Wnt ligands trigger at least three different intracellular signalling cascades: first, the canonical Wnt pathway, which converges on transcriptional regulation of its target genes by β‑catenin; second, the β‑catenin‑independent pathway involved in planar cell polarity (PCP) signalling; and third, the Wnt‑dependent calcium/protein kinase C (PKC)‑dependent pathway1,2. Here, we focus on the canonical Wnt pathway (FIG. 1).

In the absence of Wnt ligands, β‑catenin is translated and subsequently marked for degradation by phos‑phorylation at key amino‑terminal Ser and Thr residues (Ser33, Ser37, Thr41 and Ser45) by a cytoplasmic ‘destruction complex’. This complex consists of axin, the adenomatous polyposis coli (APC) tumour suppressor protein, glycogen synthase kinase 3β (GSK3β), casein kinase 1 (CK1) and protein phosphatase 2A (PP2A)2,3. In pathological conditions, β‑catenin escapes degrad‑ation when these N‑terminal Ser and Thr residues are mutated, or when components of the destruction complex are defective — such as APC, which is most commonly mutated in colon carcinoma3. When Wnt binds to the seven transmembrane domain receptors frizzled and the low density lipoprotein receptor‑related protein 5 (LRP5) or LRP6 co‑receptors, the cytoplasmic protein dishevelled (DVL) is recruited to the receptor complex, and this is suggested to cause sequestration of the rate‑limiting component axin from the degradation complex4,5. By these means, β‑catenin escapes the deg‑radation fate and is stabilized as a hypophosphorylated form with preferential nuclear localization — however, the ostensibly non‑traditional mode of transport of β‑catenin into the nucleus remains poorly understood6. Once in the nucleus, β‑catenin interacts with members

of the DNA‑binding T cell factor/lymphoid enhancer factor (TCF/LEF) family proteins to form a bipartite transcription factor.

The identification of many of its nuclear interaction partners has significantly added to our understanding of β‑catenin function as a transcription regulator. Many of these factors are involved in chromatin structure and RNA polymerase II (RNA Pol II) regulation, such as his‑tone acetylation and methylation complexes or various chromatin‑binding proteins. To obtain a comprehensive picture of nuclear β‑catenin function, all of the indiv‑idual puzzle pieces need to be assembled into a cohesive model of nuclear Wnt signalling. By reviewing our current knowledge of β‑catenin and its binding proteins in the context of chromatin and transcription, we sketch working models of nuclear β‑catenin function.

An Armadillo by the tailβ‑Catenin is the most prominent member of the Armadillo (ARM) repeat protein superfamily, featuring a central stretch of 12 imperfect ARM repeats (labelled R1–R12). Distinct N‑terminal and carboxy‑terminal domains (NTD and CTD, respectively) frame R1–R12, and a helix in the proximal CTD is positioned against the last ARM repeat7,8 (FIG. 2).

Over a decade ago, pioneering in vivo structure– function analyses assigned positive non‑redundant effec‑tor roles to different parts of the Drosophila melanogaster β‑catenin orthologue ARM protein9. Deleting the central ARM repeats completely abrogated readouts triggered by Wingless (WG), the major canonical D. melanogaster Wnt ligand. Furthermore, removal of the most N‑terminal repeats or deletion of the C‑terminal portion of the ARM

*National Research Center “Frontiers in Genetics”, Institut für Molekularbiologie, Universität Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland.‡Present address: Children’s Hospital Boston, Division of Hematology/Oncology, Harvard Stem Cell Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.Correspondence to K.B. e-mail: konrad.basler@molbio.uzh.chdoi:10.1038/nrm2654

β-Catenin hits chromatin: regulation of Wnt target gene activationChristian Mosimann*‡, George Hausmann* and Konrad Basler*

Abstract | The canonical Wnt pathway has gathered much attention in recent years owing to its fundamental contribution to metazoan development, tissue homeostasis and human malignancies. Wnt target gene transcription is regulated by nuclear β‑catenin, and genetic assays have revealed various collaborating protein cofactors. Their daunting number and diverse nature, however, make it difficult to arrange an orderly picture of the nuclear Wnt transduction events. Yet, these findings emphasize that β‑catenin‑mediated transcription affects chromatin. How does β‑catenin cope with chromatin regulation to turn on Wnt target genes?

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APCAPC

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CK1 GSK3β

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CN

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protein severely reduced signalling. This reflects the absolute requirement of the central β‑catenin region to interact with TCF, as well as the recruitment of auxiliary cofactors by the N‑ and C‑terminal regions to contribute to Wnt target gene activation (FIG. 2).

The central repeats that span R3–R10 are viewed as the core TCF interaction region10,11 (FIG. 2). Intriguingly, in the absence of a Wnt signal, TCF proteins can bind their DNA recognition sequences — referred to as Wnt response elements (WREs) — on a subset of target genes and can repress any Wnt‑independent expression12. For this purpose, TCFs recruit groucho (TLE1 in mammals)13–15, a long‑range chromatin repressor that functions with his‑tone deacetylases (HDACs) to stimulate the compression

of local chromatin and to inhibit transcription16 (BOX 1). Furthermore, C‑terminal‑binding protein (CTBP) has been implicated as a Wnt target gene repressor, but in a TCF‑independent manner17. β‑Catenin competes with groucho for TCF binding18, thus replacing the repressor with an activation scaffold. However, certain Wnt targets have stronger TCF recruitment to their enhancers after Wnt stimulation19,20 (BOX 2). This could indicate that the TCF‑binding sites at these particular WREs are only fully exposed following preparative chromatin remodelling by other, priming, trans cription factors in the vicinity. It is also possible that the DNA‑binding affinities of TCF change when bound to β‑catenin and associated complexes (FIG. 1).

Figure 1 | The β‑catenin‑dependent or canonical Wnt signalling pathway. This pathway centres on β‑catenin, which, together with the DNA‑binding T cell factor/lymphoid enhancer factor (TCF/LEF) family proteins, functions as a transcription factor to control Wnt target genes. A subset of these target genes are constitutively inhibited by pioneering nuclear TCF, which recruits transcriptional corepressors (left panel) to Wnt response elements (WREs). In the default state, β‑catenin is constitutively degraded by the cytoplasmic degradation complex, which comprises axin, adenomatous polyposis coli (APC), glycogen synthase kinase 3β (GSK3β) and casein kinase 1 (CK1). Phosphorylation of Ser and Thr residues in the β‑catenin amino terminus by this complex triggers SCF (SKP1, Cullin, F‑box)/β‑TrCP‑mediated polyubiquitylation and proteasomal degradation of β‑catenin. On Wnt ligand binding, the degradation complex is inhibited by dishevelled (DVL) and β‑catenin translocates to the nucleus, where it replaces TCF‑bound corepressors (such as groucho; middle panel) or co‑imports additional TCF to occupy WREs (right panel). Once bound to WREs through TCF, β‑catenin functions as a scaffold to recruit an auxiliary machinery of co‑activators that are involved in chromatin remodelling and control of RNA polymerase II to induce Wnt target gene expression. LGS, Legless; LRP, low‑density lipoprotein receptor‑related protein; PP2A, protein phosphatase 2A.

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TRRAP, p400 and TIP60; MED12;ISW1 (ISWI inflies; +/–)

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CTD665 781

NucleosomeAn assembly of 146 base pairs of DNA wrapped in 2 turns around an octameric complex that is composed of the core histones H2A, H2B, H3 and H4, thus forming the basic unit of chromatin.

EnhanceosomeDescribes the cooperative protein assembly that is formed at a transcription- controlling enhancer region. The enhanceosome involves gene-specific transcription factors, chromatin-remodelling factors and components of the general transcription machinery.

When TCF occupies R3–R10, the exposed N‑ and C‑terminal β‑catenin surfaces promote the recruitment of auxiliary factors. More than a decade after the seminal structure–function analyses, many exciting discoveries across several model systems have established that β‑catenin binds to various proteins that are implicated in chromatin structure and RNA Pol II regulation.

The C-terminal tail sets the transcription stageEarly work suggested that the β‑catenin CTD has auton‑omous transactivation potential when tethered to DNA21. However, the subsequent discoveries of β‑catenin inter‑action partners that bind to the region spanning R11, R12 and the proximal CTD (hereafter referred to as R11–C) have indicated that it is not the CTD alone that confirms transactivation activity to β‑catenin. Interestingly, a surplus of the R11–C fragment blocks chromatin‑dependent transcription by β‑catenin in a dominant‑negative manner in vitro22. This is consistent with the notion that the C‑terminal part of β‑catenin interacts with crucial chromatin‑dependent factors (FIG. 2).

Putting a HAT on β‑catenin. The acetylation of histone tails is the most prominent chromatin modification associated with transcriptionally active genome regions. It is thought that acetylation interferes with tight pack‑aging interactions between individual nucleosomes23 and is a key modification in the coordination of trans‑criptional control24 (BOX 1). Throughout evolution, therefore, transcription factors have acquired the means to recruit complexes with histone acetyltransferase (HAT) activity.

The prominent HAT proteins CBP (also known as CREBBP) and p300 interact with the β‑catenin region R10–C (ReF. 25,26) (FIG. 2), and, in vivo, this interaction

results in the activation of several Wnt target genes. However, the intrinsic HAT activities of CBP and p300 are not universally required25. Furthermore, both pro‑teins do not act redundantly, but instead have Wnt target gene‑selective roles as bimodal regulators that mediate activation and repression19 — the type of action might depend on an as yet elusive phosphorylation mark27,28. Pull‑down experiments also reveal an interaction between R11–C and the TRRAP–TIP60 complex, centred on the MYST family HAT TIP60 (ReF. 29). This complex is involved in chromatin regulation together with transcription factors such as E2F and p53 (ReF. 30) (FIG. 2). So far, no functional experiments have addressed this interaction.

CBP‑mediating HAT activity on WREs revealed. In a seminal study, the role of the HAT activity of CBP on the chromatin of Wnt target genes in vivo was recently anal‑ysed20. using chromatin immunoprecipitations (ChIPs) for acetylated histone H3 and H4, the study analysed the pattern of histone acetylation surrounding the WREs of the naked cuticle and Notum loci in D. melanogaster. upon pathway stimulation and in a CBP‑dependent man‑ner, nucleosomes were rapidly acetylated and saturated after 5.5 hours in a wide region (up to 30 kilobases) that surrounded the analysed WREs. This is in marked con‑trast to other previously reported metazoan genes, for which a confined acetylation island appears proximal to the promoter31. Therefore, recruitment of TCF and β‑catenin to a WRE swiftly induces widespread chrom‑atin changes — but how can β‑catenin‑recruited CBP acetylate large chromatin regions so rapidly?

An unknown factor that specifically associates with the β‑catenin–CBP complex might be responsible20. However, a simpler explanation for how such wide‑spread histone acetylation can be mediated by β‑catenin‑associated HATs is suggested by a special property of TCF proteins and the genomic distribution of their DNA‑binding sites (BOX 2). TCFs are members of the high mobility group (HMG) box protein family, which induce strong DNA bending following binding — for example, LEF1 induces an extreme 130° kink32,33. More than 15 years ago, it was postulated that this endows TCFs with the ability to serve as architectural elements to physically arrange large chromatin regions32. These new results are consistent with the binding of TCF, potentially at different sites that are distributed over a given locus, bringing distant DNA regions together and rendering them accessible to a β‑catenin‑bound HAT activity20. It will be interesting to compare the chromatin‑ remodelling dynamics at WREs with other HMG protein‑controlled target genes.

Far‑reaching and interdependent DNA conformation changes could further prepare the stage for additional cooperating transcription factors to bind in the vicinity of WREs. TCF binding could thus create a local chrom‑atin environment that, by its structure, creates a three‑dimensional specificity for enhanceosome formation. There could also be cooperativity between distant TCF sites34 (BOX 2), in particular considering the nucleosome binding of β‑catenin‑associated proteins (see below).

Figure 2 | Nuclear β‑catenin interactions. The β‑catenin molecule consists of an amino‑terminal domain (NTD), a central region that spans 12 Armadillo repeats and a carboxy‑terminal domain (CTD). Blue bars show the experimentally determined binding sites for positive β‑catenin interactors, whereas red bars show the interaction regions of negative factors and inhibitors. ISW1 (ISWI in flies) is marked as a positive or negative component, as experimental evidence to designate its sign of action is incomplete (see the main text). BRG1 is also known as SMARCA4; CBP is also known as CREBBP. APC, adenomatous polyposis coli; BRM, Brahma; HYX, Hyrax; ICAT, inhibitor of β‑catenin and TCF; LGS, Legless; MED12, mediator complex subunit 12; TCF/LEF, T cell factor/lymphoid enhancer factor.

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MLL complex(Mixed lineage leukaemia). A COMPASS (complex proteins associated with Set1)-like complex with histone methyltransferase activity, which is minimally composed of MLL, ASH2, menin, RBBP5, WDR5 and DPY30.

Histone methyltransferaseAn enzyme that catalyses the transfer of a methyl group from S-adenosylmethionine to Lys or Arg residues found in histones.

β‑Catenin controls histone rearrangements. Not only post‑translational modifications but also the nucleosome positioning along a stretch of DNA change chromatin structure. Nucleosomes can shield DNA sequences with their intimate entanglement35 (BOX 1). A class of histone‑remodelling complexes is thus required to reposition nucleosomes at target gene loci to expose interaction regions for transcription‑associated complexes. This ser‑vice is provided by the SWI/SNF family of ATPases, which shuffle or even disassemble histone octamers36. SWI/SNF proteins are increasingly being recognized as interaction partners of all sorts of transcription factors, and help to promote efficient recruitment to (and cofactor function at) their target gene loci37.

The SWI2/SNF2 family protein BRG1 (also known as SMARCA4; Brahma (BRM) in D. melanogaster) directly binds to β‑catenin at R7–R12 (FIG. 2). Genetic experi‑ments and tissue culture studies show a positive func‑tional connection between β‑catenin and BRG1 (ReF. 38). The expression of an ATPase‑deficient form of BRG1 has a dominant‑negative effect on TCF‑mediated transcrip‑tion, suggesting that BRG1 directly regulates nucleosome arrangements at Wnt‑responsive target genes. Thus, β‑catenin can localize histone‑repositioning machineries to its target gene loci. Another member of the SWI/SNF family, human ISW1, was found to co‑precipitate with β‑catenin R11–C (ReF. 29) (FIG. 2). ISW1 is involved in at least three different chromatin‑remodelling complexes, and these act in a context‑dependent manner to activate or inhibit transcription39,40. Recently, D. melanogaster ISWI has been suggested to be a cofactor of TCF, helping to mediate WG target gene repression41. However, a positive function for ISWI in association with β‑catenin remains possible.

β‑Catenin to SET the COMPASS. Mixed lineage leukaemia complexes (MLL complexes)42 interact with β‑catenin R11–C (ReF. 29) (FIG. 2). The MLL proteins contain a SET domain that has histone methyltransferase (HMT) activity (BOX 1). HMT complexes are best studied in yeast, in which the Set1 HMT enzyme assembles the COMPASS (complex proteins associated with Set1) complex to mono, di or trimethylate histone H3 tails at Lys4 (forming H3K4me1, H3K4me2 or H3K4me3), post‑translational modifications that are exclusively associated with actively transcribed genes42. H3K4me3 is an increasingly recog‑nized activation mark at transcription start sites, whereas H3K4me2 is associated with RNA Pol II‑read regions as a transcription bookmark that is placed by elong ating RNA Pol II‑associated complexes. Yet, it is unclear how HMT activity is initially brought to activ ated genes. Several COMPASS complex components have intrinsic chromatin‑binding domains that could recognize pre‑pared promoter regions43,44, so we predict that trans‑criptional activators could initially recruit and position HMT activities, which later transfer to RNA Pol II. The ability to recruit a COMPASS complex through R11–C of β‑catenin provides a simple way to bring HMT activity into the proximity of a WRE.

From yeast to humans, histone H2B monoubiquityl‑ation at Lys123 by the BRE1 E3 ubiquitin ligase and its partner RAD6 is a prerequisite for COMPASS assembly and H3K4 methylation45 (BOX 1). This sequential action highlights the considerable crosstalk and interdepend‑ence among histone modifications. BRE1 has been impli‑cated in D. melanogaster Notch‑pathway target gene induction46. Furthermore, inhibition of ubiquityl ation reactions completely blocks β‑catenin‑mediated primer extension reactions on reconstituted chromatin in vitro.

Box 1 | Chromatin and histone modifications

A human cell contains roughly 2 metres of genomic DNA packed into a tiny nucleus. Part of this packing is mediated by the intricate wrapping of the DNA around protein assemblies of the highly conserved histone family, namely histone H2A, H2B, H3 and H4, that form the nucleosomes. The nucleosome is the basic repeating unit of the genomic material, which can be stained by different methods and is referred to as chromatin (from the Greek chroma, meaning colour). Nucleosomes tightly interact with adjacent histones or other nuclear proteins through the unstructured amino-terminal histone tails that protrude from nucleosomes. This dense default chromatin packing of target gene loci creates an environment that is inaccessible for transcription. Eukaryotic transcriptional activators thus need to precisely position and stimulate chromatin-modifying protein complexes to overcome this inhibition. This reflects in the high evolutionary conservation and multitude of chromatin-remodelling mechanisms.

The position of nucleosomes can be rearranged by ATPase-dependent SWI/SNF complexes to expose regulatory DNA sequences. Furthermore, histone tails can be post-translationally modified to ease the chromatin conformation. These modifications, such as acetylation, phosphorylation, methylation, ubiquitylation or sumoylation, also create novel protein interaction surfaces.

Acetylation is a well-established chromatin modification at active genes. The modification is mediated by specific histone acetyltransferase (HAT) enzymes. HATs catalyse the acetylation of Lys residues in the histone N termini, changing their interaction properties and inducing a loosened chromatin structure. This is counteracted by histone deacetylases (HDACs), which are common transcriptional repressors. Mono, di or trimethylation of Lys or Arg residues in histone tails by histone methyltransferases (HMTs) also correlates with active (that is, H3K3me3) or repressed (that is, H3R2me1) transcription, respectively. Combinations of acetylated and methylated histone tails mark different stages during gene transcription, thus suggesting the existence of a ‘histone code’.

The histone code concept expands with various classes of nucleosome-binding domains that have differential histone modification specificities and that are found in chromatin-associating proteins. For example, bromodomains bind to acetylated histone tails, plant homology domains recognize different H3K4 methylation states, and different versions of chromodomains attach to several histone modifications. Such domains therefore link chromatin-remodelling complexes to chromatin at various stages of the transcriptional activation of a target gene.

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PAF1 complex(Polymerase-associated factor 1). A protein assembly that mediates histone modifications during initiation and elongation, minimally containing the factors PAF1, CTR9, LeO1, RTF1 and CDC73.

Although not direct evidence, this result suggests the involvement of RAD6–BRE1‑ and COMPASS‑mediated mechanisms in β‑catenin‑mediated Wnt target gene regulation29. But how can β‑catenin orchestrate this complicated histone modification sequence?

We have previously found that D. melanogaster Hyrax (HYX), as well as its human orthologue parafi‑bromin (hereafter referred to as parafibromin/HYX), functions as a positive nuclear Wnt signalling compo‑nent47. A domain in the N terminus of parafibromin/HYX interacts with R12–C of β‑catenin (FIG. 2). The C terminus, however, is homologous to yeast Cdc73, a component of the polymerase‑associated factor 1 com‑plex (PAF1 complex). This complex interacts directly with RNA Pol II through its subunits Cdc73 and Rtf1, and serves as an interaction platform to orchestrate Rad6–Bre1‑mediated H2B monoubiquitylation, as well as COMPASS recruitment45. By attracting the PAF1 com‑plex through parafibromin/HYX, the C‑terminal region of β‑catenin could anchor the H2B monoubiquityl ation and H3K4 methyl ation events that surround the WREs, as well as initiation and elongation preparation for RNA Pol II (FIG. 3). We therefore need to gain further insight into the roles and significance of RAD6–BRE1 as well as PAF1 complex function in Wnt signalling. It is tempting to speculate that other transcription factors directly recruit the PAF1 complex as a versatile platform to control histone modifications47,48.

New pieces in the BCL-9–Pygo puzzleThe most N‑terminal ARM repeat, R1, recruits BCL‑9 (Legless (LGS) in D. melanogaster)49, which in turn binds to pygopus (Pygo). This interaction is required for all WG‑dependent effects that have been analysed in D. melanogaster50–53 (FIGS 1,2). BCL‑9 interacts via its homology domain 1 (HD1) with Pygo and via the adja‑cent HD2 with β‑catenin; genetic rescue experiments have suggested that these two short domains are suffi‑cient for LGS function in D. melanogaster 53. However, BCL‑9/LGS is a long protein of over 1,400 amino acids — why has this factor maintained its length during evo‑lution? The C‑terminal tail that follows HD2 contains at least three additional short conserved domains, termed HD3, HD4 and HD5, of which HD4 and HD5 are most highly conserved in vertebrates and interspersed by Pro‑rich sequences53–55. Vertebrates have two BCL‑9 proteins (BCL‑9 and BCL‑9‑like (BCL‑9L)), causing potential redundancy in experimental setups.

Novel BCL‑9 findings. Recent work has exposed several underappreciated details about the roles of BCL‑9 (ReF. 55). Overexpressed BCL‑9 has a marked prefer‑ence to augment reporter‑based β‑catenin responses in lymphoid cells in comparison to fibroblasts, and also has a preference for particular WRE reporters, such as a Xenopus laevis‑derived siamois gene enhancer fragment. This suggests that there are unexplored post‑translational modifications of BCL‑9 or that there is interplay between BCL‑9 and cell‑type‑specific interaction partners in haemato poietic lineages. This activity is mostly Pygo‑independent, as HD1‑deleted constructs have a similar activity as wild‑type BCL‑9. This activity is mediated by the C‑terminal tail of BCL‑9, in particular by the Pro‑rich sequences55. These results clearly indicate that there is more to BCL‑9/LGS than its role as a pedestal for Pygo on β‑catenin. However, it should be noted that these experiments have been conducted by overexpres‑sion and luciferase reporter readouts in cell culture. It would be interesting to see these novel aspects further pursued in vivo.

Could Pygo act as a pioneering factor? The role of Pygo is a conundrum. Like LGS, PYGO is essential for virtu‑ally all WG‑dependent readouts in D. melanogaster. By contrast, Pygo1 and Pygo2 loss‑of‑function mice exhibit rather mild Wnt signalling defects56,57. Furthermore, loss of Pygo2 also causes defects in mouse eye lens develop‑ment58 and spermiogenesis59 that are seemingly unre‑lated to Wnt signalling. These intriguing findings suggest that, during evolution, vertebrate and invertebrate Pygo proteins have diverged in certain aspects.

Pygo consists of two distinct domains: an N‑terminal homology domain (NHD) that is exclusively found in Pygo proteins, and a C‑terminal zinc‑finger‑related plant homology domain (PHD) finger60. The NHD has intrinsic transactivation potential in D. melanogaster, but not in vertebrate, cells61. The Pygo PHD finger binds to HD1 of BCL‑9, which then tethers it to β‑catenin, leav‑ing the NHD exposed. Early studies hinted at a role for BCL‑9–Pygo in nuclear β‑catenin retention, as well as

Box 2 | What to aim for — Wnt target gene structure

Wnt signal processing by the β-catenin-dependent canonical cascade induces a specific, integrated transcriptional programme, as defined by target genes that subsequently control fundamental aspects of cellular behaviour, such as growth, differentiation or cell division. Although a growing list of Wnt-responsive genes is known across different tissues (constantly monitored on the Wnt Homepage), we have only a vague understanding of the characteristics of a Wnt-responsive gene.

Early work with T cell factor (TCF) established that the 5′ positioning of an optimal consensus binding site multimer (5′-AAGATCAAAGG-3′)96 confirms β-catenin responsiveness to a reporter97. Although this provides an important tool for Wnt research, it does not reflect the more complex architecture of endogenous TCF–β-catenin98. Furthermore, it seems that most Wnt targets are tissue- and developmental timing-specific, thereby rendering extrapolations of individual published reports difficult. Cyclin D1, for example, which is commonly analysed to check active Wnt signalling in tumours, is not always Wnt responsive99. The most promising candidate for a general target is AXIN2, the expression of which is increased in response to activated β-catenin in a broad range of tissues100. However, not all predicted TCF-binding sites in its enhancer region significantly contribute to Wnt responsiveness.

A recent chromatin immunoprecipitation (ChIP)-on-chip study revealed exciting insights into Wnt response element (WRE) distribution34. In the AXIN2 locus, TCF4-occupied sites, which can differ significantly from the above consensus sequence, dot a region of 100 kilobases 5′ as well as intronic and 3′ of the gene locus. This is only one example of the revealed pattern: WREs seem to occur in the vicinity of, as well as at locations far from, transcription start sites. Although various identified TCF4-binding sites retain their WRE potential when tested in isolation, their genome-wide occupancy suggests that WREs cooperatively or redundantly control wide-spanning chromatin organization. These exciting findings also raise the notion that β-catenin-induced chromatin remodelling could profoundly affect, or co-depend on, the activities of other transcription factors bound in a TCF-controllable genomic locus. Furthermore, it is conceivable that β-catenin requires different, or simply more, cofactors to work at endogenous WREs than on multimerized TCF-binding sites.

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Nature Reviews | Molecular Cell Biology

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β-Catenin (ARM protein in flies)

β-Catenin(ARM proteinin flies)

Polytene chromosomeA large chromosome that results from the successive replication of a homologous chromosome, without ensuing separation and cell division.

providing NHD‑based co‑transactivator function for β‑catenin62,63. So what is Pygo truly good for?

In D. melanogaster, antibody staining on polytene chromosomes and ChIP experiments revealed that PYGO colocalizes with TCF to chromatin without Wnt pathway activation in an NHD‑dependent manner61. However, there seems to be no detectable direct interaction between PYGO and TCF, and potential bridging factors, as well as direct DNA interactions, remain elusive61. Although not yet extrapolated to vertebrate cells, this intriguing set of data indicates that Pygo, using its NHD in cooperation with TCF, could serve as a pioneer factor at a particular class of WG target genes (FIG. 3a). This could promote the capture of β‑catenin by BCL‑9–Pygo at WREs that are, by default, repressed by TCF61. Other transcription factors use priming pioneer factors, such as FOXA1. As an auxiliary factor in oestrogen receptor‑α (ERα) signalling, FOXA1 localizes to the proximity of ERα response elements and

initiates chromatin‑remodelling events that enable DNA access by ERα once it is engaged in oestrogen binding64. Such a pioneering mode of action on a subset of Wnt targets could explain some, but not all, of the incomplete Wnt signalling defects of Pygo ablation in mice. It is inter‑esting to note that the β‑catenin‑dependent recruitment of the TBL1/TBL1R1 factors to the MYC and AXIN2 loci has recently been reported65. These factors have an affinity for hypoacetylated histones. This recruitment makes them further candidates for auxiliary factors that are involved in the initial recruitment of β‑catenin to a WRE.

Pygo PHD finger binding to histone tails. PHD fingers occur in many chromatin‑associated proteins and have recently been recognized to bind different H3K4 methyl‑ation states, thereby localizing chromatin‑binding factors to modified nucleosomes66,67. Specialized PHD finger proteins thus act as ‘code readers’ to localize

Figure 3 | Sequential exchange of auxiliary β‑catenin‑binding factors at Wnt response elements. Models that combine the current knowledge of protein complexes bound at the carboxy‑terminal region of β‑catenin. a | Pygopus (Pygo) localization through its amino‑terminal homology domain (NHD) to T cell factor (TCF; Pangolin (PAN) in Drosophila melanogaster)‑occupied sites in the genome through an unknown protein–protein interaction can lure β‑catenin bound to BCL‑9 (Legless (LGS) in D. melanogaster) to these loci. b | In an initial step, R11–C subsequently recruits histone acetyltransferase (HAT) activities (CBP (also known as CREBBP) and p300 or the TRRAP–TIP60 complex) as well as histone‑shuffling ATPases of the SWI/SNF family, which are represented here by BRG1 (Brahma (BRM) in D. melanogaster). When the chromatin is in an open and prepared state, the Mediator complex triggers pre‑initiation complex assembly and RNA polymerase II placement. In a later step, the polymerase‑associated factor 1 (PAF1) complex is recruited by parafibromin (Hyrax (HYX) in D. melanogaster) to recruit complexes with SET1 histone methylation activities. c,d | On histone methylation, Pygo could tether β‑catenin and its C‑terminally associated complexes to DNA by means of its homology domain 1 (HD1)‑complexed zinc‑finger‑related plant homology domain (PHD) finger. This frees the β‑catenin‑binding site on TCF and renders it accessible for competing repressors, such as groucho, which then trigger inhibitory compacting of the chromatin environment. CBD, β‑catenin‑binding domain; COMPASS, complex proteins associated with Set1; HMG, high mobility group; MED12, mediator complex subunit 12.

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Pre-initiation complexFormed minimally by the general transcription factors TFIIA, TFIIB, TFIID, TFIIe, TFIIF and TFIIH, this complex positions RNA polymerase II and prepares the necessary steps required for transcriptional initiation.

protein complexes at target gene loci in particular trans‑criptional states, ranging from inactive (unmethylated H3K4) to actively transcribed (H3K4me3)66 (BOX 1). Is the Pygo PHD finger therefore also capable of recognizing methylated H3K4?

A detailed crystallographic and biophysical analysis showed that human PYGO1 has an intrinsic affinity for all three methylation states of H3K4, particularly to H3K4me2 and H3K4me3 (ReF. 68). The structural data also revealed that BCL‑9 HD1 occupies the opposite side of the histone‑binding pocket of the PHD finger, suggesting that Pygo could simultaneously interact with BCL‑9 and methylated H3K4. Subsequent experiments not only unveiled that simultaneous HD1 binding of the Pygo PHD markedly enhances its H3K4 affinity, but also showed that D. melanogaster PYGO only interacts with H3K4 when complexed with HD1 (ReF. 68).

Novel implications for BCL‑9–Pygo function. Put into a hypothetical sequence of events, Pygo could, together with TCF, await a β‑catenin–BCL‑9 complex at a WRE (FIG. 3a) and enhance its association on increased promoter‑proximal H3K4 trimethylation during initial gene activation, as outlined above68 (FIG. 3c). This coop‑erativity would help tether TCF‑bound β‑catenin to an activated Wnt target gene. A provoking extension of such a model is that the association of β‑catenin with its target genes might become TCF independent if Pygo tethers it through BCL‑9 to methylated nucleosome tails, thus lib‑erating TCF (FIG. 3d; see below). Formation of a TCF‑free, chromatin‑bound Pygo–BCL‑9–β‑catenin complex could explain the apparent lack of a published verification of a tetrapartite Pygo–BCL‑9–β‑catenin–TCF complex in vivo, although this has been observed in vitro49.

Mediating RNA Pol II control? β‑catenin attracts HATs to open chromatin conformations, recruits SWI/SNF complexes to shuffle histones and even lures HMT activ‑ity to WREs, setting the stage for RNA Pol II to initiate transcription (FIG. 2). Yet, RNA Pol II has to be correctly orientated at a promoter by a pre-initiation complex (PIC).

The evolutionarily conserved Mediator is a modular protein complex of approximately 30 subunits that relays the presence of transcription activators to the PIC69,70. Reflecting its scaffold role, several Mediator subunits expose multiple interaction surfaces for DNA‑bound trans cription factors, the TATA‑box‑recognizing trans‑cription factor TFIID and RNA Pol II subunits. Various transcription factors, such as p53 or nuclear receptors, have been shown to recruit the Mediator complex by hooking up to distinct Mediator subunits71.

Mediator complex subunit 12 (MED12) binds directly to R12–C in vitro and positively promotes Wnt reporter activation72 (FIG. 2). However, other findings indicate inhib‑itory functions of Mediator subunits73. Loss of D. mela-nogaster Skuld (SKD; also known as MED12) and Kohtalo (KTO; also known as MED13) reduces WG target gene trans cription74. Furthermore, SKD‑ and KTO‑mutant cells no longer support the transactivation function of a DNA‑tethered, and thus completely ARM protein‑independent, PYGO NHD. Immunoprecipitation analysis has also

shown that MED12 co‑precipitates with the Pygo NHD fragment, which suggests that these proteins form a com‑plex in vivo. This finding backs up earlier work on Pygo, which assigned transactivation potential to its NHD53,75. However, transactivation experiments that involve isolated protein domains and artificial reporters have to be consid‑ered with care. As any RNA Pol II‑mediated transcription will eventually require the same factors, it is conceivable that any cryptic Pygo NHD transactivation capacity could use an overlapping set of auxiliary proteins to β‑catenin in its native context.

Heads and tails of interactor crosstalkThe potential of Pygo to interact with the β‑catenin R11–C‑ binding Mediator complex raises an intriguing ques‑tion — is there crosstalk between the β‑catenin cofactors that are recruited to its N‑ and C‑terminal tails? In vivo, HYX overexpression suppresses the dominant‑negative effect of LGS17E, which has a debilitating point mutation in the β‑catenin‑binding HD2 (ReF. 47). In another set of experiments, BCL‑9 could enhance wild‑type β‑catenin‑mediated transactivation, but only moderately boosted a C‑terminally impaired form55. Furthermore, the β‑catenin R2–R5‑binding proteins TIP48 and TIP49 (ReF. 76) are components of the R11–C‑interacting TRRAP–TIP60 complex29,77, but their significance in general β‑catenin‑dependent transcription is unclear77,78. Taken together, these findings hint at a cooperative interplay, or inter‑dependence, between the N‑ and C‑terminal β‑catenin interactors when assembled into a complex. Structure–function experiments have shown that the SWI/SNF complex, CBP and p300, as well as the Mediator com‑plex, make multiple contacts with transcriptional activa‑tors71,79,80. Also, Pygo–BCL‑9 binds β‑catenin at the same time as parafibromin/HYX47, and potentially also as other C‑terminally recruited complexes (FIG. 3b). Therefore, the Pygo NHD could provide an additional interaction sur‑face to stabilize β‑catenin R11–C‑bound cofactors, which might be of variable importance depending on the affinities of the complexes to β‑catenin itself.

A sequence of events. Many diverse factors are recruited by the C‑terminal region of β‑catenin, including the HATs CBP, p300 and TIP60, the SWI/SNF factors BRG1 (or BRM) and ISWI, the Mediator component MED12, and the PAF1 complex factor parafibromin/HYX (FIG. 2). However, it is difficult to imagine that all these bulky multi‑protein assemblies simultaneously occupy R11–C. Many transcription factors bind a similar catalogue of cofac‑tors by sequential or cycling recruitment24,80,81. A simple implication is that β‑catenin R11–C serves as a scaffold to orchestrate the recruitment and sequential exchange of chromatin‑remodelling factors at WREs29,47,55,68 (FIG. 3b).

In such a scenario, β‑catenin‑recruited HAT activity from CBP and p300, TIP60 or associated proteins, acetylates the histone tails that surround WREs to initiate chromatin remodelling, whereas ATP‑dependent histone shuffling by SWI/SNF components rearranges nucleo‑somes. Both processes could mutually enhance each other and set the stage for subsequent steps independently of the order of their action24. Mediator complex recruitment

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PHD CNN

β-Catenin

β-Catenin

CBD NN

C

CC

HD2

N

N

C

CPygo

C

N

HM

G

CBD

PHDHD1

NHD

C

N

HMG

HD1NHD

TCF

5′ WRE

Intronic or 3′ WRE

BCL-9(LGS in

flies)

TCF

Histone methylationHistone acetylation

Auxiliary cofactors andchromatin-remodelling complexes

Tissue-specifictranscription factor

Tissue-specifictranscriptionfactor

Target genepromoter

HD

2

BCL-9 (LGS in flies)

Pygo

Pygo

initiates PIC assembly, whereas binding of parafibromin/HYX brings the PAF1 complex into proximity. The PAF1 complex then facilitates H2B monoubiquitylation by RAD6–BRE1 and could be transferred to RNA Pol II — recruitment of COMPASS for H3K4 methylation and the initiation of mRNA synthesis then ensues. The whole process could repeatedly cycle to yield sustained Wnt target gene transcription (FIG. 3b).

How could such sequential cofactor exchange be cata‑lysed? We predict that the implicated factors are initially recruited by β‑catenin to WREs but do not necessarily have to remain bound to β‑catenin to carry out their functions. A connection between R11–C and the Pygo NHD, as proposed for Mediator components74, could further simplify factor exchange by providing an inter‑action ‘ping pong’ surface (FIG. 3b). Also, domains that bind modified histone tails are often found in chromatin‑ remodelling complexes: bromodomains recognize acetyl groups, and chromodomains and PHD fingers bind meth‑ylated H3 residues. All of these could facilitate a transfer of the complexes from β‑catenin to the chromatin template, freeing up R11–C for the next interaction. Chromatin modi fications could preferentially increase the affinity of a particular cofactor to a WRE, and anchoring such a cofactor triggers the assembly of a complex to catalyse the next remodelling step, and so forth — this is a highly dynamic and self‑sustaining process that is nucleated and stabilized by the C‑terminal region of β‑catenin (FIG. 4).

There could be a twist to this model, as genetic and biochemical analysis is always an ‘end point’ detection of a dynamic process. Perhaps it is not an individual, con‑stantly TCF‑bound β‑catenin molecule that sits through all the outlined steps. There could be pools of β‑catenin proteins bound to the various complexes prior to TCF recruitment. Such preloaded β‑catenin‑containing com‑plexes could then dynamically bind to TCF. This puta‑tive β‑catenin swap could be facilitated by Pygo bound to chromatin at TCF‑occupied sites in the genome. BCL‑9 would then act as an anchor to drag preloaded β‑catenin to these sites. However, there is no experimental evidence that discriminates between a chromatin‑based ping‑pong or a preloaded β‑catenin swap mechanism.

Additionally, chromatin regions that are brought into proximity by TCF‑induced DNA bending could be clamped in place by chromatin‑interacting domains recruited by β‑catenin. Thus, a collection of strategically positioned WREs could form an amenable environment for tissue‑specific enhanceosome formation (FIG. 4). This could also include Wnt‑independent transcription factors that might contribute to chromatin remodelling and RNA Pol II preparation — potentially rendering individual β‑catenin‑led chromatin‑remodeller‑recruitment steps redundant (FIG. 4). Such a function has been suggested for the bone morphogenetic protein‑mediator SMAD4 (ReF. 82), NF1, HNF4 or PPARγ34. An intriguing possibility arises from this — β‑catenin might cooperate in its chromatin‑remodelling task with tissue‑specific trans‑cription factors. It could well be that only a particular enhancer‑bound protein assembly of multiple transcrip‑tion factors is sufficient to fully induce RNA Pol II trans‑cription from particular target genes. β‑Catenin–TCF and their cofactors might therefore merely prepare the WRE‑surrounding chromatin landscape for subsequent transcription factors that are specific for a particular tissue or developmental stage. This could explain why there are few universal Wnt targets2. Furthermore, a novel class of WREs triggers repression and not TCF–ARM protein mediated activation at particular D. melanogaster target gene loci, such as Ugt36bc and Tiggrin83. As many activating chromatin‑remodelling complexes mentioned in this Review also have transcription‑inhibiting poten‑tial, it would be intriguing to analyse them for potential repressor function at these loci, and to address how the activation compared with inhibition decision is made.

Finally, several TCF‑unrelated transcription factors have been implicated in tethering β‑catenin to their target genes, such as the androgen receptor or SOX17 (ReFS 84,85). It remains to be seen whether these β‑catenin functions require the same set of cofactors as TCF‑bound Wnt target genes.

An embedded ‘off switch’ for the activation process? The chromatin binding of a Pygo PHD–BCL‑9 HD2 complex, which simultaneously binds β‑catenin through BCL‑9 HD1 (ReF. 49) (FIG. 3c), provides an intriguing hypothetical mechanism to terminate β‑catenin action, if the inter‑action affinities allow (FIG. 3d). At an active target gene, Pygo binds to methylated H3K4 by its PHD finger, which, by using BCL‑9 as an adaptor, retains β‑catenin near the

Figure 4 | cooperative mechanism of TcF‑occupied Wnt response elements. Schematic representation of a possible cooperative mechanism of T cell factor (TCF)‑occupied Wnt response elements (WREs) following β‑catenin‑triggered chromatin‑remodelling complex recruitment. The strategic positioning of TCF‑binding sites around Wnt target genes might allow for a concerted or cooperative control of DNA architecture and create an amenable three‑dimensional conformation for enhanceosome formation. Interactions of β‑catenin‑associated proteins, such as the newly recognized BCL‑9 (Legless (LGS) in Drosophila melanogaster)–pygopus (Pygo) or chromatin‑remodelling complex subunits, with modified nucleosomes could further stabilize and sculpt the gene locus. In addition, tissue‑specific Wnt‑independent transcription factors can collaborate with β‑catenin to achieve correct chromatin conformations for efficient RNA polymerase II‑mediated transcription. CBD, β‑catenin‑binding domain; HD, homology domain; HMG, high mobility group; NHD, amino‑terminal homology domain; PHD, plant homology domain.

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WRE independently of TCF. Pygo‑dependent binding of β‑catenin to methylated histones frees TCF to recruit its corepressors, such as groucho, which counteract the activating, β‑catenin‑induced chromatin‑remodelling processes (FIG. 3d). The potency of TCF‑based repres‑sors would become stronger as nuclear β‑catenin levels fall, either by active export through APC or axin86,87 (see below) or through simple stalling of fresh β‑catenin sup‑ply owing to Wnt signal termination. Thus, a constant struggle between β‑catenin and groucho for TCF bind‑ing could underlie the cessation of target gene transcrip‑tion, a fate β‑catenin gets lured into by such a potentially negative role of Pygo. Furthermore, various R11–C inter‑actors feature PHD fingers and other chromatin‑binding domains, which could also serve a similar purpose if they can simultaneously bind to modified nucleosomes and β‑catenin. Moreover, such a tethering mechanism for β‑catenin would free the central ARM repeats to bind to inhibitory proteins, as suggested for the ATP‑dependent chromatin‑remodelling factor CHD8 (ReF. 88) or APC91. Thus, the activation tools might also provide a potential off switch for the process.

Novel findings suggest that Pygo function is only required for ARM‑protein‑dependent signalling if cells also have intact Groucho89. At face value, these intrigu‑ing results could fit with the above model, whereby hypo‑thetical direct or indirect Pygo‑dependent chromatin tethering of ARM protein could liberate TCF for groucho binding (FIG. 3d). In such a scenario, when both Pygo and groucho are missing, ARM protein turnover at target gene loci could become more dependent on parameters such as the nuclear concentration of ARM protein, its binding affinity for TCF or the chromatin affinity of its cofactors at the WRE, all of which influence the duration of target gene activity (FIG. 4).

Such a mechanism could also explain earlier findings in which a version of ARM protein with a strong nuclear localization signal also overcame Pygo loss62. It will be exciting to follow future developments and their extra‑polation into vertebrate systems in this lively niche of nuclear Wnt signalling.

Experimental hints for a cycling mechanism. The cycling of β‑catenin and TCF interactors on WREs was observed when ChIP assays were applied to address the dynamics of proteins that localize at WREs following Wnt pathway activation29,90. Initial results showed that β‑catenin and cofactors, such as BCL‑9, cycle the MYC locus on and off every 60–120 min29. In a follow‑up study, β‑catenin joined LEF1, together with the TIP60 HAT and the elongation factor SSRP1 (part of the FACT complex), at the MYC and CCND1 loci 15–30 min after stimu‑lation, and oscillated on and off in intervals of 75 min90. These results, which will hopefully be further refined in future experiments, reveal a potential cyclic recruitment not only of activating cofactors, but also of β‑catenin. Curiously, following APC recruitment to the locus29, β‑catenin and its activating interactors disappeared from the analysed WREs and were replaced by the Groucho homologue TLE1 and other TCF‑associated corepres‑sors29,90. These observations link the nuclear localization

of APC with both the export of β‑catenin from the nucleus6,86 and the termination of target gene activity91.

A regulatory mechanism might involve the kinase CK2. CK2 associates with WREs that have the same dynamics as β‑catenin, and CK2 phosphorylation of LEF1 enhances its affinity for β‑catenin while decreasing TLE1 binding90. Chemical inhibition of CK2 completely blocks β‑catenin and TLE1 recruitment to WREs. Curiously, in yeast, CK2 interacts with and regulates the PAF1 complex92. Taken together, these findings indicate that CK2 phosphoryl‑ation events contribute to selective complex assembly of activators and repressors at TCF‑bound WREs.

Future directionsThe β‑catenin interactors known today represent a fairly complete set of the tools necessary for target gene activa‑tion. A number of nuclear β‑catenin‑interacting proteins were originally validated in genetic experiments, con‑firming their functional relevance in vivo19,47,74,93. We have many phenotypic and reporter readouts for Wnt activity, but we lack detailed molecular insight, such as an in‑depth knowledge of Wnt target gene architecture34. All the established Wnt readouts and known contributing factors provide an exciting platform for a closer analysis of the molecular processes that β‑catenin steers in the nucleus.

What would further enhance our understanding of nuclear Wnt processes? First, there is a surprisingly short but fortunately growing list of confirmed direct β‑catenin–TCF target genes in various tissues. These can be used to study the changes in chromatin that are triggered by pathway activation20. Second, a gap exists in our comprehension of how β‑catenin cooperates with Wnt‑independent transcription factors to control tissue‑speci fic targets, a topic that is particularly interest‑ing in the light of the DNA‑bending properties of TCF. Third, we still have little mechanistic insight into how the activity of β‑catenin can be curbed once engaged in gene activ ation. Finally, the application of sophisticated methods to probe the chromatin structure Wnt target genes, such as ChIP, is gravely needed.

Moreover, today, decades after the first association of activated Wnt signalling with cancer, we still lack effi‑cient drugs that can inhibit this pathway. Small mole‑cule inhibitors of signalling molecules are an important topic in biomedical research, yet the number of clini‑cally successful applications remains low. Nuclear Wnt signalling requires many enzymes, of which most have additional roles in other processes and are thus not Wnt specific. However, the nuclear β‑catenin assembly in its full protein interaction complexity is a tantalizing target for chemical compounds. Yet, targeting protein–protein interactions with inhibitors is a difficult task and requires well‑defined approaches94.

The exciting pioneering efforts to study β‑catenin‑mediated chromatin dynamics, as well as screens for novel components involved in these processes, suggest that the Wnt field is moving in these directions20,29,61,68,90,95. Interconnecting the known data, and of course relent‑lessly gathering novel insights, will hopefully reveal avenues to study, and therapeutically interfere with, the normal and renegade lives of β‑catenin.

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AcknowledgementsWe apologize to those colleagues whose work we do not directly cite owing to space limitations. We would also like to thank past and present members of the Basler laboratory for creating an environment that nurtured the ideas presented in this Review. This work was supported by the National Center of Competence in Research “Frontiers in Genetics,” the Swiss National Science Foundation, and the Kanton of Zürich.

DATABASESEntrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=geneAXIN2 | MYC | naked cuticle | Notum | Pygo1 | Pygo2Interpro: http://www.ebi.ac.uk/interproHMG | SETUniProtKB: http://www.uniprot.orgβ‑catenin | APC | ARM | axin | BCL‑9 | BRE1 | BRG1 | CBP | CK1 | CTBP | DVL | groucho | GSK3β | HYX | KTO | LRP5 | LRP6 | MED12 | p300 | PP2A | PYGO | RAD6 | SKD | TIP60 | TRRAP | WG

FURTHER INFORMATIONKonrad Basler’s homepage: http://www.molbio.uzh.ch/basler/research/index.htmlWnt homepage: http://www.stanford.edu/~rnusse/wntwindow.html

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