TCF/LEF transcription factors are best known for their role as mediators of Wnt signaling,
helping Wnt direct developmental transitions of stem cells in tissues or driving cell
transformation and cancer when Wnt is aberrantly active. These factors possess a High
Mobility Group DNA-binding domain that recognizes a motif called the Wnt Response
Element (WRE: 5′-CTTTGWW-3′) and an N-terminal domain that binds β-catenin (Figure
1A). β-catenin is the cytoplasmic-nuclear mediator that communicates Wnt signals from
the plasma membrane to TCF/LEFs for transcription activation (Figure 1C). The vast
majority of published studies about TCF/LEFs focus on their recruitment of this mediator
to Wnt target genes. This implies that “life” for TCF/LEFs began in 1996 when yeast
two hybrid screens identified their mutual, strong interaction [1]–[3]. In fact, discovery
of TCF/LEFs had nothing to do with Wnt and β-catenin. TCF/LEFs were first described
as DNA-binding proteins that regulated transcription of lymphocyte-specific genes
such as the T-Cell Receptor complex, and they did so by cooperating with transcription
factors bound to juxtaposed elements in enhancers [4]–[7]. The original TCF1 and LEF1
were each characterized as a set of protein isoforms, differing by the presence or
absence of N-terminal (β-catenin) and C-terminal domains, none of which were needed
for enhancer activity [8]–[12]. In this issue, Grumolato et al. [13] report that TCF1
and LEF1 have constitutive, elevated activity in leukemia and lymphoma cells. They
report that this activity is independent of β-catenin and instead involves direct
recruitment of ATF2 and related family members (Figure 1B).
10.1371/journal.pgen.1003745.g001
Figure 1
β-catenin–independent and –dependent modes of Wnt signaling.
A. The general domain structure of TCF/LEF proteins includes a highly variable Context
Regulatory Domain (CRD) and the well-conserved N-terminal β-catenin–binding (β-cat)
and High Mobility Group (“HMG”) DNA-binding domains. N-terminal truncated forms of
TCF/LEFs (left) are naturally occurring and commonly referred to as dominant negatives
(e.g., dnTCF1, dnLEF1, etc.) because they block gene regulation by displacing full-length
proteins from target genes (right). In the Grumolato study, dnTCF1 and dnLEF1 functioned
perfectly well to activate a Wnt reporter gene. B. A simplified representation of
ATF2 recruitment by TCF1 to activate transcription in a β-catenin–independent manner
(referred to as a β-catenin–independent Wnt signaling pathway in Grumolato et al.
[13]). In some contexts, displacement of weaker TCF activators such as TCF4 might
also contribute to activation. C. β-catenin–dependent Wnt signaling requires the recruitment
of β-catenin by TCF/LEFs to a Wnt Response Element (WRE) for transcriptional activation
of target genes. D. A distal enhancer for the T-Cell Receptor alpha chain gene, identified
as one of the very first targets for TCF/LEF binding (see references in text), contains
closely juxtaposed binding sites for ATF/CREB proteins (consensus binding sequence
shown below), LEF/TCFs, and ETS proteins. E. ChIP-seq studies of TCF and β-catenin
genome-wide occupancy identify significant colocalization of binding motifs for AP1
and ETS transcription factors (see text for references). Consensus sequences for AP1
and ATF/CREB sites differ by a single nucleotide (see panel D for comparison), and
ATF proteins are known to bind AP1 sites. Colocalized motifs suggest there is potential
for interaction and cooperative crosstalk between β-catenin–bound TCF/LEFs, and ATF/CREB
and ETS proteins.
Grumolato et al. describe how the TOPflash reporter for Wnt signaling, a luciferase
gene driven by a minimal promoter with multimers of WREs, has elevated constitutive
activity in leukemia cell lines. One might assume that this activity derives from
TCF/LEF recruitment of β-catenin. However, very little stabilized β-catenin could
be detected and reporter activity was recapitulated using truncated forms of TCF1
missing the N-terminal β-catenin–binding domain (isoforms labelled dominant negatives,
or dnTCF/dnLEF [Figure 1A]). In another twist, family member TCF4 could not substitute
even though it has a β-catenin–binding domain. This meant that selective action by
LEF1 and TCF1 occurred through recruitment of other transcription factors via domains
distinct from the β-catenin–binding domain. Using a candidate approach, the authors
tested for functional interactions with proteins that bind AP1 sites. AP1 factors
are homo- and heterodimerizing leucine zipper proteins of the Jun, Fos, ATF, and JDP
families [14]. Grumolato et al. report that ATF family members (especially ATF2) bind
directly to TCF1 and LEF1, not TCF4, and that interactions primarily require the Context
Dependent Regulatory domain (CRD; Figure 1A, B). That TCF1/LEF1-ATF2 interactions
are detected in multiple types of hematopoietic tumor cell lines suggests that ATF
recruitment might account for a significant portion of the “Wnt reporter activity”
in these cell types. Knockdown of ATF2 reduced cell growth and lowered expression
of TCF1 and LEF1 target genes, similar to effects from overexpression of a dominant
negative form of TCF4. Observations such as these suggest that ATF2 is integral to
the regulatory role that TCF1/LEF1 play in lymphocytes.
These discoveries highlight how TCF1/LEF1 are closely intertwined with ATF proteins.
Indeed, one of the first interactions for LEF1 and, later, TCF1 was with proteins
that bind an ATF/CREB element in the T-Cell Receptor alpha chain enhancer (Figure
1D; [4], [5]); interestingly, ATF4 was first discovered on the basis of its binding
to this element (reviewed in [15]). Additional lymphocyte-specific enhancers were
discovered as collections of ATF/CREB, TCF/LEF, and ETS elements [15]), and functional
studies showed that TCF1 and LEF1 cooperated with these proteins bound to neighboring
elements to create strong enhancers. Importantly, the β-catenin–binding domain was
entirely dispensable, its deletion enabling even greater activity in some assays [9],
[12]. Instead, it was the CRD and a strong DNA-bending function of the HMG domain
that was of primary importance; DNA bending enabling a three-way, CRD-dependent interaction
between TCF/LEFs and other enhancer factors [16]. The exact identities of the ATF/CREB
proteins were unknown and were never fully explored. The Grumolato study brings the
past back to the present by identifying specific ATF interactors for TCF1 and LEF1
for the first time, and by showing that immune system cancers possess elevated, functional
interactions. The current study does not highlight the DNA binding of ATF2 because
it appears to be recruited by TCF1/LEF1 to the Wnt reporters in a protein–protein
interaction mode.
An emerging feature of TCF/LEFs that connects with these findings is a growing recognition
of a functional split in the vertebrate family. That is, an increasing number of reports
show that TCF4 and a fourth family member, TCF3, function as repressors, or at best,
weak activators. More and more frequently it seems that TCF1 and LEF1 operate by opposing
TCF3/TCF4 repression and providing strong activation. Since TCF1 and LEF1 are strongly
active for ATF2 engagement and cooperation, and TCF4 is not, ATFs could be important
players in the push-and-pull between family members. The first study to highlight
a split in the family used morpholino knockdown and rescue experiments in Xenopus
embryos [17]. Grumolato and colleagues use the same Xenopus system to show that overexpression
of dominant negative TCF1 (dnTCF1), but not TCF4, causes axis duplication—an activity
attributed to overactive Wnt signaling. It could be, as the authors posit, that dnTCF1
was recruiting ATF proteins to WREs for gene activation. But it is also possible that
dnTCF1 was displacing endogenous, repressive TCFs such as TCF3 and/or TCF4 (Figure
1B). Of course, both mechanisms could be involved, but further studies are definitely
warranted.
This study raises other questions about TCF/LEFs and β-catenin–independent activation
of transcription. Is TOPflash the reliable indicator of Wnt signaling that its common
use implies? Or can factors such as ATF2 be recruited to activate this reporter independent
of β-catenin? The authors provide a “yes” to the latter question in their system,
but a general answer would be best addressed with strategies that avoid overexpression
of transcription factors. How much do dominant negative TCF/LEFs contribute to gene
regulation? While an exact answer is not known, it is interesting to point out that
these forms are expressed at significant levels in lymphocytes [8], [10]. In fact,
the discovery of TCF1 came from a T lymphocyte cDNA screen in which all clones were
missing the β-catenin–binding domain (the N-terminus–encoding exon discovered years
later upon inspection of genomic sequences [6], [8]). Thus, even though definitive
connections between β-catenin, TCF1, and LEF1 are clear in lymphocytes (reviewed in
[18], [19]), the discordance between their knockout phenotypes should encourage a
revisit of this issue. What about cancer? The authors point out the finding in human
sebaceous tumors in which mutations have disabled the β-catenin–binding domain of
LEF1. This mutation is proposed to be oncogenic because overexpression of dnLEF1 in
mouse skin recapitulates sebaceous tumor development [20], [21]. Perhaps β-catenin–independent
actions of TCF/LEFs are more prevalent and powerful than currently assumed. Is the
ATF/CREB and TCF/LEF interaction common? The tentative answer is yes, because almost
every ChIP-seq study of TCF/LEF binding in cancer genomes, and one study of β-catenin–binding
to the colon cancer genome, has identified the closely related, ATF-friendly, AP1
response element as a top, cosegregating motif (Figure 1E; [22]–[25], [26]). Several
studies also define cosegregating ETS elements [22], [24], [25]. Going forward, it
will be important to probe how broadly ATF2 and family members crosstalk to TCF/LEFs
(and perhaps β-catenin), and determine what the functional consequences of that crosstalk
are in terms of gene programs and cell phenotypes.
In this Perspective, we use the protein names to refer to TCF/LEFs (i.e., LEF1, TCF1,
TCF3, and TCF4). This matches the Grumolato et al. study and makes for logical reading.
However, the gene names for TCF/LEFs are different: TCF1 is encoded by the TCF7 gene,
and TCF3 and TCF4 by the TCF7L1 and TCF7L2 genes, respectively. LEF1 is the only respite.
It is encoded by the LEF1 gene.