Head-to-Tail Polymerization in the Assembly of Biomolecular Condensates.

Clustering of macromolecules is a fundamental cellular device underlying diverse biological processes that require high-avidity binding to effectors and substrates. Often, this involves a transition between diffuse and locally concentrated molecules akin to biophysical phase separation observable in vitro. One simple mechanistic paradigm underlying physiologically relevant phase transitions in cells is the reversible head-to-tail polymerization of hub proteins into filaments that are cross-linked by dimerization into dynamic three-dimensional molecular condensates. While many diverse folds and motifs can mediate dimerization, only two structurally distinct domains have been discovered so far to undergo head-to-tail polymerization, though these are widespread among all living kingdoms.

DIX Domain Polymerization Drives Assembly of Plant Cell Polarity Complexes.

Cell polarity is fundamental for tissue morphogenesis in multicellular organisms. Plants and animals evolved multicellularity independently, and it is unknown whether their polarity systems are derived from a single-celled ancestor. Planar polarity in animals is conferred by Wnt signaling, an ancient signaling pathway transduced by Dishevelled, which assembles signalosomes by dynamic head-to-tail DIX domain polymerization. In contrast, polarity-determining pathways in plants are elusive. We recently discovered Arabidopsis SOSEKI proteins, which exhibit polar localization throughout development. Here, we identify SOSEKI as ancient polar proteins across land plants. Concentration-dependent polymerization via a bona fide DIX domain allows these to recruit ANGUSTIFOLIA to polar sites, similar to the polymerization-dependent recruitment of signaling effectors by Dishevelled. Cross-kingdom domain swaps reveal functional equivalence of animal and plant DIX domains. We trace DIX domains to unicellular eukaryotes and thus show that DIX-dependent polymerization is an ancient mechanism conserved between kingdoms and central to polarity proteins.

Distinct accessory roles of Arabidopsis VEL proteins in Polycomb silencing.

Polycomb repressive complex 2 (PRC2) mediates epigenetic silencing of target genes in animals and plants. In Arabidopsis, PRC2 is required for the cold-induced epigenetic silencing of the FLC floral repressor locus to align flowering with spring. During this process, PRC2 relies on VEL accessory factors, including the constitutively expressed VRN5 and the cold-induced VIN3. The VEL proteins are physically associated with PRC2, but their individual functions remain unclear. Here, we show an intimate association between recombinant VRN5 and multiple components within a reconstituted PRC2, dependent on a compact conformation of VRN5 central domains. Key residues mediating this compact conformation are conserved among VRN5 orthologs across the plant kingdom. In contrast, VIN3 interacts with VAL1, a transcriptional repressor that binds directly to FLC These associations differentially affect their role in H3K27me deposition: Both proteins are required for H3K27me3, but only VRN5 is necessary for H3K27me2. Although originally defined as vernalization regulators, VIN3 and VRN5 coassociate with many targets in the Arabidopsis genome that are modified with H3K27me3. Our work therefore reveals the distinct accessory roles for VEL proteins in conferring cold-induced silencing on FLC, with broad relevance for PRC2 targets generally.

Wnt-Dependent Inactivation of the Groucho/TLE Co-repressor by the HECT E3 Ubiquitin Ligase Hyd/UBR5.

Extracellular signals are transduced to the cell nucleus by effectors that bind to enhancer complexes to operate transcriptional switches. For example, the Wnt enhanceosome is a multiprotein complex associated with Wnt-responsive enhancers through T cell factors (TCF) and kept silent by Groucho/TLE co-repressors. Wnt-activated ß-catenin binds to TCF to overcome this repression, but how it achieves this is unknown. Here, we discover that this process depends on the HECT E3 ubiquitin ligase Hyd/UBR5, which is required for Wnt signal responses in Drosophila and human cell lines downstream of activated Armadillo/ß-catenin. We identify Groucho/TLE as a functionally relevant substrate, whose ubiquitylation by UBR5 is induced by Wnt signaling and conferred by ß-catenin. Inactivation of TLE by UBR5-dependent ubiquitylation also involves VCP/p97, an AAA ATPase regulating the folding of various cellular substrates including ubiquitylated chromatin proteins. Thus, Groucho/TLE ubiquitylation by Hyd/UBR5 is a key prerequisite that enables Armadillo/ß-catenin to activate transcription.

Selective function of the PDZ domain of Dishevelled in noncanonical Wnt signalling.

Dishevelled is a cytoplasmic hub that transduces Wnt signals to cytoplasmic effectors, which can be broadly characterised as canonical (ß-catenin dependent) and noncanonical, to specify cell fates and behaviours during development. To transduce canonical Wnt signals, Dishevelled binds to the intracellular face of Frizzled through its DEP domain and polymerises through its DIX domain to assemble dynamic signalosomes. Dishevelled also contains a PDZ domain, whose function remains controversial. Here, we use genome editing to delete the PDZ domain-encoding region from Drosophila dishevelled. Canonical Wingless signalling is entirely normal in these deletion mutants; however, they show defects in multiple contexts controlled by noncanonical Wnt signalling, such as planar polarity. We use nuclear magnetic resonance spectroscopy to identify bona fide PDZ-binding motifs at the C termini of different polarity proteins. Although deletions of these motifs proved aphenotypic in adults, we detected changes in the proximodistal distribution of the polarity protein Flamingo (also known as Starry night) in pupal wings that suggest a modulatory role of these motifs in polarity signalling. We also provide new genetic evidence that planar polarity relies on the DEP-dependent recruitment of Dishevelled to the plasma membrane by Frizzled.

Wnt Signalosome Assembly by DEP Domain Swapping of Dishevelled.

Extracellular signals are often transduced by dynamic signaling complexes ("signalosomes") assembled by oligomerizing hub proteins following their recruitment to signal-activated transmembrane receptors. A paradigm is the Wnt signalosome, which is assembled by Dishevelled via reversible head-to-tail polymerization by its DIX domain. Its activity causes stabilization of ß-catenin, a Wnt effector with pivotal roles in animal development and cancer. How Wnt triggers signalosome assembly is unknown. Here, we use structural analysis, as well as biophysical and cell-based assays, to show that the DEP domain of Dishevelled undergoes a conformational switch, from monomeric to swapped dimer, to trigger DIX-dependent polymerization and signaling to ß-catenin. This occurs in two steps: binding of monomeric DEP to Frizzled followed by DEP domain swapping triggered by its high local concentration upon Wnt-induced recruitment into clathrin-coated pits. DEP domain swapping confers directional bias on signaling, and the dimerization provides cross-linking between Dishevelled polymers, illustrating a key principle underlying signalosome formation.

Regulation of Dishevelled DEP domain swapping by conserved phosphorylation sites.

Wnt signals bind to Frizzled receptors to trigger canonical and noncanonical signaling responses that control cell fates during animal development and tissue homeostasis. All Wnt signals are relayed by the hub protein Dishevelled. During canonical (ß-catenin-dependent) signaling, Dishevelled assembles signalosomes via dynamic head-to-tail polymerization of its Dishevelled and Axin (DIX) domain, which are cross-linked by its Dishevelled, Egl-10, and Pleckstrin (DEP) domain through a conformational switch from monomer to domain-swapped dimer. The domain-swapped conformation of DEP masks the site through which Dishevelled binds to Frizzled, implying that DEP domain swapping results in the detachment of Dishevelled from Frizzled. This would be incompatible with noncanonical Wnt signaling, which relies on long-term association between Dishevelled and Frizzled. It is therefore likely that DEP domain swapping is differentially regulated during canonical and noncanonical Wnt signaling. Here, we use NMR spectroscopy and cell-based assays to uncover intermolecular contacts in the DEP dimer that are essential for its stability and for Dishevelled function in relaying canonical Wnt signals. These contacts are mediated by an intrinsically structured sequence spanning a conserved phosphorylation site upstream of the DEP domain that serves to clamp down the swapped N-terminal α-helix onto the structural core of a reciprocal DEP molecule in the domain-swapped configuration. Mutations of this phosphorylation site and its cognate surface on the reciprocal DEP core attenuate DEP-dependent dimerization of Dishevelled and its canonical signaling activity in cells without impeding its binding to Frizzled. We propose that phosphorylation of this crucial residue could be employed to switch off canonical Wnt signaling.

Plant vernalization proteins contain unusual PHD superdomains without histone H3 binding activity.

PHD fingers are modular domains in chromatin-associated proteins that decode the methylation status of histone H3 tails. A PHD finger signature is found in plant vernalization (VEL) proteins, which function as accessory factors of the Polycomb system to control flowering in Arabidopsis through an epigenetic silencing mechanism. It has been proposed that VEL PHD fingers bind to methylated histone H3 tails to facilitate association of the Polycomb silencing machinery with target genes. Here, we use structural analysis by X-ray crystallography to show that the VEL PHD finger forms the central module of a larger compact tripartite superdomain that also contains a zinc finger and a four-helix bundle. This PHD superdomain fold is only found in one other family, the OBERON proteins, which have multiple functions in Arabidopsis meristems to control plant growth. The putative histone-binding surface of OBERON proteins exhibits the characteristic three-pronged pocket of histone-binding PHD fingers and binds to methylated histone H3 tails. However, that of VEL PHD fingers lacks this architecture and exhibits unusually high positive surface charge. This VEL PHD superdomain neither binds to unmodified nor variously modified histone H3 tails, as demonstrated by isothermal calorimetry and NMR spectroscopy. Instead, the VEL PHD superdomain interacts with negatively charged polymers. Our evidence argues for evolution of a divergent function for the PHD superdomain in vernalization that does not involve histone tail decoding.

The deubiquitinase TRABID stabilizes the K29/K48-specific E3 ubiquitin ligase HECTD1.

Ubiquitin is a versatile posttranslational modification, which is covalently attached to protein targets either as a single moiety or as a ubiquitin chain. In contrast to K48 and K63-linked chains, which have been extensively studied, the regulation and function of most atypical ubiquitin chains are only starting to emerge. The deubiquitinase TRABID/ZRANB1 is tuned for the recognition and cleavage of K29 and K33-linked chains. Yet, substrates of TRABID and the cellular functions of these atypical ubiquitin signals remain unclear. We determined the interactome of two TRABID constructs rendered catalytic dead either through a point mutation in the catalytic cysteine residue or through removal of the OTU catalytic domain. We identified 50 proteins trapped by both constructs and which therefore represent candidate substrates of TRABID. The E3 ubiquitin ligase HECTD1 was then validated as a substrate of TRABID and used UbiCREST and Ub-AQUA proteomics to show that HECTD1 preferentially assembles K29- and K48-linked ubiquitin chains. Further in vitro autoubiquitination assays using ubiquitin mutants established that while HECTD1 can assemble short homotypic K29 and K48-linked chains, it requires branching at K29/K48 in order to achieve its full ubiquitin ligase activity. We next used transient knockdown and genetic knockout of TRABID in mammalian cells in order to determine the functional relationship between TRABID and HECTD1. This revealed that upon TRABID depletion, HECTD1 is readily degraded. Thus, this study identifies HECTD1 as a mammalian E3 ligase that assembles branched K29/K48 chains and also establishes TRABID-HECTD1 as a DUB/E3 pair regulating K29 linkages.

Rotational symmetry of the structured Chip/LDB-SSDP core module of the Wnt enhanceosome.

The Chip/LIM-domain binding protein (LDB)-single-stranded DNA-binding protein (SSDP) (ChiLS) complex controls numerous cell-fate decisions in animal cells, by mediating transcription of developmental control genes via remote enhancers. ChiLS is recruited to these enhancers by lineage-specific LIM-domain proteins that bind to its Chip/LDB subunit. ChiLS recently emerged as the core module of the Wnt enhanceosome, a multiprotein complex that primes developmental control genes for timely Wnt responses. ChiLS binds to NPFxD motifs within Pygopus (Pygo) and the Osa/ARID1A subunit of the BAF chromatin remodeling complex, which could synergize with LIM proteins in tethering ChiLS to enhancers. Chip/LDB and SSDP both contain N-terminal dimerization domains that constitute the bulk of their structured cores. Here, we report the crystal structures of these dimerization domains, in part aided by DARPin chaperones. We conducted systematic surface scanning by structure-designed mutations, followed by in vitro and in vivo binding assays, to determine conserved surface residues required for binding between Chip/LDB, SSDP, and Pygo-NPFxD. Based on this, and on the 4:2 (SSDP-Chip/LDB) stoichiometry of ChiLS, we derive a highly constrained structural model for this complex, which adopts a rotationally symmetrical SSDP2-LDB2-SSDP2 architecture. Integrity of ChiLS is essential for Pygo binding, and our mutational analysis places the NPFxD pockets on either side of the Chip/LDB dimer, each flanked by an SSDP dimer. The symmetry and multivalency of ChiLS underpin its function as an enhancer module integrating Wnt signals with lineage-specific factors to operate context-dependent transcriptional switches that are pivotal for normal development and cancer.

Signalosome assembly by domains undergoing dynamic head-to-tail polymerization.

A key mechanism for guarding against inappropriate activation of signaling molecules is their weak affinity for effectors, which prevents them from undergoing accidental signal-transducing interactions due to fluctuations in their cellular concentration. The molecular devices that overcome these weak affinities are the signalosomes: dynamic clusters of transducing molecules assembled typically at signal-activated receptors. Signalosomes contain high local concentrations of protein-binding sites, and thus have a high avidity for their low-affinity ligands that generate signal responses. This review focuses on three domains - DIX (dishevelled and axin), PB1 (Phox and Bem1), and SAM (sterile alpha motif) - that undergo dynamic head-to-tail polymerization to assemble signalosomes and similar particles that require transient high local concentrations of protein-binding sites.

Essential role of the Dishevelled DEP domain in a Wnt-dependent human-cell-based complementation assay.

Dishevelled (DVL) assembles Wnt signalosomes through dynamic head-to-tail polymerisation by means of its DIX domain. It thus transduces Wnt signals to cytoplasmic effectors including ß-catenin, to control cell fates during normal development, tissue homeostasis and also in cancer. To date, most functional studies of Dishevelled relied on its Wnt-independent signalling activity resulting from overexpression, which is sufficient to trigger polymerisation, bypassing the requirement for Wnt signals. Here, we generate a human cell line devoid of endogenous Dishevelled (DVL1- DVL3), which lacks Wnt signal transduction to ß-catenin. However, Wnt responses can be restored by DVL2 stably re-expressed at near-endogenous levels. Using this assay to test mutant DVL2, we show that its DEP domain is essential, whereas its PDZ domain is dispensable, for signalling to ß-catenin. Our results imply two mutually exclusive functions of the DEP domain in Wnt signal transduction - binding to Frizzled to recruit Dishevelled to the receptor complex, and dimerising to cross-link DIX domain polymers for signalosome assembly. Our assay avoids the caveats associated with overexpressing Dishevelled, and provides a powerful tool for rigorous functional tests of this pivotal human signalling protein.

Pregnancy, Primary Aldosteronism, and Adrenal CTNNB1 Mutations.

Recent discoveries of somatic mutations permit the recognition of subtypes of aldosterone-producing adenomas with distinct clinical presentations and pathological features. Here we describe three women with hyperaldosteronism, two who presented in pregnancy and one who presented after menopause. Their aldosterone-producing adenomas harbored activating mutations of CTNNB1, encoding ß-catenin in the Wnt cell-differentiation pathway, and expressed LHCGR and GNRHR, encoding gonadal receptors, at levels that were more than 100 times as high as the levels in other aldosterone-producing adenomas. The mutations stimulate Wnt activation and cause adrenocortical cells to de-differentiate toward their common adrenal-gonadal precursor cell type. (Funded by grants from the National Institute for Health Research Cambridge Biomedical Research Centre and others.).

Decoding of methylated histone H3 tail by the Pygo-BCL9 Wnt signaling complex.

Pygo and BCL9/Legless transduce the Wnt signal by promoting the transcriptional activity of beta-catenin/Armadillo in normal and malignant cells. We show that human and Drosophila Pygo PHD fingers associate with their cognate HD1 domains from BCL9/Legless to bind specifically to the histone H3 tail methylated at lysine 4 (H3K4me). The crystal structures of ternary complexes between PHD, HD1, and two different H3K4me peptides reveal a unique mode of histone tail recognition: efficient histone binding requires HD1 association, and the PHD-HD1 complex binds preferentially to H3K4me2 while displaying insensitivity to methylation of H3R2. Therefore, this is a prime example of histone tail binding by a PHD finger (of Pygo) being modulated by a cofactor (BCL9/Legless). Rescue experiments in Drosophila indicate that Wnt signaling outputs depend on histone decoding. The specificity of this process provided by the Pygo-BCL9/Legless complex suggests that this complex facilitates an early step in the transition from gene silence to Wnt-induced transcription.

Boosting Wnt activity during colorectal cancer progression through selective hypermethylation of Wnt signaling antagonists.

BACKGROUND: There is emerging evidence that Wnt pathway activity may increase during the progression from colorectal adenoma to carcinoma and that this increase is potentially an important step towards the invasive stage. Here, we investigated whether epigenetic silencing of Wnt antagonists is the biological driver for this increased Wnt activity in human tissues and how these methylation changes correlate with MSI (Microsatelite Instability) and CIMP (CpG Island Methylator Phenotype) statuses as well as known mutations in genes driving colorectal neoplasia. METHODS: We conducted a systematic analysis by pyrosequencing, to determine the promoter methylation of CpG islands associated with 17 Wnt signaling component genes. Methylation levels were correlated with MSI and CIMP statuses and known mutations within the APC, BRAF and KRAS genes in 264 matched samples representing the progression from normal to pre-invasive adenoma to colorectal carcinoma. RESULTS: We discovered widespread hypermethylation of the Wnt antagonists SFRP1, SFRP2, SFRP5, DKK2, WIF1 and SOX17 in the transition from normal to adenoma with only the Wnt antagonists SFRP1, SFRP2, DKK2 and WIF1 showing further significant increase in methylation from adenoma to carcinoma. We show this to be accompanied by loss of expression of these Wnt antagonists, and by an increase in nuclear Wnt pathway activity. Mixed effects models revealed that mutations in APC, BRAF and KRAS occur at the transition from normal to adenoma stages whilst the hypermethylation of the Wnt antagonists continued to accumulate during the transitions from adenoma to carcinoma stages. CONCLUSION: Our study provides strong evidence for a correlation between progressive hypermethylation and silencing of several Wnt antagonists with stepping-up in Wnt pathway activity beyond the APC loss associated tumour-initiating Wnt signalling levels.

Dishevelled interacts with the DIX domain polymerization interface of Axin to interfere with its function in down-regulating ß-catenin.

Wnt/ß-catenin signaling controls numerous steps in normal animal development and can also cause cancer if inappropriately activated. In the absence of Wnt, ß-catenin is targeted continuously for proteasomal degradation by the Axin destruction complex, whose activity is blocked upon Wnt stimulation by Dishevelled, which recruits Axin to the plasma membrane and assembles it into a signalosome. This key event during Wnt signal transduction depends on dynamic head-to-tail polymerization by the DIX domain of Dishevelled. Here, we use rescue assays in Drosophila tissues and functional assays in human cells to show that polymerization-blocking mutations in the DIX domain of Axin disable its effector function in down-regulating Armadillo/ß-catenin and its response to Dishevelled during Wnt signaling. Intriguingly, NMR spectroscopy revealed that the purified DIX domains of the two proteins interact with each other directly through their polymerization interfaces, whereby the same residues mediate both homo- and heterotypic interactions. This result implies that Dishevelled has the potential to act as a "natural" dominant-negative, binding to the polymerization interface of Axin's DIX domain to interfere with its self-assembly, thereby blocking its effector function.

Inhibition of GSK3 by Wnt signalling--two contrasting models.

The key read-out of Wnt signalling is a change in the transcriptional profile of the cell, which is driven by ß-catenin. ß-catenin levels are normally kept low by a phosphorylation event that is mediated by glycogen synthase kinase 3 (GSK3, α- and ß-isoforms), which targets ß-catenin for ubiquitylation and proteasomal degradation. Wnt blocks this phosphorylation event, thereby allowing ß-catenin to accumulate and to co-activate transcription in the nucleus. Exactly how Wnt inhibits GSK3 activity towards ß-catenin is unclear and has been the focus of intensive research. Recent studies on the role of conserved PPPSPxS motifs in the cytoplasmic tail of low-density lipoprotein receptor-related protein (LRP, isoforms 5 and 6) culminated in a biochemical model: Wnt induces the phosphorylation of LRP6 PPPSPxS motifs, which consequently access the catalytic pocket of GSK3 as pseudo-substrates, thus directly blocking its activity against ß-catenin. A distinct cell-biological model was proposed more recently: Wnt proteins induce the uptake of GSK3 into multivesicular bodies (MVBs), an event that sequesters the enzyme away from newly synthesised ß-catenin substrate in the cytoplasm, thus blocking its phosphorylation. This new model is based on intriguing observations but also challenges a body of existing evidence, so will require further experimental consolidation. We shall consider whether the two models apply to different modes of Wnt signaling: acute versus chronic.

Structural basis of the interaction between BCL9-Pygo and LDB-SSBP complexes in assembling the Wnt enhanceosome.

The Wnt enhanceosome is responsible for transactivation of Wnt-responsive genes and a promising therapeutic target for treatment of numerous cancers with Adenomatous Polyposis Coli (APC) or ß-catenin mutations. How the Wnt enhanceosome is assembled remains poorly understood. Here we show that B-cell lymphoma 9 protein (BCL9), Pygopus (Pygo), LIM domain-binding protein 1 (LDB1) and single-stranded DNA-binding protein (SSBP) form a stable core complex within the Wnt enhanceosome. Their mutual interactions rely on a highly conserved N-terminal asparagine proline phenylalanine (NPF) motif of Pygo, through which the BCL9-Pygo complex binds to the LDB-SSBP core complex. Our crystal structure of a ternary complex comprising the N-terminus of human Pygo2, LDB1 and SSBP2 reveals a single LDB1-SSBP2 complex binding simultaneously to two Pygo2 molecules via their NPF motifs. These interactions critically depend on the NPF motifs which bind to a deep groove formed between LDB1 and SSBP2, potentially constituting a binding site for drugs blocking Wnt/ß-catenin signaling. Analysis of human cell lines lacking LDB or Pygo supports the functional relevance of the Pygo-LDB1-SSBP2 interaction for Wnt/ß-catenin-dependent transcription.

Stability elements in the LRP6 cytoplasmic tail confer efficient signalling upon DIX-dependent polymerization.

Wnt/beta-catenin signalling controls cell fates in development, tissue homeostasis and cancer. Wnt binding to Frizzled receptors triggers recruitment of Dishevelled to the plasma membrane and formation of a signalosome containing the LRP5/6 co-receptor, whose cytoplasmic tail (ctail) thus becomes phosphorylated at multiple PPP(S/T)Px(S/T) motifs. These then directly inhibit GSK3beta, which results in beta-catenin accumulation and signalling. Here, we revisit previous epistasis experiments, and show that Dishevelled signals through LRP5/6 in human cells and Drosophila embryos. To recapitulate this signalling event, and to define its functional elements, we fused the Dishevelled DIX domain to the LRP6 ctail, which forms cytoplasmic signalosomes with potent signalling activity mediated by its PPP(S/T)Px(S/T) motifs. Their phosphorylation and activity depends critically on DIX-mediated polymerization, and on multiple stability elements in the LRP6 ctail, including the T1479 epitope upstream of the membrane-proximal PPP(S/T)Px(S/T) motif. Thus, stable polymerization emerges as a key principle underlying the function of Dishevelled-dependent signalosomes.

The DIX domain of Dishevelled confers Wnt signaling by dynamic polymerization.

The Wnt signaling pathway controls numerous cell fates in animal development and is also a major cancer pathway. Dishevelled (Dvl) transduces the Wnt signal by interacting with the cytoplasmic Axin complex. Dvl and Axin each contain a DIX domain whose molecular properties and structure are unknown. Here, we demonstrate that the DIX domain of Dvl2 mediates dynamic polymerization, which is essential for the signaling activity of Dvl2. The purified domain polymerizes gradually, reversibly and in a concentration dependent manner, ultimately forming fibrils. The Axin DIX domain has a novel structural fold largely composed of beta-strands that engage in head-to-tail self-interaction to form filaments in the crystal. The DIX domain thus seems to mediate the formation of a dynamic interaction platform with a high local concentration of binding sites for transient Wnt signaling partners; this represents a previously uncharacterized mechanistic principle, signaling by reversible polymerization.