal epithelia.
When c-myc was first identified as a potential
transcriptional target of the Wnt/b-catenin pathway in
Wnts gone wrong: misregulated signalling in
CRC, examination of the region 5¢ of the TSS revealed
disease
the presence of conserved TCF binding sites (He et al.
Given the numerous functions of Wnt/b-catenin signal- 1998). This region of DNA could activate a minimal
ling in development and stem cell biology, it is not promoter/luciferase reporter upon Wnt/b-catenin path-
surprising that misregulation of the pathway would be way activation and thus fits the definition of a WRE.
connected to a large number of diseases. Activation of Mutation of two TCF binding sites within this WRE
Wnt/b-catenin signalling has been linked to many abolished this activation (He et al. 1998). ChIP studies
different types of human cancer, including colorectal demonstrated that this region was bound by LEF1 and
cancer (CRC) (Polakis 2000, 2007), hepatocellular b-catenin in CRC cells (Sierra et al. 2006). More
carcinomas (Armengol et al. 2009), cancers of the recently, another site enriched for b-catenin binding in
adrenal gland (El Wakil & Lalli 2011, Morris et al. CRC cells was identified downstream of the c-myc
2010), Wilm’s tumour (Tycko et al. 2007), breast transcription unit (Yochum et al. 2008). This region
cancer (Zardawi et al. 2009) and several hematological also conferred Wnt responsiveness in a reporter gene
malignancies (Ge & Wang 2010). In some cancers, for assay and contained functionally important TCF sites
example, melanoma, down-regulation of the pathway (Yochum et al. 2008). Chromatin loops between these
leads to more aggressive malignancy (Lucero et al. two WREs and the c-myc proximal promoter have been
2010). Many of these cancers contain either loss- reported (Yochum et al. 2010).
of-function mutations in the APC gene, causing stabil- While, the data described above suggest that the
ization of b-catenin, or gain-of-function mutations in WREs identified 5¢ and 3¢ of the c-myc gene contribute
b-catenin, which interfere with phosphorylation of to regulation by Wnt/b-catenin signalling, the large
b-catenin by the destruction complex (Polakis 2000, intergenic regions surrounding this Wnt target raised
2007). Loss of Axin or Axin2 is also correlated with the possibility of other WREs further removed from the
some forms of cancer (Laurent-Puig & Zucman-Rossi TSS (Fig. 6). Indeed, when a genomic fragment con-
2006). In the next section, we focus on the role of c-myc taining the 5¢ and 3¢ WREs were tested in a transgenic
activation by the Wnt/b-catenin pathway, because of mouse reporter assay, there was little transcriptional
the biologically relevance of this regulation in cancer, activity observed (Lavenu et al. 1994). An in silico
and as an example of how difficult it can be to identify search for WREs identified two regions approx. 26 and
all the DNA regulatory sequences that mediate Wnt 36 kb upstream of the c-myc TSS (Hallikas et al. 2006).
responsiveness (i.e. WRE) of a target gene. Both regions drove the expression of a reporter in
transgenic mice in a subset of the c-myc pattern, though
it is not clear whether these elements are regulated by
CRC: activation of c-myc expression by the Wnt/b-catenin
Wnt/b-catenin signalling (Hallikas et al. 2006).
pathway
Another potentially important WRE controlling
The c-myc gene encodes a basic helix-loop-helix c-myc has been identified through genome-wide associ-
(bHLH) protein that promotes cell growth in many cell ation scans for polymorphisms that increase an indi-
types (Soucek & Evan 2010). Elevated expression of vidual’s risk of developing CRC (Tomlinson et al. 2007,
c-myc has been linked to numerous human cancers Zanke et al. 2007). A single nucleotide polymorphism
(Eilers & Eisenman 2008). c-myc expression is activated (G/T; known as rs6983267) was linked with an
by Wnt/b-catenin signalling in CRC cells (He et al. increased occurrence of adenomas and CRC. While
1998, van de Wetering et al. 2002) and in the exper- control populations had an approximate 50/50 distri-
imentally induced murine epithelial hyperplasia associ- bution of the G and T allele, the G allele was found at
ated with temporally controlled deletion of APC approx. 56% in affected individuals (Tomlinson et al.
(leading to b-catenin stabilization) (Sansom et al. 2007, Zanke et al. 2007). This polymorphism is located
2004). Strikingly, simultaneous loss of APC and c-myc approx. 335 kb upstream of the c-myc TSS (Fig. 6).
in this system completely suppressed the overgrowth The realization that this polymorphism occurs in a
phenotype caused by APC removal, even though putative TCF binding site (GATGAAAGG vs. GAT-
b-catenin levels were still abnormally high (Sansom GAAAGT) suggested that this polymorphism resides
et al. 2007). Microarray analysis suggests that many of within a WRE controlling c-myc expression. Consistent
the changes in gene expression observed upon removal with this, TCF4 was highly enriched at this site
of APC are c-myc dependent (Sansom et al. 2007), compared to the other 1 Mb of DNA surrounding the
2011 The Authors

(a)
(b)
Figure 6 Complex regulation of the Wnt target gene c-myc. (a) WREs are located both near and removed from the c-myc proximal
promoter. The numbers in parentheses refer to the approximate position of the 5¢ end of the WRE in relation to the c-myc TSS.
The 5¢ ()1.2 kb) and 3¢ (+5.5 kb) WREs respond to Wnt/b-catenin signalling in cell culture (He et al. 1998, Yochum et al. 2008).
The far upstream ()335 kb) WRE is active in cell culture and transgenic mice (Tuupanen et al. 2009) and contains a polymorphism
in a TCF binding sites that correlated with increased risk of CRC (Pomerantz et al. 2009, Tuupanen et al. 2009, Wright et al.
2010). The putative WREs at )26 kb and )32 kb are expressed in transgenic mice (Hallikas et al. 2006). (b) The presence of
chromatin loops between the far upstream and the 3¢ WRE have been documented in CRC cells with high levels of Wnt/b-catenin
signalling (Pomerantz et al. 2009, Wright et al. 2010, Yochum et al. 2010). These loops are stabilized by protein complexes (data
not shown) containing TCF and b-catenin. Presumably, the large chromatin loop allows the upstream enhancer to bypass the
POU5F1 gene, though this has not been experimentally confirmed. WRE, Wnt response elements; TSS, transcription start sites;
TCF, T-cell factor; CRC, colorectal cancer.
c-myc locus (Tuupanen et al. 2009). This region had tally regulated enhancers are more than 100 kb away
WRE activity in cell culture reporter assays (Pomerantz from the nearest TSS (Visel et al. 2009). The ability to
et al. 2009, Tuupanen et al. 2009, Sotelo et al. 2010, scan the large regions of genome surrounding Wnt
Wright et al. 2010). More impressively, this region targets of interest may often be necessary to identify
drove expression of a reporter in transgenic mouse some functionally important WREs.
embryos in a pattern very similar to the majority of
endogenous c-myc expression, which was abolished by
Wnts and oxidative stress: diabetes and Alzheimer’s
mutation of the polymorphic TCF site and an adjacent
disease
site (Tuupanen et al. 2009). Consistent with the
increased risk of CRC, the G allele WRE had a greater In addition to cancers, the Wnt/b-catenin pathway has
response to Wnt/b-catenin pathway activation in cell been linked to many other diseases, including several
culture reporter assays (Pomerantz et al. 2009, Tuupa- pathologies in the kidney (Pulkkinen et al. 2008,
nen et al. 2009, Wright et al. 2010). When a CRC cell Hwang et al. 2009, Lancaster & Gleeson 2010), bone
line with a G/T genotype was examined, TCF4 and (Krishnan et al. 2006) and cardiac repair (Saraswati
b-catenin were enriched on the G allele chromatin, and et al. 2010). In this section, we briefly review the role of
this allele was more than two times more actively Wnt/b-catenin signalling in metabolic disorders and
transcribed than the T allele (Wright et al. 2010). Taken neurodegenerative diseases. While the direct links
together, these data support a model where increased between these disorders and Wnt/b-catenin signalling
recruitment of TCF4 and b-catenin to the G allele WRE are not as well established as in cancer, there are some
results in higher levels of c-myc transcription. A chro- candidate targets where misregulation of the pathway
matin loop between this distal WRE and the c-myc could underlie the pathology.
proximal promoter has been demonstrated (Pomerantz Emerging evidence has linked several players in Wnt/
et al. 2009, Wright et al. 2010), suggesting a mecha- b-catenin signalling to metabolic disorders and type 2
nism by which this element can act over such a great diabetes mellitus. Genome-wide association studies
distance (Fig. 6B). demonstrated a strong connection between diabetes
The regulation of c-myc by the Wnt/b-catenin path- type 2 risk and SNPs within the TCF7L2 (TCF4) gene,
way illustrates an inconvenient truth about studying although the cellular basis of this association is still
gene regulation in higher eukaryotes. The existence of a uncertain (Schinner et al. 2009). Mutations in the Wnt
WRE over 300 kb from the c-myc TSS should no longer receptors LRP5 and 6 have also been implicated in
cause surprise, given the existence of functional ele- obesity and type 1 diabetes and metabolic syndrome
ments acting at an even greater distance in regulating respectively (Jin 2008). Wnt/b-catenin signalling
sonic hedgehog (Jeong et al. 2006). In addition, there is appears to play roles both in the proliferation of
a growing realization that the majority of developmen- pancreatic b-cells, and in the insulin release from islet
2011 The Authors

cells. b-cell proliferation in cell culture and a transgenic target gene expression. In addition to a b-catenin
mouse models appears to be the result of Wnt activation binding domain at the N-terminus, a hallmark of this
of targets like cyclin D1 (Liu & Habener 2008, Schinner family is the presence of a highly conserved HMG
et al. 2008), as well as cyclin D2 and PitX2 (Rulifson domain, followed by a stretch of basic residues (Fig. 2).
et al. 2007). Consistent with this, knockdown of TCF4 The TCF subfamily of HMG domains is found through-
decreases b-cell proliferation and promotes apoptosis out metazoans, but not in the sister group choanofla-
(Liu & Habener 2008, Shu et al. 2008). gellates (King et al. 2008). The HMG domain contacts
In addition to promoting b-cell proliferation, the DNA largely through minor groove contacts and results
Wnt/b-catenin pathway is also required for efficient in a large bending of the double helix (Love et al. 1995).
insulin secretion (Fujino et al. 2003). The pathway may High affinity binding sites for these HMGs have been
play a role in insulin sensing by activating glucokinase determined for Drosophila TCF/Pangolin (TCF/Pan)
transcription (Schinner et al. 2008). It is interesting to and all four mammalian TCFs (Giese et al. 1991, van de
note that antagonism of Wnt signalling by oxidative Wetering et al. 1997, van Beest et al. 2000, Hallikas
stress appears to play an important role in the pathology et al. 2006, Atcha et al. 2007). While, these studies
of insulin resistance and diabetes. The forkhead box show that a site of CCTTTGATS (S = G/C) is bound
DNA-binding protein FOXO, has been shown to with highest affinity in vitro, as will be described in the
compete with TCFs for b-catenin binding (Hoogeboom following section, many functional TCF binding sites in
et al. 2008). Upregulation of FOXOs in response to WREs fit this consensus, while others diverge markedly.
oxidative stress may contribute to insulin resistance by There is a single TCF gene in almost all invertebrate
the promotion of gluconeogenesis and/or the promotion species that have been examined thus far (Figs 2 and 6),
of apoptosis and downregulation of TCF-mediated gene with TCF/Pan from flies and POP-1 from C. elegans
expression (Manolagas & Almeida 2007). being the most thoroughly characterized. In contrast,
The effects of oxidative stress may also be a factor in amphibians and mammals have four TCF genes. These
the neurodegeneration of Alzheimer’s disease (AD), are most commonly referred to as TCF1 (TCF7), LEF1
where Wnt signalling has been ascribed a neuroprotec- (LEF1), TCF3 (TCF7L1) and TCF4 (TCF7L2). The
tive role (Manolagas & Almeida 2007). Extracellular names in parentheses are from the Human Genome
accumulation of amyloid-b (A-b) has been shown to Organization (HUGO). In zebrafish, the TCF7L1 gene
bind Fz receptors and downregulate Wnt signalling, and is duplicated (TCF7L1a and TCF7L1b), giving a total
it has been hypothesized that some of the cytotoxity of five TCF genes in bony fish (Dorsky et al. 2003). As
caused by A-b may be the result of chronic suppression will be discussed below, there is evidence that some of
of Wnt/b-catenin signalling (Inestrosa & Toledo 2008). the vertebrate TCFs are more specialized in their
The protective effects of b-catenin overexpression, but function compared to their invertebrate counterparts.
not of transcriptionally inactive b-catenin, indicate that
transcriptional activation of target genes plays a role in
The TCF transcriptional switch
ameliorating A-b toxicity (Chacon et al. 2008). One
possibility is that oxidative stress may exacerbate The current working model for regulation of WREs by
cytotoxity in AD by increasing FOXO, which then TCFs proposes the existence of a transcriptional switch,
competes with TCF for b-catenin, reducing the expres- where TCF and co-repressors inhibit the target gene
sion of anti-apoptotic factors. expression in the absence of signalling, and then act
Clearly, much remains to be elucidated concerning with b-catenin and other co-activators to activate
the role Wnt/b-catenin signalling plays in neurodegen- transcription of targets (Fig. 1). Evidence for this model
eration and other diseases. Information gained from the was first obtained in the Drosophila embryo. TCF/pan
study of Wnt-mediated transcriptional regulation in mutants display mispatterning of the epidermis indica-
model systems should facilitate the identification of the tive of reduction of Wg signalling (Brunner et al. 1997,
important targets in many pathological states where van de Wetering et al. 1997, Schweizer et al. 2003).
genetics suggests that aberrant Wnt signalling plays a This defect was not as severe as that of null alleles of
causal role. Given the prominence of TCF family wg. However, a wg; TCF/pan double mutant looked
members in regulating numerous Wnt targets, the next identical to TCF/pan mutants (Cavallo et al. 1998). In
few sections will review TCF function in detail. wg mutants, there is no activation of targets and TCF
repression is intact, resulting in a severe loss of Wg
signalling phenotype. But in wg; TCF/pan double
The TCF family: major regulators of Wnt/b-
mutants, loss of repression of Wg targets allows some
catenin transcription
expression (i.e. derepression), resulting in a less severe
The TCF family of transcription factors is the best phenotype (Cavallo et al. 1998). Genetic and physical
characterized DNA-binding regulators of Wnt/b-catenin interactions between TCFs and TLE family co-repres-
2011 The Authors

sors (Cavallo et al. 1998, Roose et al. 1998) provided Wnt/b-catenin signalling. Consistent with this, TCF4 is
further support for the transcriptional model. The required for activation of Spemann organizer genes in
model has also been confirmed in fly cell culture using Xenopus (Standley et al. 2006). Conversely, loss of
a combination of RNAi and ChIP of TCF/Pan in the TCF4 can result in elevated activation of a Wnt/
absence and presence of Wg signalling (Fang et al. b-catenin signalling in CRC cells, suggesting a possible
2006). role for TCF4 as a tumour suppressor (Tang et al.
In C. elegans, the POP-1 loss-of-function phenotypes 2008). Further support for a bi-modal role for TCF4
indicate both positive and negative roles in regulating comes from studies of TCF3; TCF4 double knockouts in
Wnt targets (Phillips & Kimble 2009). In some con- the skin epithelia of mice (Nguyen et al. 2009). Loss of
texts, for example, blocking mesoderm cell fate in the both TCFs in the skin epithelia resulted in a dramatic
early embryo, repression of Wnt targets is the predom- decline in epidermal survival, which was not observed
inant effect observed (Rocheleau et al. 1997, Thorpe when b-catenin was removed (Nguyen et al. 2009).
et al. 1997). But in other stages, for example, QL Microarray profiling revealed that many genes were
neuroblast migration and stem cell specification in the repressed by TCF3 and TCF4 in a redundant manner,
somatic gonad, loss of POP-1 has a similar phenotype which were either activated or not regulated by b-cate-
as loss of other Wnt/b-catenin components (Herman nin (Nguyen et al. 2009). These data fit a model where
2001, Lam et al. 2006). Clearly, POP-1 and TCF/Pan the two TCFs are repressing gene expression in the
can both repress and activate Wnt targets. absence of Wnt/b-catenin signalling. Like TCF1, TCF4
In vertebrates, some TCF family members are more can activate or repress Wnt targets, depending on the
closely linked to either repression or activation. For context.
example, headless (hdl) mutants in zebrafish are loss of The loss-of-function studies summarized above sug-
function TCF3a alleles and display a lack of head gest a model where the transcriptional switch in
structures (Kim et al. 2000), similar to Dkk1 knockouts vertebrate WREs is mediated by two distinct TCFs.
in mice (Mukhopadhyay et al. 2001). While hdl For example, in two CRC cell lines, siRNA data fit a
mutants could be efficiently rescued with a TCF3a model, where TCF4 represses Wnt targets in the
transgene, adding the VP16 domain, a potent transcrip- absence of signalling and TCF1 works with b-catenin
tional activation domain to TCF3a, abolished rescue to activate targets (Tang et al. 2008). Given that these
activity of the transgene (Kim et al. 2000). This suggests results are not consistent with the phenotype of TCF1
that most of TCF3a’s transcriptional activity is repres- and TCF4 knockouts in mice (Korinek et al. 1998,
sive. Knockout of TCF3 in mice also resulted in Roose et al. 1999), follow-up experiments with ChIP
phenotypes associated with an increase in Wnt/b-cate- and reporter genes are required to confirm this model.
nin signalling (Merrill et al. 2004) and siRNA inhibiton In the presumptive Spemann organizer, TCF3 occupies
of TCF3 in mouse ES cells largely results in increased the WRE upstream of the siamois TSS, and its binding
expression of target genes (Cole et al. 2008). In to chromatin is reduced by Wnt/b-catenin signalling
contrast, LEF1 knockouts have phenotypes best (Hikasa et al. 2010). It will be interesting to determine
explained by a loss of Wnt/b-catenin signalling (van the occupancy of TCF4 on the siamois WRE in response
Genderen et al. 1994, Reya et al. 2000, Kratochwil to pathway activation, since this TCF is required for
et al. 2002). siamois regulation (Standley et al. 2006).
In contrast to TCF3 and LEF1, TCF1 appears to be
more versatile. Mouse embryos lacking both TCF1 and
Functional analysis of TCF-DNA recognition in WREs
LEF1 have a loss of caudal somites that is reminiscent
of Wnt3a mutants (Galceran et al. 1999). TCF1 and The DNA sequence motif (CCTTTGATS) that mediates
LEF1 also act redundantly to pattern the mesoderm in high affinity binding of TCFs in vitro (van de Wetering
Xenopus embryos, and this activity is linked to et al. 1997, van Beest et al. 2000, Hallikas et al. 2006)
transcriptional activation (Liu et al. 2005). However, has been shown to be necessary and sufficient for
TCF1 and TCF3 have also been shown to act redun- activation of TCF-b-catenin-dependent transcription.
dantly in repressing Spemann organizer genes such as This sequence motif has been found in many WREs,
siamois in ventral blastomeres (Houston et al. 2002, that is, regulatory sequences that can activate transcrip-
Standley et al. 2006). Loss of the TCF1 gene in mice tion of promoters in response to Wnt/b-catenin signal-
demonstrated that it was a tumour suppressor in the ling. Mutation of these motifs in these WREs abolished
intestine and mammary gland (Roose et al. 1999). activation by the pathway (He et al. 1998, Yamaguchi
The situation for TCF4 also indicates both positive et al. 1999, Barolo 2006, Chang et al. 2008a). In
and negative roles in regulating Wnt targets. TCF4 addition, multiple copies of this motif placed upstream
knockouts display a loss of stem cells in the intestinal of a minimal promoter confer Wnt/b-catenin respon-
crypts (Korinek et al. 1998), consistent with a loss of siveness in cell culture (Korinek et al. 1997, Lum et al.
2011 The Authors

2003, DasGupta et al. 2005) and in transgenic mice and resulted in a huge derepression of expression with no
fish (DasGupta & Fuchs 1999, Dorsky et al. 2002, loss in maximal activation (Yang et al. 2000). It appears
Maretto et al. 2003, Nakaya et al. 2005). These studies, that for this WRE, Wg/Arm signalling activates expres-
combined with the TCF loss-of-function studies des- sion by alleviating TCF/Pan repression. At the other
cribed in the previous section have led to the view extreme, mutation of TCF sites often results in loss of
that TCFs are the major transcriptional regulators of expression of the WRE reporter (Yamaguchi et al.
Wnt/b-catenin signalling in most contexts. 1999, Lee & Frasch 2000, Chang et al. 2008b). For
Testing the functionality of TCF binding sites in these WREs, there appears to be little role for TCF
WREs has suggested that the switch model for TCF repression, and regulation by the pathway occurs
transcriptional regulation is WRE-specific. In several through TCF-b-catenin mediated activation (see Fig. 7A
cases, mutation of the TCF binding sites clearly dem- and B for further explanation).
onstrated both a repressive and a positive role. When a Like many transcription factors, the TCF consensus
single TCF site is destroyed in the END-1 WRE, a site is not inviolate. Many functional TCF binding sites
reduction in activation by Wnt/b-catenin signalling was in WREs have one or more substitutions from the
observed, but the reporter was also expressed in cells consensus (Barolo 2006). Systematic analysis of TCF4-
where it was normally repressed by POP-1 (Shetty et al. DNA binding in vitro also demonstrated that single
2005). Similar phenotypes were observed when TCF substitutions from the CCTTTGATS consensus
sites were mutated in the siamois WRE in Xenopus reduced, but did not abolish, recognition by TCF4
(Brannon et al. 1997, Fan et al. 1998) as well as an eve (Hallikas et al. 2006). There is no strict correlation
WRE in flies (Knirr & Frasch 2001). In contrast, between functional significance and adherence to the
mutation of TCF sites in a WRE controlling expression TCF consensus site. For example, the TCF site that is
of decapentapleigic (dpp) in the fly visceral mesoderm polymorphic in the far upstream c-myc WRE has either
(a) (a′)
(e)
(b) (b′) (f)
(c) (c′)
(d) (d′)
Figure 7 Variations on TCF transcriptional switches. (a & b) In organisms with a single TCF gene, the protein interacts with
co-repressors in the absence of signalling, while b-catenin binding to TCF displaces co-repressors and recruits co-activators. In
WREs from the nkd and notum genes, mutation of TCF binding sites results in a loss of activation (e) presumably due to the absence
of other transcriptional activators (Chang et al. 2008b). In other WREs such as END1 the loss of a single TCF site reduces
activation by Wnt/b-catenin signalling, but there is also significant derepression of expression in cells where the WRE is not
normally active (f, middle row), presumably due to the presence of other transcriptional activators (Shetty et al. 2005). In some
cases (e.g. the visceral mesodermal dpp WRE), mutation of the TCF sites results in full activation of expression in many cells (f,
lower row), suggesting that the primary function of b-catenin is to relieve TCF repression (Yang et al. 2000). (c) In Xenopus,
where multiple TCFs reside in the same cells, the transcriptional switch may be accomplished by HIPK2-mediated phosphorylation
of the repressive TCF3, which reduces its binding to WRE chromatin, allowing TCF1 to bind and activate transcription (Hikasa &
Sokol 2011). (d) The activity of a WRE can also be blocked by dominant negative isoforms of TCF which lack the ability to bind
b-catenin (Roose et al. 1999, Hovanes et al. 2001). See the text for additional examples of WRE regulation by TCFs. WRE, Wnt
response elements; TCF, T-cell factor; HIPK2, homeodomain interacting protein kinase 2.
2011 The Authors

one (CCTTTCATG) or two (TCTTTCATG) changes can identify functional sites among the sea of irrelevant
from the consensus, with the first site having increased ones.
affinity for TCF4 (Tuupanen et al. 2009, Wright et al.
2010). Likewise, binding sites that are highly functional
Bipartite binding of some TCFs through C-clamp-Helper
in fly WREs from the naked cuticle (nkd) locus can have
site interaction
two (GCTTTGTTC) or three (GCTTTGACA) differ-
ences from the consensus (Chang et al. 2008a). In Are TCF sites the only sequence information in WREs
addition to such high affinity sites in the eve and slp1 that facilitate TCF binding? A distinct sequence motif
WREs, there were also more divergent sites (e.g. with a consensus of GCCGCCR (R = A/G) has been
ACTTCACAG) that were footprinted by TCF/Pan shown to be critical for the activation of six WREs in fly
in vitro and contributed to activation in transgenic fly cells (Chang et al. 2008b). Unlike classic TCF sites,
reporter assays (Lee & Frasch 2000, Knirr & Frasch multiple copies of this element (termed the Helper site)
2001). The heterogeneity of what constitutes a TCF are not sufficient for activation of a promoter/reporter
binding site makes locating biologically relevant WREs cassette. However, these elements greatly enhance the
by sequence analysis alone extremely difficult. ability of HMG domain binding sites to respond to
A simple analysis of randomly selected human inter- pathway activation (Chang et al. 2008b). While the
genic DNA helps to illustrate how common predicted orientation and spacing of the Helper sites are not fixed
TCF binding sites are in the genome (Table 1). Perfect in relation to HMG sites, they are predominantly within
or near perfect sites (CCTTTGAWS) are rare (1 every 6 bp of each other. It appears that the presence of a
approx. 22 kb). But if one allows a modest level Helper site nearby provides additional context to the
of degeneracy, for example, SCTTTGAWS or CTTT- TCF site to facilitate TCF/Pan activation of WREs
GWWS, the frequency increases to 1/10 300 or 1/2500 (Chang et al. 2008b).
respectively (Table 1). These sequences are well within How do Helper sites facilitate TCF-mediated activa-
the range of known functional TCF sites (see preceding tion of WREs? The breakthrough came from the
paragraph and Chang et al. 2008a, Knirr & Frasch identification of an additional DNA-binding domain
2001, Lee & Frasch 2000). When the consensus is found in some isoforms of vertebrate TCFs, for exam-
loosened to the level of the polymorphic TCF site found ple, TCF-1E and TCF-4E (Atcha et al. 2007). This
in the upstream c-myc WRE (GTTTGWWS; Pomerantz domain, coined the C-clamp, contains four highly
et al. 2009, Tuupanen et al. 2009, Wright et al. 2010), conserved Cysteine residues (Fig. 2). The presence of a
the frequency in random DNA is 1 in 645 bp (Table 1). C-clamp downstream of the HMG domain of TCF1
This analysis suggests that there are millions of enabled the protein to recognize sequences containing
potential TCF binding sites in the human genome, and the classic HMG binding site and an additional
the challenge for researchers is to determine how TCF sequence of RCCG (Atcha et al. 2007). WREs from
the LEF1 and cdx1 genes are only activated by TCFs
containing a C-clamp (Atcha et al. 2003, 2007, Hecht
Table 1 The frequency of TCF sites depends on how one & Stemmler 2003). These WREs contain RCCG motifs
defines a TCF site. An open source algorithm called Target in close proximity to HMG binding sites (Atcha et al.
Explorer (Sosinsky et al. 2003) was used to create a 2007). The RCCG motif from vertebrates is similar to
weighted matrix of high to lower quality TCF sites. This the first four nucleotides in the Helper site (GCCG). In
matrix was used to search several stretches of human intergenic
flies, the major isoform of TCF/Pan contains a C-clamp,
DNA totalling 134 kb for potential TCF sites. While TCF
and this domain is required for activation of the WREs
sites matching the high affinity consensus site (CCTTTGAWS)
containing Helper sites (Chang et al. 2008b). Further-
are relatively rare, allowing increasing degrees of degeneracy
causes a rapid increase in the number of potential sites. See text more, recombinant TCF/Pan had a dramatic increase in
for further explanation affinity for HMG sites if a Helper site was present, and
this enhanced binding was C-clamp dependent (Chang
Frequency in random
et al. 2008b). These results suggest a model where TCF/
TCF site intergenic DNA (bp)
Pan, TCF1E and TCF4E recognize DNA through a
CCTTTGAWS 1/22 300 bipartite mechanism involving HMG domain-HMG-
SCTTTGAWS 1/10 300 site and C-clamp-Helper site interactions.
CTTTGWWS 1/2 500 In contrast to vertebrates, where only some of TCF1
SCTTTGWW, SGTTTGWWS 1/1 175 and TCF4 isoforms contain a C-clamp, almost all
or SCTTTCWWS invertebrate genomes examined contain only one TCF
CTTTGWW, GTTTGWW 1/645 gene with a C-clamp. The fly TCF/pan locus is subject to
or CTTTCWW
alternative splicing but the RNA-seq profiling indicates
TCF, T-cell factor. that the two most abundant isoforms expressed
2011 The Authors

Figure 8 Phylogenetic tree showing the evolution of the TCF family. The genome sequence of Monosiga brevicollis (choanofla-
gellate) reveals no TCF family member, while that of Amphimedon queenslandica (porifera), Mnemiopsis leidyi (ctenophore),
Nematostella vectensis (cnidarian), Caenorhabditis elegans (nematode), Drosophilia melanogaster (insect) and Strongylocentrotus
purpuratus (echinoderm) have a single TCF gene encoding a protein with HMG domain, basic tail and a C-clamp. Schistosoma
mansoni (Platyhelminthes) has three TCF genes; two with a C-clamp and one without. Mammals and amphibians have four TCF
genes, two of which have C-clamp containing isoforms. These patterns suggest that the last common ancestor of all metazoans
contained a single TCF gene with a C-clamp. In the two lineages where the TCF family increased in number, the C-clamp became
dispensible in some family members. TCF, t-cell factor; HMG, high mobility group.
throughout development (van de Wetering et al. 1997), expression is altered in the cells varies from hundreds to
possess both a HMG and a C-clamp domain (see thousands (van de Wetering et al. 2002, Jackson et al.
modencode website: http://modencode.oicr.on.ca/fgb2/ 2005, Klapholz-Brown et al. 2007, Van der Flier et al.
gbrowse/fly/?name=4:87956..131430). While such de- 2007). A list of these microarray studies can be found
tailed analysis of TCF isoforms in other invertebrates on the Wnt homepage curated by the Nusse Lab at
has not yet been performed, it appears that the ancestral http://www.stanford.edu/group/nusselab/cgi-bin/wnt/
TCF gene contained both domains, and that after and also at the Stanford Microarray Database website:
amplification during the vertebrate lineage, the C-clamp http://genome-www5.stanford.edu/. One simple mes-
was lost (LEF1 and TCF3) or partially retained through sage from these data sets is the limited amount of
alternative splicing (TCF1 and TCF4) (Fig. 8). This overlap between Wnt targets in different cell types. It
model makes the prediction that Helper sites will play has been estimated that as few as 5% of targets are
an important part in specifying invertebrate WREs, as identified in all studies (Vlad et al. 2008). While
has been found in Drosophila (Chang et al. 2008b), variations in experimental protocols and signal detec-
while additional mechanisms exist for target location of tion may contribute to this low number, most of the
vertebrate TCFs lacking a C-clamp. It should be pointed cell-specific differences likely reflect the existence of
out that despite the high degree of similarity among discrete transcriptional programs. In one study, micro-
C-clamps, some of the invertebrate domains have non- array analysis of PC12 and NIH3T3 cells identified 129
conservative changes at some positions. For example, and 355 genes with alteration of expression in response
the M. leidyi C-clamp has an arginine in place of the to Wnt3a treatment respectively (Railo et al. 2009).
third cysteine (Fig. 2). Direct analysis of these C-clamps Only two genes were commonly activated in both cell
will be required to determine whether they enhance lines, one of which was axin2, often considered a
TCF binding, as is the case for TCF/Pan, TCF1E and universally induced feedback antagonist of Wnt/b-cate-
TCF4E. nin signalling (Jho et al. 2002).
While some classes of Wnt targets such as Wnt
pathway components, proliferative genes or anti-apop-
Genome-wide analysis of TCF binding and
totic genes are found in multiple studies (Longo et al.
targets
2002, Chen et al. 2007, Klapholz-Brown et al. 2007,
In an attempt to define the Wnt/b-catenin transcrip- Van der Flier et al. 2007, Railo et al. 2009), other
tome, microarray based screens have been performed in classes may be more restricted in their expression
a variety of cell types. The number of genes whose domains. For instance, angiogenic (Masckauchan et al.
2011 The Authors

2005) or osteoblastic and adipogenic targets (Jackson alternative is to use computational approaches to detect
et al. 2005) are most likely Wnt responsive only in WREs within entire genomes. For example, an algo-
permissive tissue specific contexts. rithm called the enhancer element locator utilized
One limitation of microarrays is that they do not binding site affinity matrixes and motif clustering
distinguish between direct and indirect targets. ChIP- conservation between two or more species to identify
based genomic surveys offer the potential to identify potential WREs (Hallikas et al. 2006). Several putative
regions of the genome that are enriched for a particular elements were tested in a transgenic mouse assay and
TCF or b-catenin. How many of these bound regions found to be expressed in patterns that were consistent
actually correspond to a functional WRE? For example, with positive regulation by Wnt/b-catenin signalling,
in one study of TCF4 binding sites in LS174T cells, a though this was not directly confirmed by site-directed
CRC cell line, over 6 800 high quality binding peaks mutation of the conserved TCF sites (Hallikas et al.
were identified using a ChIP-microarray approach 2006). While this method is likely to identify some
(Hatzis et al. 2008). More than 70% of the identified WREs, the challenge of sorting through the entire
peaks were over 10 kb from the nearest TSS, highlight- genome requires stringent screening parameters which
ing the tremendous amount of genomic real estate that likely miss many biologically relevant elements. While
must be examined when searching for WREs. In many one of the benefits of this algorithm is the reliance on
cases, several TCF4-bound regions were found near a conservation of motif clusters rather that strict sequence
single gene, such as the 11 peaks surrounding the Axin2 conservation, in some instances, enhancer elements in
gene. Four of these regions had WRE activity in a divergent species have been shown to retain functional
reporter assay, while 10 out of 22 other TCF4 bound conservation while losing motif clustering or locational
regions from other locations tested positive in this assay conservation (Kalay & Wittkopp 2010), and thus may
(Hatzis et al. 2008). It remains to be seen whether the elude this type of analysis.
regions that tested negative in the reporter assay are Given the likelihood that many targets of the Wnt/b-
simply non-functional binding sites for TCF4 or are catenin pathway are cell specific, it seems that a
WREs that are not active in a simple reporter assay. The combination of transcriptome analysis, physical local-
relatively small degree of overlap (12.5–20.5% depend- ization assays (e.g. ChIP-seq) and further refinement of
ing on how the comparison is made) between whether a computational approaches will be needed to efficiently
TCF4-bound region was found within 100 kb of a TSS identify WREs in all the interesting contexts where the
from a gene upregulated in adenomas suggests that pathway plays important roles. The existence of WREs
many of these Wnt targets may be indirectly regulated acting over great distances makes this undertaking even
(Hatzis et al. 2008). more challenging. These efforts should be aided by the
Another study using ChIP followed by high through- realization that many Wnt targets are controlled by
put sequencing (ChIP-seq) identified over 20 000 TCF4 TCFs in combination with other transcription factors,
bound regions in the human CRC cell line HCT116 often in direct contact with each other. Some of the
(Blahnik et al. 2010). Over 6000 of these motifs map to literature on this topic is covered in the following
putative enhancer regions with 10–100 kb of a TSS, section.
while over 9000 mapped within 2 kb of a TSS. Using
the same cell line, over 2100 regions enriched for
TCF isoforms: a complicated situation gets more so
b-catenin binding were also reported (Bottomly et al.
2010). In this study, only 47% of the peaks contained at The existence of alternative splicing and promoter
least one consensus TCF motif in the vicinity of the selection in vertebrate TCF genes results in a highly
peak. The remaining peaks may represent TCF-inde- complex and varied inventory of TCF isoforms. For
pendent b-catenin targets, peaks containing TCF bind- example, the mouse TCF4 locus contains 17 exons, and
ing motifs which diverge from the consensus, or false more than a dozen TCF4 isoforms resulting from
positives. alternative splicing have been identified (Weise et al.
The above studies indicate that TCF4 can bind to 2010). Four isoforms (E isoforms) contained a C-clamp,
regions far removed from the TSS of target genes. This though the protein sequence differed at 3 positions
suggests that the most common way of determining depending on whether exon 14 or 15 was used. Three
whether a developmental gene is directly regulated by isoforms contained a truncated C-clamp (after the third
Wnt/b-catenin signalling, that is, scanning the region cysteine – see Fig. 2) and three others contained new
immediately upstream of the candidate gene’s TSS for protein sequence after position 20 of the motif (Weise
conserved TCF binding sites, followed by site-directed et al. 2010). These six isoforms were collectively
mutagenesis, may miss many WREs. While continued referred to as S isoforms, while isoforms completely
genome-wide surveys of TCF-bound chromatin in lacking the C-clamp were labelled M isoforms (Fig. 9).
interesting developmental contexts is desirable, an Representatives from each group were compared in
2011 The Authors

Figure 9 Diversity of TCF/LEFs. Invertebrates contain a single TCF member containing the b-catenin binding (green), HMG (red),
basic tail (turquoise), and C-clamp (blue) domains. Pictured here is the most abundant isoform in Drosophila (Pan A) and the
C. elegans POP-1. In vertebrates, alternate promoter usage and alternative splicing result in a myriad of TCF isoforms with
diverse functional properties. Alternate usage of downstream promoters can result in isoforms which lack the b-catenin binding
domain, and function as natural dominant negatives, such as dnTCF1 and dnLEF1 (Roose et al. 1999, Hovanes et al. 2001).
Alternate exon usage (orange) occurs in all family members except TCF3, and the LVPQ/SXXSS motif (purple) which is invariant
in TCF3 confers repressive activity on TCF4 isoforms which contain it (as in TCF4A) (Liu et al. 2005). Inclusion of the C-clamp
motif is seen in E-tail containing isoforms TCF1E and TCF4E. M isoforms lack the C-clamp, while S isoforms contain truncated
C-clamp domains (Weise et al. 2010). Some TCF3 and TCF4 isoforms also contain CtBP binding sites. TCF, T-cell factor; LEF,
lymphoid enhancer-binding factor; HMG, high mobility group.
several assays and significant differences were found. contribute to higher levels of Wnt/b-catenin signalling
For example a TCF4E isoform could bind to and (Hovanes et al. 2001, Yokoyama et al. 2010). In
regulate a cdx1 WRE to a far greater extent than TCF4S T-helper cells, dnTCF1 is thought to regulate cell
or TCF4M isoforms (Weise et al. 2010). The biological polarization via IL4 signalling. TCF1E activates
role of the TCF4S isoforms merits further study but GATA3 expression, which then suppresses dnTCF1
there is data suggesting that TCF4E isoforms are transcription in an IL4 dependent manner. This positive
preferentially required for increased growth and prolif- feedback loop promotes T helper cell polarization
eration in some CRC cell lines (Atcha et al. 2007) and (Maier et al. 2011).
the presence of TCF4E isoforms is correlated with Other TCF isoforms influence the ability of the
increased disease progression in renal cell carcinoma proteins to act in repression or activation. In Xenopus,
(Shiina et al. 2003). TCF4A contains two motifs in the central portion of the
Another class of TCF isoforms lacks the b-catenin protein (LVPQ and SXXSS) that are missing in TCF4C
binding domain normally found at the N-terminus (Fig. 9). TCF4A can rescue embryos depleted of TCF3,
(Fig. 9). These isoforms are predicted to block Wnt/ suggesting that it can act as a repressor, while TCF4C
b-catenin signalling, since experimentally derived TCFs can rescue embryos depleted of TCF1 or LEF1,
lacking this domain act as potent dominant negatives suggesting a role in transcriptional activation (Liu et al.
(Behrens et al. 1996, Molenaar 1996, van de Wetering 2005). In CRC, expression of TCF4E isoforms clacking
et al. 1997, Kratochwil et al. 2002). For example, high a binding site for CtBP (a transcriptional co-repressor)
expression of truncated TCF1 (dnTCF1) isoforms have has been correlated with increased malignancy (Cuil-
been proposed to explain the tumour suppressor effect liere-Dartigues et al. 2006). Furthermore, these iso-
of TCF1 in mice (Roose et al. 1999). In human forms have decreased repressive activity in cell culture
intestinal epithelial cells, LEF1 can be expressed from reporter assays (Cuilliere-Dartigues et al. 2006, Tang
two distinct promoters. Transcription from the second et al. 2008).
promoter produces a LEF1 lacking the b-catenin bind- Given the complexities of understanding even a single
ing domain (Hovanes et al. 2001). In human CRC, this TCF isoform, comprehending how the entire TCF
truncated LEF1 is suppressed, which is predicted to isoform repertoire is orchestrated to influence Wnt/b-
2011 The Authors

catenin signalling is a long-term objective for the field. Interestingly, a Wnt and CaMKII-dependent efflux of
For further information on TCF isoforms, see the dnTCF1 has also been reported in human CRC cells
following review (Arce et al. 2006). (Najdi et al. 2009).
Is the NLK-mediated phosphorylation and nuclear
export found in C. elegans operating in other organ-
Post-translational modifications of TCFs
isms? In Xenopus, NLK phosphorylation of TCF4 and
Post-translational modifications of TCFs are known to LEF1 promotes ubiquitylation and degradation (Ya-
influence their subcellular localization, stability and mada et al. 2006). In Drosophila, overexpression of
their ability to bind to DNA and cofactors. These Nemo, the fly homolog of NLK, inhibits Wg/Arm
modifications can have a stimulatory or inhibitory signalling and reduction of nemo activates the pathway
effect, depending on the context. For example, the (Zeng & Verheyen 2004). Whether this occurs through
SUMO E3 ligase PIASy has been shown to facilitate nuclear efflux or degradation of TCF/Pan is not yet
sumoylation of LEF1, resulting in sequestration of the known.
protein into nuclear bodies, where it cannot activate Several other kinases have been shown to influence
Wnt targets (Sachdev et al. 2001). Conversely, PIASy TCF activity through direct phosphorylation. CKII can
sumoylates TCF4, which increases its affinity for promote Wnt target gene activation by phosphorylating
b-catenin, promoting target gene activation (Yamamoto LEF1, which reduces its affinity for TLE co-repressors
et al. 2003b). The physiological role for PIASy in the (Wang & Jones 2006, Sun & Weis 2011). The Traf2-
Wnt/b-catenin pathway is not clear, given that disrup- and Nck-interacting kinase (TNIK) is required for
tion of this gene in mice resulted in no morphological activation of TCF4-b-catenin in mammalian cells and
defects and only mild effects on some Wnt targets (Roth can form a complex with TCF4 and b-catenin (Shitash-
et al. 2004). ige et al. 2008, Mahmoudi et al. 2009). High levels of
TCFs have also been shown to be acetylated by the TNIK have also been shown to be required for maximal
invertebrate homologs of CBP and p300. These histone growth of CRC cell lines with elevated Wnt/b-catenin
acetyltransferases are normally associated with histone signalling (Shitashige et al. 2010). The positive rela-
acetylation and gene activation (Ogryzko et al. 1996). tionship between the pathway and TNIK is conserved in
Indeed, CBP/p300 is known to be recruited to WREs Xenopus, where primary axis formation and activation
through binding to b-catenin/Arm and is required for of Spemann organizer genes are dependent on TNIK
activation of Wnt targets (Mosimann et al. 2009). and its kinase activity (Satow et al. 2010). This study
Paradoxically, partial loss of fly CBP gene activity demonstrated a b-catenin-dependent recruitment of
elevated Wg/Arm signalling (Waltzer & Bienz 1998). TNIK to the siamois and other organizer WREs,
This was shown to be the result of CBP acetylation of consistent with a direct role in Wnt target gene
TCF/Pan on K25, a conserved lysine in the b-catenin/ activation (Satow et al. 2010).
Arm binding domain. This modification weakens bind- At least one other TCF phosphorylation event occurs
ing of TCF/Pan to Arm (Waltzer & Bienz 1998). An at Xenopus Wnt targets in early embryogenesis. In
inhibitory role for CBP/p300 has also been reported in Xenopus embryos, TCF3 is phosphorylated in response
mammalian cells, though the exact mechanism is not to Wnt/b-catenin signalling (Hikasa et al. 2010). Home-
clear (Li et al. 2007). In C. elegans, POP-1 can be odomain interacting protein kinase 2 (HIPK2) is a
acetylated by human p300 at K185 just N-terminal of major mediator of this Wnt-dependent phosphorylation
the HMG domain (Gay et al. 2003). Acetylation at this and requires b-catenin for this activity. Reduction of
site also occurs in worms, and was required for nuclear HIPK2 reduces TCF3 phosphorylation and inhibits
localization and biological activity of POP-1 (Gay et al. induction of the Spemann organizer (Hikasa et al.
2003). This region of POP1 is not well conserved in 2010). Importantly, expression of a TCF3 variant that
other TCFs, so it is not clear whether this mechanism cannot be modified by HIPK2 also blocks activation of
occurs in other organisms. Wnt targets. Interestingly, HIPK2-dependent phosphor-
Phosphorylation has also been linked to POP-1 ylation of TCF3 by Wnt/b-catenin signalling results in a
nuclear localization. In Wnt receiving cells, a complex reduction of TCF3 on siamois WRE chromatin (Hikasa
of the MAP kinase LIT-1 (NLK) and WRM-1 (a worm et al. 2010). These data support a model where b-cate-
b-catenin) binds to POP-1 and phosphorylates it nin promotes HIPK2 modification of TCF3, which
(Rocheleau et al. 1999). This results in nuclear export results in removal from the WRE, alleviating the
of POP-1, which is mediated by PAR-5, a 14-3-3 repressive influence of TCF3. HIPK2 also phosphory-
protein (Lo et al. 2004). This export lowers the level of lates TCFs in human cells (Hikasa & Sokol 2011), but
nuclear POP-1, facilitating the switch of POP-1 from a the functional consequence of this modification awaits
repressor to a b-catenin (SYS-1 or BAR-1) bound further study. In flies, HIPK2 promotes Wg signalling
transcriptional activator (Phillips & Kimble 2009).
2011 The Authors

through stabilization of Arm (Lee et al. 2009), though 3 or Smad 4 (Labbe et al. 2000, Nishita et al. 2000).
modification of TCF/Pan has not been excluded. These results suggest a model where a combination of
One interesting speculation is that HIPK2 and TNIK protein-DNA interactions and protein-protein interac-
act in concert to activate Wnt targets in the Xenopus tions can promote the formation of a Smad-TCF-b-
embryo. b-catenin allows HIPK2 to phosphorylate catenin complex in a signalling-dependent manner
TCF3, removing it from the WRE (Hikasa et al. 2010). (Fig. 10B).
At the same time, b-catenin recruits TNIK to TCF4,
which somehow allows the TCF4-b-catenin complex to
(a)
activate transcription (Satow et al. 2010). In this way,
b-catenin could affect a swap of the TCFs that mediate
repression and activation respectively. Recently, it has
been shown for Vent2, a Wnt target in ventral Xenopus
blastomeres, that pathway activation results in a
replacement of TCF3 by TCF1 on the Vent2 regulatory
region (Hikasa & Sokol 2011). Since TCF3 represses
and TCF1 activates Vent2 expression (Hikasa & Sokol
2011), this study provides the first direct support for a
‘TCF switch’ model where Wnt/b-catenin signalling (b)
promotes a physical change of distinct TCFs (Fig. 7C).
Whether other kinases such as TNIK are involved in this
switch remains to be examined.
Combinatorial interactions of TCFs and other

transcription factors
Given the flexibility of what constitutes a HMG binding
site, it is likely that this interaction is not sufficient for
TCFs to distinguish WREs from non-functional binding
(c)
sites that occur throughout the genome. Indeed, this
appears to be the case in Drosophila, where bipartite
binding to WREs occurs through HMG domain-HMG
site and C-clamp-Helper site interactions (Chang et al.
2008b). While the conservation of the C-clamp among
invertebrates (Figs 2 and 8) suggests that this strategy
may be prevalent in these organisms, what about the
situation in vertebrates, where most vertebrate TCF
isoforms do not possess a C-clamp? In this section,
several transcription factors are discussed that interact
with TCFs and/or b-catenin and appear to act cooper-
Figure 10 Three different mechanisms that contribute to TCF
atively with TCFs to bind to regulatory elements. target selection in the nucleus. (a) Bipartite binding of TCF/Pan
One family of transcription factors that interact with with HMG domain–HMG site and C-clamp–Helper site
TCFs on cis-regulatory elements are the Smads, which interactions at a binding site in the intronic WRE from nkd
mediate many aspects of TGF-b signalling (Moustakas (Chang et al. 2008b). This strategy increases the TCF recog-
& Heldin 2009). The Wnt/b-catenin and TGF-b path- nition site to approx. 16 basepairs. (b) Combinatorial binding
ways cross-talk at several levels (Eivers et al. 2009, between LEF1 and a Smad heterodimer on the twin WRE in
Itasaki & Hoppler 2010) and this review will limit the Xenopus (Labbe et al. 2000, Nishita et al. 2000). The adjacent
discussion to reports where both pathways appear to location of the Smad and TCF binding site again increases the
amount of basepairs required for binding. Smads and b-catenin
assemble Smad-TCF-b-catenin complexes on cis-acting
are also thought to cooperate in recruiting p300/CBP to TGFb
regulatory elements. This was first shown for the
regulated WREs (Lei et al. 2004). (c) In the case of the c-jun
regulatory region of the twin gene in Xenopus embryos
and c-myc regulatory regions, the TCF and AP-1 sites are not
(Nishita et al. 2000) and mammalian cells (Labbe et al. near each other (Nateri et al. 2005, Yochum et al. 2008),
2000). In both contexts, both Smad and HMG binding suggesting a model where DNA looping is stabilized by inter-
sites were required for maximal activation of reporter actions between c-Jun and TCF. TCF, T-cell factor; HMG,
constructs by Wnt/b-catenin and TGF-b signalling. The high mobility group; WRE, Wnt response elements; LEF,
HMG domain of LEF1 can directly interact with Smad lymphoid enhancer-binding factor.
2011 The Authors

Since these initial reports, other genes have been than a dozen sites were bound by TCF4, b-catenin and
identified that are co-regulated by TCFs and Smads. c-Jun. As previously shown for c-myc, the activation of
Most of these studies are in the context of cell culture, several Wnt targets were enhanced by serum in CRC
with regulatory elements from the Msx2 (Hussein et al. cells arrested in G0/G1 (Bottomly et al. 2010). The
2003), c-myc (Hu & Rosenblum 2005), gastrin (Lei connection between Wnt/b-catenin signalling and cell
et al. 2004, Chakladar et al. 2005), Sm22a (Shafer & cycle progression has also been noted further upstream
Towler 2009), TMEPA1 (Nakano et al. 2010) and in the pathway (Davidson & Niehrs 2010).
several osteogenic genes (Rodriguez-Carballo et al. The Wnt/b-catenin pathway-c-Jun connection has
2011). But the existence of functional Smad and TCF also been observed outside the context of intestinal
binding sites in close proximity to each other has also cells and CRC. Regulatory elements controlling either
been found in Emx2 elements active in the developing the matrilysin gene in kidney or the versican gene in
CNS of the mouse (Theil et al. 2002, Suda et al. 2010). melanoma require both TCF and AP-1 sites (Rivat et al.
While these studies mostly relied on reporter constructs, 2003, Domenzain-Reyna et al. 2009). In addition,
there is some ChIP data to suggest that Wnt/b-catenin interactions between TCF4 and c-Jun (Gan et al.
signalling can increase Smad recruitment to regulatory 2008) or b-catenin and the AP-1 complex (Toualbi
chromatin (Hussein et al. 2003, Shafer & Towler et al. 2007) can regulate Wnt targets independent of
2009). Conversely, TGF-b signalling can recruit LEF-1 AP-1 binding sites, though whether this type of regu-
or TCF4 to chromatin as well (Hussein et al. 2003, lation occurs under physiological conditions is not clear.
Nakano et al. 2010). The presence of both Smad and The Smad and c-Jun/AP-1 studies described above are
b-catenin on the chromatin has been proposed to examples where distinct signalling pathways and Wnt/
increase binding for the histone acetyltransferases b-catenin signalling converge on regulatory elements to
CBP/p300, leading to increased histone acetylation activate transcription. Such combinatorial control of
and transcription (Lei et al. 2004, Rodriguez-Carballo Wnt targets can also occur through interactions with
et al. 2011) (Fig. 10B). transcription factors not directly controlled by cell–cell
Another transcription factor linked with TCF-b-cate- signalling. One candidate for such factors is the Cdx
nin transcriptional activation is c-Jun, a basic leucine family of homeodomain proteins. As outlined in a
zipper domain protein that can bind DNA specifically previous section, cdx1 and cdx4 are known to be direct
as a homodimer or as a heterodimer with c-fos transcriptional targets of the Wnt/b-catenin pathway
(constituting AP-1) (Shaulian & Karin 2002). In CRC (Lickert et al. 2000, Ikeya & Takada 2001, Pilon et al.
cells, c-Jun, TCF4 and b-catenin cooperated in activat- 2006). In addition, there is some developmental genetic
ing the c-Jun expression (Nateri et al. 2005). Phosphor- data suggesting that the TCF-b-catenin complex may
ylated c-Jun was found to associate with TCF4 and both functionally interact with Cdx proteins (Young et al.
transcription factors occupy the c-Jun regulatory region 2009). Indeed, Cdx1 autoregulation has been shown to
(Nateri et al. 2005). These data complement genetic require a Cdx1-LEF1-b-catenin complex, through direct
interaction studies in the mouse intestine to support a interactions between the homeodomain and HMG
model, where Wnt/b-catenin signalling acts with c-Jun domains (Beland et al. 2004). More recently, a gen-
in a positive feedback loop to promote carcinogenesis ome-wide survey of Cdx2 binding in intestinal cell lines
(Nateri et al. 2005, Sancho et al. 2009). In contrast to revealed a significant overlap between Cdx2 and TCF4
most of the elements co-regulated by TCF and Smad, chromatin bound regions (Verzi et al. 2010). TCF4
the distance of the functional TCF and AP-1 site binding to chromatin was found to be partially depen-
suggests the existence of a DNA loop stabilized by dent on Cdx2 at several locations (Verzi et al. 2010).
protein-protein and protein-DNA interactions Interestingly, nested TCF-Cdx binding sites have been
(Fig. 10C). shown to be required for an intronic raldh2 enhancer
While, the Wnt/b-catenin-c-Jun autoregulatory loop that is active in the dorsal spinal cord of the chick
may be crucial for intestinal cancer in mice and CRC in (Castillo et al. 2010).
humans, additional evidence suggests that many Wnt There are other examples of TCFs interacting with
transcriptional targets in CRC cells are co-regulated by other transcription factors to regulate gene expression.
TCF4 and c-Jun. The c-myc WRE located downstream LEF1 and microphthalmia-associated transcription fac-
of the c-myc gene (Fig. 6) contains a functional AP-1 tor are thought to physically interact to regulate gene
site that is required for synergistic activation between expression in melanocyte differentiation (Yasumoto
the Wnt/b-catenin pathway and serum-derived mitogens et al. 2002). Likewise, LEF1 and the homeodomain
(Yochum et al. 2008). A genome-wide survey of chro- protein Pitx2 may interact in the developing dental
matin sites with b-catenin enrichment revealed that epithelium and other tissues (Amen et al. 2007). The
40% of the b-catenin bound regions contain both TCF short list of transcription factors that interact with TCFs
and AP-1 binding sites (Bottomly et al. 2010). More discussed here is likely only the tip of the iceberg. The
2011 The Authors

genome-wide studies of TCF4 binding patterns have (Kioussi et al. 2002). It should be pointed out that
found an enrichment for many other transcription PitX2 can also bind LEF1, suggesting that it sometimes
factor binding sites besides AP-1, including NF1, works with TCFs to regulate Wnt targets (Vadlamudi
PPARc, HNF4, Elk-1, GATA3, c-Ets-1, Bach-1 and et al. 2005, Amen et al. 2007).
FoxD-1 (Hatzis et al. 2008, Blahnik et al. 2010). One approach to rule out a role for TCFs in the
TCF-protein interactions may be the normative mech- activation of target genes by b-catenin is the use of
anism to locate WREs in the information rich nucleus, dominant negative constructs of TCF (dnTCF) that
though the binding partner is likely to be different for cannot bind b-catenin. When expressed at sufficient
different targets. It should also be noted that while the levels, such constructs should prevent the binding of
mechanisms described above have been assumed to endogenous TCFs to regulatory elements (Behrens et al.
operate for TCFs lacking C-clamps, they may also 1996, Molenaar 1996, van de Wetering et al. 1997,
function to facilitate target gene location of C-clamp Kratochwil et al. 2002). In cultured myoblasts, b-cate-
containing TCFs. It seems likely that multiple mecha- nin is required for the bHLH protein MyoD to activate
nisms acting in concert are required for TCFs to locate muscle specific genes (Kim et al. 2008). The presence of
WREs in the genome. high levels of b-catenin enhanced MyoD binding to
myogenic elements, presumably through direct binding.
This regulation was not affected by expression of
TCF-independent recruitment of b-catenin to
dnTCF (Kim et al. 2008). dnTCF also did not block
Wnt targets
the ability of b-catenin to augment transcriptional
TCFs are clearly the best characterized transcription activation of hypoxia inducible factor 1a (HIF1a) (Kaidi
factors that mediate the regulation of gene expression et al. 2007).
via Wnt/b-catenin signalling. In addition to TCFs, there In CRC cells with high endogenous levels of b-cate-
are several other DNA-binding proteins that have been nin, hypoxia (which dramatically increases HIF1a
shown to bind to b-catenin and effect transcriptional levels) results in a shift of b-catenin away from the
responses. Many nuclear receptors, including the classic Wnt target c-myc towards HIF1a targets (Kaidi
oestrogen and androgen receptors bind b-catenin and et al. 2007). It should be noted that this effect is cell
co-regulate targets of these hormones, as reviewed specific, since in several stem cells, hypoxia activates
elsewhere (Mulholland et al. 2005, Beildeck et al. Wnt/b-catenin signalling by HIF1a-dependent induction
2010). In this section, we discuss other transcription of LEF1 and TCF1 transcription and b-catenin protein
factors that serve as recruiters for nuclear b-catenin and levels (Mazumdar et al. 2010). In these cells, sufficient
how they may contribute to the enormous diversity of b-catenin is thought to allow both types of transcription
transcriptional outputs that are generated by the Wnt/ factors to activate target genes (Mazumdar et al. 2010).
b-catenin pathway. Another example where a stress-induced factor can
To conclude that a transcription factor is sufficient to divert b-catenin away from TCFs is the FOXO family
recruit b-catenin to a regulatory element, the factor of Forkhead domain transcription factors. FOXO
must bind and co-regulate targets with b-catenin. This is proteins can directly bind b-catenin and use it to
the case for Sox17, which regulates endodermal genes activate gene expression in C. elegans and mammalian
during vertebrate gastrulation. b-catenin augments cells (Essers et al. 2005). Induction of FOXO protein
Sox17¢s ability to activate target genes and the two levels during oxidative stress reduces TCF-dependent
proteins physically interact (Sinner et al. 2004). How- gene expression, due to competition for limiting levels
ever, a role for TCFs in this process was not ruled out of b-catenin (Almeida et al. 2007, Hoogeboom et al.
and in CRC cells, Sox17 has also been shown to interact 2008).
with TCFs and promote their degradation (Sinner et al. How important are non-TCFs in mediating Wnt/
2007). In another case, the homeodomain protein Prop1 b-catenin signalling during normal development? In
and b-catenin are both required for Pit1 expression in cnidarians, the only functional data thus far on TCFs
the developing mouse pituitary gland (Olson et al. suggests that in Hydra, TCF is absolutely required for
2006). These proteins directly interact and are co-local- Wnt-dependent head regeneration (Duffy et al. 2010).
ized on Pit1 regulatory chromatin. LEF1 is not required In Drosophila, Tcf/pan mutants clearly are qualitatively
for Pit1 expression, but other TCF family members were similar to wg mutants (Brunner et al. 1997, van de
not examined (Olson et al. 2006). PitX2 can also bind Wetering et al. 1997, Schweizer et al. 2003), but it is
to b-catenin and bring it to the cyclin D2 promoter in not clear that it is required for all Wg signalling. In
mouse myoblast cells (Kioussi et al. 2002). A synthetic vertebrates, the role of TCFs is much more difficult to
enhancer containing multimerized PitX2 sites is acti- assess, given the likely redundancy between the four
vated in a b-catenin-dependent manner, suggesting that TCFs. While the available data suggests that vertebrates
PitX2 is sufficient for recruiting b-catenin to DNA TCFs play a major role in mediating Wnt/b-catenin
2011 The Authors

signalling, the sum of all other transcription factors What is known about the mechanism of these
recruiting b-catenin to Wnt target genes may ultimately examples of TCF-b-catenin repression? For the stripe
be much more significant than currently appreciated. WRE, the functional TCF binding site overlaps with a
binding site for Cubitus Interruptis (Ci), which is crucial
for activation of stripe in embryos (Piepenburg et al.
Direct transcriptional repression by b-catenin
2000). This suggests that TCF/Pan-Arm may displace
This review has focused exclusively on the ability of the Ci activator. In the case of dpp, Brinker binds to the
b-catenin to activate transcription, which is reasonable WRE and somehow acts with TCF/Pan-Arm to mediate
considering the large pile of evidence for such a role. But repression in the leg imaginal disc (Theisen et al. 2007).
it is worth noting that Wnt/b-catenin signalling leads to In keratinocytes, LEF1-b-catenin works additively with
down-regulation of gene expression about as often as the Snail repressor to inhibit E-cadherin transcription
activating genes (van de Wetering et al. 2002, Jung & (Jamora et al. 2003). It is not clear in these cases or that
Kim 2005, Naishiro et al. 2005, Klapholz-Brown et al. of pINK16a where the specificity lies that allows TCF-
2007). Because of the pathway’s obvious role in b-catenin to repress, rather than activate, gene expres-
activation, it is usually assumed that these genes are sion.
indirectly repressed by Wnt/b-catenin signalling, that is, Another example of direct repression by the pathway
the pathway activates a repressor that then inhibits occurs in cultured hemocytic cells in Drosophila, where
another genes expression. While this no doubt occurs, Wg/Arm signalling represses expression of Ugt36Bc, an
there are a growing number of cases where b-catenin is enzyme deposited in the extracellular matrix (Blauwk-
thought to directly mediate repression through TCF amp et al. 2008). TCF/Pan is enriched in the same
family members. This is distinct from the well-known region upstream of the Ugt36Bc TSS that contains a
repressive role of TCFs in the absence of signalling that WRE that is repressed by the pathway. This WRE was
has been previously discussed. localized to a 178 bp region that contained no classic
What are the experimental criteria for determining TCF binding sites. However, footprinting revealed the
that a target is directly repressed by Wnt/b-catenin existence of three TCF sites, which share a consensus of
signalling? A straightforward approach is to monitor AGAWAW. These sites are functional, mediating acti-
target gene expression when the pathway is activated in vation of the WRE in the absence of signalling and Arm-
cells treated with protein synthesis inhibitors. While, dependent repression (Blauwkamp et al. 2008). Thus,
this is straightforward for many pathways, for example, Ugt36Bc is regulated by a ‘reverse transcriptional
TGFb (Kang et al. 2003), it is problematic for Wnt/ switch’ compared with the classic switch depicted in
b-catenin signalling, given the requirement for new Fig. 1.
synthesis of b-catenin once its degradation is blocked The existence of novel TCF binding sites in the Ugt
(see Fig. 1). There are hormone-inducible versions of reporter suggested that the nature of the binding site
b-catenin, created by fusing portions of a nuclear contained the specificity for the transcriptional output.
receptor to b-catenin, and these have been used to Support for this came from an experiment where the
determine activation of targets in the presence of novel sites were converted to classic ones. Strikingly,
protein synthesis inhibitors (Li et al. 2009, Elkouby this altered WRE was now activated in response to Wg/
et al. 2010). But these reagents can be tricky to utilize Arm signalling (Blauwkamp et al. 2008). The possibility
for identifying direct targets of repression (Blauwkamp of allosteric regulation of TCFs by DNA is supported by
et al. 2008). structural analysis of the LEF1 HMG domain alone and
In lieu of this approach, researchers have used a when complexed with a high affinity binding site. When
combination of site-directed mutagenesis of TCF bind- the HMG is not bound by DNA, it is partially
ing sites in repressed cis-regulatory elements to provide unstructured, particularly helix 1. Upon DNA binding,
support for direct regulation. In flies, such studies have the HMG domain is converted to a well-folded, highly
shown that WREs from the stripe and dpp genes contain ordered state (Love et al. 2004). It would not be
TCF sites that are absolutely required for repression surprising if different DNA-binding sites could influence
(Piepenburg et al. 2000, Theisen et al. 2007). In cell the conformation of TCF, which could then influence
culture, a luciferase reporter has been used in a similar the recruitment of co-activators or co-repressors by
way to demonstrate that Wnt/b-catenin signalling b-catenin/Arm.
directly represses E-cadherin in keratinocytes (Jamora Another clue to the mechanism of TCF/Arm direct
et al. 2003), and p16INK4a in melanoma (Delmas et al. repression was found through mutagenesis of the Arm
2007). In these cases, ChIP was also employed to gene. Deletion or mutation of portions of Arm known
demonstrate that TCF and b-catenin were physically to be involved in transcriptional activation created a
present on the repressed WREs (Jamora et al. 2003, variant called DisArmed. Consistent with the altera-
Delmas et al. 2007). tions, DisArmed could not regulate several WREs that
2011 The Authors

are activated by the pathway (Blauwkamp et al. 2008). for why some WREs are activated in a cell-specific
However it could still efficiently repress Ugt36Bc manner, for example, eve in cardiac mesoderm (Halfon
expression and the Ugt reporter. In addition, DisArmed et al. 2000, Lee & Frasch 2000, Knirr & Frasch 2001,
could repress several other downregulated Wg targets in Han et al. 2002). Other mechanisms, for example, cell-
cultured cells and fly hemocytes, suggesting that they specific chromatin accessibility, are thought to occur
also are directly repressed by TCF/Pan-Arm (Blauwk- (Wohrle et al. 2007), but remain relatively unexplored.
amp et al. 2008). Despite the challenge of understanding the complex-
As is the case with activated Wnt targets, there are ities of Wg target gene regulation, our current knowledge
also reports of non-TCF proteins utilizing b-catenin to is detailed enough so that it can be exploited to further
repress gene expression. The best studied is the home- understand how this pathway controls development. The
odomain protein Prop1, discussed earlier as a transcrip- realization of an ancient posterior Wnt signalling gradi-
tion factor that binds b-catenin to activate transcription ent throughout metazoans (Petersen & Reddien 2009,
(Olson et al. 2006). Like TCFs, it can also do the Niehrs 2010), provides one example. For cnidarians, we
opposite, recruiting b-catenin to a regulatory element think it likely that the potentially important targets (e.g.
controlling another transcription factor called Hesx1 Brachyury) will be bound by TCFs by a similar bipartite
(Olson et al. 2006). This repression requires reptin, a mechanism as has been shown to operate in Drosophila
transcriptional co-repressor (Olson et al. 2006). The (Chang et al. 2008b), given the presence of highly
antimetastasis gene KAI1 is also repressed by a complex conserved HMG and C-clamp domains in cnidarians
containing b-catenin and Reptin, but the DNA-binding (Figs 2 and 8). Using information from other systems
transcription factor is not known (Kim et al. 2005). may allow the dissection of the genetic networks in great
Interestingly, Reptin is known to bind to b-catenin/Arm detail, complementing the careful analysis of regulatory
in a region that is not altered in the DisArmed protein factors that are expressed temporally downstream of
(Bauer et al. 2000), though it is not known whether fly Wnts, exemplified by the study of the amphibian NC (Li
Reptin is involved in Ugt36Bc repression by Wg/Arm et al. 2009, Elkouby et al. 2010) (Fig. 4). Perhaps in the
signalling. near future, a combination of genome-wide surveys,
As with other variations from the classic regulatory WRE identification through bioinformatics and
mechanism outlined in Fig. 1, it is not yet clear how informed examination of candidate target genes can
often direct repression by b-catenin occurs, either identify important targets relatively quickly in many
through TCFs or other transcription factors. Is it a developmental systems.
rarity that plays only a small role in transcriptional This dynamic can be observed in the study of the
regulation by the Wnt/b-catenin pathway? Or has the Wnt/b-catenin pathway in CRC. The identification of a
focus on transcriptional activation and classic TCF WRE more than 335 kb removed from the c-myc TSS
binding sites obscured a currently underappreciated could have only been achieved with a combination of
aspect of Wnt gene regulation? Clearly more research in genomic approaches and a recognition of what consti-
this area is required to find out. tutes a TCF binding site (Pomerantz et al. 2009,
Tuupanen et al. 2009, Sotelo et al. 2010, Wright et al.
2010) (Fig. 6). Optimization of these approaches should
Conclusions
facilitate the identification of relevant targets for the
The first detailed characterizations of Wg/Arm signal- many other pathologies that Wnt/b-catenin signalling
ling already revealed what is now a common assump- has now been linked to.
tion, that this single pathway has a remarkable number Despite intense effort over the past 15 years, there is
of roles during fly development (Baker 1988, Peifer much to learn about how TCFs (and other transcription
et al. 1991). The appreciation of Wnt/b-catenin signal- factors) transform elevated levels of nuclear b-catenin
ling diversity has only grown in the past twenty years, into the appropriate transcriptional responses in a cell,
and this review has only covered a small fraction of the tissue and temporally specific manner. The vast size of
established roles of the pathway in metazoan develop- metazoan genomes and the great distances over which
ment. How is this diversity achieved? In vertebrates, WREs can operate make this task even more daunting.
differences among the TCF family members and the Given the importance of this pathway in normal devel-
large number of other transcription factors that recruit opment and physiology as well as numerous pathologies,
b-catenin to WREs is likely part of the answer. But in it’s clear that further investigation is worth the effort.
flies, it thus far appears that a single TCF mediates
most, if not all of the Wg/Arm pathway’s effects
Conflict of interest
(Brunner et al. 1997, van de Wetering et al. 1997,
Schweizer et al. 2003). Combinatorial inputs from The authors declare that they have no conflict of
other signalling pathways certainly provide an answer interest.
2011 The Authors

This work was support by grants from the NIH (RO1 Barembaum, M. & Bronner-Fraser, M. 2005. Early steps in
GM082994) and NSF (090348) to KMC. HCA is supported neural crest specification. Semin Cell Dev Biol 16, 642–646.
by NIH training grant T32-GM07315. Barker, N. & Clevers, H. 2010. Leucine-rich repeat-containing
G-protein-coupled receptors as markers of adult stem cells.
Gastroenterology 138, 1681–1696.
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Acta Physiol 2012, 204, 74–109

H. C. Archbold,1 Y. X. Yang,2 L. Chen2 and K. M. Cadigan1,2

Received 24 January 2011, Abstract

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orphan receptor Lgr5, which has received a great deal

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(b) (b′) (f)

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Combinatorial interactions of TCFs and other

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